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Antisense Oligonucleotide Activation via Enzymatic Antibiotic Resistance MechanismClick to copy article linkArticle link copied!
- Kristie E. DarrahKristie E. DarrahDepartment of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United StatesMore by Kristie E. Darrah
- Savannah AlbrightSavannah AlbrightDepartment of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United StatesMore by Savannah Albright
- Rohan KumbhareRohan KumbhareDepartment of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United StatesMore by Rohan Kumbhare
- Michael TsangMichael TsangDepartment of Developmental Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United StatesMore by Michael Tsang
- James K. ChenJames K. ChenDepartment of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California 94305, United StatesMore by James K. Chen
- Alexander Deiters*Alexander Deiters*Email: [email protected]Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United StatesMore by Alexander Deiters
ACS Chemical Biology
Cite this: ACS Chem. Biol. 2023, 18, 10, 2176–2182
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Abstract
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The structure and mechanism of the bacterial enzyme β-lactamase have been well-studied due to its clinical role in antibiotic resistance. β-Lactamase is known to hydrolyze the β-lactam ring of the cephalosporin scaffold, allowing a spontaneous self-immolation to occur. Previously, cephalosporin-based sensors have been developed to evaluate β-lactamase expression in both mammalian cells and zebrafish embryos. Here, we present a circular caged morpholino oligonucleotide (cMO) activated by β-lactamase-mediated cleavage of a cephalosporin motif capable of silencing the expression of T-box transcription factor Ta (tbxta), also referred to as no tail a (ntla), eliciting a distinct, observable phenotype. We explore the use of β-lactamase to elicit a biological response in aquatic embryos for the first time and expand the utility of cephalosporin as a cleavable linker beyond targeting antibiotic-resistant bacteria. The addition of β-lactamase to the current suite of enzymatic triggers presents unique opportunities for robust, orthogonal control over endogenous gene expression in a spatially resolved manner.
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Copyright © 2023 The Authors. Published by American Chemical Society
Special Issue
Published as part of ACS Chemical Biology virtual special issue “Nucleic Acid Regulation”.
Introduction
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Nucleic acid-based tools have found widespread application in the study of gene function, e.g., as antisense agents, due to their ease of synthesis, programmability, and specificity. (1−6) A drawback of oligonucleotides can be their susceptibility to nuclease-mediated degradation, which can be addressed through the incorporation of chemical modifications. (1) Morpholino oligonucleotides (MOs) are one class of modified nucleic acid analogs, containing a six-membered morpholine ring in place of the ribose and a neutral phosphoramidite linkage within the backbone. Despite the structural differences from natural nucleic acids, MOs maintain their high specificity and affinity to hybridize to the corresponding target RNA sequences. (7,8) MOs allow for gene silencing by blocking translation or mRNA splicing. (9−12)
MOs are frequently used to probe both maternal and zygotic gene function during embryonic development in zebrafish embryos, as delivery into a fertilized oocyte results in immediate and global gene silencing with effects persisting up to 5 days post-fertilization. (13−15) The development of caged MOs (cMOs) that can be activated with external triggers has broadened the utility of these tools for analysis of dynamic cellular processes with precise spatial and temporal control. This is particularly important for investigations of gene function during later developmental stages or tissue-specific cellular processes, both of which are impeded by immediate, global silencing. Upon delivery, cMOs are functionally inert and unable to engage their target sequences until activated with an external trigger, imparting conditional control over MO-induced gene silencing. (16) We and others have devised various MO caging strategies by incorporating light-, (17−22) enzyme-, (23) and more recently, small molecule-responsive elements (24) within the MO structure. Multiple designs have resulted in successful caging and subsequent conditional control of silencing function, including the incorporation of cleavable groups onto nucleobases, (18,19) short, complementary blocking oligonucleotides that form hairpins or duplexes, (17,25−27) and linkers for macrocyclization. (20−24,28−33) While all of these designs have demonstrated varying degrees of success in aquatic embryo systems, end-to-end macrocyclization has proven to be a highly modular approach that can be used with a wide range of immolative linkers (34) and commercially available MOs bearing suitable reactive handles. The curvature induced by the cyclization (and more recently bicyclization) (20) of the MO impedes target binding until a single cleavage event restores the active, linear species, which can then hybridize to its mRNA target and induce gene silencing.
Enzymatic triggers provide unique opportunities for conditional control that complement current light- and small molecule-activated cMOs. As an example, precise activation by light exposure can be complicated by the inherent structural complexity, opacity, and physical movement of individual cells within developing organisms. In the case of small molecule delivery, controlled activation of the MO can be limited by diffusion of the small molecule trigger. These shortcomings can be addressed through enzymatic activation, as the triggering enzyme can be expressed in specific cells and at controlled time points through the use of inducible or tissue-specific promoters. (35,36) The Chen lab has demonstrated this previously using an Escherichia coli nitroreductase, Nfsb, to control the activity of a cyclic cMO tethered with a 4-nitrobenzyl linker in live zebrafish embryos. (23) Additionally, the Dmochowski group has developed a caspase-3 activatable peptide nucleic acid (PNA)-based oligonucleotide sensor to probe apoptosis in cells. (37) Taken together, these reports demonstrate the versatility of enzyme-mediated strategies that can be applied to nucleic acid-based tools.
Herein, we expand upon this previous work and demonstrate for the first time the use of the bacterial enzyme β-lactamase to control oligonucleotide function. The use of β-lactamase as a triggering enzyme is particularly appealing since the structure, mechanism, and substrate specificity are well-established due to the enzyme’s prominent role in antibiotic resistance. (38,39) β-Lactamases also possess many other favorable characteristics, including stability, catalytic efficiency, and the fact that their enzymatic function is unique to bacterial systems. As a result of this biorthogonality to the mammalian cellular environment, β-lactamases have been used to spatially control the activity of various drugs in directed enzyme prodrug therapy (DEPT) strategies. (38−42) Cephalosporin motifs have been previously functionalized for β-lactamase-mediated activation of prodrugs against antibiotic resistant bacterial strains (43,44) and fluorescent sensors probing β-lactamase expression in mammalian cells and zebrafish embryos. (45,46) Here, we leverage β-lactamase-mediated antibiotic resistance mechanisms to control MO silencing function in vivo by designing and synthesizing a cyclic cMO tethered by a cephalosporin-based linker that is efficiently cleaved via β-lactamase-mediated hydrolysis.
Results and Discussion
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Upon β-lactamase catalysis, the core β-lactam ring of the linker is hydrolyzed, generating the corresponding amine, which then undergoes self-immolation and spontaneous elimination via the carbamate linkage. This elimination reaction releases the 5′-terminus of the MO to generate the active, linear MO capable of hybridizing to the mRNA target and subsequently inducing gene silencing (Figure 1).
Figure 1
Figure 1. Following enzyme-mediated hydrolysis of the β-lactam ring by β-lactamase (PDB ID 4F6H), the resulting amine intermediate undergoes self-immolation. The release of the 5′-end generates the active, linearized MO capable of hybridizing to its respective target mRNA and silencing gene expression.
To synthesize the β-lactamase-activated cMO, the linker 8 was designed using a cephalosporin core scaffold. The C-3 and C-7 positions of the core cephalosporin scaffold were synthetically modified with orthogonal azido and chloroacetamide handles, respectively, necessary for bioconjugation to the terminally modified linear MO. (39) The carbamate linkage was strategically installed at the C-3 position as it has been previously shown that leaving groups attached to the exocyclic methylene can undergo spontaneous elimination following hydrolysis of the β-lactam ring. (47,48)
The linker 8 was synthesized in four steps from commercially available 7-aminocephalosporanic acid (4) (Figure 2A). The tert-butyl ester 5 was obtained by treating 4 with tert-butyl acetate in the presence of p-toluenesulfonic acid (TsOH) and concentrated sulfuric acid. (49) The chloroacetamide at the C-7 position was installed by reacting the free amine 5 with 2-chloroacetyl chloride to form the corresponding amide 6. Selective cleavage of the acetate at the C-3 position was achieved chemoenzymatically through lipase-mediated hydrolysis of 6 using the serine hydrolase Candida antarctica lipase B (CAL B) to generate the free alcohol 7. (49) To install the azide handle, 3-azidopropan-1-amine (2) was synthesized from the corresponding alkyl chloride 1 as previously reported. (50) The amine 2 was converted to the corresponding isocyanate 3 through treatment with diphosgene. The isocyanate 3 was then added to a solution of the alcohol 7 in the presence of dibutyltin dilaurate to generate the corresponding carbamate-containing cephalosporin linker 8. It should be noted that while the canonical cephalosporin scaffold contains a carboxylic acid moiety at the C-4 position, there have been no direct studies of enzyme reactivity with any synthetic C-4-protected analogs. To test that the bulky tert-butyl ester in 8 still presents a suitable enzyme substrate, the linker (1 mM) was incubated with recombinant β-lactamase (0.5 μg), and the reaction progression was monitored by LCMS (Supporting Figure S1). Gratifyingly, nearly full cleavage of the linker 8 was observed after 3 h, and the expected cleavage product was generated. These results suggest that the presence of the tert-butyl ester at the C-4 position does not impede enzymatic cleavage of the lactam ring. With knowledge that the linker can be cleaved by β-lactamase, we moved forward with the macrocyclization of our desired MO with the functionalized linker 8.
Figure 2
Figure 2. (A) Synthetic scheme of the cephalosporin-containing linker 8. (B) Synthesis of the cephalosporin ntla cMO.
A cyclic cephalosporin cMO targeting the T-box transcription factor Ta (tbxta), also known as no tail a (ntla, 5′-GACTTGAGGCAGCATATTTCCGAT-3′, anti-start codon underlined) was prepared in three steps using the synthesized linker 8 (Figure 2B). MO-mediated silencing of ntla during early embryogenesis induces an obvious and distinct morphant phenotype that includes truncation of the embryo tail and misshapen somites. (51,52) Further, we and others have previously utilized the ntla MO as a model gene target for validating other conditionally activated cMO technologies. (17,22−24,27−29,31) The ntla MO, containing 5′-amine and 3′-disulfide handles, was subjected to macrocyclization (28,29) by first acylating the terminal amine with the NHS ester 9 to yield the corresponding alkyne suitable for bioconjugation to the azide-containing linker. The linker 8 was clicked to the alkyne-functionalized ntla MO via a copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC). (53) Lastly, the 3′-disulfide was reduced in the presence of resin-immobilized tris(2-carboxyethyl)phosphine (TCEP) to generate the free thiol, which then undergoes a spontaneous intramolecular cyclization via thioether formation with the chloroacetamide handle of 8 to generate the cyclic cephalosporin ntla cMO. Reaction progress for each step was analyzed via MALDI-TOF MS and purified by high-performance liquid chromatography (HPLC) (Supporting Figure S2). To ensure the removal of any remaining linear MO species, the final cMO product was subject to additional purification steps using iodoacetyl- and NHS-functionalized resins, as previously described. (24)
After confirmation of successful macrocyclization, we next sought to evaluate enzyme-mediated linearization and activation of the cephalosporin ntla cMO. Linearization was performed by incubating the cMO (20 μΜ) with recombinant β-lactamase for 3 h. We utilized a reticulocyte lysate-based translation system to analyze activation of the cMO. (24) The consensus ntla binding sequence (ntlaBS) was cloned directly upstream of and in frame with the coding sequence of the firefly luciferase reporter gene. Hybridization of the MO to this target sequence blocks the translational machinery from accessing the start site, thereby repressing luciferase expression (Figure 3A). Following treatment with linear ntla MO (1 μM), luciferase expression is reduced by 50%, whereas treatment with the cyclic cephalosporin ntla cMO did not impact luciferase levels. However, MO silencing function is fully restored following linearization of the cyclic cMO with β-lactamase, as evidenced by a nearly 65% reduction of reporter gene expression (Figure 3B). Taken together, this data suggests that enzymatic catalysis by β-lactamase can be used as an efficient off to on switch to conditionally control MO gene silencing activity.
Figure 3
Figure 3. (A) Luciferase reporter assay in which luciferase expression is silenced in the presence of linearized MO. (B) Luminescence signal after treatment of the indicated MO. Data represent average ± SEM from three independent experiments. (C) Gel-shift assay showing activation of the circular morpholino sequestering its complementary target DNA (cDNA) in the presence of β-lactamase. (D) Quantification of a time course analysis of cyclic cephalosporin ntla cMO cleavage following incubation with recombinant β-lactamase. Bars represent averages and error bars represent standard deviations from three independent experiments.
While switching of MO activity looked excellent, we were surprised by the overall limited silencing of luciferase expression in the in vitro translation assay and thus conducted a gel-shift analysis of the MO:target interaction. A 25-mer DNA oligonucleotide that is fully complementary to the ntla MO (cDNA, Supporting Table S2) was used as an mRNA mimic. Heteroduplex formation with the MO or cMO was visualized by native PAGE (Figure 3C). No heteroduplex formation was observed with the cMO in the absence of β-lactamase, confirming the results of the in vitro translation assay, as the curvature induced by macrocyclization of the cMO prevents target binding. It is only after incubation with the triggering enzyme that nearly full sequestration of the target cDNA is observed, as evidenced by the shift in mobility of the heteroduplex in the gel. This shift indicates successful cleavage of the linker and subsequent target hybridization by the decaged linear MO. To quantitatively determine the activated amount of cMO, the circular antisense agent was incubated with recombinant β-lactamase for up to 1 h. At defined time points, the enzyme was heat-inactivated and the MO was hybridized with an excess of cDNA. The band densities in a gel-shift assay were quantified using a standard curve (Supporting Figure S3) to determine the percentage of activated cMO. After 1 h, the cMO was 88% linearized (Figure 3D and Supporting Figure S4). The nearly complete heteroduplex formation observed by the decaged MO suggested promise for in vivo gene silencing.
Next, we sought to investigate whether β-lactamase could be used to control MO-mediated gene silencing of an endogenous gene target during early zebrafish development. β-lactamase activity can be easily evaluated using nitrocefin (10), a cephalosporin-based chromogenic substrate that changes in color from yellow to red following β-lactam hydrolysis (yielding 11; Figure 4A). (54) The utility of this substrate was validated using recombinant β-lactamase (Supporting Figure S5). We generated a β-lactamase expression construct by cloning an N-terminally HA-tagged mammalian codon-optimized β-lactamase into the pCS2+ vector, which can be used for enzyme expression in mammalian cells and as a template for in vitro mRNA production (Supporting Table S2). (55) The expression of β-lactamase was confirmed in HEK293T cells and enzymatic function was validated by monitoring cleavage of the nitrocefin substrate (Supporting Figure S6).
Figure 4
Figure 4. (A) Structure of the chromogenic β-lactamase substrate nitrocefin (10), which is hydrolyzed to 11 following β-lactamase catalysis. (B) Western blot analysis of β-lactamase expression in embryos injected with 400 pg of mRNA. (C) Zebrafish lysates analyzed for β-lactamase activity by monitoring production of 11 (absorbance at 486 nm) over time. (D) Initial velocities were determined through a linear regression analysis of the first 10 min of the data presented in (C). The error values indicate the standard deviation of values for the best fit parameters from the linear regression analysis.
In order to evaluate β-lactamase expression and activity in vivo, we next generated synthetic β-lactamase mRNA via in vitro transcription (Supporting Figure S7). The synthetic β-lactamase mRNA was injected into the yolk sac of 1- to 4-cell-stage zebrafish embryos. At 24 h post-fertilization (hpf), the injected embryos were collected, lysed, (56) and β-lactamase expression was confirmed by Western blot (Figure 4B). To confirm that β-lactamase also maintained hydrolase activity, embryos were injected with increasing amounts of mRNA, lysed, and then incubated with nitrocefin. Hydrolysis of the substrate was monitored over time. An obvious dose-dependent correlation in β-lactamase activity was observed (Figure 4C). Further, quantification of the initial rates of nitrocefin hydrolysis demonstrated a clear positive correlation between the amount of mRNA injected into the embryos and the rate of enzymatic activity (Figure 4D). These results suggest that the extent of enzymatic activity can be finely tuned with the mRNA injection amount, with maximal activity plateauing following injection of approximately 500 pg of β-lactamase mRNA.
