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Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity
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Analytical Chemistry

Cite this: Anal. Chem. 2023, 95, 38, 14308–14316
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https://doi.org/10.1021/acs.analchem.3c02552
Published September 11, 2023

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Abstract

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Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement.

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Subjects

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The first approved mRNA vaccines were deployed against SARS-CoV-2 virus. (1−3) Other therapeutic mRNA candidates are under clinical trials as vaccines, prophylactic cancer treatment, or protein replacement therapies. (4−6) The high level of interest in mRNA therapy research is motivated by mRNA technology's promising features, such as low toxicity, delivery via noninfectious agents, sequence-programmable in vivo target protein production, and inexpensive cell-free mRNA manufacturing. (7)
The first results of antitumor mRNA clinical trial were reported in 2008. (8) Since the emergency use authorization of COVID-19 mRNA vaccines on December 11, 2020, the number of mRNA clinical trials has increased significantly. Curreri and colleagues estimated that in 2021, about 222 trials employed RNA therapy; 53% were mRNA molecules. (9) Most mRNA clinical trials are in cancer treatment followed by infectious and genetic diseases. mRNA vaccines in development against infectious diseases are targeting Zika virus, rabies, respiratory syncytial virus (RSV), cytomegalovirus (CMV), and seasonal influenza. (7,9)
Rapid advances in mRNA technology require the development of novel analytical methods. USP draft guidelines (10) outline the attributes of therapeutic mRNA molecules that should be monitored. However, the application of existing separation methods is difficult due to the large size of the mRNA molecules. The length of therapeutic mRNA vaccines depends primarily on the size of the translated region encoding for the gene(s) of interest (GOI). COVID-19 mRNA vaccines coding for the SARS-CoV-2 spike protein are approximately 4400 nucleotides (nt) in length, while the vaccines encoding for two or more viral antigens or self-amplifying mRNA (saRNA) containing replicase genes (11) could be up to 12,000 nt long. This size of RNA is difficult to analyze by liquid chromatography (LC), gel electrophoresis (GE), or mass spectrometry (MS) methods. (12−14) Therefore, the analytical methods often utilize specific RNA nucleases to cut the mRNA molecules into more manageable oligonucleotides. (15−18)
The important features of therapeutic mRNA are the 5′-end-cap and 3′-end poly(A) tail. (19) The 5′-capping structure contains an N7-methylated guanosine linked to the first mRNA nucleotide via a reverse 5′ to 5′ triphosphate linkage. (20) Capping also includes 2′ O-methylation of the first mRNA nucleotide. This so-called Cap-1 structure facilitates the mRNA translation process by latching onto ribosomes in the cell. (20) 5′-capping is either performed cotranscriptionally via enzymes added to the reaction (4,20) or by incorporating the chemically synthesized Cap-1 structure post-transcriptionally. (21) The completeness of capping reaction can be measured by LC-MS of the 5′-end oligonucleotide cleaved from mRNA with RNase H or by other methods. (18,22,23)
Next-generation sequencing (NGS) techniques are applicable to the analysis of the mRNA primary vaccine sequence. (24) Alternatively, LC-MS methods were proposed for mRNA oligo mapping and confirmatory sequencing. (15−17) Messenger RNA oligo mapping utilizes specific ribonucleases that cleave mRNA into shorter oligonucleotides. The RNA sequence coverage obtained from the unique mass of oligonucleotides is relatively low (30–81%) since many noninformative (short or isobaric) oligonucleotides are produced by the digest. (15,16) More complete sequence coverage can be obtained by performing several mRNA digests with different selectivity enzymes or by combining the mRNA mapping with MS/MS oligonucleotide sequencing. Both approaches require powerful data analysis software to make this a viable technique for the routine analytical laboratories. (15,25)
The 3′-end plays a critical role in maintaining mRNA in vivo stability. mRNA vaccines are cotranscriptionally or post-transcriptionally modified with a 100–150 nt poly(A) tail to enhance their half-life in vivo. (19,21) The length and heterogeneity of the poly(A) tail analysis are one of the quality control tasks proposed for inclusion in the USP guidelines. Even when the poly(A) tail is cleaved from the mRNA molecule, the length of the target oligonucleotides presents a challenge for the state-of-the-art separation methods. Single nucleotide resolution of poly(A) species in the 100–150 nt range is achievable by capillary gel electrophoresis, (26,27) but requires a viscous high molecular weight gel matrices. Chromatographic techniques of ion-exchange (IEX) and ion-pair reversed-phase (IP RP) LC are methods of choice for separation of short oligonucleotides, (28−32) including antisense oligonucleotides (ASO) and silencing RNA (siRNA). The separation of n/n – x oligonucleotides, where x is the number of truncated nucleotides, is readily achievable for oligonucleotides up to 25 nucleotides long. A limited number of reports describe LC separation of >50 nt oligonucleotides, (31,33) while >100 nt species are extremely difficult to analyze with n/n – 1 resolution. (28,34) Separations of >100 nt single-stranded (ss) and double-stranded (ds) nucleic acids are described in the literature with IEX and IP RP LC; however, the resolution decreases with the nucleic acid length and does not afford n/n – 1 resolution. (12,13,35,36)
Size exclusion chromatography (SEC) is a method that separates the compounds according to their size. (37) The technique is widely used for the analysis or purification of polymers and proteins. (38) SEC was, to a lesser extent, applied to the separation of oligonucleotides and nucleic acids. (39−43) This may be because the SEC technique has a relatively low separation power compared to IEX or RP LC or electromigration methods used for nucleic acids analysis. (12,26,28,30,31,33,44−46) Nevertheless, SEC has been shown to be a robust and powerful method for RNA purification, (40,42) therapeutic oligonucleotide analysis, (41) and elucidation of the oligonucleotide secondary structure. (43) Because of SEC simplicity and availability of SEC columns with an improved resolution, the SEC technique can become a useful tool for the analysis of therapeutic oligonucleotides and mRNA.
In this work, we focused on the characterization of the poly(A) tail of mRNA. Our goal was to develop chromatographic methods for the analysis of the length and heterogeneity of the 100–150 nt long poly(A) tail after its cleavage from the intact mRNA molecule. The results of the developed SEC and IP RP LC methods with easy-to-implement UV detection were confirmed by the LC MS method.

Experimental Section

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Materials and Chemicals

CleanCap EPO mRNA 1 mg/mL (EPO mRNA) was purchased from TriLink Biotechnologies, San Diego, CA, USA. FLuc-beta mRNA (FlucB mRNA) 1.6 mg/mL was obtained from AmpTech, Hamburg, Germany. Optima LC MS grade acetonitrile, RNase T1 endoribonuclease, and PES 500 mL 0.1 μm filters were purchased from Thermo-Fisher Scientific, Waltham, MA USA. The enzymatic reaction was performed in 0.2 mL individual domed PCR tubes using a T100 thermal cycler (both Bio-Rad laboratories, Hercules, CA, USA) set to 37 °C. Deionized water was produced using a Milli-Q system, Millipore, St. Louis, MO, USA. Glacial acetic acid (AA), octylamine (OA; > 99.5%), sodium phosphate monobasic, sodium phosphate dibasic, 1 M solution of triethylammonium acetate (TEAA), and RNase-free water, 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, > 99.5%) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Oligodeoxyribonucleotides with adenosine (dAn) and thymidine (dTn) sequences and oligoribonucleotides with the adenosine sequence (rAn, custom synthesis) were purchased from Integrated DNA Technologies (Coralville, IA, USA). All synthetic oligonucleotides were used desalted but unpurified. 10× rCutSmart buffer and Quick CIP enzyme were purchased from New England Biolabs (Ipswich, MA, USA). The μElution Oasis HLB 96-well solid phase extraction (SPE; 2 mg sorbent/well) plate and 96-well sample collection plates (polypropylene 350 μL) were obtained from Waters Corporation (Milford, MA, USA). An Allegra X-14 centrifuge equipped with a SX4750A rotor (Beckman Coulter, Brea, CA, USA) was used for SPE sample cleanup.

LC UV and LC MS Instrument Columns and Conditions

SEC and IP RP LC UV experiments were performed using an ACQUITY UPLC H-Class premier system with an ACQUITY photodiode array detector equipped with a Titanium 5 mm detector cell (Waters Corporation, Milford, MA, USA). The SEC analysis of poly(A) tail analysis was performed with the ACQUITY premier protein SEC column, 250 Å, 1.7 μm, 4.6 × 150 mm, obtained from Waters Corporation, Milford, MA, USA. SEC method development was also performed with the 1.7 μm, 4.6 × 150 mm ACQUITY protein SEC column, 125 Å, and 2.5 μm, 4.6 × 150 mm ACQUITY protein SEC column, 450 Å. (Waters Corporation, Milford, MA, USA). The SEC mobile phase consisted of aqueous 0.1 M phosphate buffer, pH 8. The mobile phase was prepared by dissolving 6.72 g of Na2HPO4 and 0.32 g of NaH2PO4 in 500 mL of deionized Milli-Q water. The SEC mobile phase was sterilized by filtering the solution with a PES 500 mL 0.1 μm filter. The SEC separation was performed at a flow rate of 0.2 mL/min at 25 °C; the UV detection wavelength was 260 nm.
IP RP LC experiments were performed with the same ACQUITY UPLC H-Class premier system as described above. The separation column was an ACQUITY premier oligonucleotide BEH C18 column, 300 Å, 1.7 μm, 2.1 × 150 mm (Waters Corporation, Milford, MA, USA). Mobile phase A was 0.1 M octylammonium acetate (OAA) buffer in 40% acetonitrile and 1% HFIP (mix 116.13 g of deionized water with 60.86 g of acetonitrile; add 1.145 mL of glacial acetic acid, 3.305 mL of octylamine, and 2 mL of HFIP). Mobile phase B was 0.1 M OAA buffer in 90% acetonitrile and 1% HFIP (mix 19.36 g of deionized water with 136.92 g of acetonitrile; add 1.145 mL of glacial acetic acid, 3.305 mL of octylamine, and 2 mL of HFIP). The mobile phases A and B contained acetonitrile to improve the solubility of hydrophobic OA. The rationale for including HFIP (pKa 9.3) was to maintain the mobile phase pH in the mild basic range and mitigate pH shifts in the case of minor OA (pKa 10.7) and acetic acid (pKa 4.8) pipetting errors. Without addition of HFIP, the buffer pH can fluctuate in the range of several pH units. Because of the high concentration of ion-pairing buffer, the inclusion of HFIP does not make the mobile phase compatible with MS detection. (34) Mobile phase D was pure acetonitrile. The flow rate was 0.3 mL/min, column temperature was 60 °C, and UV detection wavelength was 260 nm. Mobile phase gradient was from 46% A and 54% B to 28% A, 62% B, and 10% D in 40 min. The column was then washed with pure acetonitrile mobile phase D for 1 min and equilibrated at initial gradient conditions for 15 min.
IP RP LC MS poly(A) analysis experiment was performed with a BioAccord LC MS system equipped with the ACQUITY Premier UPLC H-Class Premier system. The separation column was the ACQUITY Premier Oligonucleotide C18, 130 Å, 1.7 μm, 50 × 2.1 mm column protected by a VanGuard Premier FIT 1.7 μm, 5 × 2.1 mm cartridge (all Waters Corporation, Milford, MA, USA). The flow rate was 0.3 mL/min, column temperature is 60 °C, and UV detection wavelength is 260 nm. Mobile phase A was 8 mM N,N-diisopropyl-ethylamine (DIPEA) and 40 mM HFIP in deionized water, pH 8.8. Mobile phase B consisted of 4 mM DIPEA and 4 mM HFIP in 75% ethanol. DIPEA/HFIP is a suitable ion-pairing system for sensitive LC MS analysis. (47,48) The gradient was from 25 to 40% B in 10 min followed by 1 min wash with 85% B and 4 min equilibration at initial gradient conditions. The MS instrument was operated in negative mode, 400–5000 Da mass range, cone voltage 45 V, and 1 s MS scan.
The LC UV and LC MS data were acquired with Empower v 3.0 and MassLynx 4.2 Software, respectively. MS data deconvolution was performed with MaxEnt1 software (all Waters Corporation, Milford, MA, USA).

mRNA Digestion

EPO mRNA or FlucB mRNA samples were digested with RNase T1 using the following protocol: 10 μL of the mRNA solution was mixed with 28 μL of RNase-free water and 4 μL of 10× CutSmart buffer. 1.5 μL (1.5 units) of RNase T1 enzyme was mixed with the sample, and the cleavage reaction was carried out for 30–60 min at 37 °C. After mRNA digestion, the sample was spiked with 0.2 μL (1 unit) of Quick CIP enzyme to remove residual phosphate from the 5′ or 3′ end of the oligonucleotides. The addition of the Quick CIP enzyme was not necessary, but it improved the solution stability of the poly(A) cleaved product and synthetic oligonucleotide standards by eliminating minor (phosphorylated) peaks from SEC and IP RP LC analyses. After 20 min of Quick CIP dephosphorylation at 37 °C, the sample was transferred into a glass vial and kept in the LC instrument sample manager at 10 °C prior to analysis. Typically, 2 μL was injected for SEC. The sample was purified prior to IP RP LC analysis using a solid phase extraction (SPE) plate as described in the following section. The SPE sample purification extended the lifetime of the RP LC column.

mRNA RNase T1 Digest Sample Preparation

The RNase T1-digested mRNA sample was purified using an μElution Oasis HLB 96-well SPE plate. The sample, wash, and elution fractions were passed through the sorbent bed by centrifugation (30–180 s at 1000 g). The liquid fractions were collected into a 96-well collection plate stacked under the SPE plate. The selected SPE wells were first washed with 50 μL of acetonitrile and then equilibrated with 100 μL of 0.1 M TEAA solution. The digested mRNA sample (43.7 μL) was mixed with 4 μL of 1 M TEAA and two vials of the digest; that is, 95.4 μL was loaded on the SPE plate. The solution was collected in a clean collection plate and reloaded for the second time onto the same SPE well to reduce potential sample breakthrough. (45) The loaded SPE well was washed with 30 μL of 0.1 M TEAA and then eluted with 40 μL of 30% acetonitrile. The eluent was collected in a clean collection plate, transferred into a glass HPLC vial, and placed in the LC sample manager set to 10 °C. Ten μL of the sample was injected on the column for IP RP LC analysis.

Results

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Size Exclusion Method Development for Poly(A) Tail Length Analysis

In size exclusion chromatography, the analytes are separated according to their ability to penetrate the chromatographic sorbent pores. Smaller molecules are more retained than larger molecules, which are partially excluded from the pore volume. The molecular size range of analytes separated in SEC depends on the sorbent pore size. We used three SEC columns with different pore sizes to investigate the resolution of oligonucleotides and SEC calibration linearity. Figure 1 shows the separation of 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 120, and 150 nt oligodeoxyribonucleotide adenosine mixtures (we will refer to this sample as dA5–150). Minor peaks present in the sample are failed synthesis products or failure sequences. Because the oligo(A) species do not have a strong secondary structure, the analysis was performed at 25 °C. The flow rate 0.2 mL/min was selected as a compromise between the optimal flow rate (<0.2 mL/min) (49) and speed of analysis. We selected 0.1 M phosphate buffer, pH 8, as the mobile phase to minimize the potential nonspecific adsorption of nucleic acid to the column hardware. (50−52) Figure 1A shows that the 125 Å SEC column is more suitable for separation of short oligonucleotides; the n/n – 1 resolution was observed for dAn species up to 15 nt long oligonucleotides (see minor peaks in Figure 1A). On the other hand, the longer oligonucleotides (80–150 nt) are not resolved with the 125 Å SEC column; they elute as a single peak near the pore exclusion limit of the sorbent. The 250 Å SEC column appears to be a good choice for separation of the dA5–150 sample; separation of short oligonucleotides is partially lost, but longer oligonucleotides are clearly resolved (Figure 1B). The third experiment with the 450 Å SEC column (Figure 1C) shows that this column is suitable for separation of >150 nt oligonucleotides, while the resolution of <60 nt species is inferior compared to 125 and 250 Å SEC columns. The chromatographic data in Figure 1A–C were used to construct traditional SEC calibration plots presented in Figure 1D. The x-axis plots the oligonucleotide elution volume Vr (Vr = tr × F; tr is retention time and F is flow rate) normalized to empty column (Vc = π × d2/4 × L; d is column internal diameter and L is column length), while the y-axis is the logarithmic value of analyte molecular weight (log MW). The useful SEC separation region is near the center, where the calibration plot is linear. In the regions where the plot bends upward or downward, the analyte size approaches either the exclusion limit of the sorbent pores (Vr/Vc ∼ 0.3; bulky analytes elute in the interstitial volume between the sorbent particles) or the full inclusion limit of the sorbent pores (Vr/Vc ∼ 0.8; small analytes enter all pores unrestricted and elute in the column void volume). In both extreme cases, the SEC resolution is lost for the given class of analytes. This could be seen for calibration plots of the 125 Å SEC column for >80 nt oligonucleotides and for the 450 Å SEC plot for <10 nt oligonucleotides (Figure 1D). The 250 Å SEC columns provided the best separation of the 30–150 nt oligonucleotides. Therefore, we selected this column for poly(A) tail analysis within this corresponding range.

Figure 1

Figure 1. SEC separation of dA5–150 oligonucleotides using columns packed with 125 Å (A), 250 Å (B), and 450 Å (C) pore size sorbent. Panel (D) plots the experimental data as SEC calibration curves. 250 Å column has linear calibration for the 30–150 nt oligonucleotide range (see Figure 2A).

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SEC Calibration for RNA Poly(A) Tail Measurement

We evaluated the linearity of 250 Å SEC columns with the adenosine dA5–150 standard. For simplicity, the calibration curve was directly plotted as a function of retention time versus log N (N is the number of nucleotides). The calibration curve was linear for the dAn range 30–150 nt with a coefficient of determination R2 ∼ 0.999 (Figure 2A). This means that the length of the dAn oligonucleotides can be accurately measured from their SEC retention time. The choice of dAn oligonucleotides for SEC calibration was motivated by their easy availability. Synthetic dAn oligonucleotides are relatively inexpensive and can be purchased from vendors in high quality up to 150 nt in length. However, further experiments reveal that dAn has somewhat different retention in SEC compared to oligodeoxythymidine (dTn) standards, and an even greater retention difference was observed for oligoribonucleotide adenosines rAn (chromatograms in Figure 2B, C, and D, respectively; the dashed lines were added for retention shift visualization). This behavior is likely related to different hydrodynamic sizes of oligonucleotide samples rather than due to the ionic or hydrophobic interactions of oligonucleotides with the SEC sorbent. Addition of 10–20% of methanol to the mobile phase or change of buffer concentration did not alter the relative retention of dAn, dTn, and rAn standards. Because of the retention shift of different types of oligonucleotides, we purchased rAn standards chemically consistent with the poly(A) RNA tail and prepared oligoribonucleotide(A) calibration (Figure 2A) that can be directly used for poly(A) RNA tail length SEC measurement.

Figure 2

Figure 2. Panel (A) compares SEC calibrations obtained for RNA (rA5–120) and DNA (rA5–150) oligonucleotides. SEC chromatograms of dA5–150, dT20–100, and rA10–120 synthetic oligonucleotide standards are shown in panels (B), (C), and (D), respectively. Dotted lines highlight the observed differences in the oligonucleotide SEC retention. Data were acquired with an ACQUITY Premier Protein SEC column, 250 Å, 1.7 μm, 4.6 × 150 mm.

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SEC Poly(A) Tail Length Measurement

FLuc-beta and EPO mRNA samples obtained from vendors were used for poly(A) tail length measurement by the SEC method. The mRNA samples were digested with RNase T1 as described in the Materials and Methods section. RNase H or other nucleases can also be used to liberate the poly(A) tail from mRNA. (15,18) The advantage of RNase T1 is its high specificity compared to other enzymes. (18,22,25) RNase T1 nuclease cleaves single-stranded RNA sequences at G/N motifs, producing numerous short oligonucleotides suitable for mRNA or sgRNA mapping. (15,25) Since the poly(A) tail portion of mRNA does not contain G nucleotides, the 3′ poly(A) tail is cleaved from mRNA as a sizable 100–150 nt oligonucleotide, which significantly exceeds in length the remaining products of RNase T1 cleavage (1–30 nt species). Consequently, the poly(A) tail can be separated using an earlier described SEC method based on the 250 Å sorbent. The example of poly(A) tail SEC analysis of FLuc-beta mRNA is shown in Figure 3. The red chromatogram represents the peak of the intact FLuc-beta mRNA molecule prior to RNase T1 digestion (1970 nt long including the 120 nt poly(A) tail). The blue chromatogram represents the SEC analysis after the FLuc-beta mRNA digestion yielding 2–30 nt oligonucleotides and a 3′ poly(A) tail peak at tr = 5.007 min. For illustration, we overlaid the results with the injection of the rA120 synthetic oligonucleotide (black chromatogram). The 120 nt standard (containing a long tail of shorter synthetic impurities) elutes closely after the poly(A) tail peak. This suggests that the poly(A) tail is longer than the 120 nt. For more accurate estimation of the poly(A) tail length, we used the calibration curve log N = −0.288 × tr + 3.5507 presented in Figure 2. The experimentally measured retention time 5.007 min yields the poly(A) tail length of 128 nt. The analysis of the second EPO mRNA sample showed a poly(A) tail peak at 5.061 min, which gives a poly(A) tail length of 123 nt (for chromatogram see Figure S1).

Figure 3

Figure 3. SEC analysis of intact FLuc-beta mRNA (1970 nt, red chromatogram) and FLuc-beta mRNA digested with RNase T1 (blue chromatogram; the peak eluting near 5 min represents poly(A) tail signal). Black chromatogram is SEC analysis of the rA120 oligonucleotide; the standard is contaminated with truncated synthetic impurities eluting within a peak tail.

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Closer inspection of Figure 3 shows that the poly(A) tail peak is noticeably wider than the peak of the rA120 synthetic standard.
This is likely due to the heterogeneity of the poly(A) tail formed during the polyadenylation of mRNA. In other words, the calculated 128 nt length of poly(A) is only an average length of the multiple poly(A) tail species coeluting as a single peak because the SEC method does not have the resolution power to separate these closely related oligonucleotides. The peak broadening is a manifestation of a partial separation of the poly(A) tail species present in the sample. The investigation of heterogeneity of poly(A) tail length requires high-resolution chromatography or the MS method. The development of such a method is described in the next section.

IP RP LC Method Optimization for Separation of 100–150 nt Oligonucleotides

IP RP LC is a high-resolution method capable of n/n – 1 separation of 60 nt and potentially even longer oligonucleotides. (31,33) Gilar et al. outlined strategies for improving the oligonucleotide separation in IP RP LC; the recommendations include use of efficient columns packed with 2.5 or 1.7 μm UPLC particles, (32) shallow gradients, (53) and optimized ion-pairing mobile phases. (54) Recently, Donegan et al. (34) demonstrated that hydrophobic ion-pairing reagents such as OAA facilitate an improved oligonucleotide separation relative to hydrophilic ion-pairing systems. Based on these guidelines, we selected an efficient 150 × 2.1 mm column packed with 1.7 μm 300 Å C18 sorbent, shallow gradient, and a hydrophobic OAA ion-pairing mobile phase system. According to reports, (34,54) the oligonucleotide resolution improves with ion-pairing buffer concentration. We evaluated 50–110 mM of OAA systems and observed the corresponding improvements in resolution (data not shown). However, high concentrations of OAA led to high oligonucleotide retention and required >80% acetonitrile for elution at which point we observed the loss of signal for >120 nt oligonucleotides presumably due to sample precipitation on the column. Therefore, we limited the OAA concentration to 100 mM in mobile phase A and B (containing 40 and 90% acetonitrile, respectively) and employed a gradient of mobile phase D containing 100% acetonitrile. In the optimized method, the gradient started at 46% A, 54% B, and 0% D and concluded at 28% A, 62% B, and 10% D in 40 min. Expressed in percentage of acetonitrile, the gradient was 67% to 77% of acetonitrile in 40 min (0.25% acetonitrile/min); in terms of ion-pairing concentration, the gradient started at 100 mM and finished at 90 mM OAA in 40 min. The chosen mobile phase was optimized for oligonucleotide LC separation, but it is not compatible with MS. Two-dimensional LC could be implemented when the MS detection is desired. (55,56)

IP RP LC Study of Poly(A) Heterogeneity

The mRNA poly(A) tail sample prepared according to Materials and Methods (mRNA was digested with RNase T1 and purified by SPE) was analyzed by the IP RP LC method; the chromatograms are presented in Figure 4. Short RNA oligonucleotides were eluted before 5 min (data not shown, UV signal exceeded 1 AU), while the poly(A) tail species eluted approximately between 20 and 25 min. EPO mRNA (blue chromatogram) and FLuc-beta mRNA (red chromatogram) analyses reveal the presence of heterogeneous poly(A) tail species consisting of 5–10 dominant peaks and many minor species. The IP RP LC method does not provide a baseline separation, but the peak resolution is sufficient to visualize and quantify the poly(A) tail heterogeneity.

Figure 4

Figure 4. (A) IP RP LC analysis of the poly(A)tail released from EPO mRNA (blue chromatogram) and the FLuc-beta mRNA sample (red chromatogram) with RNase T1. IP RP LC analysis of the rA100 standard and its truncated oligonucleotides is shown as a black chromatogram. The retention data of 80, 85, 90, 95, 99, 100, and 101 nt rAn oligonucleotides were plotted as a calibration curve (panel B) and utilized to estimate the mRNA poly(A) tail species length. For additional data, see Figure S3.