We then utilized our cyclic cephalosporin ntla cMO to determine whether we could induce enzymatic activation of MO function in vivo. As mentioned previously, ntla has been established as an excellent candidate gene target for silencing using conditionally controlled MO technologies. (17,23,27−29) Silencing of ntla expression during early embryogenesis results in a distinct phenotype observable at 24 hpf, including truncation of tail length, loss of the notochord, and U-shaped somites (Figure 5A). (51,52) The quantification of embryo body length was used to evaluate the strength of the morphant phenotype (Supporting Figure S8). (24) Zebrafish embryos were injected in the yolk with 200 pg of either a negative control MO, the linear ntla MO, or our cyclic cephalosporin ntla cMO following previously established methods for zebrafish microinjections. (14,56,57) Based on the diameter (0.7 mm) and cytoplasmic bridging of early-stage embryos, we estimate the final concentration of the morpholino to be approximately 120 nM in the injected embryos. (58,59) Microinjection into the embryo yolk sac, as opposed to directly into the cell, enables a higher throughput workflow (200-300 embryos from one mating) (60) and reduces injection errors that could result in embryo-to-embryo variation. However, a caveat of this injection technique is that precise assessment of the intracellular MO concentration is difficult to discern. (59) Where indicated, 400 pg of β-lactamase mRNA was coinjected with the specified MO or cMO, and embryos were incubated until 24 hpf (Figure 5B). Embryos that were not injected or injected with the control MO developed as expected and displayed minimal signs of toxicity. However, we observed a slight increase in toxicity in embryos coinjected with MO and mRNA. To further investigate this toxicity, control MO (200 pg) was injected with increasing amounts of β-lactamase mRNA (up to 1000 pg). No trend was detected and no increase in phenotypic defects or toxicity was observed (Supporting Figure S9). Importantly, embryos injected with the cyclic cephalosporin ntla cMO alone also exhibited minimal phenotypic defects, confirming the inactivity of the cyclic cMO in vivo. However, upon coinjection of the cephalosporin ntla cMO with β-lactamase mRNA, full rescue of the ntla morphant phenotype was observed, comparable to the frequency of phenotype observed with the linear ntla MO. Gratifyingly, zebrafish coinjected with the cyclic cephalosporin ntla cMO and synthetic Nfsb mRNA did not show any ntla morphant phenotypes (Supporting Figure S10A). The same observation was made when injected embryos were exposed to 365 or 405 nm light (Supporting Figure S10B). Together, these results demonstrate the orthogonality of the two enzymatic cMO activation approaches and to the optical stimulation used for photocaged cMOs. There was a higher frequency of nonspecific phenotypic defects in Nfsb-mRNA injected embryos, showing lower toxicity of the β-lactamase expression (Supporting Figures 9A and 10A). (52) Taken together, these results support the utility of the β-lactamase enzyme for conditionally controlling cephalosporin-modified antisense oligonucleotides to investigate endogenous gene function in vivo.
Figure 5
Figure 5. (A) Representative images of ntla morphant phenotypes in zebrafish embryos at 24 hpf. Scale bar equals 1 mm. (B) Phenotypic scoring of embryos injected with the indicated morpholino and amount of β-lactamase mRNA.
Conclusion
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In summary, we leveraged enzyme-mediated antibiotic resistance mechanisms to develop a β-lactamase-triggered antisense agent. A novel linker containing a cephalosporin core-scaffold was synthesized and used to cyclize a MO. The curvature of the cyclic cMO successfully abrogated hybridization to and silencing of its mRNA target. β-lactamase-mediated cleavage of the cMO linker, MO linearization, and restoration of gene silencing were confirmed in vitro by gel-shift assay and silencing of reporter gene expression. Efficient enzyme-triggered silencing of endogenous gene expression was demonstrated in live zebrafish embryos coinjected with the cyclic cephalosporin ntla cMO and mRNA expressing β-lactamase. These results complement the current suite of conditionally controlled nucleic acids. (17−20,22−24,27−29,31) Despite only one prior example of β-lactamase use in zebrafish embryos, (45) the work presented herein demonstrates that the highly specific interaction between β-lactamase and cephalosporin provides a novel and robust means to control gene expression in a potentially tunable manner. Future studies placing β-lactamase expression under the control of tissue-specific promoters (similar to what has been shown previously with nitroreductase to enable targeted cell ablation experiments) (23) will provide spatial and temporal control of enzyme function. While numerous fish lines expressing nitroreductase have been reported, (61) transgenic fish lines stably expressing β-lactamase have not yet been generated, thereby precluding these studies. We anticipate that the enzyme activation presented here could be multiplexable with the other available orthogonal approaches for the conditional control of MO function. (19,20,22−24,28,29) Lastly, the activation of MOs demonstrates that β-lactamase expression has potential for being a broadly applicable trigger for molecular function in zebrafish, significantly expanding its utility.
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.3c00027.
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Author Information
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- Alexander Deiters - Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States;
https://orcid.org/0000-0003-0234-9209;
- James K. Chen - Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California 94305, United States;https://orcid.org/0000-0002-9220-8436
Acknowledgments
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This work was supported by the National Institutes of Health (R01GM112728) and the University of Pittsburgh.
References
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This article references 61 other publications.
- 1Khvorova, A.; Watts, J. K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. 2017, 35 (3), 238– 248, DOI: 10.1038/nbt.3765Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmtlalurc%253D&md5=d6c3f3ec136632eee79a63fc0be0bbfcThe chemical evolution of oligonucleotide therapies of clinical utilityKhvorova, Anastasia; Watts, Jonathan K.Nature Biotechnology (2017), 35 (3), 238-248CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)After nearly 40 years of development, oligonucleotide therapeutics are nearing meaningful clin. productivity. One of the key advantages of oligonucleotide drugs is that their delivery and potency are derived primarily from the chem. structure of the oligonucleotide whereas their target is defined by the base sequence. Thus, as oligonucleotides with a particular chem. design show appropriate distribution and safety profiles for clin. gene silencing in a particular tissue, this will open the door to the rapid development of addnl. drugs targeting other disease-assocd. genes in the same tissue. To achieve clin. productivity, the chem. architecture of the oligonucleotide needs to be optimized with a combination of sugar, backbone, nucleobase, and 3'- and 5'-terminal modifications. A portfolio of chemistries can be used to confer drug-like properties onto the oligonucleotide as a whole, with minor chem. changes often translating into major improvements in clin. efficacy. One outstanding challenge in oligonucleotide chem. development is the optimization of chem. architectures to ensure long-term safety. There are multiple designs that enable effective targeting of the liver, but a second challenge is to develop architectures that enable robust clin. efficacy in addnl. tissues.
- 2Chakraborty, S.; Mehtab, S.; Krishnan, Y. The predictive power of synthetic nucleic acid technologies in RNA biology. Acc. Chem. Res. 2014, 47 (6), 1710– 9, DOI: 10.1021/ar400323dGoogle Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlsl2ktro%253D&md5=4c6a94878c3542c311499ae0c3d0a1d4The Predictive Power of Synthetic Nucleic Acid Technologies in RNA BiologyChakraborty, Saikat; Mehtab, Shabana; Krishnan, YamunaAccounts of Chemical Research (2014), 47 (6), 1710-1719CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The impact of nucleic acid nanotechnol. in terms of transforming motifs from biol. in synthetic and translational ways is widely appreciated. But it is also emerging that the thinking and vision behind nucleic acids as construction material has broader implications, not just in nanotechnol. or even synthetic biol., but can feed back into our understanding of biol. itself. Physicists have treated nucleic acids as polymers and connected phys. principles to biol. by abstracting out the mol. interactions. In contrast, biologists delineate mol. players and pathways related to nucleic acids and how they may be networked. But in vitro nucleic acid nanotechnol. has provided a valuable framework for nucleic acids by connecting its biomol. interactions with its materials properties and thereby superarchitecture ultramanipulation that on multiple occasions has pre-empted the elucidation of how living cells themselves are exploiting these same structural concepts. This Account seeks to showcase the larger implications of certain architectural principles that have arisen from the field of structural DNA/RNA nanotechnol. in biol. Here we draw connections between these principles and particular mol. phenomena within living systems that have fed in to our understanding of how the cell uses nucleic acids as construction material to achieve different functions. We illustrate this by considering a few exciting and emerging examples in biol. in the context of both switchable systems and scaffolding type systems. Due to the scope of this Account, we will focus our discussion on examples of the RNA scaffold as summarized. In the context of switchable RNA architectures, the synthetic demonstration of small mols. blocking RNA translation preceded the discovery of riboswitches. In another example, it was after the description of aptazymes that the first allosteric ribozyme, glmS, was discovered. In the context of RNA architectures as structural scaffolds, there are clear parallels between DNA origami and the recently emerging mol. mechanism of heterochromatin formation by Xist RNA. Further, following the construction of well-defined 2D DNA-protein architectures, the striking observation of remarkably sculpted 2D RNA-protein hydrogel sheets in Caenorhabditis elegans speaks to the in vivo relevance of designer nucleic acid architectures. It is noteworthy that discoveries of properties in synthetic space seem to precede the uncovering of similar phenomena in vivo.
- 3Havens, M. A.; Hastings, M. L. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res. 2016, 44 (14), 6549– 63, DOI: 10.1093/nar/gkw533Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s%252FovFegsw%253D%253D&md5=09398f6508237c986d967e6658aed8e3Splice-switching antisense oligonucleotides as therapeutic drugsHavens Mallory A; Hastings Michelle LNucleic acids research (2016), 44 (14), 6549-63 ISSN:.Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials.
- 4Shen, X.; Corey, D. R. Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs. Nucleic Acids Res. 2018, 46 (4), 1584– 1600, DOI: 10.1093/nar/gkx1239Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlGisb7F&md5=b5bdec984635f12162386b6f8a6f6fe0Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAsShen, Xiulong; Corey, David R.Nucleic Acids Research (2018), 46 (4), 1584-1600CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)A review. RNA plays a central role in the expression of all genes. Because any sequence within RNA can be recognized by complementary base pairing, synthetic oligonucleotides and oligonucleotide mimics offer a general strategy for controlling processes that affect disease. The two primary antisense approaches for regulating expression through recognition of cellular RNAs are single-stranded antisense oligonucleotides and duplex RNAs. This review will discuss the chem. modifications and mol. mechanisms that make synthetic nucleic acid drugs possible. Lessons learned from recent clin. trials will be summarized. Ongoing clin. trials are likely to decisively test the adequacy of our current generation of antisense nucleic acid technologies and highlight areas where more basic research is needed.
- 5Setten, R. L.; Rossi, J. J.; Han, S. P. The current state and future directions of RNAi-based therapeutics. Nat. Rev. Drug Discovery 2019, 18 (6), 421– 446, DOI: 10.1038/s41573-019-0017-4Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXms1GjtL8%253D&md5=679994c46a9e0e4868a97001b742ce90The current state and future directions of RNAi-based therapeuticsSetten, Ryan L.; Rossi, John J.; Han, Si-pingNature Reviews Drug Discovery (2019), 18 (6), 421-446CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review. The RNA interference (RNAi) pathway regulates mRNA stability and translation in nearly all human cells. Small double-stranded RNA mols. can efficiently trigger RNAi silencing of specific genes, but their therapeutic use has faced numerous challenges involving safety and potency. However, August 2018 marked a new era for the field, with the US Food and Drug Administration approving ONPATTRO, the first RNAi-based drug. In this Review, we discuss key advances in the design and development of RNAi drugs leading up to this landmark achievement, the state of the current clin. pipeline and prospects for future advances, including novel RNAi pathway agents utilizing mechanisms beyond post-translational RNAi silencing.
- 6Rinaldi, C.; Wood, M. J. A. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nat. Rev. Neurol. 2018, 14 (1), 9– 21, DOI: 10.1038/nrneurol.2017.148Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCrs7jL&md5=a9256a0257a13c8c331e1229aecfd4aaAntisense oligonucleotides: the next frontier for treatment of neurological disordersRinaldi, Carlo; Wood, Matthew J. A.Nature Reviews Neurology (2018), 14 (1), 9-21CODEN: NRNACP; ISSN:1759-4758. (Nature Research)A review. Antisense oligonucleotides (ASOs) were first discovered to influence RNA processing and modulate protein expression over two decades ago; however, progress translating these agents into the clinic has been hampered by inadequate target engagement, insufficient biol. activity, and off-target toxic effects. Over the years, novel chem. modifications of ASOs have been employed to address these issues. These modifications, in combination with elucidation of the mechanism of action of ASOs and improved clin. trial design, have provided momentum for the translation of ASO-based strategies into therapies. Many neurol. conditions lack an effective treatment; however, as research progressively disentangles the pathogenic mechanisms of these diseases, they provide an ideal platform to test ASO-based strategies. This steady progress reached a pinnacle in the past few years with approvals of ASOs for the treatment of spinal muscular atrophy and Duchenne muscular dystrophy, which represent landmarks in a field in which disease-modifying therapies were virtually non-existent. With the rapid development of improved next-generation ASOs toward clin. application, this technol. now holds the potential to have a dramatic effect on the treatment of many neurol. conditions in the near future.
- 7Timme-Laragy, A. R.; Karchner, S. I.; Hahn, M. E. Gene knockdown by morpholino-modified oligonucleotides in the zebrafish (Danio rerio) model: applications for developmental toxicology. Methods Mol. Biol. 2012, 889, 51– 71, DOI: 10.1007/978-1-61779-867-2_5Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFGlsbbK&md5=e6a57db28fb1f79eccfcbc4335602e37Gene knockdown by morpholino-modified oligonucleotides in the zebrafish (Danio rerio) model: applications for developmental toxicologyTimme-Laragy, Alicia R.; Karchner, Sibel I.; Hahn, Mark E.Methods in Molecular Biology (New York, NY, United States) (2012), 889 (Developmental Toxicology), 51-71CODEN: MMBIED; ISSN:1064-3745. (Springer)The zebrafish (Danio rerio) has long been used as a model for developmental biol., making it an excellent model to use also in developmental toxicol. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technol. and gene knockdown via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a mol. and mechanistic level while establishing a relationship between these mol. events and morphol., behavioral, and/or physiol. effects (i.e., phenotypic anchoring). In this chapter, we address important considerations for using MOs to study developmental toxicol. in zebrafish embryos and provide a protocol for their use.
- 8Shestopalov, I. A.; Chen, J. K. Oligonucleotide-based tools for studying zebrafish development. Zebrafish 2010, 7 (1), 31– 40, DOI: 10.1089/zeb.2010.0650Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlsVSru70%253D&md5=a323a188b0dcdc1c8b3c5d10f06b81c0Oligonucleotide-Based Tools for Studying Zebrafish DevelopmentShestopalov, Ilya A.; Chen, James K.Zebrafish (2010), 7 (1), 31-40CODEN: ZEBRAL; ISSN:1545-8547. (Mary Ann Liebert, Inc.)A review. Synthetic and nonnatural oligonucleotides have been used extensively to interrogate gene function in zebrafish. In this review, we survey the capabilities and limitations of various oligonucleotide-based technologies for perturbing RNA function and tracking RNA expression. We also examine recent strategies for achieving spatiotemporal control of oligonucleotide function, particularly light-gated technologies that exploit the optical transparency of zebrafish embryos.
- 9Summerton, J.; Weller, D. Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 1997, 7 (3), 187– 95, DOI: 10.1089/oli.1.1997.7.187Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXktFWmu70%253D&md5=39f0590b00278d17744fb4567e0eb40dMorpholino antisense oligomers: design, preparation, and propertiesSummerton, James; Weller, DwightAntisense & Nucleic Acid Drug Development (1997), 7 (3), 187-195CODEN: ANADF5; ISSN:1087-2906. (Liebert)A review with 41 refs. Antisense promised major advances in treating a broad range of intractable diseases, but in recent years progress has been stymied by tech. problems, most notably inadequate specificity, ineffective delivery into the proper subcellular compartment, and unpredictable activity within cells. An overview is provided of the design, prepn., and properties of morpholino oligos, a novel antisense structural type that solves the sequence specificity problem and provides high and predictable activity in cells. Morpholino oligos also exhibit little or no nonantisense activity, afford good water soly., are immune to nucleases, and are designed to have low prodn. costs.
- 10Hardy, S.; Legagneux, V.; Audic, Y.; Paillard, L. Reverse genetics in eukaryotes. Biol. Cell 2010, 102 (10), 561– 80, DOI: 10.1042/BC20100038Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFSntb%252FL&md5=a19788a36671f2718dd18f28b30db197Reverse genetics in eukaryotesHardy, Serge; Legagneux, Vincent; Audic, Yann; Paillard, LucBiology of the Cell (2010), 102 (10), 561-580CODEN: BCELDF; ISSN:0248-4900. (Portland Press Ltd.)A review. Reverse genetics consists in the modification of the activity of a target gene to analyze the phenotypic consequences. Four main approaches are used towards this goal and will be explained in this review. Two of them are centered on genome alterations. Mutations produced by random chem. or insertional mutagenesis can be screened to recover only mutants in a specific gene of interest. Alternatively, these alterations may be specifically targeted on a gene of interest by HR (homologous recombination). The other 2 approaches are centered on mRNA. RNA interference is a powerful method to reduce the level of gene products, while MO (morpholino) antisense oligonucleotides alter mRNA metab. or translation. Some model species, such as Drosophila, are amenable to most of these approaches, whereas other model species are restricted to 1 of them. For example, in mice and yeasts, gene targeting by HR is prevalent, whereas in Xenopus and zebrafish MO oligonucleotides are mainly used. Genome-wide collections of mutants or inactivated models obtained in several species by these approaches have been made and will help decipher gene functions in the post-genomic era.
- 11Kole, R.; Krainer, A. R.; Altman, S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat. Rev. Drug Discovery 2012, 11 (2), 125– 40, DOI: 10.1038/nrd3625Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVGqt7k%253D&md5=0db21d536154c3ccd62ba0db1334e3bbRNA therapeutics: beyond RNA interference and antisense oligonucleotidesKole, Ryszard; Krainer, Adrian R.; Altman, SidneyNature Reviews Drug Discovery (2012), 11 (2), 125-140CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Here, we discuss three RNA-based therapeutic technologies exploiting various oligonucleotides that bind to RNA by base pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by inducing enzyme-dependent degrdn. of targeted mRNA. Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, steric-blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein prodn. or downregulate gene expression. Moreover, they can be extensively chem. modified to acquire more drug-like properties. The ability of RNA-blocking oligonucleotides to restore gene function makes them best suited for the treatment of genetic disorders. Pos. results from clin. trials for the treatment of Duchenne muscular dystrophy show that this technol. is close to achieving its clin. potential.
- 12Corey, D. R.; Abrams, J. M. Morpholino antisense oligonucleotides: tools for investigating vertebrate development. Genome Biol. 2001, 2 (5), reviews1015, DOI: 10.1186/gb-2001-2-5-reviews1015Google ScholarThere is no corresponding record for this reference.
- 13Ciruna, B.; Weidinger, G.; Knaut, H.; Thisse, B.; Thisse, C.; Raz, E.; Schier, A. F. Production of maternal-zygotic mutant zebrafish by germ-line replacement. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (23), 14919– 24, DOI: 10.1073/pnas.222459999Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xpt1yrtLs%253D&md5=30e39750bf35757c4e9dcfaf9a0dc7a8Production of maternal-zygotic mutant zebrafish by germ-line replacementCiruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (23), 14919-14924CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryol., genetic, genomic, and biochem. studies.
- 14Bill, B. R.; Petzold, A. M.; Clark, K. J.; Schimmenti, L. A.; Ekker, S. C. A primer for morpholino use in zebrafish. Zebrafish 2009, 6 (1), 69– 77, DOI: 10.1089/zeb.2008.0555Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXkvVGmtrs%253D&md5=7595f7f176f84ca330af855f45c75810A Primer for Morpholino Use in ZebrafishBill, Brent R.; Petzold, Andrew M.; Clark, Karl J.; Schimmenti, Lisa A.; Ekker, Stephen C.Zebrafish (2009), 6 (1), 69-77CODEN: ZEBRAL; ISSN:1545-8547. (Mary Ann Liebert, Inc.)A review. Morpholino oligonucleotides are the most common anti-sense "knockdown" technique used in zebrafish (Danio rerio). This review discusses common practices for the design, prepn., and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.
- 15Shestopalov, I. A.; Chen, J. K. Chemical technologies for probing embryonic development. Chem. Soc. Rev. 2008, 37 (7), 1294– 307, DOI: 10.1039/b703023cGoogle Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnsFejsbw%253D&md5=53344f05372a52656ff9bc50c4a138d2Chemical technologies for probing embryonic developmentShestopalov, Ilya A.; Chen, James K.Chemical Society Reviews (2008), 37 (7), 1294-1307CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Embryogenesis is a remarkable program of cell proliferation, migration, and differentiation that transforms a single fertilized egg into a complex multicellular organism. Understanding this process at the mol. and systems levels will require an interdisciplinary approach, including the concepts and technologies of chem. biol. This tutorial review provides an overview of chem. tools that have been used in developmental biol. research, focusing on methods that enable spatiotemporal control of gene function and the visualization of embryonic patterning. Limitations of current approaches and future challenges are also discussed.
- 16Darrah, K. E.; Deiters, A. Translational control of gene function through optically regulated nucleic acids. Chem. Soc. Rev. 2021, 50 (23), 13253– 13267, DOI: 10.1039/D1CS00257KGoogle Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVahtr%252FJ&md5=16aba43c93be276b4e09a6b914769710Translational control of gene function through optically regulated nucleic acidsDarrah, Kristie E.; Deiters, AlexanderChemical Society Reviews (2021), 50 (23), 13253-13267CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Translation of mRNA into protein is one of the most fundamental processes within biol. systems. Gene expression is tightly regulated both in space and time, often involving complex signaling or gene regulatory networks, as most prominently obsd. in embryo development. Thus, studies of gene function require tools with a matching level of external control. Light is an excellent conditional trigger as it is minimally invasive, can be easily tuned in wavelength and amplitude, and can be applied with excellent spatial and temporal resoln. To this end, modification of established oligonucleotide-based technologies with optical control elements, in the form of photocaging groups and photoswitches, has rendered these tools capable of navigating the dynamic regulatory pathways of mRNA translation in cellular and in vivo models. In this review, we discuss the different optochem. approaches used to generate photoresponsive nucleic acids that activate and deactivate gene expression and function at the translational level.
- 17Shestopalov, I. A.; Sinha, S.; Chen, J. K. Light-controlled gene silencing in zebrafish embryos. Nat. Chem. Biol. 2007, 3 (10), 650– 1, DOI: 10.1038/nchembio.2007.30Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVCrtrjP&md5=dc008772fa3f03e882df40d8d8b1d02eLight-controlled gene silencing in zebrafish embryosShestopalov, Ilya A.; Sinha, Surajit; Chen, James K.Nature Chemical Biology (2007), 3 (10), 650-651CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Functional genomic studies in zebrafish frequently use synthetic oligonucleotides called morpholinos that block RNA splicing or translation. However, the constitutive activity of these reagents limits their exptl. utility. We report here the synthesis of a photoactivatable morpholino targeting the no tail (ntl) gene. This caged reagent permits spatiotemporal gene regulation in vivo and the photochem. generation of functionally mosaic organisms.
- 18Deiters, A.; Garner, R. A.; Lusic, H.; Govan, J. M.; Dush, M.; Nascone-Yoder, N. M.; Yoder, J. A. Photocaged morpholino oligomers for the light-regulation of gene function in zebrafish and Xenopus embryos. J. Am. Chem. Soc. 2010, 132 (44), 15644– 50, DOI: 10.1021/ja1053863Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWmtLjI&md5=39e4e975466e4c45786de524844f5d93Photocaged Morpholino Oligomers for the Light-Regulation of Gene Function in Zebrafish and Xenopus EmbryosDeiters, Alexander; Garner, R. Aaron; Lusic, Hrvoje; Govan, Jeane M.; Dush, Mike; Nascone-Yoder, Nanette M.; Yoder, Jeffrey A.Journal of the American Chemical Society (2010), 132 (44), 15644-15650CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged (derivatized by NPOM; i.e. 6-nitropiperonyloxymethyl) morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP prodn. only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts.
- 19Bardhan, A.; Deiters, A.; Ettensohn, C. A. Conditional gene knockdowns in sea urchins using caged morpholinos. Dev. Biol. 2021, 475, 21– 29, DOI: 10.1016/j.ydbio.2021.02.014Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmslKms7o%253D&md5=977ba5021fde89085bd18ae73026078cConditional gene knockdowns in sea urchins using caged morpholinosBardhan, Anirban; Deiters, Alexander; Ettensohn, Charles A.Developmental Biology (Amsterdam, Netherlands) (2021), 475 (), 21-29CODEN: DEBIAO; ISSN:0012-1606. (Elsevier B.V.)Echinoderms are important exptl. models for analyzing embryonic development, but a lack of spatial and temporal control over gene perturbations has hindered developmental studies using these animals. Morpholino antisense oligonucleotides (MOs) have been used successfully by the echinoderm research community for almost two decades, and MOs remain the most widely used tool for acute gene knockdowns in these organisms. Echinoderm embryos develop externally and are optically transparent, making them ideally-suited to many light-based approaches for analyzing and manipulating development. Studies using zebrafish embryos have demonstrated the effectiveness of photoactivatable (caged) MOs for conditional gene knockdowns. Here we show that caged MOs, synthesized using nucleobase-caged monomers, provide light-regulated control over gene expression in sea urchin embryos. Our work provides the first robust approach for conditional gene silencing in this prominent model system.
- 20Pattanayak, S.; Sarode, B. R.; Deiters, A.; Chen, J. K. Bicyclic Caged Morpholino Oligonucleotides for Optical Gene Silencing. ChemBioChem 2022, 23 (21), e202200374, DOI: 10.1002/cbic.202200374Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFyns7jN&md5=28cd115f1602b741c2a331c2f1189a58Bicyclic Caged Morpholino Oligonucleotides for Optical Gene SilencingPattanayak, Sankha; Sarode, Bhagyesh R.; Deiters, Alexander; Chen, James K.ChemBioChem (2022), 23 (21), e202200374CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Caged morpholino oligonucleotides (cMOs) are synthetic tools that allow light-inducible gene silencing in live organisms. Previously reported cMOs have utilized hairpin, duplex, and cyclic structures, as well as caged nucleobases. While these antisense technologies enable efficient optical control of RNA splicing and translation, they can have limited dynamic range. A new caging strategy was developed where the two MO termini are conjugated to an internal position through a self-immolative trifunctional linker, thereby generating a bicyclic cMO that is conformationally resistant to RNA binding. The efficacy of this alternative cMO design has been demonstrated in zebrafish embryos and compared to linear MOs and monocyclic constructs.
- 21Yang, L.; Kim, H. B.; Sul, J. Y.; Yeldell, S. B.; Eberwine, J. H.; Dmochowski, I. J. Efficient Synthesis of Light-Triggered Circular Antisense Oligonucleotides Targeting Cellular Protein Expression. ChemBioChem 2018, 19 (12), 1250– 1254, DOI: 10.1002/cbic.201800012Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXns1Ontrg%253D&md5=d6313c9a191f06610c7f74232c4c6054Efficient Synthesis of Light-Triggered Circular Antisense Oligonucleotides Targeting Cellular Protein ExpressionYang, Linlin; Kim, Hyun Bum; Sul, Jai-Yoon; Yeldell, Sean B.; Eberwine, James H.; Dmochowski, Ivan J.ChemBioChem (2018), 19 (12), 1250-1254CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Light-activated ("caged") antisense oligonucleotides are powerful mols. for regulating gene expression at submicron spatial resoln. through the focal modulation of endogenous cellular processes. Cyclized caged oligos are particularly promising structures because of their inherent stability and similarity to naturally occurring circular DNA and RNA mols. Here, we introduce an efficient route for cyclizing an antisense oligodeoxynucleotide incorporating a photocleavable linker. Oligo cyclization was achieved for several sequences in nearly quant. yields through intramol. copper(I)-catalyzed azide-alkyne cycloaddn. (CuAAC). Caging stability and light activation were characterized by FRET efficiency, denaturing gel assay, and melting temp. measurements. Finally, a cyclized caged oligo was designed to target gfap, and it gave a tenfold redn. in glial fibrillary acidic protein upon photoactivation in astrocytes.
- 22Brown, W.; Bardhan, A.; Darrah, K.; Tsang, M.; Deiters, A. Optical Control of MicroRNA Function in Zebrafish Embryos. J. Am. Chem. Soc. 2022, 144 (37), 16819– 16826, DOI: 10.1021/jacs.2c04479Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xitlagu7jK&md5=cab3a24cf5070bfc55885da50480db6fOptical Control of MicroRNA Function in Zebrafish EmbryosBrown, Wes; Bardhan, Anirban; Darrah, Kristie; Tsang, Michael; Deiters, AlexanderJournal of the American Chemical Society (2022), 144 (37), 16819-16826CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)MicroRNAs play crucial and dynamic roles in vertebrate development and diseases. Some, like miR-430, are highly expressed during early embryo development and regulate hundreds of transcripts, which can make it difficult to study their role in the timing and location of specific developmental processes using conventional morpholino oligonucleotide (MO) knockdown or genetic deletion approaches. We demonstrate that light-activated circular morpholino oligonucleotides (cMOs) can be applied to the conditional control of microRNA function. We targeted miR-430 in zebrafish embryos to study its role in the development of the embryo body and the heart. Using 405 nm irradn., precise spatial and temporal control over miR-430 function was demonstrated, offering insight into the cell populations and developmental timepoints involved in each process.
- 23Yamazoe, S.; McQuade, L. E.; Chen, J. K. Nitroreductase-activatable morpholino oligonucleotides for in vivo gene silencing. ACS Chem. Biol. 2014, 9 (9), 1985– 90, DOI: 10.1021/cb500429uGoogle Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht1ahtrbO&md5=91d7b5afe7f92ca0ce02a573bae49bc4Nitroreductase-Activatable Morpholino Oligonucleotides for in Vivo Gene SilencingYamazoe, Sayumi; McQuade, Lindsey E.; Chen, James K.ACS Chemical Biology (2014), 9 (9), 1985-1990CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Phosphorodiamidate morpholino oligonucleotides are widely used to interrogate gene function in whole organisms, and light-activatable derivs. can reveal spatial and temporal differences in gene activity. The authors describe here a new class of caged morpholino oligonucleotides that can be activated by the bacterial nitroreductase NfsB. The authors characterize the activation kinetics of these reagents in vitro and demonstrate their efficacy in zebrafish embryos that express NfsB either ubiquitously or in defined cell populations. In combination with transgenic organisms, such enzyme-actuated antisense tools will enable gene silencing in specific cell types, including tissues that are not amenable to optical targeting.
- 24Darrah, K.; Wesalo, J.; Lukasak, B.; Tsang, M.; Chen, J. K.; Deiters, A. Small Molecule Control of Morpholino Antisense Oligonucleotide Function through Staudinger Reduction. J. Am. Chem. Soc. 2021, 143 (44), 18665– 18671, DOI: 10.1021/jacs.1c08723Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitlajur%252FF&md5=2d697c181ecf19a44cc5681fb079b47bSmall Molecule Control of Morpholino Antisense Oligonucleotide Function through Staudinger ReductionDarrah, Kristie; Wesalo, Joshua; Lukasak, Bradley; Tsang, Michael; Chen, James K.; Deiters, AlexanderJournal of the American Chemical Society (2021), 143 (44), 18665-18671CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Conditionally activated, caged morpholino antisense agents (cMOs) are tools that enable the temporal and spatial investigation of gene expression, regulation, and function during embryonic development. Cyclic MOs are conformationally gated oligonucleotide analogs that do not block gene expression until they are linearized through the application of an external trigger, such as light or enzyme activity. Here, we describe the first examples of small mol.-responsive cMOs, which undergo rapid and efficient decaging via a Staudinger redn. This is enabled by a highly flexible linker design that offers opportunities for the installation of chem. activated, self-immolative motifs. We synthesized cyclic cMOs against two distinct, developmentally relevant genes and demonstrated phosphine-triggered knockdown of gene expression in zebrafish embryos. This represents the first report of a small mol.-triggered antisense agent for gene knockdown, adding another bioorthogonal entry to the growing arsenal of gene knockdown tools.
- 25Tomasini, A. J.; Schuler, A. D.; Zebala, J. A.; Mayer, A. N. PhotoMorphs: a novel light-activated reagent for controlling gene expression in zebrafish. Genesis 2009, 47 (11), 736– 43, DOI: 10.1002/dvg.20554Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhs1Sjt7jN&md5=1cbe5279eaad8674d03dfa028acf9424PhotoMorphs: a novel light-activated reagent for controlling gene expression in zebrafishTomasini, Amber J.; Schuler, Aaron D.; Zebala, John A.; Mayer, Alan N.Genesis (Hoboken, NJ, United States) (2009), 47 (11), 736-743CODEN: GNESFY; ISSN:1526-954X. (Wiley-Liss, Inc.)Manipulating gene expression in zebrafish is crit. for exploiting the full potential of this vertebrate model organism. Morpholino oligos are the most commonly used antisense technol. for knocking down gene expression. However, morpholinos suffer from a lack of control over the timing and location of knockdown. In this report, we describe a novel light-activatable knockdown reagent called PhotoMorph. PhotoMorphs can be generated from existing morpholinos by hybridization with a complementary caging strand contg. a photocleavable linkage. The caging strand neutralizes the morpholino activity until irradn. of the PhotoMorph with UV light releases the morpholino. We generated PhotoMorphs to target genes encoding enhanced green fluorescent protein, No tail, and E-cadherin to illustrate the utility of this approach. Temporal control of gene expression with PhotoMorphs permitted us to circumvent the early lethal phenotype of E-cadherin knockdown. A splice-blocking PhotoMorph directed to the rheb gene showed light-dependent gene knockdown up to 72 hpf. PhotoMorphs thus offer a new class of lab. reagents suitable for the spatio-temporal control of gene expression in the zebrafish.