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Poly(A) tail chromatograms in Figure 4 are overlaid with the synthetic rA100 oligonucleotide (black trace). The 100 nt oligoadenosine contains a ladder of synthetic n-x impurities eluting prior to the main peak. These impurities were utilized to plot the relationship between the length of the rAn species and the retention time. This plot appears to be linear (Figure 4 inset) and could be used to extrapolate the mRNA poly(A)tail species length. According to this calibration, the prominent poly(A) tail species labeled in Figure 4 were 124 nt for EPO mRNA and 128 nt for the FLuc-beta mRNA sample. These results are consistent with the poly(A) tail length measurement performed with the SEC method described in the previous section. Additional calibration with rA100 and rA120 synthetic standards (Figure S2) or standards directly spiked to EPO and FLuc-beta mRNA digests (Figure S3) confirmed our measurement. The data could not be directly confirmed with MS because the mobile phases of both SEC and IP RP LC methods were not compatible with mass spectrometry analysis.

IP RP LC MS Analysis of Poly(A) Tail

The poly(A) tail length was investigated also with the IP RP LC-MS method with mobile phases consisting of DIPEA/HFIP aqueous buffer. This mobile phase is well suited for sensitive LC-MS analysis of oligonucleotides. (47,48) The IP RP LC mobile phases where the alkylamines are buffered by HFIP instead of acetic acid provide a significantly greater MS signal for oligonucleotide analysis. (34,57) The RNase T1-digested FLuc-beta mRNA sample was eluted from the chromatographic column with a fast 10 min gradient. Figure 5 shows the resulting chromatogram. Short RNA oligonucleotides elute before the last distinct peak at 6 min, presumably the poly(A) tail signal. The MS signal under this peak was summed, and the multiply charged MS ions were deconvoluted with MaxEnt1 software into zero charge MS spectrum presented as Figure 5C. The three most dominant MS signals in the spectrum correspond to 126, 127, and 128 nt long poly(A) species. This result is consistent with previously obtained results from SEC and IP RP LC UV analysis.

Figure 5

Figure 5. IP RP LC analysis of FLuc-beta mRNA poly(A) tail. (A) LC UV analysis, (B) LC MS LC MS total ion chromatogram (TIC), and (C) deconvolved ESI-MS spectrum of the peak eluting close to 6 min. This chromatographic peak corresponds to a mixture of heterogeneous poly(A) species with three most dominant MS signals corresponding to 126, 127, and 128 nt oligonucleotides.

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Discussion

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SEC is used in the industry mainly for analysis of synthetic polymers and proteins, and it is applicable to analysis of RNA/DNA therapeutics as well. In the initial experiment, we investigated the selection of the SEC sorbent pore size for targeted separation of poly(A) tail species from shorter oligonucleotides and intact mRNA. This evaluation was essential since the SEC sorbent pore size selection suitable for separation of the target oligonucleotide length is not well-known. Kim and colleagues (42) reported that the molecular weight range of proteins separable in SEC does not directly translate to oligonucleotides. This is because linear nucleic acids have a larger hydrodynamic radius than globular proteins. We confirmed this observation in experiments with the 250 Å SEC column (Figure S4). Indeed, large differences in log MW = a × tr + b calibrations were found between oligonucleotides and protein standards.
The sequence or secondary structure (stems, loops, duplexes, and structural polymorphisms) also has an impact on SEC retention of oligonucleotides. (43) This can be seen in the chromatograms shown in Figure 2B–D for dAn, dTn, and rAn samples. Even though the used homooligomeric samples do not have a strong secondary structure, their retention is not the same. We investigated whether the differences are due to secondary ionic or hydrophobic interactions of analytes with the SEC sorbent. Neither the addition of methanol (10 or 20%) nor the change in the buffer type and concentration yielded the retention strictly according to the oligonucleotide length. We concluded that retention differences were most likely a manifestation of the different oligonucleotide hydrodynamic sizes. This unfortunately means that it is necessary to use expensive rAn standards as calibrants for poly(A) tail RNA measurement instead of more affordable dAn, as we originally planned. This problem with calibration standards could be circumvented, as illustrated in Figure 2A. Two linear calibrations were constructed for the same column using dAn and rAn standards. The calibrations have essentially the same slope with a small difference in the intercept. This, in principle, means that a retention time correction factor can be applied to superimpose the dAn calibration onto the rAn calibration curve (see Figure S5). The concept of using dAn standards as surrogates for rAn calibrants in SEC was validated with two UPLC systems and three SEC columns. The experiments confirmed that the dAn and rAn calibrations are parallel. When the correction factor Δtr = 0.306 min is used to adjust experimental dAn retention times, both dAn and rAn SEC calibration curves become virtually identical (Figure S5B). Table S1 lists the retention data of four SEC experiments with dAn and rAn calibrants. The average retention difference between rAn and dAn represents the proposed correction factor Δtr of 0.306 min. While the tubing volume and injection loop size of LC instrument affect the absolute retention times of calibrants in SEC, the relative retention difference between rAn and dAn standards remains constant (Table S1). Therefore, it is possible to utilize inexpensive, readily available, and chemically stable dAn oligonucleotides as surrogates for the rAn calibration standards. In the simplest implementation, the analyst can add the Δtr = 0.306 min correction factor to the measured dAn retention times (Figure S5) prior to plotting the calibration curve log N = a × tr + b. The resulting SEC calibration is directly applicable for mRNA poly(A) tail measurement.
We investigated the comparability of rAn and dAn calibrations for poly(A) tail analysis of EPO and Fluc-beta mRNA samples. Table S2 lists the retention data of four experiments and their interpretation using rAn and corrected dAn SEC calibrations (using a 0.306 min correction factor). Both calibrations gave similar poly(A) tail length measurement within one nucleotide length difference for calibrations obtained with 40–120 nt standards. When we used 40–150 nt dAn calibration standards, the calculated length of the poly(A) tail was approximately 3 nt longer. This is due to the change in the calibration slope caused by the 150 nt data point deviating slightly from the linear trend. 150 nt dA standard appears to lie at the upper range of the calibration linearity (Figures 2A and S5B).
The 250 Å column chosen for SEC analysis has metal hardware modified with ethylene-bridged siloxane-modified metal surface chemistry known as hybrid surface technology (HST). (50,58) HST has been shown to mitigate the nonspecific adsorption of nucleic acid on LC hardware, which can have an adverse effect on oligonucleotide analysis. (51,52,59) The 100 mM phosphate buffer and basic pH were shown to reduce the nonspecific adsorption, (50) but HST columns are preferred for nucleic acid analysis when available.
The IP RP LC method is a high-resolution method capable of simultaneously measuring poly(A) tail length and heterogeneity, which makes the SEC method seemingly redundant. However, IP RP LC is significantly more complex and less robust than the isocratic SEC method. (60) The method described in this work is optimized for the separation of 100–150 nt poly(A) tail oligonucleotides. In IP RP LC, a small drift of retention times is sometimes detected. (61) This is due to the evaporation of semivolatile ion-pairing mobile phases, incomplete thermal equilibration of the column, and inconsistent column equilibration time between injections. Such a retention shift can affect the poly(A) length measurement accuracy. Calibration with size standards should be performed prior or after the sample analysis (Figure 4) or the rAn standards can be spiked to the sample as shown in Figure S3.
RNase T1 utilized in this work cleaves mRNA at the G/N motifs. Due to cleavage selectivity, the liberated poly(A) tail sequence may contain one or several additional nucleotides at the 5′ end other than G. The sequence of interrogated mRNA is typically known; this knowledge should be utilized in the poly(A) tail length measurement as was the case with FLuc-beta mRNA analysis in this study. The poly(A) tail fragment created with RNase T1 cleavage contained a single additional cytidine at the 5′ end (see Figure S6). The poly(A) analysis of the second sample, EPO mRNA, is complicated by the fact that the full sequence of EPO mRNA was not provided by the manufacturer. The exact number of additional nucleotides at the 5′ end of the EPO mRNA poly(A) tail after RNase T1 cleavage is not known. The length of the EPO mRNA poly(A) tail reported in Figures S1 and S3 (123 nt) should be treated as an approximation. This result was consistent with EPO mRNA poly(A) LC MS measurement (Figure S7).
We should also mention that the applicability of RNase T1 for digestion of BioNTech/Pfizer SARS-Cov-2 mRNA vaccine is complicated by the fact that the poly(A) tail includes a 10 nt long motif including G nucleotides. In this case, RNase T1 will generate two sections of the poly(A) tail (Figure S8). The mRNA sequence should be always consulted prior to measurement; other enzymes can be used for mRNA poly(A) tail cleavage if desired. (15,17,18,22,23)

Conclusions

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We demonstrate that the SEC column with 250 Å pore size is suitable for the separation of 40–150 nt long oligonucleotides with log N vs tr calibration linearity. The developed method was applied to robust and repeatable measurement of the poly(A) tail length after its cleavage form mRNA. Because synthetic poly(A) RNA standards are scarce, we evaluated the utility of oligo(A) DNA surrogate standards for SEC calibration. After we applied an experimentally derived retention correction, the surrogate SEC standard calibration provided virtually identical poly(A) tail measurement results as those of RNA poly(A) tail calibrants (Tables S1 and S2).
The SEC method provides information about an average poly(A) tail length, but it cannot directly measure the microheterogeneity of the mRNA 3′-end. The microheterogeneity analysis of the poly(A) tail was performed with a high-resolution IP RP LC method based on the OAA ion-pairing system. This method can resolve poly(A) tail species differing in a single nucleotide up to approximately 150 nt in length. SEC and IP RP LC methods use simple UV detection, which makes them suitable for routine quality control of mRNA analysis.
The performance of the SEC and IP RP LC methods for poly(A) tail characterization was verified with LC-MS (Figures 5, S7, and S9). The LC-MS method resolves short RNA oligonucleotides RNase T1 digestion products from poly(A) tail in a 10 min gradient using the MS compatible mobile phase. The summed and deconvoluted MS data of the poly(A) tail peak (Figures 5C, S7, and S9) reveal dominant MS signals corresponding to 126, 127, and 128 nt long oligonucleotides for the Fluc-beta mRNA sample (125–127 nt long rAn sequences plus one cytidine) and 122–124 nt long oligonucleotides for the EPO mRNA sample. The LC-MS results are consistent with IP RP LC UV and SEC UV measurements presented in Figures 3, S1, and S3. This finding confirms that the developed SEC and IP R P LC methods for poly(A) tail length measurement provide accurate and useful results and are applicable to mRNA quality control. The LC-MS method utilized more expensive high-resolution MS instrument and deconvolution software, but it provides data-rich results, including the direct molecular weight measurement of poly(A) tail species and the molecular weight difference between the observed poly(A) species (329.2 Da mass differences in the MS spectrum presented in Figure 5C confirm that the microheterogeneity is due to different numbers of A mononucleotides in the poly(A) tail). We expect that LC MS will be a preferred method for mRNA analysis in biopharma research.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.3c02552.

  • Chromatograms, IP RP LC calibration curve, SEC calibration curve, mass spectrometry spectra, and sequences of mRNA (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Catalin Doneanu - Discovery and Development, Waters Corporation, Milford, Massachusetts 01757, United States
    • Maissa M. Gaye - Consumables Research, Waters Corporation, Milford, Massachusetts 01757, United States
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare the following competing financial interest(s): The authors are employee of Waters Corporation, manufacturer of some consumables and instruments used in this study.

    ACQUITY, UPLC, BEH, Empower, MassLynx, BioAccord, VanGuard, and Oasis are trademarks of Waters Technologies Corporation. CleanCap is a trademark of TriLink BioTechnologies, LCC. rCutSmart is a trademark of New England Biolabs. Mill-Q is a trademark of Merck KGaA.

Acknowledgments

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We thank Matthew Lauber and Joseph Fredette for their comments to the manuscript.

References

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This article references 61 other publications.

  1. 1
    Castells, M. C.; Phillips, E. J. Maintaining Safety with SARS-CoV-2 Vaccines. N. Engl. J. Med. 2021, 384 (7), 643– 649,  DOI: 10.1056/NEJMra2035343
    Google Scholar
    1
    Maintaining safety with SARS-CoV-2 vaccines
    Castells, Mariana C.; Phillips, Elizabeth J.
    New England Journal of Medicine (2021), 384 (7), 643-649CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
    A review. Careful vaccine-safety surveillance over time, paired with elucidation of mechanisms of adverse events across different SARS-CoV-2 vaccine platforms, will be needed to inform a strategic and systematic approach to vaccine safety. Indeed, mRNA vaccines are a promising new technol., and demonstration of their safety is relevant to the development of vaccines against several other viruses of global importance and many cancers. For the immediate future, during a pandemic that is still increasing, it is crit. that we focus on safe and efficient approaches to implementing mass vaccination.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXkvVGhurw%253D&md5=a42f84b5b88c4702df8e6668b928a426
  2. 2
    Gote, V.; Bolla, P. K.; Kommineni, N.; Butreddy, A.; Nukala, P. K.; Palakurthi, S. S.; Khan, W. A Comprehensive Review of mRNA Vaccines. Int. J. Mol. Sci. 2023, 24 (3), 2700,  DOI: 10.3390/ijms24032700
    Google Scholar
    2
    A Comprehensive Review of mRNA Vaccines
    Gote, Vrinda; Bolla, Pradeep Kumar; Kommineni, Nagavendra; Butreddy, Arun; Nukala, Pavan Kumar; Palakurthi, Sushesh Srivatsa; Khan, Wahid
    International Journal of Molecular Sciences (2023), 24 (3), 2700CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. MRNA vaccines have been demonstrated as a powerful alternative to traditional conventional vaccines because of their high potency, safety and efficacy, capacity for rapid clin. development, and potential for rapid, low-cost manufg. These vaccines have progressed from being a mere curiosity to emerging as COVID-19 pandemic vaccine front-runners. The advancements in the field of nanotechnol. for developing delivery vehicles for mRNA vaccines are highly significant. In this review we have summarized each and every aspect of the mRNA vaccine. The article describes the mRNA structure, its pharmacol. function of immunity induction, lipid nanoparticles (LNPs), and the upstream, downstream, and formulation process of mRNA vaccine manufg. Addnl., mRNA vaccines in clin. trials are also described. A deep dive into the future perspectives of mRNA vaccines, such as its freeze-drying, delivery systems, and LNPs targeting antigen-presenting cells and dendritic cells, are also summarized.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXjsFajtLk%253D&md5=3f1a47449a68bc378e2c1f7a2fc0c678
  3. 3
    Watanabe, A.; Kani, R.; Iwagami, M.; Takagi, H.; Yasuhara, J.; Kuno, T. Assessment of Efficacy and Safety of mRNA COVID-19 Vaccines in Children Aged 5 to 11 Years: A Systematic Review and Meta-analysis. JAMA Pediatr. 2023, 177 (4), 384– 394,  DOI: 10.1001/jamapediatrics.2022.6243
    Google Scholar
    3
    Assessment of Efficacy and Safety of mRNA COVID-19 Vaccines in Children Aged 5 to 11 Years: A Systematic Review and Meta-analysis
    Watanabe Atsuyuki; Kani Ryoma; Iwagami Masao; Iwagami Masao; Takagi Hisato; Yasuhara Jun; Kuno Toshiki
    JAMA pediatrics (2023), 177 (4), 384-394 ISSN:.
    Importance: Evidence of the efficacy and safety of messenger RNA (mRNA) COVID-19 vaccines in children aged 5 to 11 years has been emerging. Collecting these data will inform clinicians, families, and policy makers. Objective: To evaluate the efficacy and safety of mRNA COVID-19 vaccines in children aged 5 to 11 years in a systematic review and meta-analysis. Data Sources: PubMed and Embase databases were searched on September 29, 2022, without language restrictions. Study Selection: Randomized clinical trials and observational studies comparing vaccinated vs unvaccinated children aged 5 to 11 years and reporting efficacy or safety outcomes were included. Studies reporting safety outcomes in vaccinated children only (ie, no control group) were also included. Data Extraction and Synthesis: Two investigators independently extracted relevant data from each study. Odds ratios (ORs) for efficacy and safety outcomes and incidences of adverse events (AEs) following vaccination were synthesized using a random-effects model. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses and Meta-analysis of Observational Studies in Epidemiology reporting guidelines. Main Outcomes and Measures: The primary outcome was SARS-CoV-2 infections with or without symptoms. The secondary outcomes included symptomatic SARS-CoV-2 infections, hospitalizations, and multisystem inflammatory syndrome in children. The incidences of each AE following vaccination were also evaluated. Results: Two randomized clinical trials and 15 observational studies involving 10 935 541 vaccinated children (median or mean age range, 8.0-9.5 years) and 2 635 251 unvaccinated children (median or mean age range, 7.0-9.5 years) were included. Two-dose mRNA COVID-19 vaccination compared with no vaccination was associated with lower risks of SARS-CoV-2 infections with or without symptoms (OR, 0.47; 95% CI, 0.35-0.64), symptomatic SARS-CoV-2 infections (OR, 0.53; 95% CI, 0.41-0.70), hospitalizations (OR, 0.32; 95% CI, 0.15-0.68), and multisystem inflammatory syndrome in children (OR, 0.05; 95% CI, 0.02-0.10). Two randomized clinical trials and 5 observational studies investigated AEs among vaccinated children. Most vaccinated children experienced at least 1 local AE following the first injection (32 494 of 55 959 [86.3%]) and second injection (28 135 of 46 447 [86.3%]). Vaccination was associated with a higher risk of any AEs compared with placebo (OR, 1.92; 95% CI, 1.26-2.91). The incidence of AEs that prevented normal daily activities was 8.8% (95% CI, 5.4%-14.2%) and that of myocarditis was estimated to be 1.8 per million (95% CI, 0.000%-0.001%) following the second injection. Conclusions and Relevance: In this systematic review and meta-analysis, COVID-19 mRNA vaccines among children aged 5 to 11 years were associated with measures of efficacy in preventing SARS-CoV-2 infection and severe COVID-19-related illnesses. While most children developed local AEs, severe AEs were rare, and most of AEs resolved within several days. These data provide evidence for future recommendations.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB28jjvVOisg%253D%253D&md5=3d8a45144c72acfe80fccfea19b20ed4
  4. 4
    Chaudhary, N.; Weissman, D.; Whitehead, K. A. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat. Rev. Drug Discovery 2021, 20 (11), 817– 838,  DOI: 10.1038/s41573-021-00283-5
    Google Scholar
    4
    mRNA vaccines for infectious diseases: principles, delivery and clinical translation
    Chaudhary, Namit; Weissman, Drew; Whitehead, Kathryn A.
    Nature Reviews Drug Discovery (2021), 20 (11), 817-838CODEN: NRDDAG; ISSN:1474-1776. (Nature Portfolio)
    A review. Abstr.: Over the past several decades, mRNA (mRNA) vaccines have progressed from a scepticism-inducing idea to clin. reality. In 2020, the COVID-19 pandemic catalyzed the most rapid vaccine development in history, with mRNA vaccines at the forefront of those efforts. Although it is now clear that mRNA vaccines can rapidly and safely protect patients from infectious disease, addnl. research is required to optimize mRNA design, intracellular delivery and applications beyond SARS-CoV-2 prophylaxis. In this Review, we describe the technologies that underlie mRNA vaccines, with an emphasis on lipid nanoparticles and other non-viral delivery vehicles. We also overview the pipeline of mRNA vaccines against various infectious disease pathogens and discuss key questions for the future application of this breakthrough vaccine platform.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVygtL%252FP&md5=6e3a059338985252dc07eeb71f7ae6ae
  5. 5
    Magadum, A.; Kaur, K.; Zangi, L. mRNA-Based Protein Replacement Therapy for the Heart. Mol. Ther. 2019, 27 (4), 785– 793,  DOI: 10.1016/j.ymthe.2018.11.018
    Google Scholar
    5
    mRNA-Based Protein Replacement Therapy for the Heart
    Magadum, Ajit; Kaur, Keerat; Zangi, Lior
    Molecular Therapy (2019), 27 (4), 785-793CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)
    A review:. Myocardial infarction (MI) and heart failure (HF) are the leading causes of death in the United States and in most other industrialized nations. MI leads to a massive loss of cardiomyocytes (CMs), which are replaced with non-CM cells, leading to scarring and, in most cases, HF. The adult mammalian heart has a low intrinsic regenerative capacity, mainly because of cell-cycle arrest in CMs. No effective treatment promoting heart regeneration is currently available. Recent efforts to use DNA-based or viral gene therapy approaches to induce cardiac regeneration post-MI or in HF conditions have encountered major challenges, mostly because of the poor and uncontrolled delivery of the introduced genes. Modified mRNA (modRNA) is a safe, non-immunogenic, efficient, transient, local, and controlled nucleic acid delivery system that can overcome the obstacles to DNA-based or viral approaches for cardiac gene delivery. We here review the use of modRNA in cardiac therapy, to induce cardioprotection and vascular or cardiac regeneration after MI. We discuss the current challenges in modRNA-based cardiac treatment, which will need to be overcome for the application of such treatment to ischemic heart disease.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXntFOnsb8%253D&md5=cc917f7fadd692cb7696a481798b9b75
  6. 6
    Miao, L.; Zhang, Y.; Huang, L. mRNA vaccine for cancer immunotherapy. Mol. Cancer 2021, 20 (1), 41,  DOI: 10.1186/s12943-021-01335-5
    Google Scholar
    6
    mRNA vaccine for cancer immunotherapy
    Miao, Lei; Zhang, Yu; Huang, Leaf
    Molecular Cancer (2021), 20 (1), 41CODEN: MCOACG; ISSN:1476-4598. (BioMed Central Ltd.)
    A review. MRNA vaccines have become a promising platform for cancer immunotherapy. During vaccination, naked or vehicle loaded mRNA vaccines efficiently express tumor antigens in antigen-presenting cells (APCs), facilitate APC activation and innate/adaptive immune stimulation. mRNA cancer vaccine precedes other conventional vaccine platforms due to high potency, safe administration, rapid development potentials, and cost-effective manufg. However, mRNA vaccine applications have been limited by instability, innate immunogenicity, and inefficient in vivo delivery. Appropriate mRNA structure modifications (i.e., codon optimizations, nucleotide modifications, self-amplifying mRNAs, etc.) and formulation methods (i.e., lipid nanoparticles (LNPs), polymers, peptides, etc.) have been investigated to overcome these issues. Tuning the administration routes and co-delivery of multiple mRNA vaccines with other immunotherapeutic agents (e.g., checkpoint inhibitors) have further boosted the host anti-tumor immunity and increased the likelihood of tumor cell eradication. With the recent U. S. Food and Drug Administration (FDA) approvals of LNP-loaded mRNA vaccines for the prevention of COVID-19 and the promising therapeutic outcomes of mRNA cancer vaccines achieved in several clin. trials against multiple aggressive solid tumors, we envision the rapid advancing of mRNA vaccines for cancer immunotherapy in the near future. This review provides a detailed overview of the recent progress and existing challenges of mRNA cancer vaccines and future considerations of applying mRNA vaccine for cancer immunotherapies.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXht1Smsr7K&md5=8475d10d81d634deda80801e6dbb20a6
  7. 7
    Lorentzen, C. L.; Haanen, J. B.; Met, Ö.; Svane, I. M. Clinical advances and ongoing trials on mRNA vaccines for cancer treatment. Lancet Oncol. 2022, 23 (10), e450– e458,  DOI: 10.1016/S1470-2045(22)00372-2
    Google Scholar
    7
    Clinical advances and ongoing trials on mRNA vaccines for cancer treatment
    Lorentzen, Cathrine Lund; Haanen, John B.; Met, Ozcan; Svane, Inge Marie
    Lancet Oncology (2022), 23 (10), e450-e458CODEN: LOANBN; ISSN:1470-2045. (Elsevier Ltd.)
    Years of research exploring mRNA vaccines for cancer treatment in preclin. and clin. trials have set the stage for the rapid development of mRNA vaccines during the COVID-19 pandemic. Therapeutic cancer vaccines based on mRNA are well tolerated, and the inherent advantage in ease of prodn., which rivals the best available conventional vaccine manuf. methods, renders mRNA vaccines a promising option for cancer immunotherapy. Technol. advances have optimized mRNA-based vaccine stability, structure, and delivery methods, and multiple clin. trials investigating mRNA vaccine therapy are now enrolling patients with various cancer diagnoses. Although therapeutic mRNA-based cancer vaccines have not yet been approved for std. treatment, encouraging results from early clin. trials with mRNA vaccines as monotherapy and in combination with checkpoint inhibitors have been obtained. This Review summarises the latest clin. advances in mRNA-based vaccines for cancer treatment and reflects on future perspectives and challenges for this new and promising treatment approach.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFygtrzN&md5=676d862ef4ac7a9934fba6afb546517a
  8. 8
    Weide, B.; Carralot, J. P.; Reese, A.; Scheel, B.; Eigentler, T. K.; Hoerr, I.; Rammensee, H. G.; Garbe, C.; Pascolo, S. Results of the first phase I/II clinical vaccination trial with direct injection of mRNA. J. Immunother. 2008, 31 (2), 180– 188,  DOI: 10.1097/CJI.0b013e31815ce501
    Google Scholar
    8
    Results of the First Phase I/II Clinical Vaccination Trial With Direct Injection of mRNA
    Weide, Benjamin; Carralot, Jean-Philippe; Reese, Anne; Scheel, Birgit; Eigentler, Thomas Kurt; Hoerr, Ingmar; Rammensee, Hans-Georg; Garbe, Claus; Pascolo, Steve
    Journal of Immunotherapy (2008), 31 (2), 180-188CODEN: JOIMF8; ISSN:1524-9557. (Lippincott Williams & Wilkins)
    Vaccination against tumor antigens has been shown to be a safe and efficacious prophylactic and therapeutic antitumor treatment in many animal models. Clin. studies in humans indicate that specific immunotherapy can also result in clin. benefits. The active pharmaceutical ingredient in such vaccines can be DNA, RNA, protein, or peptide and can be administered naked, encapsulated, or after delivery in vitro into cells that are then adoptively transferred. One of the easiest, most versatile and theor. safest technologies relies on the direct injection of naked mRNA (mRNA) that code for tumor antigens. We and others have shown in mice that intradermal application of naked mRNA results in protein expression and the development of an immune response. We used this protocol to vaccinate 15 melanoma patients. For each patient a growing metastasis was removed, total RNA was extd., reverse-transcribed, amplified, and cloned. Libraries of cDNA were transcribed to produce unlimited amts. of copy mRNA. Autologous prepns. were applied intradermally in combination with granulocyte macrophage colony-stimulating factor as adjuvant. We demonstrate here that such treatment is feasible and safe (phase 1 criteria). Furthermore, an increase in antitumor humoral immune response was seen in some patients. However, a demonstration of clin. effectiveness of direct injection of copy mRNA for antitumor immunotherapy was not shown in this study and must be evaluated in subsequent trials.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjt1Cju7c%253D&md5=6146d1459c80d66622dc52db8722bc60
  9. 9
    Curreri, A.; Sankholkar, D.; Mitragotri, S.; Zhao, Z. RNA therapeutics in the clinic. Bioeng. Transl. Med. 2023, 8 (1), e10374  DOI: 10.1002/btm2.10374
    Google Scholar
    9
    RNA therapeutics in the clinic
    Curreri, Alexander; Sankholkar, Disha; Mitragotri, Samir; Zhao, Zongmin
    Bioengineering & Translational Medicine (2023), 8 (1), e10374CODEN: BTMIAQ; ISSN:2380-6761. (John Wiley & Sons, Inc.)
    A review. RNA (RNA) therapeutics are being actively researched as a therapeutic modality in preclin. and clin. studies. They have become one of the most ubiquitously known and discussed therapeutics in recent years in part due to the ongoing coronavirus pandemic. Since the first approval in 1998, research on RNA therapeutics has progressed to discovering new therapeutic targets and delivery strategies to enhance their safety and efficacy. Here, we provide an overview of the current clin. relevant RNA therapeutics, mechanistic basis of their function, and strategies to improve their clin. use. We discuss the 17 approved RNA therapeutics and perform an in-depth anal. of the 222 ongoing clin. trials, with an emphasis on their resp. mechanisms and disease areas. We also provide perspectives on the challenges for clin. translation of RNA therapeutics and suggest potential strategies to address these challenges.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xit1Wrs7fK&md5=ac3bbae01052423a845b25369836176b
  10. 10
    USP, Analytical Procedures for mRNA Vaccine Quality, 2nd draft. USP Draft Guidelines 2023, EA966W_2022–03.
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Bloom, K.; van den Berg, F.; Arbuthnot, P. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 2021, 28 (3–4), 117– 129,  DOI: 10.1038/s41434-020-00204-y
    Google Scholar
    11
    Self-amplifying RNA vaccines for infectious diseases
    Bloom, Kristie; van den Berg, Fiona; Arbuthnot, Patrick
    Gene Therapy (2021), 28 (3-4), 117-129CODEN: GETHEC; ISSN:0969-7128. (Nature Portfolio)
    A review. Vaccinol. is shifting toward synthetic RNA platforms which allow for rapid, scalable, and cell-free manufg. of prophylactic and therapeutic vaccines. The simple development pipeline is based on in vitro transcription of antigen-encoding sequences or immunotherapies as synthetic RNA transcripts, which are then formulated for delivery. This approach may enable a quicker response to emerging disease outbreaks, as is evident from the swift pursuit of RNA vaccine candidates for the global SARS-CoV-2 pandemic. Both conventional and self-amplifying RNAs have shown protective immunization in preclin. studies against multiple infectious diseases including influenza, RSV, Rabies, Ebola, and HIV-1. Self-amplifying RNAs have shown enhanced antigen expression at lower doses compared to conventional mRNA, suggesting this technol. may improve immunization. This review will explore how self-amplifying RNAs are emerging as important vaccine candidates for infectious diseases, the advantages of synthetic manufg. approaches, and their potential for preventing and treating chronic infections.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1ersLzK&md5=8d46530246635e8a97616fcd7d6be25f
  12. 12
    Kanavarioti, A. HPLC methods for purity evaluation of man-made single-stranded RNAs. Sci. Rep. 2019, 9 (1), 1019,  DOI: 10.1038/s41598-018-37642-z
    Google Scholar
    12
    HPLC methods for purity evaluation of man-made single-stranded RNAs
    Kanavarioti Anastassia
    Scientific reports (2019), 9 (1), 1019 ISSN:.
    Synthetic RNA oligos exhibit purity decreasing as a function of length, because the efficiency of the total synthesis is the numerical product of the individual step efficiencies, typically below 98%. Analytical methods for RNAs up to the 60 nucleotides (nt) have been reported, but they fall short for purity evaluation of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuticals. In an attempt to exploit a single HPLC method and obtain both identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at high temperature in the presence of an organic cosolvent is the current analytical strategy. Here we report that IP-RP is less suitable compared to the conventional ion-exchange (IEX) for analysis of 100nt RNAs. We demonstrate the relative stability of RNA in the denaturing/basic IEX mobile phase, lay out a protocol to determine the on-the-column stability of any RNA, and establish the applicability of this method for quality testing of sgRNA, tRNA, and mRNA. Unless well resolving HPLC methods are used for batch-to-batch evaluation of man-made RNAs, process development will remain shortsighted, and observed off-target effects in-vitro or in-vivo may be partially related to low purity and the presence of shorter sequences.
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  13. 13
    Fekete, S.; Yang, H.; Wyndham, K.; Lauber, M. Salt gradient and ion-pair mediated anion exchange of intact messenger ribonucleic acids. J. Chromatogr. Open 2022, 2, 100031  DOI: 10.1016/j.jcoa.2022.100031
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    There is no corresponding record for this reference.
  14. 14
    Packer, M.; Gyawali, D.; Yerabolu, R.; Schariter, J.; White, P. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems. Nat. Commun. 2021, 12 (1), 6777,  DOI: 10.1038/s41467-021-26926-0
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    14
    A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems
    Packer, Meredith; Gyawali, Dipendra; Yerabolu, Ravikiran; Schariter, Joseph; White, Phil
    Nature Communications (2021), 12 (1), 6777CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liq. chromatog. (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity anal. techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidn. and subsequent hydrolysis of the tertiary amine. It thus remains crit. to ensure robust anal. methods and stringent manufg. control to ensure mRNA stability and high activity in LNP delivery systems.
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  15. 15
    Jiang, T.; Yu, N.; Kim, J.; Murgo, J. R.; Kissai, M.; Ravichandran, K.; Miracco, E. J.; Presnyak, V.; Hua, S. Oligonucleotide Sequence Mapping of Large Therapeutic mRNAs via Parallel Ribonuclease Digestions and LC-MS/MS. Anal. Chem. 2019, 91 (13), 8500– 8506,  DOI: 10.1021/acs.analchem.9b01664
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    15
    Oligonucleotide sequence mapping of large therapeutic mRNAs via parallel ribonuclease digestions and LC-MS/MS
    Jiang, Tao; Yu, Ningxi; Kim, Jaeah; Murgo, John-Ross; Kissai, Mildred; Ravichandran, Kanchana; Miracco, Edward J.; Presnyak, Vladimir; Hua, Serenus
    Analytical Chemistry (Washington, DC, United States) (2019), 91 (13), 8500-8506CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Characterization of mRNA sequences is a crit. aspect of mRNA drug development and regulatory filing. Herein, we developed a novel bottom-up oligonucleotide sequence mapping workflow combining multiple endonucleases that cleave mRNA at different frequencies. RNase T1, colicin E5, and mazF were applied in parallel to provide complementary sequence coverage for large mRNAs. Combined use of multiple endonucleases resulted in significantly improved sequence coverage: greater than 70% sequence coverage was achieved on mRNAs near 3000 nucleotides long. Oligonucleotide mapping simulations with large human RNA databases demonstrate that the proposed workflow can pos. identify a single correct sequence from hundreds of similarly sized sequences. In addn., the workflow is sensitive and specific enough to detect minor sequence impurities such as single nucleotide polymorphisms (SNPs) with a sensitivity of less than 1%. LC-MS/MS-based oligonucleotide sequence mapping can serve as an orthogonal sequence characterization method to techniques such as Sanger sequencing or next-generation sequencing (NGS), providing high-throughput sequence identification and sensitive impurity detection.
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  16. 16
    Wolf, E. J.; Grünberg, S.; Dai, N.; Chen, T. H.; Roy, B.; Yigit, E.; Corrêa, I. R. Human RNase 4 improves mRNA sequence characterization by LC-MS/MS. Nucleic Acids Res. 2022, 50 (18), e106  DOI: 10.1093/nar/gkac632
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    16
    Human RNase 4 improves mRNA sequence characterization by LC-MS/MS
    Wolf, Eric J.; Grunberg, Sebastian; Dai, Nan; Chen, Tien-Hao; Roy, Bijoyita; Yigit, Erbay; Correa, Ivan R., Jr.
    Nucleic Acids Research (2022), 50 (18), e106CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    With the rapid growth of synthetic mRNA (mRNA)-based therapeutics and vaccines, the development of anal. tools for characterization of long, complex RNAs has become essential. Tandem liq. chromatog.-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence anal. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine-two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5' cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS.
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  17. 17
    Nwokeoji, A. O.; Earll, M. E.; Kilby, P. M.; Portwood, D. E.; Dickman, M. J. High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2019, 1104, 212– 219,  DOI: 10.1016/j.jchromb.2018.11.027
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    17
    High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography
    Nwokeoji, Alison O.; Earll, Mark E.; Kilby, Peter M.; Portwood, David E.; Dickman, Mark J.
    Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2019), 1104 (), 212-219CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
    The emergence of new sustainable approaches for insect management using RNA interference (RNAi) based insecticides has created the demand for high throughput anal. techniques to fully characterize and accurately quantify double stranded RNA (dsRNA) prior to downstream RNAi applications. In this study we have developed a method for the rapid characterization of single stranded and double stranded RNA using high resoln. RNase mapping in conjunction with ion-pair reverse-phase chromatog. utilizing a column with superficially porous particles. The high resoln. oligoribonucleotide map provides an important 'fingerprint' for identity testing and bioprocess monitoring. Reproducible RNA mapping chromatograms were generated from replicate analyses. Moreover, this approach was used to provide a method to rapidly distinguish different RNA sequences of the same size, based on differences in the resulting chromatograms. Principal components anal. of the high resoln. RNA mapping data enabled us to rapidly compare multiple HPLC chromatograms and distinguish two dsRNA sequences of different size which share 72% sequence homol. We used the high resoln. RNase mapping method to rapidly fingerprint biomanufd. dsRNA across a no. of different batches. The resulting chromatograms in conjunction with principal components anal. demonstrated high similarity in the dsRNA produced across the different batches highlighting the potential ability of this method to provide information for batch release in a high throughput manner.
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  18. 18
    Beverly, M.; Dell, A.; Parmar, P.; Houghton, L. Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS. Anal. Bioanal. Chem. 2016, 408 (18), 5021– 5030,  DOI: 10.1007/s00216-016-9605-x
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    18
    Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS
    Beverly, Michael; Dell, Amy; Parmar, Parul; Houghton, Leslie
    Analytical and Bioanalytical Chemistry (2016), 408 (18), 5021-5030CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    A label-free method for detg. the 5'-end cap identity and orientation of a mRNA is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quant. and qual. information on the 5' cap was detd. from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA prepns. was detd. to be between 88 and 98 % depending on the modification type and length of the mRNA. MRNA of 2.2K and 9K nucleotides in length and contg. the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping.
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  19. 19
    Jia, L.; Qian, S. B. Therapeutic mRNA Engineering from Head to Tail. Acc. Chem. Res. 2021, 54 (23), 4272– 4282,  DOI: 10.1021/acs.accounts.1c00541
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    19
    Therapeutic mRNA Engineering from Head to Tail
    Jia, Longfei; Qian, Shu-Bing
    Accounts of Chemical Research (2021), 54 (23), 4272-4282CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. Synthetic mRNA, once delivered into cells, can be readily translated into proteins by ribosomes, which do not distinguish exogenous mRNAs from endogenous transcripts. Until recently, the intrinsic instability and immunostimulatory property of exogenous RNAs largely hindered the therapeutic application of synthetic mRNAs. Thanks to major technol. innovations, such as introduction of chem. modified nucleosides, synthetic mRNAs have become programmable therapeutic reagents. Compared to DNA or protein-based therapeutic reagents, synthetic mRNAs bear several advantages: flexible design, easy optimization, low-cost prepn., and scalable synthesis. Therapeutic mRNAs are commonly designed to encode specific antigens to elicit organismal immune response to pathogens like viruses, express functional proteins to replace defective ones inside cells, or introduce novel enzymes to achieve unique functions like genome editing. Recent years have witnessed stunning progress on the development of mRNA vaccines against SARS-CoV-2. This success is built upon our fundamental understanding of mRNA metab. and translational control, a knowledge accumulated during the past several decades. Given the astronomical no. of sequence combinations of 4 nucleotides, sequence-dependent control of mRNA translation remains incompletely understood. Rational design of synthetic mRNAs with robust translation and optimal stability remains challenging. Massively paralleled reporter assay (MPRA) has been proven to be powerful in identifying sequence elements in controlling mRNA translatability and stability. Indeed, a completely randomized sequence in 5' untranslated region (5'UTR) drives a wide range of translational outputs. We will discuss general principles of mRNA translation in eukaryotic cells and elucidate the role of coding and noncoding regions in the translational regulation. From the therapeutic perspective, we will highlight the unique features of 5' cap, 5'UTR, coding region (CDS), stop codon, 3'UTR, and poly(A) tail. By focusing on the design strategies in mRNA engineering, we hope this Account will contribute to the rational design of synthetic mRNAs with broad therapeutic potential.
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  20. 20
    Ramanathan, A.; Robb, G. B.; Chan, S. H. mRNA capping: biological functions and applications. Nucleic Acids Res. 2016, 44 (16), 7511– 7526,  DOI: 10.1093/nar/gkw551
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    20
    mRNA capping: biological functions and applications
    Ramanathan Anand; Robb G Brett; Chan Siu-Hong
    Nucleic acids research (2016), 44 (16), 7511-26 ISSN:.
    The 5' m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2'O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose.
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  21. 21