- 26Tallafuss, A.; Gibson, D.; Morcos, P.; Li, Y.; Seredick, S.; Eisen, J.; Washbourne, P. Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafish. Development 2012, 139 (9), 1691– 9, DOI: 10.1242/dev.072702Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XotlKisb0%253D&md5=a458a3387ad59c5371e323ac01205d63Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafishTallafuss, Alexandra; Gibson, Dan; Morcos, Paul; Li, Yongfu; Seredick, Steve; Eisen, Judith; Washbourne, PhilipDevelopment (Cambridge, United Kingdom) (2012), 139 (9), 1691-1699CODEN: DEVPED; ISSN:0950-1991. (Company of Biologists Ltd.)To understand the mol. mechanisms of development it is essential to be able to turn genes on and off at will and in a spatially restricted fashion. Morpholino oligonucleotides (MOs) are very common tools used in several model organisms with which it is possible to block gene expression. Recently developed photo-activated MOs allow control over the onset of MO activity. However, deactivation of photo-cleavable MO activity has remained elusive. Here, we describe photo-cleavable MOs with which it is possible to activate or de-activate MO function by UV exposure in a temporal and spatial manner. We show, using several different genes as examples, that it is possible to turn gene expression on or off both in the entire zebrafish embryo and in single cells. We use these tools to demonstrate the sufficiency of no tail expression as late as tailbud stage to drive medial precursor cells towards the notochord cell fate. As a broader approach for the use of photo-cleavable MOs, we show temporal control over gal4 function, which has many potential applications in multiple transgenic lines. We demonstrate temporal manipulation of Gal4 transgene expression in only primary motoneurons and not secondary motoneurons, heretofore impossible with conventional transgenic approaches. In another example, we follow and analyze neural crest cells that regained sox10 function after deactivation of a photo-cleavable sox10-MO at different time points. Our results suggest that sox10 function might not be crit. during neural crest formation.
- 27Ouyang, X.; Shestopalov, I. A.; Sinha, S.; Zheng, G.; Pitt, C. L.; Li, W. H.; Olson, A. J.; Chen, J. K. Versatile synthesis and rational design of caged morpholinos. J. Am. Chem. Soc. 2009, 131 (37), 13255– 69, DOI: 10.1021/ja809933hGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVClsr7M&md5=17ab8c99c360c0d8cd8a6de312b5bb7fVersatile Synthesis and Rational Design of Caged MorpholinosOuyang, Xiaohu; Shestopalov, Ilya A.; Sinha, Surajit; Zheng, Genhua; Pitt, Cameron L. W.; Li, Wen-Hong; Olson, Andrew J.; Chen, James K.Journal of the American Chemical Society (2009), 131 (37), 13255-13269CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these mol. mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chem. technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from com. available reagents. Using this simplified procedure, we have systematically prepd. cMOs with differing structural configurations and investigated how the in vitro thermodn. properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prep. a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chem. probes in vivo for functional genomic studies.
- 28Yamazoe, S.; Shestopalov, I. A.; Provost, E.; Leach, S. D.; Chen, J. K. Cyclic caged morpholinos: conformationally gated probes of embryonic gene function. Angew. Chem., Int. Ed. 2012, 51 (28), 6908– 11, DOI: 10.1002/anie.201201690Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xot12itLk%253D&md5=4a5d186014dd009513db6ad4e3dfffd3Cyclic caged morpholinos: conformationally gated probes of embryonic gene functionYamazoe, Sayumi; Shestopalov, Ilya A.; Provost, Elayne; Leach, Steven D.; Chen, James K.Angewandte Chemie, International Edition (2012), 51 (28), 6908-6911, S6908/1-S6908/28CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Next-generation cyclic caged morpholino-based antisense oligonucleotides were used as effective reverse-genetics tools in zebrafish embryos to study the timing of exocrine fate commitment in developing pancreas. The zebrafish pancreas transcription factor 1 alpha (ptf1a) gene was targeted.
- 29Yamazoe, S.; Liu, Q.; McQuade, L. E.; Deiters, A.; Chen, J. K. Sequential gene silencing using wavelength-selective caged morpholino oligonucleotides. Angew. Chem., Int. Ed. 2014, 53 (38), 10114– 8, DOI: 10.1002/anie.201405355Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht1GqsLbI&md5=840d1f876b958ebcc25e66250eb89e7aSequential gene silencing using wavelength-selective caged morpholino oligonucleotidesYamazoe, Sayumi; Liu, Qingyang; McQuade, Lindsey E.; Deiters, Alexander; Chen, James K.Angewandte Chemie, International Edition (2014), 53 (38), 10114-10118CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Spectrally differentiated caged morpholino oligonucleotides (cMOs) and wavelength-selective illumination have been used to sequentially inactivate organismal gene function. The efficacy of these reverse-genetic chem. probes has been demonstrated in zebrafish embryos, and these reagents have been employed to examine the mechanisms of mesoderm patterning.
- 30Wang, Y.; Wu, L.; Wang, P.; Lv, C.; Yang, Z.; Tang, X. Manipulation of gene expression in zebrafish using caged circular morpholino oligomers. Nucleic Acids Res. 2012, 40 (21), 11155– 62, DOI: 10.1093/nar/gks840Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhslaksr7F&md5=6934d98ed5b46d1771872fea33a7227aManipulation of gene expression in zebrafish using caged circular morpholino oligomersWang, Yuan; Wu, Li; Wang, Peng; Lv, Cong; Yang, Zhenjun; Tang, XinjingNucleic Acids Research (2012), 40 (21), 11155-11162CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Morpholino oligomers (MOs) have been widely used to knock down specific genes in zebrafish, but their constitutive activities limit their exptl. applications for studying a gene with multiple functions or within a gene network. We report herein a new design and synthesis of caged circular MOs (caged cMOs) with two ends linked by a photocleavable moiety. These caged cMOs were successfully used to photomodulate β-catenin-2 and no tail expression in zebrafish embryos.
- 31Griepenburg, J. C.; Rapp, T. L.; Carroll, P. J.; Eberwine, J.; Dmochowski, I. J. Ruthenium-caged antisense morpholinos for regulating gene expression in zebrafish embryos. Chem. Sci. 2015, 6 (4), 2342– 2346, DOI: 10.1039/C4SC03990DGoogle Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvVCju70%253D&md5=7093c6ea7d35f8840bf655ac9d264833Ruthenium-caged antisense morpholinos for regulating gene expression in zebrafish embryosGriepenburg, Julianne C.; Rapp, Teresa L.; Carroll, Patrick J.; Eberwine, James; Dmochowski, Ivan J.Chemical Science (2015), 6 (4), 2342-2346CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Photochem. approaches afford high spatiotemporal control over mol. structure and function, for broad applications in materials and biol. science. Here, we present the first example of a visible light responsive ruthenium-based photolinker, Ru(bipyridine)2(3-ethynylpyridine)2 (RuBEP), which was reacted stoichiometrically with a 25mer DNA or morpholino (MO) oligonucleotide functionalized with 3' and 5' terminal azides, via Cu(I)-mediated [3+2] Huisgen cycloaddn. reactions. RuBEP-caged circular morpholinos (Ru-MOs) targeting two early developmental zebrafish genes, chordin and notail, were synthesized and tested in vivo. One-cell-stage zebrafish embryos microinjected with Ru-MO and incubated in the dark for 24 h developed normally, consistent with caging, whereas irradn. at 450 nm dissocd. one 3-ethynylpyridine ligand (Φ = 0.33) and uncaged the MO to achieve gene knockdown. As demonstrated, Ru photolinkers provide a versatile method for controlling structure and function of biopolymers.
- 32O’Connor, M. J.; Beebe, L. L.; Deodato, D.; Ball, R. E.; Page, A. T.; VanLeuven, A. J.; Harris, K. T.; Park, S.; Hariharan, V.; Lauderdale, J. D.; Dore, T. M. Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino. ACS Chem. Neurosci. 2019, 10 (1), 266– 278, DOI: 10.1021/acschemneuro.8b00231Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c3ntlOgtQ%253D%253D&md5=70a92f468408c45b048281e01b2cfc75Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker MorpholinoO'Connor Matthew J; Deodato Davide; Dore Timothy M; Beebe Lindsey L; Park Sungdae; Ball Rebecca E; Page A Tyler; VanLeuven Ariel J; Hariharan Vani; Lauderdale James D; Harris Kyle T; Dore Timothy M; Lauderdale James DACS chemical neuroscience (2019), 10 (1), 266-278 ISSN:.γ-Amino butyric acid (GABA) mediated signaling is critical in the central and enteric nervous systems, pancreas, lungs, and other tissues. It is associated with many neurological disorders and craniofacial development. Glutamic acid decarboxylase (GAD) synthesizes GABA from glutamate, and knockdown of the gad1 gene results in craniofacial defects that are lethal in zebrafish. To bypass this and enable observation of the neurological defects resulting from knocking down gad1 expression, a photoactivatable morpholino oligonucleotide (MO) against gad1 was prepared by cyclization with a photocleavable linker rendering the MO inactive. The cyclized MO was stable in the dark and toward degradative enzymes and was completely linearized upon brief exposure to 405 nm light. In the course of investigating the function of the ccMOs in zebrafish, we discovered that zebrafish possess paralogous gad1 genes, gad1a and gad1b. A gad1b MO injected at the 1-4 cell stage caused severe morphological defects in head development, which could be bypassed, enabling the fish to develop normally, if the fish were injected with a photoactivatable, cyclized gad1b MO and grown in the dark. At 1 day post fertilization (dpf), light activation of the gad1b MO followed by observation at 3 and 7 dpf led to increased and abnormal electrophysiological brain activity compared to wild type animals. The photocleavable linker can be used to cyclize and inactivate any MO, and represents a general strategy to parse the function of developmentally important genes in a spatiotemporal manner.
- 33Wu, L.; Wang, Y.; Wu, J.; Lv, C.; Wang, J.; Tang, X. Caged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cells. Nucleic Acids Res. 2013, 41 (1), 677– 86, DOI: 10.1093/nar/gks996Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFaitA%253D%253D&md5=1b29cf249753e4103f213da0d746723fCaged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cellsWu, Li; Wang, Yuan; Wu, Junzhou; Lv, Cong; Wang, Jie; Tang, XinjingNucleic Acids Research (2013), 41 (1), 677-686CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)We synthesized three 20mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10mer oligodeoxynucleotides (ODNs). With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by RNase H were readily photomodulated. RNA cleavage rates were upregulated ∼43-, 25- and 15-fold for R20, R20B2 and R20B4, resp., upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amt. of RNA digestion was still obsd. The loss of binding ability indicated promising gene photoregulation through a non-enzymic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2'-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both RNase H and non-enzyme involved antisense strategies.
- 34Alouane, A.; Labruère, R.; Le Saux, T.; Schmidt, F.; Jullien, L. Self-Immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications. Angew. Chem., Int. Ed. 2015, 54 (26), 7492– 7509, DOI: 10.1002/anie.201500088Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXps1eguro%253D&md5=fe98dd1aff7066a9a58073ee3c57f688Self-immolative spacers: kinetic aspects, structure-property relationships, and applicationsAlouane, Ahmed; Labruere, Raphael; Le Saux, Thomas; Schmidt, Frederic; Jullien, LudovicAngewandte Chemie, International Edition (2015), 54 (26), 7492-7509CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Self-immolative spacers are covalent assemblies tailored to correlate the cleavage of two chem. bonds after activation of a protective part in a precursor: Upon stimulation, the protective moiety is removed, which generates a cascade of disassembling reactions leading to the temporally sequential release of smaller mols. Originally introduced to overcome limitations for drug delivery, self-immolative spacers have gained wide interest in medicinal chem., anal. chem., and material science. For most applications, the kinetics of the disassembly of the activated self-immolative spacer governs functional properties. This Review addresses kinetic aspects of self-immolation. It provides information for selecting a particular self-immolative motif for a specific demand. Moreover, it should help researchers design kinetic expts. and fully exploit the rich perspectives of self-immolative spacers.
- 35Yazawa, R.; Hirono, I.; Aoki, T. Characterization of promoter activities of four different Japanese flounder promoters in transgenic zebrafish. Mar. Biotechnol. (NY) 2005, 7 (6), 625– 33, DOI: 10.1007/s10126-005-0011-1Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2MnjtVGjtA%253D%253D&md5=7d0ab93150e29fc93c052363f4fe91dcCharacterization of promoter activities of four different Japanese flounder promoters in transgenic zebrafishYazawa Ryosuke; Hirono Ikuo; Aoki TakashiMarine biotechnology (New York, N.Y.) (2005), 7 (6), 625-33 ISSN:1436-2228.An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5' flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish.
- 36Udvadia, A. J.; Linney, E. Windows into development: historic, current, and future perspectives on transgenic zebrafish. Dev. Biol. 2003, 256 (1), 1– 17, DOI: 10.1016/S0012-1606(02)00083-0Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXitlWhurc%253D&md5=0c78aa9cd90ab01349053d9bd929ae96Windows into development: historic, current, and future perspectives on transgenic zebrafishUdvadia, Ava J.; Linney, ElwoodDevelopmental Biology (Orlando, FL, United States) (2003), 256 (1), 1-17CODEN: DEBIAO; ISSN:0012-1606. (Elsevier Science)A review. The recent explosion of transgenic zebrafish lines in the literature demonstrates the value of this model system for detailed in vivo anal. of gene regulation and morphogenetic movements. The optical clarity and rapid early development of zebrafish provides the ability to follow these events as they occur in live, developing embryos. This article will review the development of transgenic technol. in zebrafish as well as the current and future uses of transgenic zebrafish to explore the dynamic environment of the developing vertebrate embryo.
- 37Yang, L.; Eberwine, J. H.; Dmochowski, I. J. Caspase-Activated Oligonucleotide Probe. Bioconjugate Chem. 2020, 31 (9), 2172– 2178, DOI: 10.1021/acs.bioconjchem.0c00362Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGju7zL&md5=adc5d603692003b1dedd34ca19d0b2c1Caspase-Activated Oligonucleotide ProbeYang, Linlin; Eberwine, James H.; Dmochowski, Ivan J.Bioconjugate Chemistry (2020), 31 (9), 2172-2178CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Light-activated ("caged") oligonucleotides provide a strategy for modulating the activity of antisense oligos, siRNA, miRNA, aptamers, DNAzymes, and mRNA-capturing probes with high spatiotemporal resoln. However, the near-UV and visible wavelengths that promote these bond-breaking reactions poorly penetrate living tissue, which limits some biol. applications. To address this issue, we describe the first example of a protease-activated oligonucleotide probe, capable of reporting on caspase-3 during cellular apoptosis. The 2'-F RNA-peptide substrate-peptide nucleic acid (PNA) hairpin structure was generated in 30% yield in a single bioconjugation step.
- 38Bonomo, R. A. β-Lactamases: A Focus on Current Challenges. Cold Spring Harbor Perspect. Med. 2017, 7 (1), a025239, DOI: 10.1101/cshperspect.a025239Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFKms7rK&md5=ecbc5d8ec1265dab4c924a9724cee6b7β-lactamases: a focus on current challengesBonomo, Robert A.Cold Spring Harbor Perspectives in Medicine (2017), 7 (1), a025239/1-a025239/16CODEN: CSHPFV; ISSN:2157-1422. (Cold Spring Harbor Laboratory Press)β-Lactamases, the enzymes that hydrolyze β-lactam antibiotics, remain the greatest threat to the usage of these agents. In this review, the mechanism of hydrolysis is discussed for both those enzymes that use serine at the active site and those that require divalent zinc ions for hydrolysis. The β-lactamases now include >2000 unique, naturally occurring amino acid sequences. Some of the clin. most important of these are the class A penicillinases, the extended-spectrum β-lactamases (ESBLs), the AmpC cephalosporinases, and the carbapenem-hydrolyzing enzymes in both the serine and metalloenzyme groups. Because of the versatility of these enzymes to evolve as ne β-lactams are used therapeutically, new approaches to antimicrobial therapy may be required.