    Shanmugasundaram, M.; Senthilvelan, A.; Kore, A. R. Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines. Chem. Rec. 2022, 22 (8), e202200005  DOI: 10.1002/tcr.202200005
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    21
    Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines
    Shanmugasundaram, Muthian; Senthilvelan, Annamalai; Kore, Anilkumar R.
    Chemical Record (2022), 22 (8), e202200005CODEN: CRHEAK; ISSN:1528-0691. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review on The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS-CoV-2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chem. modified mRNA cap analogs contain a unique cap structure, m7G[5']ppp[5']N (where N=G, A, C or U), present at the 5'-end of many eukaryotic cellular and viral RNAs and several non-coding RNAs. The chem. modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide contg. a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog contg. N1 modification, cap analog contg. N2 modification, dinucleotide contg. fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addn., the biol. applications of these novel cap analogs are delineated.
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  22. 22
    Chan, S. H.; Whipple, J. M.; Dai, N.; Kelley, T. M.; Withers, K.; Tzertzinis, G.; Corrêa, I. R., Jr.; Robb, G. B. RNase H-based analysis of synthetic mRNA 5′ cap incorporation. RNA 2022, 28 (8), 1144– 1155,  DOI: 10.1261/rna.079173.122
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    22
    RNase H-based analysis of synthetic mRNA 5' cap incorporation
    Chan, S. Hong; Whipple, Joseph M.; Dai, Nan; Kelley, Theresa M.; Withers, Kathryn; Tzertzinis, George; Correa, Ivan R., Jr.; Robb, G. Brett
    RNA (2022), 28 (8), 1144-1155CODEN: RNARFU; ISSN:1469-9001. (Cold Spring Harbor Laboratory Press)
    Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the crit. quality attributes in mRNA manufg. RNA cap anal. involves multiple steps: generation of predefined short fragments from the 5' end of the kilobase-long synthetic mRNA mols. using RNase H, a ribozyme or a DNAzyme, enrichment of the 5' cleavage products, and LC-MS intact mass anal. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purifn. using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5' cap anal. using less resource-intensive anal. methods, we hope to make RNA cap anal. more accessible to the scientific community.
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  23. 23
    Lapham, J.; Crothers, D. M. RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation. RNA 1996, 2 (3), 289– 296
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    23
    RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation
    Lapham, Jon; Crothers, Donald M.
    RNA (1996), 2 (3), 289-96CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
    Large quantities of RNA for study by NMR and x-ray crystallog. can be produced by transcription reactions in vitro using T7 bacteriophage RNA polymerase. A limitation on producing RNA with this polymerase has been the strong dependence of the yield of the transcription reaction on the sequence at the 5' end of the RNA produced. We report a procedure for obtaining large quantities of enzymically synthesized RNA from T7 RNA polymerase that has no dependence on the 5' end sequence of the target RNA. RNase H has been shown previously (Inoue H, Hayase Y, Iwai S, Ohtsuka E, 1987, FEBS Lett 215:327-330) to cleave RNA site specifically using 2'-O-Me RNA/DNA chimeras to direct the cleavage site. We show that 2'-O-Me RNA nucleotides on the 5'-side of the DNA nucleotides in the chimera are not essential for site-specific cleavage. This allowed us to design the method such that the same 2'-O-Me chimera may be used to process any RNA sequence. We have adapted this reaction to the cleavage of NMR-scale quantities of RNA at high yield. RNA is synthesized using T7 RNA polymerase with a 15-nt high-yielding leader sequence at the 5' end, and then this sequence is cleaved off with the RNase H cleavage reaction. The cleaved RNA has 3'-hydroxyl and 5'-phosphate ends, so that the products can be used directly as substrates for ligation by T4 DNA ligase. We show that the cleavage reaction occurs efficiently in soln. and on a solid streptavidin/agarose matrix. We report an example in which we are able to improve transcription yield by more than five-fold using this technique in the synthesis of a 15N isotopically labeled hairpin found in the Crithidia fasciculata spliced leader RNA. We were able to obtain a 0.5-mM NMR sample from this inherently poorly transcribing sequence, while minimizing the amt. of isotopically labeled rNTPs used to produce it. The NMR spectroscopic results are consistent with the predicted RNA secondary structure.
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  24. 24
    Zhang, H.; He, L.; Cai, L. Transcriptome Sequencing: RNA-Seq. Methods Mol. Biol. 2018, 1754, 15– 27,  DOI: 10.1007/978-1-4939-7717-8_2
    Google Scholar
    24
    Transcriptome sequencing: RNA-seq
    Zhang, Hong; He, Lin; Cai, Lei
    Methods in Molecular Biology (New York, NY, United States) (2018), 1754 (Computational Systems Biology), 15-27CODEN: MMBIED; ISSN:1940-6029. (Springer)
    A review. RNA sequencing (RNA-seq) can not only be used to identify the expression of common or rare transcripts but also in the identification of other abnormal events, such as alternative splicing, novel transcripts, and fusion genes. In principle, RNA-seq can be carried out by almost all of the next-generation sequencing (NGS) platforms, but the libraries of different platforms are not exactly the same; each platform has its own kit to meet the special requirements of the instrument design.
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  25. 25
    Goyon, A.; Scott, B.; Kurita, K.; Maschinot, C.; Meyer, K.; Yehl, P.; Zhang, K. On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC-MS System. Anal. Chem. 2022, 94 (2), 1169– 1177,  DOI: 10.1021/acs.analchem.1c04350
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    25
    On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC-MS System
    Goyon, Alexandre; Scott, Brandon; Kurita, Kenji; Maschinot, Chad; Meyer, Kevin; Yehl, Peter; Zhang, Kelly
    Analytical Chemistry (Washington, DC, United States) (2022), 94 (2), 1169-1177CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    In this study, for the first time, the automated digestion and sequencing of an RNA mol. via the use of immobilized RNase cartridges attached to a multidimensional liq. chromatog. (LC)-mass spectrometry (MS) system are presented. We first developed an online digestion-HILIC two-dimensional (2D)-LC-MS method in order to sequence CRISPR guide RNAs for gene editing. Three RNases (T1, A, and U2) were immobilized on polyetheretherketone cartridges, and their performance was evaluated. Ultrafast digestions were performed within 2.3 min with the online approach vs. 30 min via the conventional off-line approach. The higher sequence coverage was achieved by the RNase T1 (71%), which is the same as the off-line mode. A 20-fold redn. in the gRNA sample amt. was achieved with the online digestion approach (6.5μg) in comparison to that with the off-line approach (130μg). In the second step, a three-dimensional (3D)-LC-MS method was developed for the sequencing of fractions collected online across the main peak and the partially sepd. tail by the ref. ion-pairing RPLC method. Addnl. insights were gained in order to better understand the cause of the main peak tailing.
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  26. 26
    Carrilho, E.; Ruiz-Martinez, M. C.; Berka, J.; Smirnov, I.; Goetzinger, W.; Miller, A. W.; Brady, D.; Karger, B. L. Rapid DNA sequencing of more than 1000 bases per run by capillary electrophoresis using replaceable linear polyacrylamide solutions. Anal. Chem. 1996, 68 (19), 3305– 3313,  DOI: 10.1021/ac960411r
    Google Scholar
    26
    Rapid DNA Sequencing of More Than 1000 Bases per Run by Capillary Electrophoresis Using Replaceable Linear Polyacrylamide Solutions
    Carrilho, Emanuel; Ruiz-Martinez, Marie C.; Berka, Jan; Smirnov, Igor; Goetzinger, Wolfgang; Miller, Arthur W.; Brady, David; Karger, Barry L.
    Analytical Chemistry (1996), 68 (19), 3305-3313CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solns. has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and sepn. conditions, and enhanced base-calling software. The influences of LPA mol. wt. and concn. on sepn. were investigated. Addnl., the sepn. buffer, column temp., and elec. field were adjusted to increase the no. of resolvable DNA fragments per run while maintaining an enhanced sepn. speed. Using low concns. [2% (w/v)] of high mol. wt. LPA polymers (>5.5 × 106 Da), elevated column temp. (50 °C) and moderately high field (150 V/cm), rapid sequencing anal. for more than 1000 bases on a Model ssM13mp18 template was obtained with 96.8% accuracy. Base-calling software contributed to the high accuracy at these read lengths.
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  27. 27
    Kleparnik, K.; Foret, F.; Berka, J.; Goetzinger, W.; Miller, A. W.; Karger, B. L. The use of elevated column temperature to extend DNA sequencing read lengths in capillary electrophoresis with replaceable polymer matrices. Electrophoresis 1996, 17 (12), 1860– 1866,  DOI: 10.1002/elps.1150171210
    Google Scholar
    27
    The use of elevated column temperature to extend DNA sequencing read lengths in capillary electrophoresis with replaceable polymer matrixes
    Kleparnik, Karel; Foret, Frantisek; Berka, Jan; Goetzinger, Wolfgang; Miller, Arthur W.; Karger, Barry L.
    Electrophoresis (1996), 17 (12), 1860-1866CODEN: ELCTDN; ISSN:0173-0835. (VCH)
    Capillary electrophoresis with a replaceable linear polyacrylamide matrix operated at elevated column temps. of 55° and 60° was used to extend the sepn. of DNA sequencing fragments to lengths greater than 800 bases. A solid-state heater was employed to provide stable, uniform temp. control over a significant portion of the capillary. The polymer matrix, 3% w/v linear polyacrylamide in a denaturing buffer, was replaced in the capillary after each run. Using dye-labeled primers and Sequenase chem. on a M13mp18 single-stranded template, four-color sepns. for the sequencing products were obtained, with read lengths in excess of 800 bases. This paper also briefly discusses the effects of buffer denaturants and capillary temp. on sepn. speed, resoln., and gel compression.
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  28. 28
    Huber, C. G.; Stimpf, E.; Oefner, P. J.; Bonn, G. K. A comparison of micropellicular anion-exchange and reversed-phase stationary phases for HPLC analysis of oligonucleotides. LC-GC 1996, 14 (February), 114– 127
    Google Scholar
    28
    A comparison of micropellicular anion-exchange and reversed-phase stationary phases for HPLC analysis of oligonucleotides
    Huber, C. G.; Stimpf, E.; Oefner, P. J.; Bonn, G. K.
    LC-GC (1996), 14 (2), 114, 116, 118, 120, 124, 127CODEN: LCGCE7; ISSN:0888-9090. (Advanstar)
    To exploit the benefits of fast mass transfer, high column efficiency, and short anal. times of chromatog. sepns. of oligonucleotides with micropellicular packings, the authors used monodisperse 2-μd dp silica particles and 2.3-μm dp poly(styrene-divinylbenzene) (PS-DVB) particles as supports for anion-exchange and reversed-phase packing materials. They synthesized anion exchangers by amination and subsequent quaternization of PS-DVB or by coating silica gel with polyethylenimine. The alkylation of PS-DVB yielded a highly efficient packing material for ion-pair reversed-phase chromatog. of oligonucleotides that permitted single-base resoln. of homooligonucleotides with lengths of at least 80 nucleotides. They found that oligonucleotide retention depended on both its size and base compn. in anion-exchange and ion-pair reversed-phase modes. The authors also evaluated different packings designed for the anal. of synthetic and radiolabeled oligonucleotides.
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  29. 29
    Roussis, S. G.; Pearce, M.; Rentel, C. Small alkyl amines as ion-pair reagents for the separation of positional isomers of impurities in phosphate diester oligonucleotides. J. Chromatogr. A 2019, 1594, 105– 111,  DOI: 10.1016/j.chroma.2019.02.026
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    29
    Small alkyl amines as ion-pair reagents for the separation of positional isomers of impurities in phosphate diester oligonucleotides
    Roussis, Stilianos G.; Pearce, Megan; Rentel, Claus
    Journal of Chromatography A (2019), 1594 (), 105-111CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
    In an effort to improve sepn. of impurities in oligonucleotide drugs, alkyl amines of different length and carbon content were evaluated as reagents in ion pair-reversed phase (IP-RP) HPLC with mass spectrometric detection. A range of columns was tested in combination with different buffers, ion-pair modifiers and varying pH adjustments. For phosphorothioate oligonucleotides, larger amines, like tri-Bu and hexyl amine provided the best chromatog., as small amines tended to broaden peaks due to the sepn. of diastereoisomers. For phosphate diester oligonucleotides, the best sepns. were obtained using small alkyl amines, like propyl-, isopropyl- and diethylamine. Conditions optimized for oligonucleotide sequence and type of impurity enabled full sepn. of the individual components of composite impurities, such as n-1, N3-(2-cyanoethyl)thymine (CNET), deaminated and 3-(2-oxopropyl)imidazopyrimidinone (OPC) impurities. The addn. of long-chain alkyl acids like hexanoic acid to the IP buffer resulted in further improvements in peak sepn.
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  30. 30
    Thayer, J. R.; Barreto, V.; Rao, S.; Pohl, C. Control of oligonucleotide retention on a pH-stabilized strong anion exchange column. Anal. Biochem. 2005, 338 (1), 39– 47,  DOI: 10.1016/j.ab.2004.11.013
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    30
    Control of oligonucleotide retention on a pH-stabilized strong anion exchange column
    Thayer, J. R.; Barreto, Victor; Rao, Srinivasa; Pohl, Christopher
    Analytical Biochemistry (2005), 338 (1), 39-47CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
    Strong anion exchange columns are preferred for oligonucleotide analyses due to their ability to effectively control secondary structure and poly(G) interactions. Methacrylate-based anion exchange phases minimize hydrophobic interactions with oligonucleotides, but they also tend to hydrolyze under alk. conditions. In this article, we report the use of an anion exchange column prepd. from a new class of methacrylate monomers designed to improve hydrolytic stability. This column is used to show predictable adjustment of oligonucleotide retention by eluent pH and compn. Features of the new column include (i) large, predictable, pH-dependent retention shifts (varying with specific changes in 5' or 3' terminal bases with NaCl-based eluents); (ii) reduced retention when solvent is added to NaCl-based eluents; and (iii) suppression of much of the column's hydrophobic interactions when CH3CN is used with NaClO4-based eluents at a neutral pH (i.e., this eluent system separates oligonucleotides primarily in order of their length). These observations will aid the development of elution conditions for both size-dependent and base sequence-dependent (or base compn.-dependent) sepns.
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  31. 31
    Thayer, J. R., High-Resolution Separation of Oligonucleotides on a Pellicular Anion-Exchange Column. Thermo Scientific Application note 21996 , 2014.
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    There is no corresponding record for this reference.
  32. 32
    Gilar, M.; Fountain, K. J.; Budman, Y.; Neue, U. D.; Yardley, K. R.; Rainville, P. D.; Russell, R. J., 2nd; Gebler, J. C. Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction. J. Chromatogr. A 2002, 958 (1–2), 167– 182,  DOI: 10.1016/S0021-9673(02)00306-0
    Google Scholar
    32
    Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: Retention prediction
    Gilar, Martin; Fountain, Kenneth J.; Budman, Yeva; Neue, Uwe D.; Yardley, Kurt R.; Rainville, Paul D.; Russell, Reb J., II; Gebler, John C.
    Journal of Chromatography A (2002), 958 (1-2), 167-182CODEN: JCRAEY; ISSN:0021-9673. (Elsevier Science B.V.)
    An ion-pair reversed-phase HPLC method was evaluated for the sepn. of synthetic oligonucleotides. Mass transfer in the stationary phase is a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 μm), elevated temp. and a relatively slow flow-rate were used to enhance mass transfer. A short 50 mm column allows for an efficient sepn. up to 30mer oligonucleotides. The sepn. strategy consists of a shallow linear gradient of org. modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases were traditionally used for oligonucleotide sepns. with good result. However, the oligonucleotide retention is affected by its nucleotide compn. The authors developed a math. model for the prediction of oligonucleotide retention from sequence and length. The authors used the model successfully to select the optimal initial gradient strength for fast HPLC purifn. of synthetic oligonucleotides. The authors also used ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aq. buffers are useful for a highly efficient and less sequence-dependent sepn. of heterooligonucleotides.
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  33. 33
    Gilar, M. Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection. Anal. Biochem. 2001, 298 (2), 196– 206,  DOI: 10.1006/abio.2001.5386
    Google Scholar
    33
    Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection
    Gilar, Martin
    Analytical Biochemistry (2001), 298 (2), 196-206CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
    Native and modified synthetic oligonucleotides were purified by reversed-phase HPLC using volatile ion-pairing mobile phases. Purifn. of 10-90 nmol of oligonucleotides in a single injection was demonstrated using a 4.6×75-mm HPLC column packed with porous 2.5 μm C18 sorbent. Sepn. of target products from N-1 failure fragments was achieved for oligonucleotides in the 4- to 60-mer size range. We employed a combination of absorbance and mass spectrometry detection to identify byproducts of oligonucleotide synthesis. This method was also employed for anal. and purifn. of fluorescently labeled oligonucleotides. (c) 2001 Academic Press.
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  34. 34
    Donegan, M.; Nguyen, J. M.; Gilar, M. Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides. J. Chromatogr. A 2022, 1666, 462860  DOI: 10.1016/j.chroma.2022.462860
    Google Scholar
    34
    Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides
    Donegan, Michael; Nguyen, Jennifer M.; Gilar, Martin
    Journal of Chromatography A (2022), 1666 (), 462860CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
    We performed a systematic study of thirteen alkylamines used as ion-pairing reagents for ion-pair reversed-phase liq. chromatog. (IP RP LC) sepns. of oligonucleotides on a C18 column. We proposed a method to classify the hydrophobicity of alkylamines by their retention in RP LC. The IP reagent hydrophobicity correlated with the retention and resoln. of oligonucleotides in the corresponding IP mobile phases. The baseline resoln. was achieved up to 30 mer for hydrophilic, or up to 50 mer for hydrophobic IP reagents. Hydrophobic alkylamines permitted useful oligonucleotide sepns. at relatively low buffer concns., such as 5-10 mM alkylamine-acetate IP systems. These buffers were compatible with mass spectrometry detection, however, replacement of acetic acid with hexafluoroisopropanol in the mobile phase improved the MS signal by 2-3 orders of magnitude. Expts. with native and chem. modified oligonucleotides highlighted the mixed-mode nature of IP RP LC. When using hydrophobic IP reagents, the ionic retention mechanism of oligonucleotides is enhanced while hydrophobic retention is diminished.
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  35. 35
    Huber, C. G.; Oefner, P. J.; Bonn, G. K. Rapid and Accurate Sizing of DNA Fragments by Ion-Pair Chromatography on Alkylated Nonporous Poly(styrene-divinylbenzene) Particles. Anal. Chem. 1995, 67, 578– 585,  DOI: 10.1021/ac00099a015
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    35
    Rapid and Accurate Sizing of DNA Fragments by Ion-Pair Chromatography on Alkylated Nonporous Poly(styrene-divinylbenzene) Particles
    Huber, Christian G.; Oefner, Peter J.; Bonn, Guenther K.
    Analytical Chemistry (1995), 67 (3), 578-85CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Ion-pair reversed-phase HPLC on nonporous alkylated poly(styrene-divinylbenzene) particles allowed the sepn. of double-stranded DNA fragments in a few minutes with a resoln. comparable to that obtained in slab or capillary gel electrophoresis. Using gradients of acetonitrile in 0.1M triethylammonium acetate, DNA fragments differing in length only by 2-3% could be resolved. Synthesis of the stationary phase was very reproducible, and equilibration of a freshly packed column for at least 2 h proved to be necessary for optimum column performance, whereas column regeneration for 1-5 min was adequate between gradient runs. Various DNA mol. wt. markers ranging from 51 to 2176 base pairs in length were used to study the size dependence of retention. A plot of capacity factors vs. logarithm of mol. wts. revealed a clear correlation between size and retention. Both a local and a global approxn. method, which differed with regard to the no. of known std. fragments used below and above the fragments with unknown length to create a sizing curve based on simple linear regression, gave essentially identical results, with the inaccuracy of length measurement being 0.05-3.2%. This compares favorably with the accuracy obtained in gel electrophoresis.
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  36. 36
    Kuwayama, T.; Ozaki, M.; Shimotsuma, M.; Hirose, T. Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores. Anal. Sci. 2023, 39 (3), 417– 425,  DOI: 10.1007/s44211-022-00253-w
    Google Scholar
    36
    Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores
    Kuwayama, Tomomi; Ozaki, Makoto; Shimotsuma, Motoshi; Hirose, Tsunehisa
    Analytical Sciences (2023), 39 (3), 417-425CODEN: ANSCEN; ISSN:1348-2246. (Springer)
    Messenger ribonucleic acids (mRNAs) have been used in vaccines for various diseases and are attracting attention as a new pharmaceutical paradigm. The purifn. of mRNAs is necessary because various impurities, such as template DNAs and transcription enzymes, remain in the crude product after mRNA synthesis. Among the various purifn. methods, reversed-phase high-performance liq. chromatog. (RP-HPLC) is currently attracting attention. Herein, we optimized the pore size of the packing materials, the mobile phase compn., and the temp. of the process; we also evaluated changes in the sepn. patterns of RNA strands of various lengths via RP-HPLC. Addnl., single-stranded (50-1000 nucleotides in length) and double-stranded (80-500 base pairs in length) RNAs were sepd. while their non-denatured states were maintained by performing the anal. at 60°C using triethylammonium acetate as the mobile phase and octadecyl-based RNA-RP1 with super-wide pores (> 30 nm) as the column. Furthermore, impurities in a long-stranded RNA of several thousand nucleotides synthesized by in vitro transcription were successfully sepd. using an RNA-RP1 column. The columns used in this study are expected to sep. various RNA strands and the impurities contained in them.
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  37. 37
    Barth, H. G.; Boyes, B. E.; Jackson, C. Size exclusion chromatography. Anal. Chem. 1994, 66 (12), 595– 620,  DOI: 10.1021/ac00084a022
    Google Scholar
    37
    Size Exclusion Chromatography
    Barth, Howard G.; Jackson, Christian; Boyes, Barry E.
    Analytical Chemistry (1994), 66 (12), 595R-620RCODEN: ANCHAM; ISSN:0003-2700.
    A review, with many refs., is given on band broadening, non-size exclusion chromatog., detectors, packings, compositional heterogeneity, physicochem. studies, microcolumn SEC, preparative SEC, coupled column/column switching, automation/quality control, and selected applications.
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  38. 38
    Irvine, G. B.; Irvine, G. B., Determination of molecular size by size-exclusion chromatography (gel filtration). In Current Protocols in Cell Biology; Wiley, 2001, Chapter 5, Unit 5.5.
    Google Scholar
    There is no corresponding record for this reference.
  39. 39
    Munholland, J. M.; Bright, K. A.; Nazar, R. N. Use of a volatile buffer system in ion-exchange high-performance liquid chromatography of oligonucleotides. Anal. Biochem. 1989, 178 (2), 320– 323,  DOI: 10.1016/0003-2697(89)90645-3
    Google Scholar
    39
    Use of a volatile buffer system in ion-exchange high-performance liquid chromatography of oligonucleotides
    Munholland, Janet M.; Bright, Karen A.; Nazar, Ross N.
    Analytical Biochemistry (1989), 178 (2), 320-3CODEN: ANBCA2; ISSN:0003-2697.
    A volatile buffer was adapted for use with ion-exchange HPLC in the anal. and prepn. of oligonucleotides. The system employs a com. weakly basic anion-exchange column contg. DEAE-derivatized silica gel and eluted with a volatile buffer gradient of triethylamine acetate and acetonitrile. Nucleic acid digests and oligonucleotides synthesized by chem. or enzymic methods can be analyzed or purified with nearly quant. recovery following solvent volatilization.
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  40. 40
    Lukavsky, P. J.; Puglisi, J. D. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 2004, 10 (5), 889– 893,  DOI: 10.1261/rna.5264804
    Google Scholar
    40
    Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides
    Lukavsky, Peter J.; Puglisi, Joseph D.
    RNA (2004), 10 (5), 889-893CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
    We present a fast and simple protocol for large-scale prepn. and purifn. of RNA oligonucleotides. RNA oligonucleotides are prepd. by in vitro transcription with T7 RNA polymerase from linearized plasmid DNA templates constructed by PCR. In place of denaturing polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatog. is employed to purify the RNA oligonucleotide from the transcription mixt. yielding >99% pure RNA product. In contrast to PAGE-based purifn., the gel filtration method does not require denaturation of the RNA oligonucleotide, which is desirable for larger RNAs, and the product is free of low-mol.-wt. acrylamide contaminants, which greatly benefits NMR, crystallog., and other biophys. studies of large RNAs and RNA-protein complexes.
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  41. 41
    Shimoyama, A.; Fujisaka, A.; Obika, S. Evaluation of size-exclusion chromatography for the analysis of phosphorothioate oligonucleotides. J. Pharm. Biomed. Anal. 2017, 136, 55– 65,  DOI: 10.1016/j.jpba.2016.12.036
    Google Scholar
    41
    Evaluation of size-exclusion chromatography for the analysis of phosphorothioate oligonucleotides
    Shimoyama, Atsuko; Fujisaka, Aki; Obika, Satoshi
    Journal of Pharmaceutical and Biomedical Analysis (2017), 136 (), 55-65CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
    We evaluated size exclusion chromatog. (SEC) for the detection of high-order structure of phosphorothioate oligonucleotides (PS-oligo). Because of strong interaction between PS-oligo and column packing material, peaks were broader and elution time was longer than those of the corresponding natural DNA oligonucleotides. However, single- and double-stranded structures of PS-oligo were clearly sepd. and discriminated, while single-stranded with high-order structures such as G-quadruplex and hairpin structure were not distinguished from each other.
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  42. 42
    Kim, I.; McKenna, S. A.; Viani Puglisi, E.; Puglisi, J. D. Rapid purification of RNAs using fast performance liquid chromatography (FPLC). RNA 2007, 13 (2), 289– 894,  DOI: 10.1261/rna.342607
    Google Scholar
    42
    Rapid purification of RNAs using fast performance liquid chromatography (FPLC)
    Kim, Insil; Mckenna, Sean A.; Puglisi, Elisabetta Viani; Puglisi, Joseph D.
    RNA (2007), 13 (2), 289-294CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
    We present here an improved RNA purifn. method using fast performance liq. chromatog. (FPLC) size-exclusion chromatog. in place of denaturing PAGE. The method allows prepn. of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present std. curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can sep. monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodol. allows simple and rapid purifn. of RNA oligonucleotides for structural and biophys. studies.
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  43. 43
    Largy, E.; Mergny, J. L. Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism. Nucleic Acids Res. 2014, 42 (19), e149  DOI: 10.1093/nar/gku751
    Google Scholar
    43
    Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism
    Largy, Eric; Mergny, Jean-Louis
    Nucleic Acids Research (2014), 42 (19), e149/1-e149/15CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    In recent years, an increasing no. of reports have been focused on the structure and biol. role of non-canonical nucleic acid secondary structures. Many of these studies involve the use of oligonucleotides that can often adopt a variety of structures depending on the exptl. conditions, and hence change the outcome of an assay. The knowledge of the structure(s) formed by oligonucleotides is thus crit. to correctly interpret the results, and gain insight into the biol. role of these particular sequences. Herein we demonstrate that size-exclusion HPLC (SE-HPLC) is a simple yet surprisingly powerful tool to quickly and effortlessly assess the secondary structure(s) formed by oligonucleotides. For the first time, an extensive calibration and validation of the use of SE-HPLC to confidently detect the presence of different species displaying various structure and/or molecularity, involving >110 oligonucleotides forming a variety of secondary structures (antiparallel, parallel, A-tract bent and mismatched duplexes, triplexes, G-quadruplexes and i-motifs, RNA stem loops), is performed. Moreover, we introduce simple metrics that allow the use of SE-HPLC without the need for a tedious calibration work. We show that the remarkable versatility of the method allows to quickly establish the influence of a no. of exptl. parameters on nucleic acid structuration and to operate on a wide range of oligonucleotide concns. Case studies are provided to clearly illustrate the all-terrain capabilities of SE-HPLC for oligonucleotide secondary structure anal. Finally, this manuscript features a no. of important observations contributing to a better understanding of nucleic acid structural polymorphism.