- 39Tooke, C. L.; Hinchliffe, P.; Bragginton, E. C.; Colenso, C. K.; Hirvonen, V. H. A.; Takebayashi, Y.; Spencer, J. beta-Lactamases and beta-Lactamase Inhibitors in the 21st Century. J. Mol. Biol. 2019, 431 (18), 3472– 3500, DOI: 10.1016/j.jmb.2019.04.002Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnt1Cmur8%253D&md5=abb0775c85a735c83a96dabac95a7ca5β-Lactamases and β-Lactamase Inhibitors in the 21st CenturyTooke, Catherine L.; Hinchliffe, Philip; Bragginton, Eilis C.; Colenso, Charlotte K.; Hirvonen, Viivi H. A.; Takebayashi, Yuiko; Spencer, JamesJournal of Molecular Biology (2019), 431 (18), 3472-3500CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)A review. The β-lactams retain a central place in the antibacterial armamentarium. In Gram-neg. bacteria, β-lactamase enzymes that hydrolyze the amide bond of the four-membered β-lactam ring are the primary resistance mechanism, with multiple enzymes disseminating on mobile genetic elements across opportunistic pathogens such as Enterobacteriaceae (e.g., Escherichia coli) and non-fermenting organisms (e.g., Pseudomonas aeruginosa). β-Lactamases divide into four classes; the active-site serine β-lactamases (classes A, C and D) and the zinc-dependent or metallo-β-lactamases (MBLs; class B). Here we review recent advances in mechanistic understanding of each class, focusing upon how growing nos. of crystal structures, in particular for β-lactam complexes, and methods such as neutron diffraction and mol. simulations, have improved understanding of the biochem. of β-lactam breakdown. A second focus is β-lactamase interactions with carbapenems, as carbapenem-resistant bacteria are of grave clin. concern and carbapenem-hydrolyzing enzymes such as KPC (class A) NDM (class B) and OXA-48 (class D) are proliferating worldwide. An overview is provided of the changing landscape of β-lactamase inhibitors, exemplified by the introduction to the clinic of combinations of β-lactams with diazabicyclooctanone and cyclic boronate serine β-lactamase inhibitors, and of progress and strategies toward clin. useful MBL inhibitors. Despite the long history of β-lactamase research, we contend that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallog.; the need for new inhibitors, particularly for MBLs; the likely impact of new β-lactam:inhibitor combinations and the continuing clin. importance of β-lactams mean that this remains a rewarding research area.
- 40Bagshawe, K. D. Antibody-directed enzyme prodrug therapy (ADEPT) for cancer. Expert Rev. Anticancer Ther 2006, 6 (10), 1421– 31, DOI: 10.1586/14737140.6.10.1421Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFeru7zL&md5=a937f4361e244e2d34f63f605891bbcaAntibody-directed enzyme prodrug therapy (ADEPT) for cancerBagshawe, Kenneth D.Expert Review of Anticancer Therapy (2006), 6 (10), 1421-1431CODEN: ERATBJ; ISSN:1473-7140. (Future Drugs Ltd.)A review. Antibody-directed enzyme prodrug therapy was conceived as a means of restricting the action of cytotoxic drugs to tumor sites. Since antigenic targets were a central component of the approach, colonic cancer, with its virtually universal expression of carcinoembryonic antigen at the cellular level, presented an obvious starting point. The principle of antibody-directed enzyme prodrug therapy is to use an antibody directed at a tumor-assocd. antigen to vector an enzyme to tumor sites. The enzyme should be retained at tumor sites after it has cleared from blood and normal tissues. A nontoxic prodrug, a substrate for the enzyme, is then given and, by cleaving an inactivating component from the prodrug, a potent cytotoxic agent is generated. One of the potential advantages of such a system is that a small cytotoxic agent, generated within a tumor site, is much more diffusible than a large antibody mol. Moreover, failure to express the target antigen by cancer cells does not protect them from the bystander action of the cytotoxic agent. This review will primarily consider the studies of the London group since this is the only group that has so far reported clin. trials and it is only through clin. trials that the requirements of a successful antibody-directed enzyme prodrug therapy system can be identified.
- 41Zhang, J.; Kale, V.; Chen, M. Gene-directed enzyme prodrug therapy. AAPS J. 2015, 17 (1), 102– 10, DOI: 10.1208/s12248-014-9675-7Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVequ73L&md5=5d088566e4305bb80be167d2bc8656d7Gene-Directed Enzyme Prodrug TherapyZhang, Jin; Kale, Vijay; Chen, MingnanAAPS Journal (2015), 17 (1), 102-110CODEN: AJAOB6; ISSN:1550-7416. (Springer)A review. As one targeting strategy of prodrug delivery, gene-directed enzyme prodrug therapy (GDEPT) promises to realize the targeting through its three key features in cancer therapy-cell-specific gene delivery and expression, controlled conversion of prodrugs to drugs in target cells, and expanded toxicity to the target cells' neighbors through bystander effects. After over 20 years of development, multiple GDEPT systems have advanced into clin. trials. However, no GDEPT product is currently marketed as a drug, suggesting that there are still barriers to overcome before GDEPT becomes a std. therapy. In this review, we first provide a general introduction of this prodrug targeting strategy. Then, we utilize the four most thoroughly studied systems to illustrate components, mechanisms, preclin. and clin. results, and further development directions of GDEPT. These four systems are herpes simplex virus thymidine kinase/ganciclovir, cytosine deaminase/5-fluorocytosine, cytochrome P 450/oxazaphosphorines, and nitroreductase/CB1954 system. Later, we focus our discussion on bystander effects including local and distant bystander effects. Lastly, we discuss carriers that are used to deliver genes for GDEPT including virus carriers and non-virus carriers. Among these carriers, the stem cell-based gene delivery system represents one of the newest carriers under development, and may brought about a breakthrough to the gene delivery issue of GDEPT.
- 42Schellmann, N.; Deckert, P. M.; Bachran, D.; Fuchs, H.; Bachran, C. Targeted enzyme prodrug therapies. Mini-Rev. Med. Chem. 2010, 10 (10), 887– 904, DOI: 10.2174/138955710792007196Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFensLjN&md5=c147e612acd4569b5388ae8b08eafe53Targeted enzyme prodrug therapiesSchellmann, N.; Deckert, P. M.; Bachran, D.; Fuchs, H.; Bachran, C.Mini-Reviews in Medicinal Chemistry (2010), 10 (10), 887-904CODEN: MMCIAE; ISSN:1389-5575. (Bentham Science Publishers Ltd.)A review. The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a no. of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A no. of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview of the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.
- 43Pereira, M. P.; Shi, J.; Kelley, S. O. Peptide Targeting of an Antibiotic Prodrug toward Phagosome-Entrapped Mycobacteria. ACS Infect Dis 2015, 1 (12), 586– 92, DOI: 10.1021/acsinfecdis.5b00099Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1OhurjJ&md5=d664a0d74e81e7152516326435669291Peptide targeting of an antibiotic prodrug toward phagosome-entrapped mycobacteriaPereira, Mark P.; Shi, Julie; Kelley, Shana O.ACS Infectious Diseases (2015), 1 (12), 586-592CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)Mycobacterial infections are difficult to treat due to the bacterium's slow growth, ability to reside in intracellular compartments within macrophages, and resistance mechanisms that limit the effectiveness of conventional antibiotics. Developing antibiotics that overcome these challenges is therefore crit. to providing a pipeline of effective antimicrobial agents. Here, the authors describe the synthesis and testing of a unique peptide-drug conjugate that exhibits high levels of antimicrobial activity against M. smegmatis and M. tuberculosis as well as clearance of intracellular mycobacteria from cultured macrophages. Using an engineered peptide sequence, they deliver a potent DHFR inhibitor and target the intracellular phagosomes where mycobacteria reside and also incorporate a β-lactamase-cleavable cephalosporin linker to enhance the targeting of quiescent intracellular β-lactam-resistant mycobacteria. By using this type of prodrug approach to target intracellular mycobacterial infections, the emergence of antibacterial resistance mechanisms could be minimized.
- 44Liu, R.; Miller, P. A.; Vakulenko, S. B.; Stewart, N. K.; Boggess, W. C.; Miller, M. J. A Synthetic Dual Drug Sideromycin Induces Gram-Negative Bacteria To Commit Suicide with a Gram-Positive Antibiotic. J. Med. Chem. 2018, 61 (9), 3845– 3854, DOI: 10.1021/acs.jmedchem.8b00218Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltV2rsbk%253D&md5=0c3db140a8cd4e7c9f533120a2407cf6A Synthetic Dual Drug Sideromycin Induces Gram-Negative Bacteria To Commit Suicide with a Gram-Positive AntibioticLiu, Rui; Miller, Patricia A.; Vakulenko, Sergei B.; Stewart, Nichole K.; Boggess, William C.; Miller, Marvin J.Journal of Medicinal Chemistry (2018), 61 (9), 3845-3854CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Many antibiotics lack activity against Gram-neg. bacteria because they cannot permeate the outer membrane or suffer from efflux and, in the case of β-lactams, are degraded by β-lactamases. Herein, we describe the synthesis and studies of a dual drug conjugate (1) of a siderophore linked to a cephalosporin with an attached oxazolidinone. The cephalosporin component of 1 is rapidly hydrolyzed by purified ADC-1 β-lactamase to release the oxazolidinone. Conjugate 1 is active against clin. isolates of Acinetobacter baumannii as well as strains producing large amts. of ADC-1 β-lactamase. Overall, the results are consistent with siderophore-mediated active uptake, inherent activity of the delivered dual drug, and in the presence of β-lactamases, intracellular release of the oxazolidinone upon cleavage of the cephalosporin to allow the freed oxazolidinone to inactivate its target. The ultimate result demonstrates that Gram-pos. oxazolidinone antibiotics can be made to be effective against Gram-neg. bacteria by β-lactamase triggered release.
- 45Raz, E.; Zlokarnik, G.; Tsien, R. Y.; Driever, W. beta-lactamase as a marker for gene expression in live zebrafish embryos. Dev. Biol. 1998, 203 (2), 290– 4, DOI: 10.1006/dbio.1998.8999Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXnslyqtr8%253D&md5=229fe38f5712323c662d951118fda525β-Lactamase as a marker for gene expression in live zebrafish embryosRaz, Erez; Zlokarnik, Gregor; Tsien, Roger Y.; Driever, WolfgangDevelopmental Biology (1998), 203 (2), 290-294CODEN: DEBIAO; ISSN:0012-1606. (Academic Press)In this report the authors describe the development of a sensitive assay for gene expression in zebrafish embryos using β-lactamase as a reporter gene. The authors show that injection of a green fluorescent substrate for β-lactamase allows the detection of reporter gene expression in live embryos. The β-lactamase enzyme catalyzes the hydrolysis of the substrate, thereby disrupting fluorescence resonance energy transfer from the donor to the acceptor dye in the mol. As a result, a blue fluorescent product is produced and retained specifically in cells within which the enzyme is expressed. β-Lactamase is therefore suitable for monitoring spatially restricted patterns of gene expression in the early embryo. The authors suggest that this new reporter system provides a major advancement in sensitivity over the existing methods for monitoring gene expression in vivo during early embryogenesis. (c) 1998 Academic Press.
- 46Zlokarnik, G.; Negulescu, P. A.; Knapp, T. E.; Mere, L.; Burres, N.; Feng, L.; Whitney, M.; Roemer, K.; Tsien, R. Y. Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 1998, 279 (5347), 84– 8, DOI: 10.1126/science.279.5347.84Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjtVSjsg%253D%253D&md5=25d3beee86614403847e2ec7b26aed18Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporterZlokarnik, Gregor; Negulescu, Paul A.; Knapp, Thomas E.; Mere, Lora; Burres, Neal; Feng, Luxin; Whitney, Michael; Roemer, Klaus; Tsien, Roger Y.Science (Washington, D. C.) (1998), 279 (5347), 84-88CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Gene expression was visualized in single living mammalian cells with β-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each enzyme mol. changed the fluorescence of many substrate mols. from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual cells contg. less than 100 β-lactamase mols. The robust change in emission ratio reveals quant. heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.
- 47Boyd, D. B. Electronic structures of cephalosporins and penicillins. 15. Inductive effect of the 3-position side chain in cephalosporins. J. Med. Chem. 1984, 27 (1), 63– 6, DOI: 10.1021/jm00367a012Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXitVSruw%253D%253D&md5=10aa695ae86dd0b1d9c5f3fbca297555Electronic structures of cephalosporins and penicillins. 15. Inductive effect of the 3-position side chain in cephalosporinsBoyd, Donald B.Journal of Medicinal Chemistry (1984), 27 (1), 63-6CODEN: JMCMAR; ISSN:0022-2623.A discussion with 39 refs. Induction appears to be the primary means by which the side chain at position 3 of the cephem nucleus influences the chem. reactivity of the β-lactam ring. In vitro antibacterial activity data suggest that when the cephalosporin is in the active site of the target bacterial enzymes, the presence of a leaving group in the side chain can promote inhibition.
- 48Faraci, W. S.; Pratt, R. F. Elimination of a good leaving group from the 3′-position of a cephalosporin need not be concerted with β-lactam ring opening: TEM-2 β-lactamase-catalyzed hydrolysis of pyridine-2-azo-4′-(N′,N′-dimethylaniline) cephalosporin (PADAC) and of cephaloridine. J. Am. Chem. Soc. 1984, 106 (5), 1489– 1490, DOI: 10.1021/ja00317a053Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXpvFShtw%253D%253D&md5=343a8f7fde7a9cc798ca696e793f07a0Elimination of a good leaving group from the 3'-position of a cephalosporin need not be concerted with β-lactam ring opening: TEM-2 β-lactamase-catalyzed hydrolysis of pyridine-2-azo-4'-(N',N'-dimethylaniline) cephalosporin (PADAC) and of cephaloridineFaraci, W. Stephen; Pratt, R. F.Journal of the American Chemical Society (1984), 106 (5), 1489-90CODEN: JACSAT; ISSN:0002-7863.The hydrolysis of the chromogenic cephalosporin PADAC and of cephaloridine in the presence of TEM-2 β-lactamase occurs in 2 steps, the 1st enzyme-catalyzed and the 2nd not. It is proposed that the enzyme-catalyzed reaction involves β-lactam ring opening, whereas the 2nd step involves spontaneous elimination of the 3-substituent from the cephalosporate thereby formed. Thus, release of good leaving groups from the 3'-position of cephalosporins need not be concerted with β-lactam ring opening. Other enzymes, e.g., Bacillus β-lactamase II, may catalyze elimination as well as β-lactam hydrolysis, either in a concerted or nonconcerted reaction at the enzyme surface.
- 49Patterson, L. D.; Miller, M. J. Enzymatic deprotection of the cephalosporin 3′-acetoxy group using Candida antarctica lipase B. J. Org. Chem. 2010, 75 (4), 1289– 92, DOI: 10.1021/jo902406bGoogle Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVSitrk%253D&md5=21f4867040cea21848961d8c27477339Enzymatic Deprotection of the Cephalosporin 3'-Acetoxy Group Using Candida antarctica Lipase BPatterson, Leslie D.; Miller, Marvin J.Journal of Organic Chemistry (2010), 75 (4), 1289-1292CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)Cephalosporins remain one of the most important classes of antibiotics. A useful site for derivatization involves generation of and chem. at the 3'-hydroxymethyl position. While 3'-acetoxymethyl-substituted cephalosporins are readily available, deacetylation to access the free 3'-hydroxymethyl group is problematic when the carboxylic acid is protected as an ester. Herein we report that this important transformation has been efficiently accomplished using Candida antarctica lipase B. Although this transformation is difficult to carry out using chem. methods, the enzymic deacetylation has been successful on gram scale, when the cephalosporin is protected as either the benzhydryl or tert-Bu esters and on the corresponding sulfoxide and sulfone of the tert-Bu ester.
- 50Sarkar, K.; Madras, G.; Chatterjee, K. Dendron conjugation to graphene oxide using click chemistry for efficient gene delivery. RSC Adv. 2015, 5 (62), 50196– 50211, DOI: 10.1039/C5RA07004JGoogle Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpt1Ojtbc%253D&md5=e3ee0d99f8c8880373d492ffe3d42a16Dendron conjugation to graphene oxide using click chemistry for efficient gene deliverySarkar, Kishor; Madras, Giridhar; Chatterjee, KaushikRSC Advances (2015), 5 (62), 50196-50211CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)Owing to its large surface area and rapid cellular uptake, graphene oxide (GO) is emerging as an attractive candidate material for delivery of drugs and genes. The inherent sp2 π-π interaction of GO helps to carry drugs and single stranded RNA (ssRNA) but there is no such interaction with double stranded DNA (dsDNA). In this work, a polyamidoamine (PAMAM) dendron was conjugated with nano GO (nGO) through "click" chem. to improve the DNA complexation capability of GO as well as its transfection efficiency. The DNA complexation capability of GO was significantly enhanced after dendronization of GO yielding spherical nanosized (250-350 nm) particles of the dendronized GO (DGO)/pDNA complex with a pos. zeta potential. The transfection efficiency of GO dramatically increased after conjugation of the PAMAM dendron. Transfection efficiency of 51% in HeLa cells with cell viability of 80% was obsd. The transfection efficiency was significantly higher than that of polyethyleneimine 25 kDa (27% efficiency) and also surpassed that of lipofectamine 2000 (47% efficiency). The uptake of the DGO/pDNA complex by the caveolae mediated endocytosis pathway may significantly contribute to the high transfection efficiency. Thus, dendronized GO is shown to be an efficient gene carrier with minimal toxicity and is a promising candidate for use as a nonviral carrier for gene therapy.