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  44. 44
    Gilar, M.; Belenky, A.; Budman, Y.; Smisek, D. L.; Cohen, A. S. Study of phosphorothioate-modified oligonucleotide resistance to 3′-exonuclease using capillary electrophoresis. J. Chromatogr. B Biomed. Sci. Appl. 1998, 714 (1), 13– 20,  DOI: 10.1016/S0378-4347(98)00160-1
    Google Scholar
    44
    Study of phosphorothioate-modified oligonucleotide resistance to 3'-exonuclease using capillary electrophoresis
    Gilar, Martin; Belenky, Alexei; Budman, Yeva; Smisek, David L.; Cohen, Aharon S.
    Journal of Chromatography B: Biomedical Sciences and Applications (1998), 714 (1), 13-20CODEN: JCBBEP; ISSN:0378-4347. (Elsevier Science B.V.)
    The effect of phosphorothioate (PS) internucleotide linkages on the stability of phosphodiester oligodeoxyribonucleotides (ODNs) was investigated using 25-mer ODNs contg. single or multiple PS backbone modifications. The in vitro stability of the oligomers was measured both in 3'-exonuclease soln. and in plasma. For the sepn. of ODNs, capillary electrophoresis with a replaceable polymer sepn. matrix was used. As expected, DNA fragments with PS linkages at the 3'-end were found to be more resistant to 3'-exonuclease hydrolysis. Also increasing exonuclease resistance was the non-specific adsorption of phosphorothioate ODNs to enzyme.
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  45. 45
    Gilar, M.; Bouvier, E. S. P. Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography. J. Chromatogr. A 2000, 890 (1), 167– 177,  DOI: 10.1016/S0021-9673(00)00521-5
    Google Scholar
    45
    Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography
    Gilar, M.; Bouvier, E. S. P.
    Journal of Chromatography A (2000), 890 (1), 167-177CODEN: JCRAEY; ISSN:0021-9673. (Elsevier Science B.V.)
    Purifn. of target oligodeoxyribonucleotides from failure sequence byproducts of synthesis is often required for polymerase chain reaction primers, DNA sequencing and other oligonucleotide applications. We have developed purifn. protocols based on a reversed-phase mechanism ("trityl on" purifn.) using a 96-well Oasis HLB extn. plate. The Oasis HLB sorbent combines excellent pH stability with a high loading capacity allowing for single-step purifn. of 0.2 μM scale synthesis. After sample loading and washing, the oligonucleotide trityl group is cleaved on the plate with 2% trifluoroacetic acid. Target DNA is eluted with acetonitrile-0.36 mM triethylamine acetate, pH 11.3 (10:90, vol./vol.). Typical yield of purified product is 60-95%. Final purity, measured by capillary gel electrophoresis, was found to be 90% or greater. Alternatively, highly pure oligonucleotides can be obtained by a RP-HPLC "trityl off" method using an XTerra C18 column. The use of volatile triethylamine acetate buffer as an ion-pair for RP-HPLC eliminates the need for further desalting.
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  46. 46
    Thayer, J. R.; Wu, Y.; Hansen, E.; Angelino, M. D.; Rao, S. Separation of oligonucleotide phosphorothioate diastereoisomers by Pellicular anion-exchange chromatography. J. Chromatogr. A 2011, 1218 (6), 802– 808,  DOI: 10.1016/j.chroma.2010.12.051
    Google Scholar
    46
    Separation of oligonucleotide phosphorothioate diastereoisomers by pellicular anion-exchange chromatography
    Thayer, James R.; Wu, Yansheng; Hansen, Erik; Angelino, Mark D.; Rao, Srinivasa
    Journal of Chromatography A (2011), 1218 (6), 802-808CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
    Synthetic oligonucleotides (ONs) are often prepd. for development of therapeutic candidates. Among the modifications most often incorporated into therapeutic ONs are phosphorothioate (PT) linkages. The PT linkage introduces an addnl. chiral center at phosphorus to the chiral centers in D-ribose (and 2-deoxy-D-ribose) of the nucleic acid. Therefore, modified linkages can produce a diastereoisomer pair ([Rp] and [Sp]) at each PT linkage. These isomers are of identical length, sequence, charge and mass, and are not reliably sepd. by most chromatog. approaches (e.g., reversed phase chromatog.) unless the ON is very short. Further these isomers are not distinguishable by single-stage mass spectrometry. During chromatog. of a purified anti-NGF (nerve growth factor) aptamer contg. 37 bases with 2 PT linkages by monolithic pellicular anion-exchange (pAE) column, we obsd. four components. The four components were postulated to be: (i) distinct folding conformations; (ii) fully and partially athioated aptamers; or (iii) PT diastereoisomers. Fractionation of the components, followed by de- and re- naturation failed to produce the original forms by refolding, eliminating option (i). Mass spectrometry of the fractionated, desalted samples revealed no significant mass differences, eliminating option (ii). Oxidative conversion of the PT to phosphodiester (PO) linkages in each of the purified components produced a single chromatog. peak, co-eluting with authentic PO aptamer, and having the PO aptamer mass. We conclude that the components resolved by pAE chromatog. are diastereoisomers arising from the two PT linkages. Hence, pAE chromatog. further enhances characterization of ON therapeutics harboring limited PT linkages and having up to 37 bases.
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  47. 47
    Liu, R.; Ruan, Y.; Liu, Z.; Gong, L. The role of fluoroalcohols as counter anions for ion-pairing reversed-phase liquid chromatography/high-resolution electrospray ionization mass spectrometry analysis of oligonucleotides. Rapid Commun. Mass Spectrom. 2019, 33 (7), 697– 709,  DOI: 10.1002/rcm.8386
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    47
    The role of fluoroalcohols as counter anions for ion-pairing reversed-phase liquid chromatography/high-resolution electrospray ionization mass spectrometry analysis of oligonucleotides
    Liu, Rong; Ruan, Yanjiao; Liu, Zhongqiu; Gong, Lingzhi
    Rapid Communications in Mass Spectrometry (2019), 33 (7), 697-709CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Rationale : Hexafluoroisopropanol (HFIP) has been widely used as a counter anion in the mobile phase for ion-pairing reversed-phase liq. chromatog./mass spectrometry (IP-RP-LC/MS) anal. of oligonucleotides. However, researchers are still searching for improvements to counter anions for LC/MS anal. of oligonucleotides. This study aimed to find alternatives to HFIP for analyzing oligonucleotides. Methods : The study was performed using an Agilent 1290 ultra-high-performance liq. chromatog. (UHPLC) system coupled to an Agilent 6540 mass spectrometer by using an oligonucleotide BEH C18 column (100 × 2.1 mm, 1.7 μm). Buffer systems contg. ion-pairing reagents (triethylamine, tripropylamine, hexylamine, dimethylbutylamine, diisopropylethylamine, N,N-dimethylcyclohexylamine, and octylamine) and fluoroalcs. (HFIP and hexafluoro-2-methyl-2-propanol (HFTP)) were compared chromatog. and mass spectrometrically. Results : Results showed that HFTP has better desalting ability than HFIP, but both HFIP and HFTP have comparable effects on the sepn. of oligonucleotides sized from 10mer to 40mer for most of ion-pairing reagents, with the exception of triethylamine and N,N-dimethylcyclohexylamine, where HFIP performed better than HFTP. Conclusions : The choice of fluoroalcs. in IP-RP-LC/MS anal. of oligonucleotides depends on the type of ion-pairing reagents used in the mobile phase. As a guideline, we would recommend to use either HA-HFIP or HA-HFTP for small oligonucleotides, but TPA-HFTP for large oligonucleotides for IP-RP-LC/MS anal. of synthetic oligonucleotides.
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  48. 48
    Li, N.; El Zahar, N. M.; Saad, J. G.; van der Hage, E. R. E.; Bartlett, M. G. Alkylamine ion-pairing reagents and the chromatographic separation of oligonucleotides. J. Chromatogr. A 2018, 1580, 110– 119,  DOI: 10.1016/j.chroma.2018.10.040
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    48
    Alkylamine ion-pairing reagents and the chromatographic separation of oligonucleotides
    Li, Ning; El Zahar, N. M.; Saad, Jack G.; van der Hage, Erik R. E.; Bartlett, Michael G.
    Journal of Chromatography A (2018), 1580 (), 110-119CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
    Alkylamines are commonly used to improve both chromatog. and mass spectral performance of electrospray ionization liq. chromatog. mass spectrometry based methods for the anal. of oligonucleotides. Recently several new alkylamines have been introduced to enhance the electrospray mass spectral response for oligonucleotides; however, the chromatog. properties of these new alkylamines have not been rigorously assessed. We have investigated the retention, peak width, resoln. and general chromatog. performance of fifteen different alkylamines for the sepn. of a model DNA, RNA and an antisense therapeutic oligonucleotide. Eleven of the fifteen alkylamines were shown to provide similar chromatog. performance across all three classes of oligonucleotides. Based on these findings, a model for the mechanism of retention of oligonucleotides using alkylamines and hexafluoroisopropanol mobile phases is proposed. Depending on the concns. of alkylamines and pH adjustment, oligonucleotides can be retained by micellar chromatog. and not the generally held ion-pairing mechanism. This conclusion is supported by light scattering, transmission electron microscopy and ion mobility expts. detecting three micron aggregates in the mobile phase at concns. that are routinely used for LC-MS anal. of oligonucleotides. These aggregates are not detected at lower alkylamine concns. where the retention mechanism follows an ion-pairing mechanism. The formation of these aggregates appears to be dependent on the pH of the mobile phase.
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  49. 49
    Neue, U. D. HPLC columns: theory, technology, and practice. Wiley-VCH: New York, 1997.
    Google Scholar
    There is no corresponding record for this reference.
  50. 50
    Gilar, M.; DeLano, M.; Gritti, F. Mitigation of analyte loss on metal surfaces in liquid chromatography. J. Chromatogr. A 2021, 1650, 462247  DOI: 10.1016/j.chroma.2021.462247
    Google Scholar
    50
    Mitigation of analyte loss on metal surfaces in liquid chromatography
    Gilar, Martin; DeLano, Mathew; Gritti, Fabrice
    Journal of Chromatography A (2021), 1650 (), 462247CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
    The adsorptive loss of acidic analytes in liq. chromatog. was investigated using metal frits. Repetitive injections of acidic small mols. or an oligonucleotide were made on individual 2.1 or 4.6 mm i.d. column frits. Losses were obsd. for adenosine 5'-(α,β-methylene) diphosphate, 2-pyridinol 1-oxide and the 25-mer phosphorothioate oligonucleotide Trecovirsen (GEM91) on stainless steel and titanium frits. Analyte adsorption was greatest at acidic pH due to the pos. charge on the metal oxide surface. Analyte recovery increased when a series of injections was performed; this effect is known as sample conditioning. Nearly complete recovery was achieved when the metal adsorptive sites were satd. with the analyte. A similar effect was achieved by conditioning the frits with phosphoric, citric or etidronic acids, or their buffered solns. These procedures can be utilized to mitigate analyte loss. However, the effect is temporary, as the conditioning agent is gradually removed by the running mobile phase. Metal frits modified with hybrid org./inorg. surface technol. were shown to mitigate analyte-to-metal surface interactions and improve recovery of acidic analytes. Quant. recovery of a 15-35 mer oligodeoxythymidine mixt. was achieved using column hardware modified with hybrid surface technol., without a need for column conditioning prior to anal.
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  51. 51
    Guimaraes, G. J.; Sutton, J. M.; Gilar, M.; Donegan, M.; Bartlett, M. G. Impact of Nonspecific Adsorption to Metal Surfaces in Ion Pair-RP LC-MS Impurity Analysis of Oligonucleotides. J. Pharm. Biomed. Anal. 2022, 208, 114439  DOI: 10.1016/j.jpba.2021.114439
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    51
    Impact of Nonspecific Adsorption to Metal Surfaces in Ion Pair-RP LC-MS Impurity Analysis of Oligonucleotides
    Guimaraes, Guilherme J.; Sutton, J. Michael; Gilar, Martin; Donegan, Michael; Bartlett, Michael G.
    Journal of Pharmaceutical and Biomedical Analysis (2022), 208 (), 114439CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
    Nonspecific adsorption has been a consistent challenge in the anal. of oligonucleotides. Nonspecific adsorption is a result of interactions between charged acidic analytes and adsorption sites present in metallic surfaces located in the fluidic path of chromatog. systems. Due to their high surface area, adsorption to column frits is esp. concerning. Poor peak shape, low recovery and compromised LOQ have been assocd. with this phenomenon. Alternative methods including substitution of stainless steel for different hardware materials and mobile phase additives have been explored in an attempt to minimize this issue. Chem. modification of metal surfaces using hybrid surface technol. (HST) by-passes the limitation of stainless steel construction material by forming a hybrid org./inorg. layer that acts as a barrier and limits nonspecific interactions. In this study we explore the implications of this new technol. in sensitive anal. and detn. of relative impurity levels of oligonucleotides. Higher relative impurity levels and better reproducibility were obtained with columns using HST.
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  52. 52
    Tuytten, R.; Lemière, F.; Witters, E.; Van Dongen, W.; Slegers, H.; Newton, R. P.; Van Onckelen, H.; Esmans, E. L. Stainless steel electrospray probe: a dead end for phosphorylated organic compounds?. J. Chromatogr. A 2006, 1104 (1–2), 209– 221,  DOI: 10.1016/j.chroma.2005.12.004
    Google Scholar
    52
    Stainless steel electrospray probe: A dead end for phosphorylated organic compounds?
    Tuytten, R.; Lemiere, F.; Witters, E.; Van Dongen, W.; Slegers, H.; Newton, R. P.; Van Onckelen, H.; Esmans, E. L.
    Journal of Chromatography A (2006), 1104 (1-2), 209-221CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
    A study of the interaction of phosphorylated org. compds. with the stainless components of a liq. chromatog.-electrospray ionization-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten) likely pitfall in the LC-ESI-MS anal. of phosphorylated compds. The retention behavior of some representative compds. of different important classes of phosphorylated biomols. such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compds. when these compds. were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ∼pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realize adsorption on a stainless surface. Furthermore, the no. of phosphate moieties seemed to enhance the adsorption properties of the mols. and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was an important factor in the course of these adsorption phenomena.
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  53. 53
    Gilar, M.; Neue, U. D. Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides. J. Chromatogr. A 2007, 1169 (1–2), 139– 150,  DOI: 10.1016/j.chroma.2007.09.005
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    53
    Peak capacity in gradient reversed-phase liquid chromatography of biopolymers
    Gilar, Martin; Neue, Uwe D.
    Journal of Chromatography A (2007), 1169 (1-2), 139-150CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
    Reversed-phase ultra-performance liq. chromatog. was used for biopolymer sepns. in isocratic and gradient mode. The gradient elution mode was employed to est. the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 μm C18 sorbent is below 0.2 mL/min for our analytes. However, the max. peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the mol. wt. of the analyte. The isocratic sepn. mode was utilized to measure the dependence of the retention factor on the mobile phase compn. Consts. derived from isocratic expts. were utilized in a math. model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to exptl. results.
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  54. 54
    Fountain, K. J.; Gilar, M.; Gebler, J. C. Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17 (7), 646– 653,  DOI: 10.1002/rcm.959
    Google Scholar
    54
    Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry
    Fountain, Kenneth J.; Gilar, Martin; Gebler, John C.
    Rapid Communications in Mass Spectrometry (2003), 17 (7), 646-653CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Ion-pair reversed-phase high-performance liq. chromatog. (IP-RP-HPLC) was utilized in tandem with neg.-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the anal. of native and chem. modified oligonucleotides. Sepn. was performed on a 1.0 × 50 mm column packed with porous C18 sorbent with a particle size of 2.5 μm and an av. pore diam. of 140 Å. A method was developed which maximizes both chromatog. sepn. and mass spectrometric sensitivity using an optimized buffer system contg. triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol with a methanol gradient. The ESI-TOFMS tuning parameters were also optimized in order to minimize in-source fragmentation and achieve the best sensitivity. Analyses of native, phosphorothioate, and guanine-rich oligonucleotides were performed by LC/MS. Detection limits were at sub-picomole levels with an av. mass accuracy of 125 ppm. The described method allowed for the LC/MS anal. of oligonucleotides up to 110mer in length with little alkali cation adduction. Since sensitive detection of oligonucleotides was achieved with UV detection, we utilized a combination of UV-MS for quantitation (UV) and characterization (MS) of oligonucleotides and their failure sequence fragments/metabolites.
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  55. 55
    Koshel, B.; Birdsall, R.; Chen, W. Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2020, 1137, 121906  DOI: 10.1016/j.jchromb.2019.121906
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    55
    Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides
    Koshel, Brooke; Birdsall, Robert; Chen, Weibin
    Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2020), 1137 (), 121906CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
    Two-dimensional liq. chromatog. coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide anal. have been relatively unexplored. When analyzing oligonucleotides in one-dimension, selecting an ion-pairing agent often requires a balance between acceptable chromatog. and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the sepn. mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based sepn., but MS response is compromised due to ion suppression. 2D-LC/MS was used to show that dye-conjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional anal. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatog. in both the first and second dimension (IP-RP - IP-RP) was used to transfer the n-1 impurity from a length-based sepn. in the first-dimension to a second-dimension anal. for identity confirmation using a single quadrupole detector. Identical C18 column chem. was used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the two-dimensional anal. can be detected at low nanogram levels, comparable to results achieved in a one-dimensional diln. series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resoln. instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques.
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  56. 56
    Goyon, A.; Zhang, K. Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup. Anal. Chem. 2020, 92 (8), 5944– 5951,  DOI: 10.1021/acs.analchem.0c00114
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    56
    Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup
    Goyon, Alexandre; Zhang, Kelly
    Analytical Chemistry (Washington, DC, United States) (2020), 92 (8), 5944-5951CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Detn. of phosphorothioate oligonucleotide purity and impurity profile is commonly performed by ion-pairing reversed-phase liq. chromatog. (IPRP) with a mobile phase contg. triethylamine (TEA) and hexafluoro-2-propanol (HFIP). However, ion-suppressing effects of TEA hamper mass spectrometry (MS) instrumentation sensitivity and HFIP can affect the robustness of the mass spectrometer due to its corrosive nature. Anion exchange chromatog. (AEX) is an orthogonal sepn. mode to IPRP but typically cannot be directly coupled to MS. In this work, we developed a multiple heart-cutting IPRP-, AEX-hydrophilic interaction liq. chromatog.(HILIC)/MS method for quantification and high sensitivity identification of antisense oligonucleotide (ASO) impurities using a Q-Exactive mass spectrometer. Notably, both AEX-HILIC and IPRP-HILIC modes could be operated on a versatile two-dimensional liq. chromatog. (2D-LC) setup including several column selectors. The HILIC mobile phase contained 25 mM ammonium acetate and allowed identifying impurities at levels down to 0.3%. Careful selection of the sample loop vol. and the 2D HILIC column dimension allowed straightforward coupling of HILIC for both IPRP and AEX without the need to use any solvent modulation. Overall, the 2D HILIC allowed online desalting of AEX and IPRP modes and further sepn. of addnl. impurities.
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  57. 57
    Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Analysis of oligonucleotides by HPLC-electrospray ionization mass spectrometry. Anal. Chem. 1997, 69, 1320– 1325,  DOI: 10.1021/ac960916h
    Google Scholar
    57
    Analysis of Oligonucleotides by HPLC-Electrospray Ionization Mass Spectrometry
    Apffel, Alex; Chakel, John A.; Fischer, Steven; Lichtenwalter, Kay; Hancock, William S.
    Analytical Chemistry (1997), 69 (7), 1320-1325CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A new interface procedure was developed that allows, for the first time, the high-efficiency anal. of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and online electrospray-ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resoln. can be obtained with low levels of cation adduct formation in the neg. ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC sepn. and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compds. such as phosphorothioates and their metabolites, as well as of materials used in clin. trials.
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  58. 58
    DeLano, M.; Walter, T. H.; Lauber, M. A.; Gilar, M.; Jung, M. C.; Nguyen, J. M.; Boissel, C.; Patel, A. V.; Bates-Harrison, A.; Wyndham, K. D. Using Hybrid Organic-Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC. Anal. Chem. 2021, 93 (14), 5773– 5781,  DOI: 10.1021/acs.analchem.0c05203
    Google Scholar
    58
    Using Hybrid Organic-Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC
    DeLano, Mathew; Walter, Thomas H.; Lauber, Matthew A.; Gilar, Martin; Jung, Moon Chul; Nguyen, Jennifer M.; Boissel, Cheryl; Patel, Amit V.; Bates-Harrison, Andrew; Wyndham, Kevin D.
    Analytical Chemistry (Washington, DC, United States) (2021), 93 (14), 5773-5781CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Interactions of analytes with metal surfaces in high-performance liq. chromatog. (HPLC) instruments and columns have been reported to cause deleterious effects ranging from peak tailing to a complete loss of the analyte signal. These effects are due to the adsorption of certain analytes on the metal oxide layer on the surface of the metal components. We have developed a novel surface modification technol. and applied it to the metal components in ultra-HPLC (UHPLC) instruments and columns to mitigate these interactions. A hybrid org.-inorg. surface, based on an ethylene-bridged siloxane chem., was developed for use with reversed-phase and hydrophilic interaction chromatog. We have characterized the performance of UHPLC instruments and columns that incorporate this surface technol. and compared the results with those obtained using their conventional counterparts. We demonstrate improved performance when using the hybrid surface technol. for sepns. of nucleotides, a phosphopeptide, and an oligonucleotide. The hybrid surface technol. was found to result in higher and more consistent analyte peak areas and improved peak shape, particularly when using low analyte mass loads and acidic mobile phases. Reduced abundances of iron adducts in the mass spectrum of a peptide were also obsd. when using UHPLC systems and columns that incorporate hybrid surface technol. These results suggest that this technol. will be particularly beneficial in UHPLC/mass spectrometry investigations of metal-sensitive analytes.
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  59. 59
    Nguyen, J. M.; Gilar, M.; Koshel, B.; Donegan, M.; MacLean, J.; Li, Z.; Lauber, M. A. Assessing the impact of nonspecific binding on oligonucleotide bioanalysis. Bioanalysis 2021, 13, 1233,  DOI: 10.4155/bio-2021-0115
    Google Scholar
    59
    Assessing the impact of nonspecific binding on oligonucleotide bioanalysis
    Nguyen, Jennifer M.; Gilar, Martin; Koshel, Brooke; Donegan, Michael; MacLean, Jason; Li, Zhimin; Lauber, Matthew A.
    Bioanalysis (2021), 13 (16), 1233-1244CODEN: BIOAB4; ISSN:1757-6180. (Newlands Press Ltd.)
    Accurate and reliable quantification of oligonucleotides can be difficult, which has led to an increased focus on bioanal. methods for more robust analyses. Recent advances toward mitigating sample losses on liq. chromatog. (LC) systems have produced recovery advantages for oligonucleotide sepns. LC instruments and columns constructed from MP35N metal alloy and stainless steel columns were compared against LC hardware modified with hybrid inorg.-org. silica surfaces. Designed to minimize metal-analyte adsorption, these surfaces demonstrated a 73% increase in 25-mer phosphorothioate oligonucleotide recovery using ion-pairing reversed-phase LC vs. std. LC surfaces, most particularly upon initial use. Hybrid silica chromatog. surfaces improve the performance, detection limits and reproducibility of oligonucleotide bioanal. assays.
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  60. 60
    Birdsall, R. E.; Gilar, M.; Shion, H.; Yu, Y. Q.; Chen, W. Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/mass spectrometry analysis. Rapid Commun. Mass Spectrom. 2016, 30 (14), 1667– 1679,  DOI: 10.1002/rcm.7596
    Google Scholar
    60
    Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/mass spectrometry analysis
    Birdsall, Robert E.; Gilar, Martin; Shion, Henry; Yu, Ying Qing; Chen, Weibin
    Rapid Communications in Mass Spectrometry (2016), 30 (14), 1667-1679CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Electrospray ionization mass spectrometry (ESI-MS)-based techniques commonly used in oligonucleotide analyses are known to be sensitive to alkali metal adduct formation. Adducts directly impact the sensitivity of MS-based analyses as the available charge is distributed across the parent peak and adduct(s). The current study systematically evaluated common liq. chromatog. (LC) components in LC/ESI-MS configurations used in oligonucleotide anal. to identify metal adduct contributions from LC instrumentation. A UPLC liq. chromatog. system was configured with a single quadrupole MS detector (ACQUITY QDa, Waters Corp.) to monitor adduct formation in oligonucleotide sepns. An ion-pairing mobile phase comprised of 15 mM triethylamine and 400 mM hexafluoro-2-propanol was used in conjunction with an oligonucleotide sepn. column (Waters OST BEH C18, 2.1 mm × 50 mm) for all sepns. A 10-min method was used to provide statistical figures of merit and evaluate adduct formation over time. Trace alkali metal salts in the mobile phase and reagents are the main source of metal salt adducts in LC/ESI-MS-based configurations. Non-specific adsorption sites located throughout the fluidic path contribute to adduct formation in oligonucleotide analyses. Ion-pairing mobile phases prepd. at neutral or slightly basic pH result in up to a 57% loss of spectral abundance to adduct formation in the current study. Implementation of a short low pH reconditioning step was obsd. to effectively displace trace metal salts non-specifically adsorbed to surfaces in the fluidic path and was able to maintain an av. MS spectral abundance ≥94% with a high degree of repeatability (relative std. deviation 0.8%) over an extended time study. The proposed method offers the ability to rapidly regenerate adsorption sites with minimal impact on productivity while retaining assay sensitivity afforded by MS detection with reduced adduct formation.
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  61. 61
    Birdsall, R. E.; Kellett, J.; Yu, Y. Q.; Chen, W. Application of mobile phase additives to reduce metal-ion mediated adsorption of non-phosphorylated peptides in RPLC/MS-based assays. J. Chromatogr. B Analyt. Technol. Biomed. Life. Sci. 2019, 1126–1127, 121773  DOI: 10.1016/j.jchromb.2019.121773
    Google Scholar
    61
    Application of mobile phase additives to reduce metal-ion mediated adsorption of non-phosphorylated peptides in RPLC/MS-based assays
    Birdsall, Robert E.; Kellett, Jacob; Yu, Ying Qing; Chen, Weibin
    Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2019), 1126-1127 (), 121773CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
    Metal-ion mediated adsorption in liq. chromatog. has been identified as a contributing factor in poor peak shape, tailing, and diminished recovery of compds. prone to cation exchange-like interaction with metal-based activity sites. Peptides that exhibit neg. charge-bearing amino acids such as aspartic acid and glutamic acid are particularly sensitive to metal-ion mediated adsorption in RPLC/MS-based sepns. when using weak acids (e.g. formic acid) as mobile phase additives. Citric acid and medronic acid as metal complexing mobile phase additives were evaluated for their ability to mitigate metal-ion mediated adsorption in RPLC/MS-based peptide mapping assays. Chromatog. performance was stabilized with peak tailing for peptides of interest reduced by as much as 40% in the presence of a chelator at a mobile phase concn. of 1 ppm. Performance gains are stable over a 67-h time study with an av. USP tailing factor of 1.00, % relative std. deviation = 0.64. The stabilizing effect of the chelator improved peptide mapping assay robustness with relative peak areas for target impurities calcd. at 2.28% (% relative std. deviation = 2.36) and 2.40% (% relative std. deviation = 2.37). Collectively this study demonstrates that chelators as mobile phase additives offers a means to improve chromatog. performance for biomols. sensitive to metal-ion mediated adsorption under formic acid-based RPLC conditions.
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  • Abstract

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    Figure 1

    Figure 1. SEC separation of dA5–150 oligonucleotides using columns packed with 125 Å (A), 250 Å (B), and 450 Å (C) pore size sorbent. Panel (D) plots the experimental data as SEC calibration curves. 250 Å column has linear calibration for the 30–150 nt oligonucleotide range (see Figure 2A).

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    Figure 2

    Figure 2. Panel (A) compares SEC calibrations obtained for RNA (rA5–120) and DNA (rA5–150) oligonucleotides. SEC chromatograms of dA5–150, dT20–100, and rA10–120 synthetic oligonucleotide standards are shown in panels (B), (C), and (D), respectively. Dotted lines highlight the observed differences in the oligonucleotide SEC retention. Data were acquired with an ACQUITY Premier Protein SEC column, 250 Å, 1.7 μm, 4.6 × 150 mm.

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    Figure 3

    Figure 3. SEC analysis of intact FLuc-beta mRNA (1970 nt, red chromatogram) and FLuc-beta mRNA digested with RNase T1 (blue chromatogram; the peak eluting near 5 min represents poly(A) tail signal). Black chromatogram is SEC analysis of the rA120 oligonucleotide; the standard is contaminated with truncated synthetic impurities eluting within a peak tail.

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    Figure 4

    Figure 4. (A) IP RP LC analysis of the poly(A)tail released from EPO mRNA (blue chromatogram) and the FLuc-beta mRNA sample (red chromatogram) with RNase T1. IP RP LC analysis of the rA100 standard and its truncated oligonucleotides is shown as a black chromatogram. The retention data of 80, 85, 90, 95, 99, 100, and 101 nt rAn oligonucleotides were plotted as a calibration curve (panel B) and utilized to estimate the mRNA poly(A) tail species length. For additional data, see Figure S3.

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    Figure 5

    Figure 5. IP RP LC analysis of FLuc-beta mRNA poly(A) tail. (A) LC UV analysis, (B) LC MS LC MS total ion chromatogram (TIC), and (C) deconvolved ESI-MS spectrum of the peak eluting close to 6 min. This chromatographic peak corresponds to a mixture of heterogeneous poly(A) species with three most dominant MS signals corresponding to 126, 127, and 128 nt oligonucleotides.

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  • References

    This article references 61 other publications.