- 51Halpern, M. E.; Ho, R. K.; Walker, C.; Kimmel, C. B. Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation. Cell 1993, 75 (1), 99– 111, DOI: 10.1016/S0092-8674(05)80087-XGoogle Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2c%252Fgs1Gmsg%253D%253D&md5=1858c7da3d2edc4ecd63ea7e91d22680Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutationHalpern M E; Ho R K; Walker C; Kimmel C BCell (1993), 75 (1), 99-111 ISSN:0092-8674.Dorsal mesoderm is thought to provide important signals for axis formation and neural differentiation in vertebrate embryos. We have examined induction and patterning in a zebrafish mutant, no tail, that lacks a derivative of dorsal mesoderm, the notochord. Despite the absence of a differentiated notochord, development of the central nervous system including floor plate appears normal, likely owing to the presence of notochord precursor cells. In contrast, somites are misshapen, and muscle pioneer cells are absent. Wild-type cells transplanted into mutant hosts can autonomously differentiate into notochord and thereby rescue somitic defects, suggesting that interactions between notochord and paraxial mesoderm are necessary for proper somite patterning. Thus, cells derived from dorsal mesoderm may have multiple signaling functions during zebrafish embryogenesis.
- 52Schulte-Merker, S.; van Eeden, F. J. M.; Halpern, M. E.; Kimmel, C. B.; Nüsslein-Volhard, C. no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene. Development 1994, 120 (4), 1009– 15, DOI: 10.1242/dev.120.4.1009Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmsleltr0%253D&md5=322293a463aa41ce475b68f2efd9fc8ano tail (ntl) is the zebrafish homolog of the mouse T (Brachyury) geneSchulte-Merker, S.; van Eeden, F. J. M.; Halpern, M. E.; Kimmel, C. B.; Nusslein-Volhard, C.Development (Cambridge, United Kingdom) (1994), 120 (4), 1009-15CODEN: DEVPED; ISSN:0950-1991.The mouse T (Brachyury) gene is required for normal mesoderm development and the extension of the body axis. Recently, two mutant alleles of a zebrafish gene, no tail (ntl), have been isolated (Halpern, M. E., Ho., R. K., Walker, C. and Kimmel, C. B. (1993) Cell 75, 99-111). Ntl mutant embryos resemble mouse T/T mutant embryos in that they lack a differentiated notochord and the caudal region of their bodies. We report here that this phenotype is caused by mutation of the zebrafish homolog of the T gene. While ntl embryos express mutant mRNA, they show no nuclear protein product. Later, expression of mRNA in mutants, but not in wild types, is greatly reduced along the dorsal midline where the notochord normally forms. This suggests that the protein is required for maintaining transcription of its own gene.
- 53Shabanpoor, F.; Gait, M. J. Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry. Chem. Commun. 2013, 49 (87), 10260– 2, DOI: 10.1039/C3CC46067CGoogle Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFynsLzI&md5=fd9873978cb407be3fe54c4ab8ecaa7cDevelopment of a general methodology for labeling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistryShabanpoor, Fazel; Gait, Michael J.Chemical Communications (Cambridge, United Kingdom) (2013), 49 (87), 10260-10262CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)We describe a general methodol. for fluorescent labeling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labeling with a fluorescent compd. (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labeled PMOs was seen in a biol. assay.
- 54O’Callaghan, C. H.; Morris, A.; Kirby, S. M.; Shingler, A. H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1972, 1 (4), 283– 8, DOI: 10.1128/AAC.1.4.283Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaE2c7ns1agsg%253D%253D&md5=dc646a87f692cf4413e81d22218da743Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrateO'Callaghan C H; Morris A; Kirby S M; Shingler A HAntimicrobial agents and chemotherapy (1972), 1 (4), 283-8 ISSN:0066-4804.A new cephalosporin with a highly reactive beta-lactam ring was found to give an immediate color change in the presence of beta-lactamases from many bacteria, including staphylococci, Bacillus species, Enterobacteriaceae, and Pseudomonas. The reaction is confined to organisms producing beta-lactamases, but it is sufficiently sensitive to indicate the presence of this enzyme is small amounts in strains previously considered not to produce it. The compound has an unusual ultraviolet spectrum, and the color change can be followed quantitatively by measuring changes in absorption which occur in the 380- to 500-nm region, where cephalosporins normally have no absorption. The development of color is thought to be a consequence of the beta-lactam ring being unusually highly conjugated with the 3-substituent. Although in the bacteria only beta-lactamases produce this color change, it was found that serum and tissues from experimental animals also rapidly produced the colored breakdown product, which was then excreted in the urine. The mechanism of the mammalian breakdown was considered to be different from that found in bacteria.
- 55D’Astolfo, D. S.; Pagliero, R. J.; Pras, A.; Karthaus, W. R.; Clevers, H.; Prasad, V.; Lebbink, R. J.; Rehmann, H.; Geijsen, N. Efficient intracellular delivery of native proteins. Cell 2015, 161 (3), 674– 690, DOI: 10.1016/j.cell.2015.03.028Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXnt1emtr0%253D&md5=6d1ec33e3e73be57383e1d964e4235b2Efficient intracellular delivery of native proteinsD'Astolfo, Diego S.; Pagliero, Romina J.; Pras, Anita; Karthaus, Wouter R.; Clevers, Hans; Prasad, Vikram; Lebbink, Robert Jan; Rehmann, Holger; Geijsen, NielsCell (Cambridge, MA, United States) (2015), 161 (3), 674-690CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compd. (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner.
- 56Yuan, S.; Sun, Z. Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. J. Visualized Exp. 2009, 27, e1113, DOI: 10.3791/1113Google ScholarThere is no corresponding record for this reference.
- 57Rosen, J. N.; Sweeney, M. F.; Mably, J. D. Microinjection of zebrafish embryos to analyze gene function. J. Visualized Exp. 2009, 25, 1115, DOI: 10.3791/1115Google ScholarThere is no corresponding record for this reference.
- 58Kimmel, C. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.; Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 1995, 203 (3), 253– 310, DOI: 10.1002/aja.1002030302Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK287nt12qsw%253D%253D&md5=629ae03ffdeb5b0a9f0e5ea52fb3133cStages of embryonic development of the zebrafishKimmel C B; Ballard W W; Kimmel S R; Ullmann B; Schilling T FDevelopmental dynamics : an official publication of the American Association of Anatomists (1995), 203 (3), 253-310 ISSN:1058-8388.We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis--the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts.
- 59Kimmel, C. B.; Law, R. D. Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells. Dev. Biol. 1985, 108 (1), 78– 85, DOI: 10.1016/0012-1606(85)90010-7Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL2M7isl2ksQ%253D%253D&md5=b8b4fa9b5be9054a0e388421ce93f6b9Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cellsKimmel C B; Law R DDevelopmental biology (1985), 108 (1), 78-85 ISSN:0012-1606.Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomere's lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cell's sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.
- 60Hoo, J. Y.; Kumari, Y.; Shaikh, M. F.; Hue, S. M.; Goh, B. H. Zebrafish: A Versatile Animal Model for Fertility Research. Biomed Res. Int. 2016, 2016, 9732780, DOI: 10.1155/2016/9732780Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2szjvVCnsQ%253D%253D&md5=5353875da64e8611a62b4df23b952d9dZebrafish: A Versatile Animal Model for Fertility ResearchHoo Jing Ying; Kumari Yatinesh; Shaikh Mohd Farooq; Hue Seow Mun; Goh Bey HingBioMed research international (2016), 2016 (), 9732780 ISSN:.The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research.
- 61Curado, S.; Stainier, D. Y.; Anderson, R. M. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat. Protoc 2008, 3 (6), 948– 54, DOI: 10.1038/nprot.2008.58Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmvVemtrg%253D&md5=57fd17f5871cd47ca3443a83f203f7b1Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studiesCurado, Silvia; Stainier, Didier Y. R.; Anderson, Ryan M.Nature Protocols (2008), 3 (6), 948-954CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chem. and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, the authors provide a step-by-step protocol for a hybrid chem.-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the redn. of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR+ cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.
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Abstract
Figure 1
Figure 1. Following enzyme-mediated hydrolysis of the β-lactam ring by β-lactamase (PDB ID 4F6H), the resulting amine intermediate undergoes self-immolation. The release of the 5′-end generates the active, linearized MO capable of hybridizing to its respective target mRNA and silencing gene expression.
Figure 2
Figure 2. (A) Synthetic scheme of the cephalosporin-containing linker 8. (B) Synthesis of the cephalosporin ntla cMO.
Figure 3
Figure 3. (A) Luciferase reporter assay in which luciferase expression is silenced in the presence of linearized MO. (B) Luminescence signal after treatment of the indicated MO. Data represent average ± SEM from three independent experiments. (C) Gel-shift assay showing activation of the circular morpholino sequestering its complementary target DNA (cDNA) in the presence of β-lactamase. (D) Quantification of a time course analysis of cyclic cephalosporin ntla cMO cleavage following incubation with recombinant β-lactamase. Bars represent averages and error bars represent standard deviations from three independent experiments.
Figure 4
Figure 4. (A) Structure of the chromogenic β-lactamase substrate nitrocefin (10), which is hydrolyzed to 11 following β-lactamase catalysis. (B) Western blot analysis of β-lactamase expression in embryos injected with 400 pg of mRNA. (C) Zebrafish lysates analyzed for β-lactamase activity by monitoring production of 11 (absorbance at 486 nm) over time. (D) Initial velocities were determined through a linear regression analysis of the first 10 min of the data presented in (C). The error values indicate the standard deviation of values for the best fit parameters from the linear regression analysis.
Figure 5
Figure 5. (A) Representative images of ntla morphant phenotypes in zebrafish embryos at 24 hpf. Scale bar equals 1 mm. (B) Phenotypic scoring of embryos injected with the indicated morpholino and amount of β-lactamase mRNA.
References
This article references 61 other publications.
- 1Khvorova, A.; Watts, J. K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. 2017, 35 (3), 238– 248, DOI: 10.1038/nbt.37651https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmtlalurc%253D&md5=d6c3f3ec136632eee79a63fc0be0bbfcThe chemical evolution of oligonucleotide therapies of clinical utilityKhvorova, Anastasia; Watts, Jonathan K.Nature Biotechnology (2017), 35 (3), 238-248CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)After nearly 40 years of development, oligonucleotide therapeutics are nearing meaningful clin. productivity. One of the key advantages of oligonucleotide drugs is that their delivery and potency are derived primarily from the chem. structure of the oligonucleotide whereas their target is defined by the base sequence. Thus, as oligonucleotides with a particular chem. design show appropriate distribution and safety profiles for clin. gene silencing in a particular tissue, this will open the door to the rapid development of addnl. drugs targeting other disease-assocd. genes in the same tissue. To achieve clin. productivity, the chem. architecture of the oligonucleotide needs to be optimized with a combination of sugar, backbone, nucleobase, and 3'- and 5'-terminal modifications. A portfolio of chemistries can be used to confer drug-like properties onto the oligonucleotide as a whole, with minor chem. changes often translating into major improvements in clin. efficacy. One outstanding challenge in oligonucleotide chem. development is the optimization of chem. architectures to ensure long-term safety. There are multiple designs that enable effective targeting of the liver, but a second challenge is to develop architectures that enable robust clin. efficacy in addnl. tissues.
- 2Chakraborty, S.; Mehtab, S.; Krishnan, Y. The predictive power of synthetic nucleic acid technologies in RNA biology. Acc. Chem. Res. 2014, 47 (6), 1710– 9, DOI: 10.1021/ar400323d2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlsl2ktro%253D&md5=4c6a94878c3542c311499ae0c3d0a1d4The Predictive Power of Synthetic Nucleic Acid Technologies in RNA BiologyChakraborty, Saikat; Mehtab, Shabana; Krishnan, YamunaAccounts of Chemical Research (2014), 47 (6), 1710-1719CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The impact of nucleic acid nanotechnol. in terms of transforming motifs from biol. in synthetic and translational ways is widely appreciated. But it is also emerging that the thinking and vision behind nucleic acids as construction material has broader implications, not just in nanotechnol. or even synthetic biol., but can feed back into our understanding of biol. itself. Physicists have treated nucleic acids as polymers and connected phys. principles to biol. by abstracting out the mol. interactions. In contrast, biologists delineate mol. players and pathways related to nucleic acids and how they may be networked. But in vitro nucleic acid nanotechnol. has provided a valuable framework for nucleic acids by connecting its biomol. interactions with its materials properties and thereby superarchitecture ultramanipulation that on multiple occasions has pre-empted the elucidation of how living cells themselves are exploiting these same structural concepts. This Account seeks to showcase the larger implications of certain architectural principles that have arisen from the field of structural DNA/RNA nanotechnol. in biol. Here we draw connections between these principles and particular mol. phenomena within living systems that have fed in to our understanding of how the cell uses nucleic acids as construction material to achieve different functions. We illustrate this by considering a few exciting and emerging examples in biol. in the context of both switchable systems and scaffolding type systems. Due to the scope of this Account, we will focus our discussion on examples of the RNA scaffold as summarized. In the context of switchable RNA architectures, the synthetic demonstration of small mols. blocking RNA translation preceded the discovery of riboswitches. In another example, it was after the description of aptazymes that the first allosteric ribozyme, glmS, was discovered. In the context of RNA architectures as structural scaffolds, there are clear parallels between DNA origami and the recently emerging mol. mechanism of heterochromatin formation by Xist RNA. Further, following the construction of well-defined 2D DNA-protein architectures, the striking observation of remarkably sculpted 2D RNA-protein hydrogel sheets in Caenorhabditis elegans speaks to the in vivo relevance of designer nucleic acid architectures. It is noteworthy that discoveries of properties in synthetic space seem to precede the uncovering of similar phenomena in vivo.
- 3Havens, M. A.; Hastings, M. L. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res. 2016, 44 (14), 6549– 63, DOI: 10.1093/nar/gkw5333https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s%252FovFegsw%253D%253D&md5=09398f6508237c986d967e6658aed8e3Splice-switching antisense oligonucleotides as therapeutic drugsHavens Mallory A; Hastings Michelle LNucleic acids research (2016), 44 (14), 6549-63 ISSN:.Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials.
- 4Shen, X.; Corey, D. R. Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs. Nucleic Acids Res. 2018, 46 (4), 1584– 1600, DOI: 10.1093/nar/gkx12394https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlGisb7F&md5=b5bdec984635f12162386b6f8a6f6fe0Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAsShen, Xiulong; Corey, David R.Nucleic Acids Research (2018), 46 (4), 1584-1600CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)A review. RNA plays a central role in the expression of all genes. Because any sequence within RNA can be recognized by complementary base pairing, synthetic oligonucleotides and oligonucleotide mimics offer a general strategy for controlling processes that affect disease. The two primary antisense approaches for regulating expression through recognition of cellular RNAs are single-stranded antisense oligonucleotides and duplex RNAs. This review will discuss the chem. modifications and mol. mechanisms that make synthetic nucleic acid drugs possible. Lessons learned from recent clin. trials will be summarized. Ongoing clin. trials are likely to decisively test the adequacy of our current generation of antisense nucleic acid technologies and highlight areas where more basic research is needed.
- 5Setten, R. L.; Rossi, J. J.; Han, S. P. The current state and future directions of RNAi-based therapeutics. Nat. Rev. Drug Discovery 2019, 18 (6), 421– 446, DOI: 10.1038/s41573-019-0017-45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXms1GjtL8%253D&md5=679994c46a9e0e4868a97001b742ce90The current state and future directions of RNAi-based therapeuticsSetten, Ryan L.; Rossi, John J.; Han, Si-pingNature Reviews Drug Discovery (2019), 18 (6), 421-446CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review. The RNA interference (RNAi) pathway regulates mRNA stability and translation in nearly all human cells. Small double-stranded RNA mols. can efficiently trigger RNAi silencing of specific genes, but their therapeutic use has faced numerous challenges involving safety and potency. However, August 2018 marked a new era for the field, with the US Food and Drug Administration approving ONPATTRO, the first RNAi-based drug. In this Review, we discuss key advances in the design and development of RNAi drugs leading up to this landmark achievement, the state of the current clin. pipeline and prospects for future advances, including novel RNAi pathway agents utilizing mechanisms beyond post-translational RNAi silencing.