    1. 1
      Castells, M. C.; Phillips, E. J. Maintaining Safety with SARS-CoV-2 Vaccines. N. Engl. J. Med. 2021, 384 (7), 643– 649,  DOI: 10.1056/NEJMra2035343
      1
      Maintaining safety with SARS-CoV-2 vaccines
      Castells, Mariana C.; Phillips, Elizabeth J.
      New England Journal of Medicine (2021), 384 (7), 643-649CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
      A review. Careful vaccine-safety surveillance over time, paired with elucidation of mechanisms of adverse events across different SARS-CoV-2 vaccine platforms, will be needed to inform a strategic and systematic approach to vaccine safety. Indeed, mRNA vaccines are a promising new technol., and demonstration of their safety is relevant to the development of vaccines against several other viruses of global importance and many cancers. For the immediate future, during a pandemic that is still increasing, it is crit. that we focus on safe and efficient approaches to implementing mass vaccination.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXkvVGhurw%253D&md5=a42f84b5b88c4702df8e6668b928a426
    2. 2
      Gote, V.; Bolla, P. K.; Kommineni, N.; Butreddy, A.; Nukala, P. K.; Palakurthi, S. S.; Khan, W. A Comprehensive Review of mRNA Vaccines. Int. J. Mol. Sci. 2023, 24 (3), 2700,  DOI: 10.3390/ijms24032700
      2
      A Comprehensive Review of mRNA Vaccines
      Gote, Vrinda; Bolla, Pradeep Kumar; Kommineni, Nagavendra; Butreddy, Arun; Nukala, Pavan Kumar; Palakurthi, Sushesh Srivatsa; Khan, Wahid
      International Journal of Molecular Sciences (2023), 24 (3), 2700CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      A review. MRNA vaccines have been demonstrated as a powerful alternative to traditional conventional vaccines because of their high potency, safety and efficacy, capacity for rapid clin. development, and potential for rapid, low-cost manufg. These vaccines have progressed from being a mere curiosity to emerging as COVID-19 pandemic vaccine front-runners. The advancements in the field of nanotechnol. for developing delivery vehicles for mRNA vaccines are highly significant. In this review we have summarized each and every aspect of the mRNA vaccine. The article describes the mRNA structure, its pharmacol. function of immunity induction, lipid nanoparticles (LNPs), and the upstream, downstream, and formulation process of mRNA vaccine manufg. Addnl., mRNA vaccines in clin. trials are also described. A deep dive into the future perspectives of mRNA vaccines, such as its freeze-drying, delivery systems, and LNPs targeting antigen-presenting cells and dendritic cells, are also summarized.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXjsFajtLk%253D&md5=3f1a47449a68bc378e2c1f7a2fc0c678
    3. 3
      Watanabe, A.; Kani, R.; Iwagami, M.; Takagi, H.; Yasuhara, J.; Kuno, T. Assessment of Efficacy and Safety of mRNA COVID-19 Vaccines in Children Aged 5 to 11 Years: A Systematic Review and Meta-analysis. JAMA Pediatr. 2023, 177 (4), 384– 394,  DOI: 10.1001/jamapediatrics.2022.6243
      3
      Assessment of Efficacy and Safety of mRNA COVID-19 Vaccines in Children Aged 5 to 11 Years: A Systematic Review and Meta-analysis
      Watanabe Atsuyuki; Kani Ryoma; Iwagami Masao; Iwagami Masao; Takagi Hisato; Yasuhara Jun; Kuno Toshiki
      JAMA pediatrics (2023), 177 (4), 384-394 ISSN:.
      Importance: Evidence of the efficacy and safety of messenger RNA (mRNA) COVID-19 vaccines in children aged 5 to 11 years has been emerging. Collecting these data will inform clinicians, families, and policy makers. Objective: To evaluate the efficacy and safety of mRNA COVID-19 vaccines in children aged 5 to 11 years in a systematic review and meta-analysis. Data Sources: PubMed and Embase databases were searched on September 29, 2022, without language restrictions. Study Selection: Randomized clinical trials and observational studies comparing vaccinated vs unvaccinated children aged 5 to 11 years and reporting efficacy or safety outcomes were included. Studies reporting safety outcomes in vaccinated children only (ie, no control group) were also included. Data Extraction and Synthesis: Two investigators independently extracted relevant data from each study. Odds ratios (ORs) for efficacy and safety outcomes and incidences of adverse events (AEs) following vaccination were synthesized using a random-effects model. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses and Meta-analysis of Observational Studies in Epidemiology reporting guidelines. Main Outcomes and Measures: The primary outcome was SARS-CoV-2 infections with or without symptoms. The secondary outcomes included symptomatic SARS-CoV-2 infections, hospitalizations, and multisystem inflammatory syndrome in children. The incidences of each AE following vaccination were also evaluated. Results: Two randomized clinical trials and 15 observational studies involving 10 935 541 vaccinated children (median or mean age range, 8.0-9.5 years) and 2 635 251 unvaccinated children (median or mean age range, 7.0-9.5 years) were included. Two-dose mRNA COVID-19 vaccination compared with no vaccination was associated with lower risks of SARS-CoV-2 infections with or without symptoms (OR, 0.47; 95% CI, 0.35-0.64), symptomatic SARS-CoV-2 infections (OR, 0.53; 95% CI, 0.41-0.70), hospitalizations (OR, 0.32; 95% CI, 0.15-0.68), and multisystem inflammatory syndrome in children (OR, 0.05; 95% CI, 0.02-0.10). Two randomized clinical trials and 5 observational studies investigated AEs among vaccinated children. Most vaccinated children experienced at least 1 local AE following the first injection (32 494 of 55 959 [86.3%]) and second injection (28 135 of 46 447 [86.3%]). Vaccination was associated with a higher risk of any AEs compared with placebo (OR, 1.92; 95% CI, 1.26-2.91). The incidence of AEs that prevented normal daily activities was 8.8% (95% CI, 5.4%-14.2%) and that of myocarditis was estimated to be 1.8 per million (95% CI, 0.000%-0.001%) following the second injection. Conclusions and Relevance: In this systematic review and meta-analysis, COVID-19 mRNA vaccines among children aged 5 to 11 years were associated with measures of efficacy in preventing SARS-CoV-2 infection and severe COVID-19-related illnesses. While most children developed local AEs, severe AEs were rare, and most of AEs resolved within several days. These data provide evidence for future recommendations.
      >> More from SciFinder ®
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    4. 4
      Chaudhary, N.; Weissman, D.; Whitehead, K. A. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat. Rev. Drug Discovery 2021, 20 (11), 817– 838,  DOI: 10.1038/s41573-021-00283-5
      4
      mRNA vaccines for infectious diseases: principles, delivery and clinical translation
      Chaudhary, Namit; Weissman, Drew; Whitehead, Kathryn A.
      Nature Reviews Drug Discovery (2021), 20 (11), 817-838CODEN: NRDDAG; ISSN:1474-1776. (Nature Portfolio)
      A review. Abstr.: Over the past several decades, mRNA (mRNA) vaccines have progressed from a scepticism-inducing idea to clin. reality. In 2020, the COVID-19 pandemic catalyzed the most rapid vaccine development in history, with mRNA vaccines at the forefront of those efforts. Although it is now clear that mRNA vaccines can rapidly and safely protect patients from infectious disease, addnl. research is required to optimize mRNA design, intracellular delivery and applications beyond SARS-CoV-2 prophylaxis. In this Review, we describe the technologies that underlie mRNA vaccines, with an emphasis on lipid nanoparticles and other non-viral delivery vehicles. We also overview the pipeline of mRNA vaccines against various infectious disease pathogens and discuss key questions for the future application of this breakthrough vaccine platform.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVygtL%252FP&md5=6e3a059338985252dc07eeb71f7ae6ae
    5. 5
      Magadum, A.; Kaur, K.; Zangi, L. mRNA-Based Protein Replacement Therapy for the Heart. Mol. Ther. 2019, 27 (4), 785– 793,  DOI: 10.1016/j.ymthe.2018.11.018
      5
      mRNA-Based Protein Replacement Therapy for the Heart
      Magadum, Ajit; Kaur, Keerat; Zangi, Lior
      Molecular Therapy (2019), 27 (4), 785-793CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)
      A review:. Myocardial infarction (MI) and heart failure (HF) are the leading causes of death in the United States and in most other industrialized nations. MI leads to a massive loss of cardiomyocytes (CMs), which are replaced with non-CM cells, leading to scarring and, in most cases, HF. The adult mammalian heart has a low intrinsic regenerative capacity, mainly because of cell-cycle arrest in CMs. No effective treatment promoting heart regeneration is currently available. Recent efforts to use DNA-based or viral gene therapy approaches to induce cardiac regeneration post-MI or in HF conditions have encountered major challenges, mostly because of the poor and uncontrolled delivery of the introduced genes. Modified mRNA (modRNA) is a safe, non-immunogenic, efficient, transient, local, and controlled nucleic acid delivery system that can overcome the obstacles to DNA-based or viral approaches for cardiac gene delivery. We here review the use of modRNA in cardiac therapy, to induce cardioprotection and vascular or cardiac regeneration after MI. We discuss the current challenges in modRNA-based cardiac treatment, which will need to be overcome for the application of such treatment to ischemic heart disease.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXntFOnsb8%253D&md5=cc917f7fadd692cb7696a481798b9b75
    6. 6
      Miao, L.; Zhang, Y.; Huang, L. mRNA vaccine for cancer immunotherapy. Mol. Cancer 2021, 20 (1), 41,  DOI: 10.1186/s12943-021-01335-5
      6
      mRNA vaccine for cancer immunotherapy
      Miao, Lei; Zhang, Yu; Huang, Leaf
      Molecular Cancer (2021), 20 (1), 41CODEN: MCOACG; ISSN:1476-4598. (BioMed Central Ltd.)
      A review. MRNA vaccines have become a promising platform for cancer immunotherapy. During vaccination, naked or vehicle loaded mRNA vaccines efficiently express tumor antigens in antigen-presenting cells (APCs), facilitate APC activation and innate/adaptive immune stimulation. mRNA cancer vaccine precedes other conventional vaccine platforms due to high potency, safe administration, rapid development potentials, and cost-effective manufg. However, mRNA vaccine applications have been limited by instability, innate immunogenicity, and inefficient in vivo delivery. Appropriate mRNA structure modifications (i.e., codon optimizations, nucleotide modifications, self-amplifying mRNAs, etc.) and formulation methods (i.e., lipid nanoparticles (LNPs), polymers, peptides, etc.) have been investigated to overcome these issues. Tuning the administration routes and co-delivery of multiple mRNA vaccines with other immunotherapeutic agents (e.g., checkpoint inhibitors) have further boosted the host anti-tumor immunity and increased the likelihood of tumor cell eradication. With the recent U. S. Food and Drug Administration (FDA) approvals of LNP-loaded mRNA vaccines for the prevention of COVID-19 and the promising therapeutic outcomes of mRNA cancer vaccines achieved in several clin. trials against multiple aggressive solid tumors, we envision the rapid advancing of mRNA vaccines for cancer immunotherapy in the near future. This review provides a detailed overview of the recent progress and existing challenges of mRNA cancer vaccines and future considerations of applying mRNA vaccine for cancer immunotherapies.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXht1Smsr7K&md5=8475d10d81d634deda80801e6dbb20a6
    7. 7
      Lorentzen, C. L.; Haanen, J. B.; Met, Ö.; Svane, I. M. Clinical advances and ongoing trials on mRNA vaccines for cancer treatment. Lancet Oncol. 2022, 23 (10), e450– e458,  DOI: 10.1016/S1470-2045(22)00372-2
      7
      Clinical advances and ongoing trials on mRNA vaccines for cancer treatment
      Lorentzen, Cathrine Lund; Haanen, John B.; Met, Ozcan; Svane, Inge Marie
      Lancet Oncology (2022), 23 (10), e450-e458CODEN: LOANBN; ISSN:1470-2045. (Elsevier Ltd.)
      Years of research exploring mRNA vaccines for cancer treatment in preclin. and clin. trials have set the stage for the rapid development of mRNA vaccines during the COVID-19 pandemic. Therapeutic cancer vaccines based on mRNA are well tolerated, and the inherent advantage in ease of prodn., which rivals the best available conventional vaccine manuf. methods, renders mRNA vaccines a promising option for cancer immunotherapy. Technol. advances have optimized mRNA-based vaccine stability, structure, and delivery methods, and multiple clin. trials investigating mRNA vaccine therapy are now enrolling patients with various cancer diagnoses. Although therapeutic mRNA-based cancer vaccines have not yet been approved for std. treatment, encouraging results from early clin. trials with mRNA vaccines as monotherapy and in combination with checkpoint inhibitors have been obtained. This Review summarises the latest clin. advances in mRNA-based vaccines for cancer treatment and reflects on future perspectives and challenges for this new and promising treatment approach.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFygtrzN&md5=676d862ef4ac7a9934fba6afb546517a
    8. 8
      Weide, B.; Carralot, J. P.; Reese, A.; Scheel, B.; Eigentler, T. K.; Hoerr, I.; Rammensee, H. G.; Garbe, C.; Pascolo, S. Results of the first phase I/II clinical vaccination trial with direct injection of mRNA. J. Immunother. 2008, 31 (2), 180– 188,  DOI: 10.1097/CJI.0b013e31815ce501
      8
      Results of the First Phase I/II Clinical Vaccination Trial With Direct Injection of mRNA
      Weide, Benjamin; Carralot, Jean-Philippe; Reese, Anne; Scheel, Birgit; Eigentler, Thomas Kurt; Hoerr, Ingmar; Rammensee, Hans-Georg; Garbe, Claus; Pascolo, Steve
      Journal of Immunotherapy (2008), 31 (2), 180-188CODEN: JOIMF8; ISSN:1524-9557. (Lippincott Williams & Wilkins)
      Vaccination against tumor antigens has been shown to be a safe and efficacious prophylactic and therapeutic antitumor treatment in many animal models. Clin. studies in humans indicate that specific immunotherapy can also result in clin. benefits. The active pharmaceutical ingredient in such vaccines can be DNA, RNA, protein, or peptide and can be administered naked, encapsulated, or after delivery in vitro into cells that are then adoptively transferred. One of the easiest, most versatile and theor. safest technologies relies on the direct injection of naked mRNA (mRNA) that code for tumor antigens. We and others have shown in mice that intradermal application of naked mRNA results in protein expression and the development of an immune response. We used this protocol to vaccinate 15 melanoma patients. For each patient a growing metastasis was removed, total RNA was extd., reverse-transcribed, amplified, and cloned. Libraries of cDNA were transcribed to produce unlimited amts. of copy mRNA. Autologous prepns. were applied intradermally in combination with granulocyte macrophage colony-stimulating factor as adjuvant. We demonstrate here that such treatment is feasible and safe (phase 1 criteria). Furthermore, an increase in antitumor humoral immune response was seen in some patients. However, a demonstration of clin. effectiveness of direct injection of copy mRNA for antitumor immunotherapy was not shown in this study and must be evaluated in subsequent trials.
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    9. 9
      Curreri, A.; Sankholkar, D.; Mitragotri, S.; Zhao, Z. RNA therapeutics in the clinic. Bioeng. Transl. Med. 2023, 8 (1), e10374  DOI: 10.1002/btm2.10374
      9
      RNA therapeutics in the clinic
      Curreri, Alexander; Sankholkar, Disha; Mitragotri, Samir; Zhao, Zongmin
      Bioengineering & Translational Medicine (2023), 8 (1), e10374CODEN: BTMIAQ; ISSN:2380-6761. (John Wiley & Sons, Inc.)
      A review. RNA (RNA) therapeutics are being actively researched as a therapeutic modality in preclin. and clin. studies. They have become one of the most ubiquitously known and discussed therapeutics in recent years in part due to the ongoing coronavirus pandemic. Since the first approval in 1998, research on RNA therapeutics has progressed to discovering new therapeutic targets and delivery strategies to enhance their safety and efficacy. Here, we provide an overview of the current clin. relevant RNA therapeutics, mechanistic basis of their function, and strategies to improve their clin. use. We discuss the 17 approved RNA therapeutics and perform an in-depth anal. of the 222 ongoing clin. trials, with an emphasis on their resp. mechanisms and disease areas. We also provide perspectives on the challenges for clin. translation of RNA therapeutics and suggest potential strategies to address these challenges.
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    10. 10
      USP, Analytical Procedures for mRNA Vaccine Quality, 2nd draft. USP Draft Guidelines 2023, EA966W_2022–03.
      There is no corresponding record for this reference.
    11. 11
      Bloom, K.; van den Berg, F.; Arbuthnot, P. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 2021, 28 (3–4), 117– 129,  DOI: 10.1038/s41434-020-00204-y
      11
      Self-amplifying RNA vaccines for infectious diseases
      Bloom, Kristie; van den Berg, Fiona; Arbuthnot, Patrick
      Gene Therapy (2021), 28 (3-4), 117-129CODEN: GETHEC; ISSN:0969-7128. (Nature Portfolio)
      A review. Vaccinol. is shifting toward synthetic RNA platforms which allow for rapid, scalable, and cell-free manufg. of prophylactic and therapeutic vaccines. The simple development pipeline is based on in vitro transcription of antigen-encoding sequences or immunotherapies as synthetic RNA transcripts, which are then formulated for delivery. This approach may enable a quicker response to emerging disease outbreaks, as is evident from the swift pursuit of RNA vaccine candidates for the global SARS-CoV-2 pandemic. Both conventional and self-amplifying RNAs have shown protective immunization in preclin. studies against multiple infectious diseases including influenza, RSV, Rabies, Ebola, and HIV-1. Self-amplifying RNAs have shown enhanced antigen expression at lower doses compared to conventional mRNA, suggesting this technol. may improve immunization. This review will explore how self-amplifying RNAs are emerging as important vaccine candidates for infectious diseases, the advantages of synthetic manufg. approaches, and their potential for preventing and treating chronic infections.
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    12. 12
      Kanavarioti, A. HPLC methods for purity evaluation of man-made single-stranded RNAs. Sci. Rep. 2019, 9 (1), 1019,  DOI: 10.1038/s41598-018-37642-z
      12
      HPLC methods for purity evaluation of man-made single-stranded RNAs
      Kanavarioti Anastassia
      Scientific reports (2019), 9 (1), 1019 ISSN:.
      Synthetic RNA oligos exhibit purity decreasing as a function of length, because the efficiency of the total synthesis is the numerical product of the individual step efficiencies, typically below 98%. Analytical methods for RNAs up to the 60 nucleotides (nt) have been reported, but they fall short for purity evaluation of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuticals. In an attempt to exploit a single HPLC method and obtain both identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at high temperature in the presence of an organic cosolvent is the current analytical strategy. Here we report that IP-RP is less suitable compared to the conventional ion-exchange (IEX) for analysis of 100nt RNAs. We demonstrate the relative stability of RNA in the denaturing/basic IEX mobile phase, lay out a protocol to determine the on-the-column stability of any RNA, and establish the applicability of this method for quality testing of sgRNA, tRNA, and mRNA. Unless well resolving HPLC methods are used for batch-to-batch evaluation of man-made RNAs, process development will remain shortsighted, and observed off-target effects in-vitro or in-vivo may be partially related to low purity and the presence of shorter sequences.
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    13. 13
      Fekete, S.; Yang, H.; Wyndham, K.; Lauber, M. Salt gradient and ion-pair mediated anion exchange of intact messenger ribonucleic acids. J. Chromatogr. Open 2022, 2, 100031  DOI: 10.1016/j.jcoa.2022.100031
      There is no corresponding record for this reference.
    14. 14
      Packer, M.; Gyawali, D.; Yerabolu, R.; Schariter, J.; White, P. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems. Nat. Commun. 2021, 12 (1), 6777,  DOI: 10.1038/s41467-021-26926-0
      14
      A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems
      Packer, Meredith; Gyawali, Dipendra; Yerabolu, Ravikiran; Schariter, Joseph; White, Phil
      Nature Communications (2021), 12 (1), 6777CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liq. chromatog. (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity anal. techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidn. and subsequent hydrolysis of the tertiary amine. It thus remains crit. to ensure robust anal. methods and stringent manufg. control to ensure mRNA stability and high activity in LNP delivery systems.
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    15. 15
      Jiang, T.; Yu, N.; Kim, J.; Murgo, J. R.; Kissai, M.; Ravichandran, K.; Miracco, E. J.; Presnyak, V.; Hua, S. Oligonucleotide Sequence Mapping of Large Therapeutic mRNAs via Parallel Ribonuclease Digestions and LC-MS/MS. Anal. Chem. 2019, 91 (13), 8500– 8506,  DOI: 10.1021/acs.analchem.9b01664
      15
      Oligonucleotide sequence mapping of large therapeutic mRNAs via parallel ribonuclease digestions and LC-MS/MS
      Jiang, Tao; Yu, Ningxi; Kim, Jaeah; Murgo, John-Ross; Kissai, Mildred; Ravichandran, Kanchana; Miracco, Edward J.; Presnyak, Vladimir; Hua, Serenus
      Analytical Chemistry (Washington, DC, United States) (2019), 91 (13), 8500-8506CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Characterization of mRNA sequences is a crit. aspect of mRNA drug development and regulatory filing. Herein, we developed a novel bottom-up oligonucleotide sequence mapping workflow combining multiple endonucleases that cleave mRNA at different frequencies. RNase T1, colicin E5, and mazF were applied in parallel to provide complementary sequence coverage for large mRNAs. Combined use of multiple endonucleases resulted in significantly improved sequence coverage: greater than 70% sequence coverage was achieved on mRNAs near 3000 nucleotides long. Oligonucleotide mapping simulations with large human RNA databases demonstrate that the proposed workflow can pos. identify a single correct sequence from hundreds of similarly sized sequences. In addn., the workflow is sensitive and specific enough to detect minor sequence impurities such as single nucleotide polymorphisms (SNPs) with a sensitivity of less than 1%. LC-MS/MS-based oligonucleotide sequence mapping can serve as an orthogonal sequence characterization method to techniques such as Sanger sequencing or next-generation sequencing (NGS), providing high-throughput sequence identification and sensitive impurity detection.
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    16. 16
      Wolf, E. J.; Grünberg, S.; Dai, N.; Chen, T. H.; Roy, B.; Yigit, E.; Corrêa, I. R. Human RNase 4 improves mRNA sequence characterization by LC-MS/MS. Nucleic Acids Res. 2022, 50 (18), e106  DOI: 10.1093/nar/gkac632
      16
      Human RNase 4 improves mRNA sequence characterization by LC-MS/MS
      Wolf, Eric J.; Grunberg, Sebastian; Dai, Nan; Chen, Tien-Hao; Roy, Bijoyita; Yigit, Erbay; Correa, Ivan R., Jr.
      Nucleic Acids Research (2022), 50 (18), e106CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      With the rapid growth of synthetic mRNA (mRNA)-based therapeutics and vaccines, the development of anal. tools for characterization of long, complex RNAs has become essential. Tandem liq. chromatog.-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence anal. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine-two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5' cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS.
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    17. 17
      Nwokeoji, A. O.; Earll, M. E.; Kilby, P. M.; Portwood, D. E.; Dickman, M. J. High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2019, 1104, 212– 219,  DOI: 10.1016/j.jchromb.2018.11.027
      17
      High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography
      Nwokeoji, Alison O.; Earll, Mark E.; Kilby, Peter M.; Portwood, David E.; Dickman, Mark J.
      Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2019), 1104 (), 212-219CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
      The emergence of new sustainable approaches for insect management using RNA interference (RNAi) based insecticides has created the demand for high throughput anal. techniques to fully characterize and accurately quantify double stranded RNA (dsRNA) prior to downstream RNAi applications. In this study we have developed a method for the rapid characterization of single stranded and double stranded RNA using high resoln. RNase mapping in conjunction with ion-pair reverse-phase chromatog. utilizing a column with superficially porous particles. The high resoln. oligoribonucleotide map provides an important 'fingerprint' for identity testing and bioprocess monitoring. Reproducible RNA mapping chromatograms were generated from replicate analyses. Moreover, this approach was used to provide a method to rapidly distinguish different RNA sequences of the same size, based on differences in the resulting chromatograms. Principal components anal. of the high resoln. RNA mapping data enabled us to rapidly compare multiple HPLC chromatograms and distinguish two dsRNA sequences of different size which share 72% sequence homol. We used the high resoln. RNase mapping method to rapidly fingerprint biomanufd. dsRNA across a no. of different batches. The resulting chromatograms in conjunction with principal components anal. demonstrated high similarity in the dsRNA produced across the different batches highlighting the potential ability of this method to provide information for batch release in a high throughput manner.
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    18. 18
      Beverly, M.; Dell, A.; Parmar, P.; Houghton, L. Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS. Anal. Bioanal. Chem. 2016, 408 (18), 5021– 5030,  DOI: 10.1007/s00216-016-9605-x
      18
      Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS
      Beverly, Michael; Dell, Amy; Parmar, Parul; Houghton, Leslie
      Analytical and Bioanalytical Chemistry (2016), 408 (18), 5021-5030CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      A label-free method for detg. the 5'-end cap identity and orientation of a mRNA is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quant. and qual. information on the 5' cap was detd. from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA prepns. was detd. to be between 88 and 98 % depending on the modification type and length of the mRNA. MRNA of 2.2K and 9K nucleotides in length and contg. the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping.
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    19. 19
      Jia, L.; Qian, S. B. Therapeutic mRNA Engineering from Head to Tail. Acc. Chem. Res. 2021, 54 (23), 4272– 4282,  DOI: 10.1021/acs.accounts.1c00541
      19
      Therapeutic mRNA Engineering from Head to Tail
      Jia, Longfei; Qian, Shu-Bing
      Accounts of Chemical Research (2021), 54 (23), 4272-4282CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Synthetic mRNA, once delivered into cells, can be readily translated into proteins by ribosomes, which do not distinguish exogenous mRNAs from endogenous transcripts. Until recently, the intrinsic instability and immunostimulatory property of exogenous RNAs largely hindered the therapeutic application of synthetic mRNAs. Thanks to major technol. innovations, such as introduction of chem. modified nucleosides, synthetic mRNAs have become programmable therapeutic reagents. Compared to DNA or protein-based therapeutic reagents, synthetic mRNAs bear several advantages: flexible design, easy optimization, low-cost prepn., and scalable synthesis. Therapeutic mRNAs are commonly designed to encode specific antigens to elicit organismal immune response to pathogens like viruses, express functional proteins to replace defective ones inside cells, or introduce novel enzymes to achieve unique functions like genome editing. Recent years have witnessed stunning progress on the development of mRNA vaccines against SARS-CoV-2. This success is built upon our fundamental understanding of mRNA metab. and translational control, a knowledge accumulated during the past several decades. Given the astronomical no. of sequence combinations of 4 nucleotides, sequence-dependent control of mRNA translation remains incompletely understood. Rational design of synthetic mRNAs with robust translation and optimal stability remains challenging. Massively paralleled reporter assay (MPRA) has been proven to be powerful in identifying sequence elements in controlling mRNA translatability and stability. Indeed, a completely randomized sequence in 5' untranslated region (5'UTR) drives a wide range of translational outputs. We will discuss general principles of mRNA translation in eukaryotic cells and elucidate the role of coding and noncoding regions in the translational regulation. From the therapeutic perspective, we will highlight the unique features of 5' cap, 5'UTR, coding region (CDS), stop codon, 3'UTR, and poly(A) tail. By focusing on the design strategies in mRNA engineering, we hope this Account will contribute to the rational design of synthetic mRNAs with broad therapeutic potential.
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    20. 20
      Ramanathan, A.; Robb, G. B.; Chan, S. H. mRNA capping: biological functions and applications. Nucleic Acids Res. 2016, 44 (16), 7511– 7526,  DOI: 10.1093/nar/gkw551
      20
      mRNA capping: biological functions and applications
      Ramanathan Anand; Robb G Brett; Chan Siu-Hong
      Nucleic acids research (2016), 44 (16), 7511-26 ISSN:.
      The 5' m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2'O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose.
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    21. 21
      Shanmugasundaram, M.; Senthilvelan, A.; Kore, A. R. Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines. Chem. Rec. 2022, 22 (8), e202200005  DOI: 10.1002/tcr.202200005
      21
      Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines
      Shanmugasundaram, Muthian; Senthilvelan, Annamalai; Kore, Anilkumar R.
      Chemical Record (2022), 22 (8), e202200005CODEN: CRHEAK; ISSN:1528-0691. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review on The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS-CoV-2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chem. modified mRNA cap analogs contain a unique cap structure, m7G[5']ppp[5']N (where N=G, A, C or U), present at the 5'-end of many eukaryotic cellular and viral RNAs and several non-coding RNAs. The chem. modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide contg. a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog contg. N1 modification, cap analog contg. N2 modification, dinucleotide contg. fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addn., the biol. applications of these novel cap analogs are delineated.
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    22. 22
      Chan, S. H.; Whipple, J. M.; Dai, N.; Kelley, T. M.; Withers, K.; Tzertzinis, G.; Corrêa, I. R., Jr.; Robb, G. B. RNase H-based analysis of synthetic mRNA 5′ cap incorporation. RNA 2022, 28 (8), 1144– 1155,  DOI: 10.1261/rna.079173.122
      22
      RNase H-based analysis of synthetic mRNA 5' cap incorporation
      Chan, S. Hong; Whipple, Joseph M.; Dai, Nan; Kelley, Theresa M.; Withers, Kathryn; Tzertzinis, George; Correa, Ivan R., Jr.; Robb, G. Brett
      RNA (2022), 28 (8), 1144-1155CODEN: RNARFU; ISSN:1469-9001. (Cold Spring Harbor Laboratory Press)
      Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the crit. quality attributes in mRNA manufg. RNA cap anal. involves multiple steps: generation of predefined short fragments from the 5' end of the kilobase-long synthetic mRNA mols. using RNase H, a ribozyme or a DNAzyme, enrichment of the 5' cleavage products, and LC-MS intact mass anal. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purifn. using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5' cap anal. using less resource-intensive anal. methods, we hope to make RNA cap anal. more accessible to the scientific community.
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    23. 23
      Lapham, J.; Crothers, D. M. RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation. RNA 1996, 2 (3), 289– 296
      23
      RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation
      Lapham, Jon; Crothers, Donald M.
      RNA (1996), 2 (3), 289-96CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
      Large quantities of RNA for study by NMR and x-ray crystallog. can be produced by transcription reactions in vitro using T7 bacteriophage RNA polymerase. A limitation on producing RNA with this polymerase has been the strong dependence of the yield of the transcription reaction on the sequence at the 5' end of the RNA produced. We report a procedure for obtaining large quantities of enzymically synthesized RNA from T7 RNA polymerase that has no dependence on the 5' end sequence of the target RNA. RNase H has been shown previously (Inoue H, Hayase Y, Iwai S, Ohtsuka E, 1987, FEBS Lett 215:327-330) to cleave RNA site specifically using 2'-O-Me RNA/DNA chimeras to direct the cleavage site. We show that 2'-O-Me RNA nucleotides on the 5'-side of the DNA nucleotides in the chimera are not essential for site-specific cleavage. This allowed us to design the method such that the same 2'-O-Me chimera may be used to process any RNA sequence. We have adapted this reaction to the cleavage of NMR-scale quantities of RNA at high yield. RNA is synthesized using T7 RNA polymerase with a 15-nt high-yielding leader sequence at the 5' end, and then this sequence is cleaved off with the RNase H cleavage reaction. The cleaved RNA has 3'-hydroxyl and 5'-phosphate ends, so that the products can be used directly as substrates for ligation by T4 DNA ligase. We show that the cleavage reaction occurs efficiently in soln. and on a solid streptavidin/agarose matrix. We report an example in which we are able to improve transcription yield by more than five-fold using this technique in the synthesis of a 15N isotopically labeled hairpin found in the Crithidia fasciculata spliced leader RNA. We were able to obtain a 0.5-mM NMR sample from this inherently poorly transcribing sequence, while minimizing the amt. of isotopically labeled rNTPs used to produce it. The NMR spectroscopic results are consistent with the predicted RNA secondary structure.
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    24. 24
      Zhang, H.; He, L.; Cai, L. Transcriptome Sequencing: RNA-Seq. Methods Mol. Biol. 2018, 1754, 15– 27,  DOI: 10.1007/978-1-4939-7717-8_2
      24
      Transcriptome sequencing: RNA-seq
      Zhang, Hong; He, Lin; Cai, Lei
      Methods in Molecular Biology (New York, NY, United States) (2018), 1754 (Computational Systems Biology), 15-27CODEN: MMBIED; ISSN:1940-6029. (Springer)
      A review. RNA sequencing (RNA-seq) can not only be used to identify the expression of common or rare transcripts but also in the identification of other abnormal events, such as alternative splicing, novel transcripts, and fusion genes. In principle, RNA-seq can be carried out by almost all of the next-generation sequencing (NGS) platforms, but the libraries of different platforms are not exactly the same; each platform has its own kit to meet the special requirements of the instrument design.
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    25. 25
      Goyon, A.; Scott, B.; Kurita, K.; Maschinot, C.; Meyer, K.; Yehl, P.; Zhang, K. On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC-MS System. Anal. Chem. 2022, 94 (2), 1169– 1177,  DOI: 10.1021/acs.analchem.1c04350
      25
      On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC-MS System
      Goyon, Alexandre; Scott, Brandon; Kurita, Kenji; Maschinot, Chad; Meyer, Kevin; Yehl, Peter; Zhang, Kelly
      Analytical Chemistry (Washington, DC, United States) (2022), 94 (2), 1169-1177CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      In this study, for the first time, the automated digestion and sequencing of an RNA mol. via the use of immobilized RNase cartridges attached to a multidimensional liq. chromatog. (LC)-mass spectrometry (MS) system are presented. We first developed an online digestion-HILIC two-dimensional (2D)-LC-MS method in order to sequence CRISPR guide RNAs for gene editing. Three RNases (T1, A, and U2) were immobilized on polyetheretherketone cartridges, and their performance was evaluated. Ultrafast digestions were performed within 2.3 min with the online approach vs. 30 min via the conventional off-line approach. The higher sequence coverage was achieved by the RNase T1 (71%), which is the same as the off-line mode. A 20-fold redn. in the gRNA sample amt. was achieved with the online digestion approach (6.5μg) in comparison to that with the off-line approach (130μg). In the second step, a three-dimensional (3D)-LC-MS method was developed for the sequencing of fractions collected online across the main peak and the partially sepd. tail by the ref. ion-pairing RPLC method. Addnl. insights were gained in order to better understand the cause of the main peak tailing.
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    26. 26
      Carrilho, E.; Ruiz-Martinez, M. C.; Berka, J.; Smirnov, I.; Goetzinger, W.; Miller, A. W.; Brady, D.; Karger, B. L. Rapid DNA sequencing of more than 1000 bases per run by capillary electrophoresis using replaceable linear polyacrylamide solutions. Anal. Chem. 1996, 68 (19), 3305– 3313,  DOI: 10.1021/ac960411r
      26
      Rapid DNA Sequencing of More Than 1000 Bases per Run by Capillary Electrophoresis Using Replaceable Linear Polyacrylamide Solutions
      Carrilho, Emanuel; Ruiz-Martinez, Marie C.; Berka, Jan; Smirnov, Igor; Goetzinger, Wolfgang; Miller, Arthur W.; Brady, David; Karger, Barry L.
      Analytical Chemistry (1996), 68 (19), 3305-3313CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solns. has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and sepn. conditions, and enhanced base-calling software. The influences of LPA mol. wt. and concn. on sepn. were investigated. Addnl., the sepn. buffer, column temp., and elec. field were adjusted to increase the no. of resolvable DNA fragments per run while maintaining an enhanced sepn. speed. Using low concns. [2% (w/v)] of high mol. wt. LPA polymers (>5.5 × 106 Da), elevated column temp. (50 °C) and moderately high field (150 V/cm), rapid sequencing anal. for more than 1000 bases on a Model ssM13mp18 template was obtained with 96.8% accuracy. Base-calling software contributed to the high accuracy at these read lengths.
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    27. 27
      Kleparnik, K.; Foret, F.; Berka, J.; Goetzinger, W.; Miller, A. W.; Karger, B. L. The use of elevated column temperature to extend DNA sequencing read lengths in capillary electrophoresis with replaceable polymer matrices. Electrophoresis 1996, 17 (12), 1860– 1866,  DOI: 10.1002/elps.1150171210
      27
      The use of elevated column temperature to extend DNA sequencing read lengths in capillary electrophoresis with replaceable polymer matrixes
      Kleparnik, Karel; Foret, Frantisek; Berka, Jan; Goetzinger, Wolfgang; Miller, Arthur W.; Karger, Barry L.
      Electrophoresis (1996), 17 (12), 1860-1866CODEN: ELCTDN; ISSN:0173-0835. (VCH)
      Capillary electrophoresis with a replaceable linear polyacrylamide matrix operated at elevated column temps. of 55° and 60° was used to extend the sepn. of DNA sequencing fragments to lengths greater than 800 bases. A solid-state heater was employed to provide stable, uniform temp. control over a significant portion of the capillary. The polymer matrix, 3% w/v linear polyacrylamide in a denaturing buffer, was replaced in the capillary after each run. Using dye-labeled primers and Sequenase chem. on a M13mp18 single-stranded template, four-color sepns. for the sequencing products were obtained, with read lengths in excess of 800 bases. This paper also briefly discusses the effects of buffer denaturants and capillary temp. on sepn. speed, resoln., and gel compression.
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    28. 28
      Huber, C. G.; Stimpf, E.; Oefner, P. J.; Bonn, G. K. A comparison of micropellicular anion-exchange and reversed-phase stationary phases for HPLC analysis of oligonucleotides. LC-GC 1996, 14 (February), 114– 127
      28
      A comparison of micropellicular anion-exchange and reversed-phase stationary phases for HPLC analysis of oligonucleotides
      Huber, C. G.; Stimpf, E.; Oefner, P. J.; Bonn, G. K.
      LC-GC (1996), 14 (2), 114, 116, 118, 120, 124, 127CODEN: LCGCE7; ISSN:0888-9090. (Advanstar)
      To exploit the benefits of fast mass transfer, high column efficiency, and short anal. times of chromatog. sepns. of oligonucleotides with micropellicular packings, the authors used monodisperse 2-μd dp silica particles and 2.3-μm dp poly(styrene-divinylbenzene) (PS-DVB) particles as supports for anion-exchange and reversed-phase packing materials. They synthesized anion exchangers by amination and subsequent quaternization of PS-DVB or by coating silica gel with polyethylenimine. The alkylation of PS-DVB yielded a highly efficient packing material for ion-pair reversed-phase chromatog. of oligonucleotides that permitted single-base resoln. of homooligonucleotides with lengths of at least 80 nucleotides. They found that oligonucleotide retention depended on both its size and base compn. in anion-exchange and ion-pair reversed-phase modes. The authors also evaluated different packings designed for the anal. of synthetic and radiolabeled oligonucleotides.
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    29. 29
      Roussis, S. G.; Pearce, M.; Rentel, C. Small alkyl amines as ion-pair reagents for the separation of positional isomers of impurities in phosphate diester oligonucleotides. J. Chromatogr. A 2019, 1594, 105– 111,  DOI: 10.1016/j.chroma.2019.02.026
      29
      Small alkyl amines as ion-pair reagents for the separation of positional isomers of impurities in phosphate diester oligonucleotides
      Roussis, Stilianos G.; Pearce, Megan; Rentel, Claus
      Journal of Chromatography A (2019), 1594 (), 105-111CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      In an effort to improve sepn. of impurities in oligonucleotide drugs, alkyl amines of different length and carbon content were evaluated as reagents in ion pair-reversed phase (IP-RP) HPLC with mass spectrometric detection. A range of columns was tested in combination with different buffers, ion-pair modifiers and varying pH adjustments. For phosphorothioate oligonucleotides, larger amines, like tri-Bu and hexyl amine provided the best chromatog., as small amines tended to broaden peaks due to the sepn. of diastereoisomers. For phosphate diester oligonucleotides, the best sepns. were obtained using small alkyl amines, like propyl-, isopropyl- and diethylamine. Conditions optimized for oligonucleotide sequence and type of impurity enabled full sepn. of the individual components of composite impurities, such as n-1, N3-(2-cyanoethyl)thymine (CNET), deaminated and 3-(2-oxopropyl)imidazopyrimidinone (OPC) impurities. The addn. of long-chain alkyl acids like hexanoic acid to the IP buffer resulted in further improvements in peak sepn.
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    30. 30
      Thayer, J. R.; Barreto, V.; Rao, S.; Pohl, C. Control of oligonucleotide retention on a pH-stabilized strong anion exchange column. Anal. Biochem. 2005, 338 (1), 39– 47,  DOI: 10.1016/j.ab.2004.11.013
      30
      Control of oligonucleotide retention on a pH-stabilized strong anion exchange column
      Thayer, J. R.; Barreto, Victor; Rao, Srinivasa; Pohl, Christopher
      Analytical Biochemistry (2005), 338 (1), 39-47CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      Strong anion exchange columns are preferred for oligonucleotide analyses due to their ability to effectively control secondary structure and poly(G) interactions. Methacrylate-based anion exchange phases minimize hydrophobic interactions with oligonucleotides, but they also tend to hydrolyze under alk. conditions. In this article, we report the use of an anion exchange column prepd. from a new class of methacrylate monomers designed to improve hydrolytic stability. This column is used to show predictable adjustment of oligonucleotide retention by eluent pH and compn. Features of the new column include (i) large, predictable, pH-dependent retention shifts (varying with specific changes in 5' or 3' terminal bases with NaCl-based eluents); (ii) reduced retention when solvent is added to NaCl-based eluents; and (iii) suppression of much of the column's hydrophobic interactions when CH3CN is used with NaClO4-based eluents at a neutral pH (i.e., this eluent system separates oligonucleotides primarily in order of their length). These observations will aid the development of elution conditions for both size-dependent and base sequence-dependent (or base compn.-dependent) sepns.
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    31. 31
      Thayer, J. R., High-Resolution Separation of Oligonucleotides on a Pellicular Anion-Exchange Column. Thermo Scientific Application note 21996 , 2014.
      There is no corresponding record for this reference.
    32. 32