- 6Rinaldi, C.; Wood, M. J. A. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nat. Rev. Neurol. 2018, 14 (1), 9– 21, DOI: 10.1038/nrneurol.2017.1486https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCrs7jL&md5=a9256a0257a13c8c331e1229aecfd4aaAntisense oligonucleotides: the next frontier for treatment of neurological disordersRinaldi, Carlo; Wood, Matthew J. A.Nature Reviews Neurology (2018), 14 (1), 9-21CODEN: NRNACP; ISSN:1759-4758. (Nature Research)A review. Antisense oligonucleotides (ASOs) were first discovered to influence RNA processing and modulate protein expression over two decades ago; however, progress translating these agents into the clinic has been hampered by inadequate target engagement, insufficient biol. activity, and off-target toxic effects. Over the years, novel chem. modifications of ASOs have been employed to address these issues. These modifications, in combination with elucidation of the mechanism of action of ASOs and improved clin. trial design, have provided momentum for the translation of ASO-based strategies into therapies. Many neurol. conditions lack an effective treatment; however, as research progressively disentangles the pathogenic mechanisms of these diseases, they provide an ideal platform to test ASO-based strategies. This steady progress reached a pinnacle in the past few years with approvals of ASOs for the treatment of spinal muscular atrophy and Duchenne muscular dystrophy, which represent landmarks in a field in which disease-modifying therapies were virtually non-existent. With the rapid development of improved next-generation ASOs toward clin. application, this technol. now holds the potential to have a dramatic effect on the treatment of many neurol. conditions in the near future.
- 7Timme-Laragy, A. R.; Karchner, S. I.; Hahn, M. E. Gene knockdown by morpholino-modified oligonucleotides in the zebrafish (Danio rerio) model: applications for developmental toxicology. Methods Mol. Biol. 2012, 889, 51– 71, DOI: 10.1007/978-1-61779-867-2_57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFGlsbbK&md5=e6a57db28fb1f79eccfcbc4335602e37Gene knockdown by morpholino-modified oligonucleotides in the zebrafish (Danio rerio) model: applications for developmental toxicologyTimme-Laragy, Alicia R.; Karchner, Sibel I.; Hahn, Mark E.Methods in Molecular Biology (New York, NY, United States) (2012), 889 (Developmental Toxicology), 51-71CODEN: MMBIED; ISSN:1064-3745. (Springer)The zebrafish (Danio rerio) has long been used as a model for developmental biol., making it an excellent model to use also in developmental toxicol. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technol. and gene knockdown via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a mol. and mechanistic level while establishing a relationship between these mol. events and morphol., behavioral, and/or physiol. effects (i.e., phenotypic anchoring). In this chapter, we address important considerations for using MOs to study developmental toxicol. in zebrafish embryos and provide a protocol for their use.
- 8Shestopalov, I. A.; Chen, J. K. Oligonucleotide-based tools for studying zebrafish development. Zebrafish 2010, 7 (1), 31– 40, DOI: 10.1089/zeb.2010.06508https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlsVSru70%253D&md5=a323a188b0dcdc1c8b3c5d10f06b81c0Oligonucleotide-Based Tools for Studying Zebrafish DevelopmentShestopalov, Ilya A.; Chen, James K.Zebrafish (2010), 7 (1), 31-40CODEN: ZEBRAL; ISSN:1545-8547. (Mary Ann Liebert, Inc.)A review. Synthetic and nonnatural oligonucleotides have been used extensively to interrogate gene function in zebrafish. In this review, we survey the capabilities and limitations of various oligonucleotide-based technologies for perturbing RNA function and tracking RNA expression. We also examine recent strategies for achieving spatiotemporal control of oligonucleotide function, particularly light-gated technologies that exploit the optical transparency of zebrafish embryos.
- 9Summerton, J.; Weller, D. Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 1997, 7 (3), 187– 95, DOI: 10.1089/oli.1.1997.7.1879https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXktFWmu70%253D&md5=39f0590b00278d17744fb4567e0eb40dMorpholino antisense oligomers: design, preparation, and propertiesSummerton, James; Weller, DwightAntisense & Nucleic Acid Drug Development (1997), 7 (3), 187-195CODEN: ANADF5; ISSN:1087-2906. (Liebert)A review with 41 refs. Antisense promised major advances in treating a broad range of intractable diseases, but in recent years progress has been stymied by tech. problems, most notably inadequate specificity, ineffective delivery into the proper subcellular compartment, and unpredictable activity within cells. An overview is provided of the design, prepn., and properties of morpholino oligos, a novel antisense structural type that solves the sequence specificity problem and provides high and predictable activity in cells. Morpholino oligos also exhibit little or no nonantisense activity, afford good water soly., are immune to nucleases, and are designed to have low prodn. costs.
- 10Hardy, S.; Legagneux, V.; Audic, Y.; Paillard, L. Reverse genetics in eukaryotes. Biol. Cell 2010, 102 (10), 561– 80, DOI: 10.1042/BC2010003810https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFSntb%252FL&md5=a19788a36671f2718dd18f28b30db197Reverse genetics in eukaryotesHardy, Serge; Legagneux, Vincent; Audic, Yann; Paillard, LucBiology of the Cell (2010), 102 (10), 561-580CODEN: BCELDF; ISSN:0248-4900. (Portland Press Ltd.)A review. Reverse genetics consists in the modification of the activity of a target gene to analyze the phenotypic consequences. Four main approaches are used towards this goal and will be explained in this review. Two of them are centered on genome alterations. Mutations produced by random chem. or insertional mutagenesis can be screened to recover only mutants in a specific gene of interest. Alternatively, these alterations may be specifically targeted on a gene of interest by HR (homologous recombination). The other 2 approaches are centered on mRNA. RNA interference is a powerful method to reduce the level of gene products, while MO (morpholino) antisense oligonucleotides alter mRNA metab. or translation. Some model species, such as Drosophila, are amenable to most of these approaches, whereas other model species are restricted to 1 of them. For example, in mice and yeasts, gene targeting by HR is prevalent, whereas in Xenopus and zebrafish MO oligonucleotides are mainly used. Genome-wide collections of mutants or inactivated models obtained in several species by these approaches have been made and will help decipher gene functions in the post-genomic era.
- 11Kole, R.; Krainer, A. R.; Altman, S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat. Rev. Drug Discovery 2012, 11 (2), 125– 40, DOI: 10.1038/nrd362511https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVGqt7k%253D&md5=0db21d536154c3ccd62ba0db1334e3bbRNA therapeutics: beyond RNA interference and antisense oligonucleotidesKole, Ryszard; Krainer, Adrian R.; Altman, SidneyNature Reviews Drug Discovery (2012), 11 (2), 125-140CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Here, we discuss three RNA-based therapeutic technologies exploiting various oligonucleotides that bind to RNA by base pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by inducing enzyme-dependent degrdn. of targeted mRNA. Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, steric-blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein prodn. or downregulate gene expression. Moreover, they can be extensively chem. modified to acquire more drug-like properties. The ability of RNA-blocking oligonucleotides to restore gene function makes them best suited for the treatment of genetic disorders. Pos. results from clin. trials for the treatment of Duchenne muscular dystrophy show that this technol. is close to achieving its clin. potential.
- 12Corey, D. R.; Abrams, J. M. Morpholino antisense oligonucleotides: tools for investigating vertebrate development. Genome Biol. 2001, 2 (5), reviews1015, DOI: 10.1186/gb-2001-2-5-reviews1015There is no corresponding record for this reference.
- 13Ciruna, B.; Weidinger, G.; Knaut, H.; Thisse, B.; Thisse, C.; Raz, E.; Schier, A. F. Production of maternal-zygotic mutant zebrafish by germ-line replacement. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (23), 14919– 24, DOI: 10.1073/pnas.22245999913https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xpt1yrtLs%253D&md5=30e39750bf35757c4e9dcfaf9a0dc7a8Production of maternal-zygotic mutant zebrafish by germ-line replacementCiruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (23), 14919-14924CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryol., genetic, genomic, and biochem. studies.
- 14Bill, B. R.; Petzold, A. M.; Clark, K. J.; Schimmenti, L. A.; Ekker, S. C. A primer for morpholino use in zebrafish. Zebrafish 2009, 6 (1), 69– 77, DOI: 10.1089/zeb.2008.055514https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXkvVGmtrs%253D&md5=7595f7f176f84ca330af855f45c75810A Primer for Morpholino Use in ZebrafishBill, Brent R.; Petzold, Andrew M.; Clark, Karl J.; Schimmenti, Lisa A.; Ekker, Stephen C.Zebrafish (2009), 6 (1), 69-77CODEN: ZEBRAL; ISSN:1545-8547. (Mary Ann Liebert, Inc.)A review. Morpholino oligonucleotides are the most common anti-sense "knockdown" technique used in zebrafish (Danio rerio). This review discusses common practices for the design, prepn., and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.
- 15Shestopalov, I. A.; Chen, J. K. Chemical technologies for probing embryonic development. Chem. Soc. Rev. 2008, 37 (7), 1294– 307, DOI: 10.1039/b703023c15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnsFejsbw%253D&md5=53344f05372a52656ff9bc50c4a138d2Chemical technologies for probing embryonic developmentShestopalov, Ilya A.; Chen, James K.Chemical Society Reviews (2008), 37 (7), 1294-1307CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Embryogenesis is a remarkable program of cell proliferation, migration, and differentiation that transforms a single fertilized egg into a complex multicellular organism. Understanding this process at the mol. and systems levels will require an interdisciplinary approach, including the concepts and technologies of chem. biol. This tutorial review provides an overview of chem. tools that have been used in developmental biol. research, focusing on methods that enable spatiotemporal control of gene function and the visualization of embryonic patterning. Limitations of current approaches and future challenges are also discussed.
- 16Darrah, K. E.; Deiters, A. Translational control of gene function through optically regulated nucleic acids. Chem. Soc. Rev. 2021, 50 (23), 13253– 13267, DOI: 10.1039/D1CS00257K16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVahtr%252FJ&md5=16aba43c93be276b4e09a6b914769710Translational control of gene function through optically regulated nucleic acidsDarrah, Kristie E.; Deiters, AlexanderChemical Society Reviews (2021), 50 (23), 13253-13267CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Translation of mRNA into protein is one of the most fundamental processes within biol. systems. Gene expression is tightly regulated both in space and time, often involving complex signaling or gene regulatory networks, as most prominently obsd. in embryo development. Thus, studies of gene function require tools with a matching level of external control. Light is an excellent conditional trigger as it is minimally invasive, can be easily tuned in wavelength and amplitude, and can be applied with excellent spatial and temporal resoln. To this end, modification of established oligonucleotide-based technologies with optical control elements, in the form of photocaging groups and photoswitches, has rendered these tools capable of navigating the dynamic regulatory pathways of mRNA translation in cellular and in vivo models. In this review, we discuss the different optochem. approaches used to generate photoresponsive nucleic acids that activate and deactivate gene expression and function at the translational level.
- 17Shestopalov, I. A.; Sinha, S.; Chen, J. K. Light-controlled gene silencing in zebrafish embryos. Nat. Chem. Biol. 2007, 3 (10), 650– 1, DOI: 10.1038/nchembio.2007.3017https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVCrtrjP&md5=dc008772fa3f03e882df40d8d8b1d02eLight-controlled gene silencing in zebrafish embryosShestopalov, Ilya A.; Sinha, Surajit; Chen, James K.Nature Chemical Biology (2007), 3 (10), 650-651CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Functional genomic studies in zebrafish frequently use synthetic oligonucleotides called morpholinos that block RNA splicing or translation. However, the constitutive activity of these reagents limits their exptl. utility. We report here the synthesis of a photoactivatable morpholino targeting the no tail (ntl) gene. This caged reagent permits spatiotemporal gene regulation in vivo and the photochem. generation of functionally mosaic organisms.
- 18Deiters, A.; Garner, R. A.; Lusic, H.; Govan, J. M.; Dush, M.; Nascone-Yoder, N. M.; Yoder, J. A. Photocaged morpholino oligomers for the light-regulation of gene function in zebrafish and Xenopus embryos. J. Am. Chem. Soc. 2010, 132 (44), 15644– 50, DOI: 10.1021/ja105386318https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWmtLjI&md5=39e4e975466e4c45786de524844f5d93Photocaged Morpholino Oligomers for the Light-Regulation of Gene Function in Zebrafish and Xenopus EmbryosDeiters, Alexander; Garner, R. Aaron; Lusic, Hrvoje; Govan, Jeane M.; Dush, Mike; Nascone-Yoder, Nanette M.; Yoder, Jeffrey A.Journal of the American Chemical Society (2010), 132 (44), 15644-15650CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged (derivatized by NPOM; i.e. 6-nitropiperonyloxymethyl) morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP prodn. only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts.
- 19Bardhan, A.; Deiters, A.; Ettensohn, C. A. Conditional gene knockdowns in sea urchins using caged morpholinos. Dev. Biol. 2021, 475, 21– 29, DOI: 10.1016/j.ydbio.2021.02.01419https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmslKms7o%253D&md5=977ba5021fde89085bd18ae73026078cConditional gene knockdowns in sea urchins using caged morpholinosBardhan, Anirban; Deiters, Alexander; Ettensohn, Charles A.Developmental Biology (Amsterdam, Netherlands) (2021), 475 (), 21-29CODEN: DEBIAO; ISSN:0012-1606. (Elsevier B.V.)Echinoderms are important exptl. models for analyzing embryonic development, but a lack of spatial and temporal control over gene perturbations has hindered developmental studies using these animals. Morpholino antisense oligonucleotides (MOs) have been used successfully by the echinoderm research community for almost two decades, and MOs remain the most widely used tool for acute gene knockdowns in these organisms. Echinoderm embryos develop externally and are optically transparent, making them ideally-suited to many light-based approaches for analyzing and manipulating development. Studies using zebrafish embryos have demonstrated the effectiveness of photoactivatable (caged) MOs for conditional gene knockdowns. Here we show that caged MOs, synthesized using nucleobase-caged monomers, provide light-regulated control over gene expression in sea urchin embryos. Our work provides the first robust approach for conditional gene silencing in this prominent model system.
- 20Pattanayak, S.; Sarode, B. R.; Deiters, A.; Chen, J. K. Bicyclic Caged Morpholino Oligonucleotides for Optical Gene Silencing. ChemBioChem 2022, 23 (21), e202200374, DOI: 10.1002/cbic.20220037420https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFyns7jN&md5=28cd115f1602b741c2a331c2f1189a58Bicyclic Caged Morpholino Oligonucleotides for Optical Gene SilencingPattanayak, Sankha; Sarode, Bhagyesh R.; Deiters, Alexander; Chen, James K.ChemBioChem (2022), 23 (21), e202200374CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Caged morpholino oligonucleotides (cMOs) are synthetic tools that allow light-inducible gene silencing in live organisms. Previously reported cMOs have utilized hairpin, duplex, and cyclic structures, as well as caged nucleobases. While these antisense technologies enable efficient optical control of RNA splicing and translation, they can have limited dynamic range. A new caging strategy was developed where the two MO termini are conjugated to an internal position through a self-immolative trifunctional linker, thereby generating a bicyclic cMO that is conformationally resistant to RNA binding. The efficacy of this alternative cMO design has been demonstrated in zebrafish embryos and compared to linear MOs and monocyclic constructs.
- 21Yang, L.; Kim, H. B.; Sul, J. Y.; Yeldell, S. B.; Eberwine, J. H.; Dmochowski, I. J. Efficient Synthesis of Light-Triggered Circular Antisense Oligonucleotides Targeting Cellular Protein Expression. ChemBioChem 2018, 19 (12), 1250– 1254, DOI: 10.1002/cbic.20180001221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXns1Ontrg%253D&md5=d6313c9a191f06610c7f74232c4c6054Efficient Synthesis of Light-Triggered Circular Antisense Oligonucleotides Targeting Cellular Protein ExpressionYang, Linlin; Kim, Hyun Bum; Sul, Jai-Yoon; Yeldell, Sean B.; Eberwine, James H.; Dmochowski, Ivan J.ChemBioChem (2018), 19 (12), 1250-1254CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Light-activated ("caged") antisense oligonucleotides are powerful mols. for regulating gene expression at submicron spatial resoln. through the focal modulation of endogenous cellular processes. Cyclized caged oligos are particularly promising structures because of their inherent stability and similarity to naturally occurring circular DNA and RNA mols. Here, we introduce an efficient route for cyclizing an antisense oligodeoxynucleotide incorporating a photocleavable linker. Oligo cyclization was achieved for several sequences in nearly quant. yields through intramol. copper(I)-catalyzed azide-alkyne cycloaddn. (CuAAC). Caging stability and light activation were characterized by FRET efficiency, denaturing gel assay, and melting temp. measurements. Finally, a cyclized caged oligo was designed to target gfap, and it gave a tenfold redn. in glial fibrillary acidic protein upon photoactivation in astrocytes.