      Gilar, M.; Fountain, K. J.; Budman, Y.; Neue, U. D.; Yardley, K. R.; Rainville, P. D.; Russell, R. J., 2nd; Gebler, J. C. Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction. J. Chromatogr. A 2002, 958 (1–2), 167– 182,  DOI: 10.1016/S0021-9673(02)00306-0
      32
      Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: Retention prediction
      Gilar, Martin; Fountain, Kenneth J.; Budman, Yeva; Neue, Uwe D.; Yardley, Kurt R.; Rainville, Paul D.; Russell, Reb J., II; Gebler, John C.
      Journal of Chromatography A (2002), 958 (1-2), 167-182CODEN: JCRAEY; ISSN:0021-9673. (Elsevier Science B.V.)
      An ion-pair reversed-phase HPLC method was evaluated for the sepn. of synthetic oligonucleotides. Mass transfer in the stationary phase is a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 μm), elevated temp. and a relatively slow flow-rate were used to enhance mass transfer. A short 50 mm column allows for an efficient sepn. up to 30mer oligonucleotides. The sepn. strategy consists of a shallow linear gradient of org. modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases were traditionally used for oligonucleotide sepns. with good result. However, the oligonucleotide retention is affected by its nucleotide compn. The authors developed a math. model for the prediction of oligonucleotide retention from sequence and length. The authors used the model successfully to select the optimal initial gradient strength for fast HPLC purifn. of synthetic oligonucleotides. The authors also used ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aq. buffers are useful for a highly efficient and less sequence-dependent sepn. of heterooligonucleotides.
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    33. 33
      Gilar, M. Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection. Anal. Biochem. 2001, 298 (2), 196– 206,  DOI: 10.1006/abio.2001.5386
      33
      Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection
      Gilar, Martin
      Analytical Biochemistry (2001), 298 (2), 196-206CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
      Native and modified synthetic oligonucleotides were purified by reversed-phase HPLC using volatile ion-pairing mobile phases. Purifn. of 10-90 nmol of oligonucleotides in a single injection was demonstrated using a 4.6×75-mm HPLC column packed with porous 2.5 μm C18 sorbent. Sepn. of target products from N-1 failure fragments was achieved for oligonucleotides in the 4- to 60-mer size range. We employed a combination of absorbance and mass spectrometry detection to identify byproducts of oligonucleotide synthesis. This method was also employed for anal. and purifn. of fluorescently labeled oligonucleotides. (c) 2001 Academic Press.
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    34. 34
      Donegan, M.; Nguyen, J. M.; Gilar, M. Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides. J. Chromatogr. A 2022, 1666, 462860  DOI: 10.1016/j.chroma.2022.462860
      34
      Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides
      Donegan, Michael; Nguyen, Jennifer M.; Gilar, Martin
      Journal of Chromatography A (2022), 1666 (), 462860CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      We performed a systematic study of thirteen alkylamines used as ion-pairing reagents for ion-pair reversed-phase liq. chromatog. (IP RP LC) sepns. of oligonucleotides on a C18 column. We proposed a method to classify the hydrophobicity of alkylamines by their retention in RP LC. The IP reagent hydrophobicity correlated with the retention and resoln. of oligonucleotides in the corresponding IP mobile phases. The baseline resoln. was achieved up to 30 mer for hydrophilic, or up to 50 mer for hydrophobic IP reagents. Hydrophobic alkylamines permitted useful oligonucleotide sepns. at relatively low buffer concns., such as 5-10 mM alkylamine-acetate IP systems. These buffers were compatible with mass spectrometry detection, however, replacement of acetic acid with hexafluoroisopropanol in the mobile phase improved the MS signal by 2-3 orders of magnitude. Expts. with native and chem. modified oligonucleotides highlighted the mixed-mode nature of IP RP LC. When using hydrophobic IP reagents, the ionic retention mechanism of oligonucleotides is enhanced while hydrophobic retention is diminished.
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    35. 35
      Huber, C. G.; Oefner, P. J.; Bonn, G. K. Rapid and Accurate Sizing of DNA Fragments by Ion-Pair Chromatography on Alkylated Nonporous Poly(styrene-divinylbenzene) Particles. Anal. Chem. 1995, 67, 578– 585,  DOI: 10.1021/ac00099a015
      35
      Rapid and Accurate Sizing of DNA Fragments by Ion-Pair Chromatography on Alkylated Nonporous Poly(styrene-divinylbenzene) Particles
      Huber, Christian G.; Oefner, Peter J.; Bonn, Guenther K.
      Analytical Chemistry (1995), 67 (3), 578-85CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Ion-pair reversed-phase HPLC on nonporous alkylated poly(styrene-divinylbenzene) particles allowed the sepn. of double-stranded DNA fragments in a few minutes with a resoln. comparable to that obtained in slab or capillary gel electrophoresis. Using gradients of acetonitrile in 0.1M triethylammonium acetate, DNA fragments differing in length only by 2-3% could be resolved. Synthesis of the stationary phase was very reproducible, and equilibration of a freshly packed column for at least 2 h proved to be necessary for optimum column performance, whereas column regeneration for 1-5 min was adequate between gradient runs. Various DNA mol. wt. markers ranging from 51 to 2176 base pairs in length were used to study the size dependence of retention. A plot of capacity factors vs. logarithm of mol. wts. revealed a clear correlation between size and retention. Both a local and a global approxn. method, which differed with regard to the no. of known std. fragments used below and above the fragments with unknown length to create a sizing curve based on simple linear regression, gave essentially identical results, with the inaccuracy of length measurement being 0.05-3.2%. This compares favorably with the accuracy obtained in gel electrophoresis.
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    36. 36
      Kuwayama, T.; Ozaki, M.; Shimotsuma, M.; Hirose, T. Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores. Anal. Sci. 2023, 39 (3), 417– 425,  DOI: 10.1007/s44211-022-00253-w
      36
      Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores
      Kuwayama, Tomomi; Ozaki, Makoto; Shimotsuma, Motoshi; Hirose, Tsunehisa
      Analytical Sciences (2023), 39 (3), 417-425CODEN: ANSCEN; ISSN:1348-2246. (Springer)
      Messenger ribonucleic acids (mRNAs) have been used in vaccines for various diseases and are attracting attention as a new pharmaceutical paradigm. The purifn. of mRNAs is necessary because various impurities, such as template DNAs and transcription enzymes, remain in the crude product after mRNA synthesis. Among the various purifn. methods, reversed-phase high-performance liq. chromatog. (RP-HPLC) is currently attracting attention. Herein, we optimized the pore size of the packing materials, the mobile phase compn., and the temp. of the process; we also evaluated changes in the sepn. patterns of RNA strands of various lengths via RP-HPLC. Addnl., single-stranded (50-1000 nucleotides in length) and double-stranded (80-500 base pairs in length) RNAs were sepd. while their non-denatured states were maintained by performing the anal. at 60°C using triethylammonium acetate as the mobile phase and octadecyl-based RNA-RP1 with super-wide pores (> 30 nm) as the column. Furthermore, impurities in a long-stranded RNA of several thousand nucleotides synthesized by in vitro transcription were successfully sepd. using an RNA-RP1 column. The columns used in this study are expected to sep. various RNA strands and the impurities contained in them.
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    37. 37
      Barth, H. G.; Boyes, B. E.; Jackson, C. Size exclusion chromatography. Anal. Chem. 1994, 66 (12), 595– 620,  DOI: 10.1021/ac00084a022
      37
      Size Exclusion Chromatography
      Barth, Howard G.; Jackson, Christian; Boyes, Barry E.
      Analytical Chemistry (1994), 66 (12), 595R-620RCODEN: ANCHAM; ISSN:0003-2700.
      A review, with many refs., is given on band broadening, non-size exclusion chromatog., detectors, packings, compositional heterogeneity, physicochem. studies, microcolumn SEC, preparative SEC, coupled column/column switching, automation/quality control, and selected applications.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXjtlyjsLo%253D&md5=ca59c2d98d85927137d352ce12d237e0
    38. 38
      Irvine, G. B.; Irvine, G. B., Determination of molecular size by size-exclusion chromatography (gel filtration). In Current Protocols in Cell Biology; Wiley, 2001, Chapter 5, Unit 5.5.
      There is no corresponding record for this reference.
    39. 39
      Munholland, J. M.; Bright, K. A.; Nazar, R. N. Use of a volatile buffer system in ion-exchange high-performance liquid chromatography of oligonucleotides. Anal. Biochem. 1989, 178 (2), 320– 323,  DOI: 10.1016/0003-2697(89)90645-3
      39
      Use of a volatile buffer system in ion-exchange high-performance liquid chromatography of oligonucleotides
      Munholland, Janet M.; Bright, Karen A.; Nazar, Ross N.
      Analytical Biochemistry (1989), 178 (2), 320-3CODEN: ANBCA2; ISSN:0003-2697.
      A volatile buffer was adapted for use with ion-exchange HPLC in the anal. and prepn. of oligonucleotides. The system employs a com. weakly basic anion-exchange column contg. DEAE-derivatized silica gel and eluted with a volatile buffer gradient of triethylamine acetate and acetonitrile. Nucleic acid digests and oligonucleotides synthesized by chem. or enzymic methods can be analyzed or purified with nearly quant. recovery following solvent volatilization.
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    40. 40
      Lukavsky, P. J.; Puglisi, J. D. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 2004, 10 (5), 889– 893,  DOI: 10.1261/rna.5264804
      40
      Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides
      Lukavsky, Peter J.; Puglisi, Joseph D.
      RNA (2004), 10 (5), 889-893CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
      We present a fast and simple protocol for large-scale prepn. and purifn. of RNA oligonucleotides. RNA oligonucleotides are prepd. by in vitro transcription with T7 RNA polymerase from linearized plasmid DNA templates constructed by PCR. In place of denaturing polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatog. is employed to purify the RNA oligonucleotide from the transcription mixt. yielding >99% pure RNA product. In contrast to PAGE-based purifn., the gel filtration method does not require denaturation of the RNA oligonucleotide, which is desirable for larger RNAs, and the product is free of low-mol.-wt. acrylamide contaminants, which greatly benefits NMR, crystallog., and other biophys. studies of large RNAs and RNA-protein complexes.
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    41. 41
      Shimoyama, A.; Fujisaka, A.; Obika, S. Evaluation of size-exclusion chromatography for the analysis of phosphorothioate oligonucleotides. J. Pharm. Biomed. Anal. 2017, 136, 55– 65,  DOI: 10.1016/j.jpba.2016.12.036
      41
      Evaluation of size-exclusion chromatography for the analysis of phosphorothioate oligonucleotides
      Shimoyama, Atsuko; Fujisaka, Aki; Obika, Satoshi
      Journal of Pharmaceutical and Biomedical Analysis (2017), 136 (), 55-65CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
      We evaluated size exclusion chromatog. (SEC) for the detection of high-order structure of phosphorothioate oligonucleotides (PS-oligo). Because of strong interaction between PS-oligo and column packing material, peaks were broader and elution time was longer than those of the corresponding natural DNA oligonucleotides. However, single- and double-stranded structures of PS-oligo were clearly sepd. and discriminated, while single-stranded with high-order structures such as G-quadruplex and hairpin structure were not distinguished from each other.
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    42. 42
      Kim, I.; McKenna, S. A.; Viani Puglisi, E.; Puglisi, J. D. Rapid purification of RNAs using fast performance liquid chromatography (FPLC). RNA 2007, 13 (2), 289– 894,  DOI: 10.1261/rna.342607
      42
      Rapid purification of RNAs using fast performance liquid chromatography (FPLC)
      Kim, Insil; Mckenna, Sean A.; Puglisi, Elisabetta Viani; Puglisi, Joseph D.
      RNA (2007), 13 (2), 289-294CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
      We present here an improved RNA purifn. method using fast performance liq. chromatog. (FPLC) size-exclusion chromatog. in place of denaturing PAGE. The method allows prepn. of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present std. curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can sep. monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodol. allows simple and rapid purifn. of RNA oligonucleotides for structural and biophys. studies.
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    43. 43
      Largy, E.; Mergny, J. L. Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism. Nucleic Acids Res. 2014, 42 (19), e149  DOI: 10.1093/nar/gku751
      43
      Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism
      Largy, Eric; Mergny, Jean-Louis
      Nucleic Acids Research (2014), 42 (19), e149/1-e149/15CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      In recent years, an increasing no. of reports have been focused on the structure and biol. role of non-canonical nucleic acid secondary structures. Many of these studies involve the use of oligonucleotides that can often adopt a variety of structures depending on the exptl. conditions, and hence change the outcome of an assay. The knowledge of the structure(s) formed by oligonucleotides is thus crit. to correctly interpret the results, and gain insight into the biol. role of these particular sequences. Herein we demonstrate that size-exclusion HPLC (SE-HPLC) is a simple yet surprisingly powerful tool to quickly and effortlessly assess the secondary structure(s) formed by oligonucleotides. For the first time, an extensive calibration and validation of the use of SE-HPLC to confidently detect the presence of different species displaying various structure and/or molecularity, involving >110 oligonucleotides forming a variety of secondary structures (antiparallel, parallel, A-tract bent and mismatched duplexes, triplexes, G-quadruplexes and i-motifs, RNA stem loops), is performed. Moreover, we introduce simple metrics that allow the use of SE-HPLC without the need for a tedious calibration work. We show that the remarkable versatility of the method allows to quickly establish the influence of a no. of exptl. parameters on nucleic acid structuration and to operate on a wide range of oligonucleotide concns. Case studies are provided to clearly illustrate the all-terrain capabilities of SE-HPLC for oligonucleotide secondary structure anal. Finally, this manuscript features a no. of important observations contributing to a better understanding of nucleic acid structural polymorphism.
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    44. 44
      Gilar, M.; Belenky, A.; Budman, Y.; Smisek, D. L.; Cohen, A. S. Study of phosphorothioate-modified oligonucleotide resistance to 3′-exonuclease using capillary electrophoresis. J. Chromatogr. B Biomed. Sci. Appl. 1998, 714 (1), 13– 20,  DOI: 10.1016/S0378-4347(98)00160-1
      44
      Study of phosphorothioate-modified oligonucleotide resistance to 3'-exonuclease using capillary electrophoresis
      Gilar, Martin; Belenky, Alexei; Budman, Yeva; Smisek, David L.; Cohen, Aharon S.
      Journal of Chromatography B: Biomedical Sciences and Applications (1998), 714 (1), 13-20CODEN: JCBBEP; ISSN:0378-4347. (Elsevier Science B.V.)
      The effect of phosphorothioate (PS) internucleotide linkages on the stability of phosphodiester oligodeoxyribonucleotides (ODNs) was investigated using 25-mer ODNs contg. single or multiple PS backbone modifications. The in vitro stability of the oligomers was measured both in 3'-exonuclease soln. and in plasma. For the sepn. of ODNs, capillary electrophoresis with a replaceable polymer sepn. matrix was used. As expected, DNA fragments with PS linkages at the 3'-end were found to be more resistant to 3'-exonuclease hydrolysis. Also increasing exonuclease resistance was the non-specific adsorption of phosphorothioate ODNs to enzyme.
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    45. 45
      Gilar, M.; Bouvier, E. S. P. Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography. J. Chromatogr. A 2000, 890 (1), 167– 177,  DOI: 10.1016/S0021-9673(00)00521-5
      45
      Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography
      Gilar, M.; Bouvier, E. S. P.
      Journal of Chromatography A (2000), 890 (1), 167-177CODEN: JCRAEY; ISSN:0021-9673. (Elsevier Science B.V.)
      Purifn. of target oligodeoxyribonucleotides from failure sequence byproducts of synthesis is often required for polymerase chain reaction primers, DNA sequencing and other oligonucleotide applications. We have developed purifn. protocols based on a reversed-phase mechanism ("trityl on" purifn.) using a 96-well Oasis HLB extn. plate. The Oasis HLB sorbent combines excellent pH stability with a high loading capacity allowing for single-step purifn. of 0.2 μM scale synthesis. After sample loading and washing, the oligonucleotide trityl group is cleaved on the plate with 2% trifluoroacetic acid. Target DNA is eluted with acetonitrile-0.36 mM triethylamine acetate, pH 11.3 (10:90, vol./vol.). Typical yield of purified product is 60-95%. Final purity, measured by capillary gel electrophoresis, was found to be 90% or greater. Alternatively, highly pure oligonucleotides can be obtained by a RP-HPLC "trityl off" method using an XTerra C18 column. The use of volatile triethylamine acetate buffer as an ion-pair for RP-HPLC eliminates the need for further desalting.
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    46. 46
      Thayer, J. R.; Wu, Y.; Hansen, E.; Angelino, M. D.; Rao, S. Separation of oligonucleotide phosphorothioate diastereoisomers by Pellicular anion-exchange chromatography. J. Chromatogr. A 2011, 1218 (6), 802– 808,  DOI: 10.1016/j.chroma.2010.12.051
      46
      Separation of oligonucleotide phosphorothioate diastereoisomers by pellicular anion-exchange chromatography
      Thayer, James R.; Wu, Yansheng; Hansen, Erik; Angelino, Mark D.; Rao, Srinivasa
      Journal of Chromatography A (2011), 1218 (6), 802-808CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      Synthetic oligonucleotides (ONs) are often prepd. for development of therapeutic candidates. Among the modifications most often incorporated into therapeutic ONs are phosphorothioate (PT) linkages. The PT linkage introduces an addnl. chiral center at phosphorus to the chiral centers in D-ribose (and 2-deoxy-D-ribose) of the nucleic acid. Therefore, modified linkages can produce a diastereoisomer pair ([Rp] and [Sp]) at each PT linkage. These isomers are of identical length, sequence, charge and mass, and are not reliably sepd. by most chromatog. approaches (e.g., reversed phase chromatog.) unless the ON is very short. Further these isomers are not distinguishable by single-stage mass spectrometry. During chromatog. of a purified anti-NGF (nerve growth factor) aptamer contg. 37 bases with 2 PT linkages by monolithic pellicular anion-exchange (pAE) column, we obsd. four components. The four components were postulated to be: (i) distinct folding conformations; (ii) fully and partially athioated aptamers; or (iii) PT diastereoisomers. Fractionation of the components, followed by de- and re- naturation failed to produce the original forms by refolding, eliminating option (i). Mass spectrometry of the fractionated, desalted samples revealed no significant mass differences, eliminating option (ii). Oxidative conversion of the PT to phosphodiester (PO) linkages in each of the purified components produced a single chromatog. peak, co-eluting with authentic PO aptamer, and having the PO aptamer mass. We conclude that the components resolved by pAE chromatog. are diastereoisomers arising from the two PT linkages. Hence, pAE chromatog. further enhances characterization of ON therapeutics harboring limited PT linkages and having up to 37 bases.
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    47. 47
      Liu, R.; Ruan, Y.; Liu, Z.; Gong, L. The role of fluoroalcohols as counter anions for ion-pairing reversed-phase liquid chromatography/high-resolution electrospray ionization mass spectrometry analysis of oligonucleotides. Rapid Commun. Mass Spectrom. 2019, 33 (7), 697– 709,  DOI: 10.1002/rcm.8386
      47
      The role of fluoroalcohols as counter anions for ion-pairing reversed-phase liquid chromatography/high-resolution electrospray ionization mass spectrometry analysis of oligonucleotides
      Liu, Rong; Ruan, Yanjiao; Liu, Zhongqiu; Gong, Lingzhi
      Rapid Communications in Mass Spectrometry (2019), 33 (7), 697-709CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
      Rationale : Hexafluoroisopropanol (HFIP) has been widely used as a counter anion in the mobile phase for ion-pairing reversed-phase liq. chromatog./mass spectrometry (IP-RP-LC/MS) anal. of oligonucleotides. However, researchers are still searching for improvements to counter anions for LC/MS anal. of oligonucleotides. This study aimed to find alternatives to HFIP for analyzing oligonucleotides. Methods : The study was performed using an Agilent 1290 ultra-high-performance liq. chromatog. (UHPLC) system coupled to an Agilent 6540 mass spectrometer by using an oligonucleotide BEH C18 column (100 × 2.1 mm, 1.7 μm). Buffer systems contg. ion-pairing reagents (triethylamine, tripropylamine, hexylamine, dimethylbutylamine, diisopropylethylamine, N,N-dimethylcyclohexylamine, and octylamine) and fluoroalcs. (HFIP and hexafluoro-2-methyl-2-propanol (HFTP)) were compared chromatog. and mass spectrometrically. Results : Results showed that HFTP has better desalting ability than HFIP, but both HFIP and HFTP have comparable effects on the sepn. of oligonucleotides sized from 10mer to 40mer for most of ion-pairing reagents, with the exception of triethylamine and N,N-dimethylcyclohexylamine, where HFIP performed better than HFTP. Conclusions : The choice of fluoroalcs. in IP-RP-LC/MS anal. of oligonucleotides depends on the type of ion-pairing reagents used in the mobile phase. As a guideline, we would recommend to use either HA-HFIP or HA-HFTP for small oligonucleotides, but TPA-HFTP for large oligonucleotides for IP-RP-LC/MS anal. of synthetic oligonucleotides.
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    48. 48
      Li, N.; El Zahar, N. M.; Saad, J. G.; van der Hage, E. R. E.; Bartlett, M. G. Alkylamine ion-pairing reagents and the chromatographic separation of oligonucleotides. J. Chromatogr. A 2018, 1580, 110– 119,  DOI: 10.1016/j.chroma.2018.10.040
      48
      Alkylamine ion-pairing reagents and the chromatographic separation of oligonucleotides
      Li, Ning; El Zahar, N. M.; Saad, Jack G.; van der Hage, Erik R. E.; Bartlett, Michael G.
      Journal of Chromatography A (2018), 1580 (), 110-119CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      Alkylamines are commonly used to improve both chromatog. and mass spectral performance of electrospray ionization liq. chromatog. mass spectrometry based methods for the anal. of oligonucleotides. Recently several new alkylamines have been introduced to enhance the electrospray mass spectral response for oligonucleotides; however, the chromatog. properties of these new alkylamines have not been rigorously assessed. We have investigated the retention, peak width, resoln. and general chromatog. performance of fifteen different alkylamines for the sepn. of a model DNA, RNA and an antisense therapeutic oligonucleotide. Eleven of the fifteen alkylamines were shown to provide similar chromatog. performance across all three classes of oligonucleotides. Based on these findings, a model for the mechanism of retention of oligonucleotides using alkylamines and hexafluoroisopropanol mobile phases is proposed. Depending on the concns. of alkylamines and pH adjustment, oligonucleotides can be retained by micellar chromatog. and not the generally held ion-pairing mechanism. This conclusion is supported by light scattering, transmission electron microscopy and ion mobility expts. detecting three micron aggregates in the mobile phase at concns. that are routinely used for LC-MS anal. of oligonucleotides. These aggregates are not detected at lower alkylamine concns. where the retention mechanism follows an ion-pairing mechanism. The formation of these aggregates appears to be dependent on the pH of the mobile phase.
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    49. 49
      Neue, U. D. HPLC columns: theory, technology, and practice. Wiley-VCH: New York, 1997.
      There is no corresponding record for this reference.
    50. 50
      Gilar, M.; DeLano, M.; Gritti, F. Mitigation of analyte loss on metal surfaces in liquid chromatography. J. Chromatogr. A 2021, 1650, 462247  DOI: 10.1016/j.chroma.2021.462247
      50
      Mitigation of analyte loss on metal surfaces in liquid chromatography
      Gilar, Martin; DeLano, Mathew; Gritti, Fabrice
      Journal of Chromatography A (2021), 1650 (), 462247CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      The adsorptive loss of acidic analytes in liq. chromatog. was investigated using metal frits. Repetitive injections of acidic small mols. or an oligonucleotide were made on individual 2.1 or 4.6 mm i.d. column frits. Losses were obsd. for adenosine 5'-(α,β-methylene) diphosphate, 2-pyridinol 1-oxide and the 25-mer phosphorothioate oligonucleotide Trecovirsen (GEM91) on stainless steel and titanium frits. Analyte adsorption was greatest at acidic pH due to the pos. charge on the metal oxide surface. Analyte recovery increased when a series of injections was performed; this effect is known as sample conditioning. Nearly complete recovery was achieved when the metal adsorptive sites were satd. with the analyte. A similar effect was achieved by conditioning the frits with phosphoric, citric or etidronic acids, or their buffered solns. These procedures can be utilized to mitigate analyte loss. However, the effect is temporary, as the conditioning agent is gradually removed by the running mobile phase. Metal frits modified with hybrid org./inorg. surface technol. were shown to mitigate analyte-to-metal surface interactions and improve recovery of acidic analytes. Quant. recovery of a 15-35 mer oligodeoxythymidine mixt. was achieved using column hardware modified with hybrid surface technol., without a need for column conditioning prior to anal.
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    51. 51
      Guimaraes, G. J.; Sutton, J. M.; Gilar, M.; Donegan, M.; Bartlett, M. G. Impact of Nonspecific Adsorption to Metal Surfaces in Ion Pair-RP LC-MS Impurity Analysis of Oligonucleotides. J. Pharm. Biomed. Anal. 2022, 208, 114439  DOI: 10.1016/j.jpba.2021.114439
      51
      Impact of Nonspecific Adsorption to Metal Surfaces in Ion Pair-RP LC-MS Impurity Analysis of Oligonucleotides
      Guimaraes, Guilherme J.; Sutton, J. Michael; Gilar, Martin; Donegan, Michael; Bartlett, Michael G.
      Journal of Pharmaceutical and Biomedical Analysis (2022), 208 (), 114439CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
      Nonspecific adsorption has been a consistent challenge in the anal. of oligonucleotides. Nonspecific adsorption is a result of interactions between charged acidic analytes and adsorption sites present in metallic surfaces located in the fluidic path of chromatog. systems. Due to their high surface area, adsorption to column frits is esp. concerning. Poor peak shape, low recovery and compromised LOQ have been assocd. with this phenomenon. Alternative methods including substitution of stainless steel for different hardware materials and mobile phase additives have been explored in an attempt to minimize this issue. Chem. modification of metal surfaces using hybrid surface technol. (HST) by-passes the limitation of stainless steel construction material by forming a hybrid org./inorg. layer that acts as a barrier and limits nonspecific interactions. In this study we explore the implications of this new technol. in sensitive anal. and detn. of relative impurity levels of oligonucleotides. Higher relative impurity levels and better reproducibility were obtained with columns using HST.
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    52. 52
      Tuytten, R.; Lemière, F.; Witters, E.; Van Dongen, W.; Slegers, H.; Newton, R. P.; Van Onckelen, H.; Esmans, E. L. Stainless steel electrospray probe: a dead end for phosphorylated organic compounds?. J. Chromatogr. A 2006, 1104 (1–2), 209– 221,  DOI: 10.1016/j.chroma.2005.12.004
      52
      Stainless steel electrospray probe: A dead end for phosphorylated organic compounds?
      Tuytten, R.; Lemiere, F.; Witters, E.; Van Dongen, W.; Slegers, H.; Newton, R. P.; Van Onckelen, H.; Esmans, E. L.
      Journal of Chromatography A (2006), 1104 (1-2), 209-221CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      A study of the interaction of phosphorylated org. compds. with the stainless components of a liq. chromatog.-electrospray ionization-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten) likely pitfall in the LC-ESI-MS anal. of phosphorylated compds. The retention behavior of some representative compds. of different important classes of phosphorylated biomols. such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compds. when these compds. were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ∼pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realize adsorption on a stainless surface. Furthermore, the no. of phosphate moieties seemed to enhance the adsorption properties of the mols. and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was an important factor in the course of these adsorption phenomena.
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    53. 53
      Gilar, M.; Neue, U. D. Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides. J. Chromatogr. A 2007, 1169 (1–2), 139– 150,  DOI: 10.1016/j.chroma.2007.09.005
      53
      Peak capacity in gradient reversed-phase liquid chromatography of biopolymers
      Gilar, Martin; Neue, Uwe D.
      Journal of Chromatography A (2007), 1169 (1-2), 139-150CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      Reversed-phase ultra-performance liq. chromatog. was used for biopolymer sepns. in isocratic and gradient mode. The gradient elution mode was employed to est. the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 μm C18 sorbent is below 0.2 mL/min for our analytes. However, the max. peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the mol. wt. of the analyte. The isocratic sepn. mode was utilized to measure the dependence of the retention factor on the mobile phase compn. Consts. derived from isocratic expts. were utilized in a math. model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to exptl. results.
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    54. 54
      Fountain, K. J.; Gilar, M.; Gebler, J. C. Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17 (7), 646– 653,  DOI: 10.1002/rcm.959
      54
      Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry
      Fountain, Kenneth J.; Gilar, Martin; Gebler, John C.
      Rapid Communications in Mass Spectrometry (2003), 17 (7), 646-653CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
      Ion-pair reversed-phase high-performance liq. chromatog. (IP-RP-HPLC) was utilized in tandem with neg.-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the anal. of native and chem. modified oligonucleotides. Sepn. was performed on a 1.0 × 50 mm column packed with porous C18 sorbent with a particle size of 2.5 μm and an av. pore diam. of 140 Å. A method was developed which maximizes both chromatog. sepn. and mass spectrometric sensitivity using an optimized buffer system contg. triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol with a methanol gradient. The ESI-TOFMS tuning parameters were also optimized in order to minimize in-source fragmentation and achieve the best sensitivity. Analyses of native, phosphorothioate, and guanine-rich oligonucleotides were performed by LC/MS. Detection limits were at sub-picomole levels with an av. mass accuracy of 125 ppm. The described method allowed for the LC/MS anal. of oligonucleotides up to 110mer in length with little alkali cation adduction. Since sensitive detection of oligonucleotides was achieved with UV detection, we utilized a combination of UV-MS for quantitation (UV) and characterization (MS) of oligonucleotides and their failure sequence fragments/metabolites.
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    55. 55
      Koshel, B.; Birdsall, R.; Chen, W. Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2020, 1137, 121906  DOI: 10.1016/j.jchromb.2019.121906
      55
      Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides
      Koshel, Brooke; Birdsall, Robert; Chen, Weibin
      Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2020), 1137 (), 121906CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
      Two-dimensional liq. chromatog. coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide anal. have been relatively unexplored. When analyzing oligonucleotides in one-dimension, selecting an ion-pairing agent often requires a balance between acceptable chromatog. and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the sepn. mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based sepn., but MS response is compromised due to ion suppression. 2D-LC/MS was used to show that dye-conjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional anal. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatog. in both the first and second dimension (IP-RP - IP-RP) was used to transfer the n-1 impurity from a length-based sepn. in the first-dimension to a second-dimension anal. for identity confirmation using a single quadrupole detector. Identical C18 column chem. was used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the two-dimensional anal. can be detected at low nanogram levels, comparable to results achieved in a one-dimensional diln. series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resoln. instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques.
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    56. 56
      Goyon, A.; Zhang, K. Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup. Anal. Chem. 2020, 92 (8), 5944– 5951,  DOI: 10.1021/acs.analchem.0c00114
      56
      Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup
      Goyon, Alexandre; Zhang, Kelly
      Analytical Chemistry (Washington, DC, United States) (2020), 92 (8), 5944-5951CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Detn. of phosphorothioate oligonucleotide purity and impurity profile is commonly performed by ion-pairing reversed-phase liq. chromatog. (IPRP) with a mobile phase contg. triethylamine (TEA) and hexafluoro-2-propanol (HFIP). However, ion-suppressing effects of TEA hamper mass spectrometry (MS) instrumentation sensitivity and HFIP can affect the robustness of the mass spectrometer due to its corrosive nature. Anion exchange chromatog. (AEX) is an orthogonal sepn. mode to IPRP but typically cannot be directly coupled to MS. In this work, we developed a multiple heart-cutting IPRP-, AEX-hydrophilic interaction liq. chromatog.(HILIC)/MS method for quantification and high sensitivity identification of antisense oligonucleotide (ASO) impurities using a Q-Exactive mass spectrometer. Notably, both AEX-HILIC and IPRP-HILIC modes could be operated on a versatile two-dimensional liq. chromatog. (2D-LC) setup including several column selectors. The HILIC mobile phase contained 25 mM ammonium acetate and allowed identifying impurities at levels down to 0.3%. Careful selection of the sample loop vol. and the 2D HILIC column dimension allowed straightforward coupling of HILIC for both IPRP and AEX without the need to use any solvent modulation. Overall, the 2D HILIC allowed online desalting of AEX and IPRP modes and further sepn. of addnl. impurities.
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    57. 57
      Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Analysis of oligonucleotides by HPLC-electrospray ionization mass spectrometry. Anal. Chem. 1997, 69, 1320– 1325,  DOI: 10.1021/ac960916h
      57
      Analysis of Oligonucleotides by HPLC-Electrospray Ionization Mass Spectrometry
      Apffel, Alex; Chakel, John A.; Fischer, Steven; Lichtenwalter, Kay; Hancock, William S.
      Analytical Chemistry (1997), 69 (7), 1320-1325CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A new interface procedure was developed that allows, for the first time, the high-efficiency anal. of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and online electrospray-ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resoln. can be obtained with low levels of cation adduct formation in the neg. ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC sepn. and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compds. such as phosphorothioates and their metabolites, as well as of materials used in clin. trials.
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    58. 58
      DeLano, M.; Walter, T. H.; Lauber, M. A.; Gilar, M.; Jung, M. C.; Nguyen, J. M.; Boissel, C.; Patel, A. V.; Bates-Harrison, A.; Wyndham, K. D. Using Hybrid Organic-Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC. Anal. Chem. 2021, 93 (14), 5773– 5781,  DOI: 10.1021/acs.analchem.0c05203
      58
      Using Hybrid Organic-Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC
      DeLano, Mathew; Walter, Thomas H.; Lauber, Matthew A.; Gilar, Martin; Jung, Moon Chul; Nguyen, Jennifer M.; Boissel, Cheryl; Patel, Amit V.; Bates-Harrison, Andrew; Wyndham, Kevin D.
      Analytical Chemistry (Washington, DC, United States) (2021), 93 (14), 5773-5781CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Interactions of analytes with metal surfaces in high-performance liq. chromatog. (HPLC) instruments and columns have been reported to cause deleterious effects ranging from peak tailing to a complete loss of the analyte signal. These effects are due to the adsorption of certain analytes on the metal oxide layer on the surface of the metal components. We have developed a novel surface modification technol. and applied it to the metal components in ultra-HPLC (UHPLC) instruments and columns to mitigate these interactions. A hybrid org.-inorg. surface, based on an ethylene-bridged siloxane chem., was developed for use with reversed-phase and hydrophilic interaction chromatog. We have characterized the performance of UHPLC instruments and columns that incorporate this surface technol. and compared the results with those obtained using their conventional counterparts. We demonstrate improved performance when using the hybrid surface technol. for sepns. of nucleotides, a phosphopeptide, and an oligonucleotide. The hybrid surface technol. was found to result in higher and more consistent analyte peak areas and improved peak shape, particularly when using low analyte mass loads and acidic mobile phases. Reduced abundances of iron adducts in the mass spectrum of a peptide were also obsd. when using UHPLC systems and columns that incorporate hybrid surface technol. These results suggest that this technol. will be particularly beneficial in UHPLC/mass spectrometry investigations of metal-sensitive analytes.
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    59. 59
      Nguyen, J. M.; Gilar, M.; Koshel, B.; Donegan, M.; MacLean, J.; Li, Z.; Lauber, M. A. Assessing the impact of nonspecific binding on oligonucleotide bioanalysis. Bioanalysis 2021, 13, 1233,  DOI: 10.4155/bio-2021-0115
      59
      Assessing the impact of nonspecific binding on oligonucleotide bioanalysis
      Nguyen, Jennifer M.; Gilar, Martin; Koshel, Brooke; Donegan, Michael; MacLean, Jason; Li, Zhimin; Lauber, Matthew A.
      Bioanalysis (2021), 13 (16), 1233-1244CODEN: BIOAB4; ISSN:1757-6180. (Newlands Press Ltd.)
      Accurate and reliable quantification of oligonucleotides can be difficult, which has led to an increased focus on bioanal. methods for more robust analyses. Recent advances toward mitigating sample losses on liq. chromatog. (LC) systems have produced recovery advantages for oligonucleotide sepns. LC instruments and columns constructed from MP35N metal alloy and stainless steel columns were compared against LC hardware modified with hybrid inorg.-org. silica surfaces. Designed to minimize metal-analyte adsorption, these surfaces demonstrated a 73% increase in 25-mer phosphorothioate oligonucleotide recovery using ion-pairing reversed-phase LC vs. std. LC surfaces, most particularly upon initial use. Hybrid silica chromatog. surfaces improve the performance, detection limits and reproducibility of oligonucleotide bioanal. assays.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvFeitb%252FJ&md5=8d0b4e76b05b5c074f20c1e49c616e82
    60. 60
      Birdsall, R. E.; Gilar, M.; Shion, H.; Yu, Y. Q.; Chen, W. Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/mass spectrometry analysis. Rapid Commun. Mass Spectrom. 2016, 30 (14), 1667– 1679,  DOI: 10.1002/rcm.7596
      60
      Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/mass spectrometry analysis
      Birdsall, Robert E.; Gilar, Martin; Shion, Henry; Yu, Ying Qing; Chen, Weibin
      Rapid Communications in Mass Spectrometry (2016), 30 (14), 1667-1679CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
      Electrospray ionization mass spectrometry (ESI-MS)-based techniques commonly used in oligonucleotide analyses are known to be sensitive to alkali metal adduct formation. Adducts directly impact the sensitivity of MS-based analyses as the available charge is distributed across the parent peak and adduct(s). The current study systematically evaluated common liq. chromatog. (LC) components in LC/ESI-MS configurations used in oligonucleotide anal. to identify metal adduct contributions from LC instrumentation. A UPLC liq. chromatog. system was configured with a single quadrupole MS detector (ACQUITY QDa, Waters Corp.) to monitor adduct formation in oligonucleotide sepns. An ion-pairing mobile phase comprised of 15 mM triethylamine and 400 mM hexafluoro-2-propanol was used in conjunction with an oligonucleotide sepn. column (Waters OST BEH C18, 2.1 mm × 50 mm) for all sepns. A 10-min method was used to provide statistical figures of merit and evaluate adduct formation over time. Trace alkali metal salts in the mobile phase and reagents are the main source of metal salt adducts in LC/ESI-MS-based configurations. Non-specific adsorption sites located throughout the fluidic path contribute to adduct formation in oligonucleotide analyses. Ion-pairing mobile phases prepd. at neutral or slightly basic pH result in up to a 57% loss of spectral abundance to adduct formation in the current study. Implementation of a short low pH reconditioning step was obsd. to effectively displace trace metal salts non-specifically adsorbed to surfaces in the fluidic path and was able to maintain an av. MS spectral abundance ≥94% with a high degree of repeatability (relative std. deviation 0.8%) over an extended time study. The proposed method offers the ability to rapidly regenerate adsorption sites with minimal impact on productivity while retaining assay sensitivity afforded by MS detection with reduced adduct formation.
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    61. 61
      Birdsall, R. E.; Kellett, J.; Yu, Y. Q.; Chen, W. Application of mobile phase additives to reduce metal-ion mediated adsorption of non-phosphorylated peptides in RPLC/MS-based assays. J. Chromatogr. B Analyt. Technol. Biomed. Life. Sci. 2019, 1126–1127, 121773  DOI: 10.1016/j.jchromb.2019.121773
      61
      Application of mobile phase additives to reduce metal-ion mediated adsorption of non-phosphorylated peptides in RPLC/MS-based assays
      Birdsall, Robert E.; Kellett, Jacob; Yu, Ying Qing; Chen, Weibin
      Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2019), 1126-1127 (), 121773CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
      Metal-ion mediated adsorption in liq. chromatog. has been identified as a contributing factor in poor peak shape, tailing, and diminished recovery of compds. prone to cation exchange-like interaction with metal-based activity sites. Peptides that exhibit neg. charge-bearing amino acids such as aspartic acid and glutamic acid are particularly sensitive to metal-ion mediated adsorption in RPLC/MS-based sepns. when using weak acids (e.g. formic acid) as mobile phase additives. Citric acid and medronic acid as metal complexing mobile phase additives were evaluated for their ability to mitigate metal-ion mediated adsorption in RPLC/MS-based peptide mapping assays. Chromatog. performance was stabilized with peak tailing for peptides of interest reduced by as much as 40% in the presence of a chelator at a mobile phase concn. of 1 ppm. Performance gains are stable over a 67-h time study with an av. USP tailing factor of 1.00, % relative std. deviation = 0.64. The stabilizing effect of the chelator improved peptide mapping assay robustness with relative peak areas for target impurities calcd. at 2.28% (% relative std. deviation = 2.36) and 2.40% (% relative std. deviation = 2.37). Collectively this study demonstrates that chelators as mobile phase additives offers a means to improve chromatog. performance for biomols. sensitive to metal-ion mediated adsorption under formic acid-based RPLC conditions.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.3c02552.

    • Chromatograms, IP RP LC calibration curve, SEC calibration curve, mass spectrometry spectra, and sequences of mRNA (PDF)


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