- 22Brown, W.; Bardhan, A.; Darrah, K.; Tsang, M.; Deiters, A. Optical Control of MicroRNA Function in Zebrafish Embryos. J. Am. Chem. Soc. 2022, 144 (37), 16819– 16826, DOI: 10.1021/jacs.2c0447922https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xitlagu7jK&md5=cab3a24cf5070bfc55885da50480db6fOptical Control of MicroRNA Function in Zebrafish EmbryosBrown, Wes; Bardhan, Anirban; Darrah, Kristie; Tsang, Michael; Deiters, AlexanderJournal of the American Chemical Society (2022), 144 (37), 16819-16826CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)MicroRNAs play crucial and dynamic roles in vertebrate development and diseases. Some, like miR-430, are highly expressed during early embryo development and regulate hundreds of transcripts, which can make it difficult to study their role in the timing and location of specific developmental processes using conventional morpholino oligonucleotide (MO) knockdown or genetic deletion approaches. We demonstrate that light-activated circular morpholino oligonucleotides (cMOs) can be applied to the conditional control of microRNA function. We targeted miR-430 in zebrafish embryos to study its role in the development of the embryo body and the heart. Using 405 nm irradn., precise spatial and temporal control over miR-430 function was demonstrated, offering insight into the cell populations and developmental timepoints involved in each process.
- 23Yamazoe, S.; McQuade, L. E.; Chen, J. K. Nitroreductase-activatable morpholino oligonucleotides for in vivo gene silencing. ACS Chem. Biol. 2014, 9 (9), 1985– 90, DOI: 10.1021/cb500429u23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht1ahtrbO&md5=91d7b5afe7f92ca0ce02a573bae49bc4Nitroreductase-Activatable Morpholino Oligonucleotides for in Vivo Gene SilencingYamazoe, Sayumi; McQuade, Lindsey E.; Chen, James K.ACS Chemical Biology (2014), 9 (9), 1985-1990CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Phosphorodiamidate morpholino oligonucleotides are widely used to interrogate gene function in whole organisms, and light-activatable derivs. can reveal spatial and temporal differences in gene activity. The authors describe here a new class of caged morpholino oligonucleotides that can be activated by the bacterial nitroreductase NfsB. The authors characterize the activation kinetics of these reagents in vitro and demonstrate their efficacy in zebrafish embryos that express NfsB either ubiquitously or in defined cell populations. In combination with transgenic organisms, such enzyme-actuated antisense tools will enable gene silencing in specific cell types, including tissues that are not amenable to optical targeting.
- 24Darrah, K.; Wesalo, J.; Lukasak, B.; Tsang, M.; Chen, J. K.; Deiters, A. Small Molecule Control of Morpholino Antisense Oligonucleotide Function through Staudinger Reduction. J. Am. Chem. Soc. 2021, 143 (44), 18665– 18671, DOI: 10.1021/jacs.1c0872324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitlajur%252FF&md5=2d697c181ecf19a44cc5681fb079b47bSmall Molecule Control of Morpholino Antisense Oligonucleotide Function through Staudinger ReductionDarrah, Kristie; Wesalo, Joshua; Lukasak, Bradley; Tsang, Michael; Chen, James K.; Deiters, AlexanderJournal of the American Chemical Society (2021), 143 (44), 18665-18671CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Conditionally activated, caged morpholino antisense agents (cMOs) are tools that enable the temporal and spatial investigation of gene expression, regulation, and function during embryonic development. Cyclic MOs are conformationally gated oligonucleotide analogs that do not block gene expression until they are linearized through the application of an external trigger, such as light or enzyme activity. Here, we describe the first examples of small mol.-responsive cMOs, which undergo rapid and efficient decaging via a Staudinger redn. This is enabled by a highly flexible linker design that offers opportunities for the installation of chem. activated, self-immolative motifs. We synthesized cyclic cMOs against two distinct, developmentally relevant genes and demonstrated phosphine-triggered knockdown of gene expression in zebrafish embryos. This represents the first report of a small mol.-triggered antisense agent for gene knockdown, adding another bioorthogonal entry to the growing arsenal of gene knockdown tools.
- 25Tomasini, A. J.; Schuler, A. D.; Zebala, J. A.; Mayer, A. N. PhotoMorphs: a novel light-activated reagent for controlling gene expression in zebrafish. Genesis 2009, 47 (11), 736– 43, DOI: 10.1002/dvg.2055425https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhs1Sjt7jN&md5=1cbe5279eaad8674d03dfa028acf9424PhotoMorphs: a novel light-activated reagent for controlling gene expression in zebrafishTomasini, Amber J.; Schuler, Aaron D.; Zebala, John A.; Mayer, Alan N.Genesis (Hoboken, NJ, United States) (2009), 47 (11), 736-743CODEN: GNESFY; ISSN:1526-954X. (Wiley-Liss, Inc.)Manipulating gene expression in zebrafish is crit. for exploiting the full potential of this vertebrate model organism. Morpholino oligos are the most commonly used antisense technol. for knocking down gene expression. However, morpholinos suffer from a lack of control over the timing and location of knockdown. In this report, we describe a novel light-activatable knockdown reagent called PhotoMorph. PhotoMorphs can be generated from existing morpholinos by hybridization with a complementary caging strand contg. a photocleavable linkage. The caging strand neutralizes the morpholino activity until irradn. of the PhotoMorph with UV light releases the morpholino. We generated PhotoMorphs to target genes encoding enhanced green fluorescent protein, No tail, and E-cadherin to illustrate the utility of this approach. Temporal control of gene expression with PhotoMorphs permitted us to circumvent the early lethal phenotype of E-cadherin knockdown. A splice-blocking PhotoMorph directed to the rheb gene showed light-dependent gene knockdown up to 72 hpf. PhotoMorphs thus offer a new class of lab. reagents suitable for the spatio-temporal control of gene expression in the zebrafish.
- 26Tallafuss, A.; Gibson, D.; Morcos, P.; Li, Y.; Seredick, S.; Eisen, J.; Washbourne, P. Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafish. Development 2012, 139 (9), 1691– 9, DOI: 10.1242/dev.07270226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XotlKisb0%253D&md5=a458a3387ad59c5371e323ac01205d63Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafishTallafuss, Alexandra; Gibson, Dan; Morcos, Paul; Li, Yongfu; Seredick, Steve; Eisen, Judith; Washbourne, PhilipDevelopment (Cambridge, United Kingdom) (2012), 139 (9), 1691-1699CODEN: DEVPED; ISSN:0950-1991. (Company of Biologists Ltd.)To understand the mol. mechanisms of development it is essential to be able to turn genes on and off at will and in a spatially restricted fashion. Morpholino oligonucleotides (MOs) are very common tools used in several model organisms with which it is possible to block gene expression. Recently developed photo-activated MOs allow control over the onset of MO activity. However, deactivation of photo-cleavable MO activity has remained elusive. Here, we describe photo-cleavable MOs with which it is possible to activate or de-activate MO function by UV exposure in a temporal and spatial manner. We show, using several different genes as examples, that it is possible to turn gene expression on or off both in the entire zebrafish embryo and in single cells. We use these tools to demonstrate the sufficiency of no tail expression as late as tailbud stage to drive medial precursor cells towards the notochord cell fate. As a broader approach for the use of photo-cleavable MOs, we show temporal control over gal4 function, which has many potential applications in multiple transgenic lines. We demonstrate temporal manipulation of Gal4 transgene expression in only primary motoneurons and not secondary motoneurons, heretofore impossible with conventional transgenic approaches. In another example, we follow and analyze neural crest cells that regained sox10 function after deactivation of a photo-cleavable sox10-MO at different time points. Our results suggest that sox10 function might not be crit. during neural crest formation.
- 27Ouyang, X.; Shestopalov, I. A.; Sinha, S.; Zheng, G.; Pitt, C. L.; Li, W. H.; Olson, A. J.; Chen, J. K. Versatile synthesis and rational design of caged morpholinos. J. Am. Chem. Soc. 2009, 131 (37), 13255– 69, DOI: 10.1021/ja809933h27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVClsr7M&md5=17ab8c99c360c0d8cd8a6de312b5bb7fVersatile Synthesis and Rational Design of Caged MorpholinosOuyang, Xiaohu; Shestopalov, Ilya A.; Sinha, Surajit; Zheng, Genhua; Pitt, Cameron L. W.; Li, Wen-Hong; Olson, Andrew J.; Chen, James K.Journal of the American Chemical Society (2009), 131 (37), 13255-13269CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these mol. mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chem. technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from com. available reagents. Using this simplified procedure, we have systematically prepd. cMOs with differing structural configurations and investigated how the in vitro thermodn. properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prep. a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chem. probes in vivo for functional genomic studies.
- 28Yamazoe, S.; Shestopalov, I. A.; Provost, E.; Leach, S. D.; Chen, J. K. Cyclic caged morpholinos: conformationally gated probes of embryonic gene function. Angew. Chem., Int. Ed. 2012, 51 (28), 6908– 11, DOI: 10.1002/anie.20120169028https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xot12itLk%253D&md5=4a5d186014dd009513db6ad4e3dfffd3Cyclic caged morpholinos: conformationally gated probes of embryonic gene functionYamazoe, Sayumi; Shestopalov, Ilya A.; Provost, Elayne; Leach, Steven D.; Chen, James K.Angewandte Chemie, International Edition (2012), 51 (28), 6908-6911, S6908/1-S6908/28CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Next-generation cyclic caged morpholino-based antisense oligonucleotides were used as effective reverse-genetics tools in zebrafish embryos to study the timing of exocrine fate commitment in developing pancreas. The zebrafish pancreas transcription factor 1 alpha (ptf1a) gene was targeted.
- 29Yamazoe, S.; Liu, Q.; McQuade, L. E.; Deiters, A.; Chen, J. K. Sequential gene silencing using wavelength-selective caged morpholino oligonucleotides. Angew. Chem., Int. Ed. 2014, 53 (38), 10114– 8, DOI: 10.1002/anie.20140535529https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht1GqsLbI&md5=840d1f876b958ebcc25e66250eb89e7aSequential gene silencing using wavelength-selective caged morpholino oligonucleotidesYamazoe, Sayumi; Liu, Qingyang; McQuade, Lindsey E.; Deiters, Alexander; Chen, James K.Angewandte Chemie, International Edition (2014), 53 (38), 10114-10118CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Spectrally differentiated caged morpholino oligonucleotides (cMOs) and wavelength-selective illumination have been used to sequentially inactivate organismal gene function. The efficacy of these reverse-genetic chem. probes has been demonstrated in zebrafish embryos, and these reagents have been employed to examine the mechanisms of mesoderm patterning.
- 30Wang, Y.; Wu, L.; Wang, P.; Lv, C.; Yang, Z.; Tang, X. Manipulation of gene expression in zebrafish using caged circular morpholino oligomers. Nucleic Acids Res. 2012, 40 (21), 11155– 62, DOI: 10.1093/nar/gks84030https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhslaksr7F&md5=6934d98ed5b46d1771872fea33a7227aManipulation of gene expression in zebrafish using caged circular morpholino oligomersWang, Yuan; Wu, Li; Wang, Peng; Lv, Cong; Yang, Zhenjun; Tang, XinjingNucleic Acids Research (2012), 40 (21), 11155-11162CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Morpholino oligomers (MOs) have been widely used to knock down specific genes in zebrafish, but their constitutive activities limit their exptl. applications for studying a gene with multiple functions or within a gene network. We report herein a new design and synthesis of caged circular MOs (caged cMOs) with two ends linked by a photocleavable moiety. These caged cMOs were successfully used to photomodulate β-catenin-2 and no tail expression in zebrafish embryos.
- 31Griepenburg, J. C.; Rapp, T. L.; Carroll, P. J.; Eberwine, J.; Dmochowski, I. J. Ruthenium-caged antisense morpholinos for regulating gene expression in zebrafish embryos. Chem. Sci. 2015, 6 (4), 2342– 2346, DOI: 10.1039/C4SC03990D31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvVCju70%253D&md5=7093c6ea7d35f8840bf655ac9d264833Ruthenium-caged antisense morpholinos for regulating gene expression in zebrafish embryosGriepenburg, Julianne C.; Rapp, Teresa L.; Carroll, Patrick J.; Eberwine, James; Dmochowski, Ivan J.Chemical Science (2015), 6 (4), 2342-2346CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Photochem. approaches afford high spatiotemporal control over mol. structure and function, for broad applications in materials and biol. science. Here, we present the first example of a visible light responsive ruthenium-based photolinker, Ru(bipyridine)2(3-ethynylpyridine)2 (RuBEP), which was reacted stoichiometrically with a 25mer DNA or morpholino (MO) oligonucleotide functionalized with 3' and 5' terminal azides, via Cu(I)-mediated [3+2] Huisgen cycloaddn. reactions. RuBEP-caged circular morpholinos (Ru-MOs) targeting two early developmental zebrafish genes, chordin and notail, were synthesized and tested in vivo. One-cell-stage zebrafish embryos microinjected with Ru-MO and incubated in the dark for 24 h developed normally, consistent with caging, whereas irradn. at 450 nm dissocd. one 3-ethynylpyridine ligand (Φ = 0.33) and uncaged the MO to achieve gene knockdown. As demonstrated, Ru photolinkers provide a versatile method for controlling structure and function of biopolymers.
- 32O’Connor, M. J.; Beebe, L. L.; Deodato, D.; Ball, R. E.; Page, A. T.; VanLeuven, A. J.; Harris, K. T.; Park, S.; Hariharan, V.; Lauderdale, J. D.; Dore, T. M. Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino. ACS Chem. Neurosci. 2019, 10 (1), 266– 278, DOI: 10.1021/acschemneuro.8b0023132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c3ntlOgtQ%253D%253D&md5=70a92f468408c45b048281e01b2cfc75Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker MorpholinoO'Connor Matthew J; Deodato Davide; Dore Timothy M; Beebe Lindsey L; Park Sungdae; Ball Rebecca E; Page A Tyler; VanLeuven Ariel J; Hariharan Vani; Lauderdale James D; Harris Kyle T; Dore Timothy M; Lauderdale James DACS chemical neuroscience (2019), 10 (1), 266-278 ISSN:.γ-Amino butyric acid (GABA) mediated signaling is critical in the central and enteric nervous systems, pancreas, lungs, and other tissues. It is associated with many neurological disorders and craniofacial development. Glutamic acid decarboxylase (GAD) synthesizes GABA from glutamate, and knockdown of the gad1 gene results in craniofacial defects that are lethal in zebrafish. To bypass this and enable observation of the neurological defects resulting from knocking down gad1 expression, a photoactivatable morpholino oligonucleotide (MO) against gad1 was prepared by cyclization with a photocleavable linker rendering the MO inactive. The cyclized MO was stable in the dark and toward degradative enzymes and was completely linearized upon brief exposure to 405 nm light. In the course of investigating the function of the ccMOs in zebrafish, we discovered that zebrafish possess paralogous gad1 genes, gad1a and gad1b. A gad1b MO injected at the 1-4 cell stage caused severe morphological defects in head development, which could be bypassed, enabling the fish to develop normally, if the fish were injected with a photoactivatable, cyclized gad1b MO and grown in the dark. At 1 day post fertilization (dpf), light activation of the gad1b MO followed by observation at 3 and 7 dpf led to increased and abnormal electrophysiological brain activity compared to wild type animals. The photocleavable linker can be used to cyclize and inactivate any MO, and represents a general strategy to parse the function of developmentally important genes in a spatiotemporal manner.
- 33Wu, L.; Wang, Y.; Wu, J.; Lv, C.; Wang, J.; Tang, X. Caged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cells. Nucleic Acids Res. 2013, 41 (1), 677– 86, DOI: 10.1093/nar/gks99633https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFaitA%253D%253D&md5=1b29cf249753e4103f213da0d746723fCaged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cellsWu, Li; Wang, Yuan; Wu, Junzhou; Lv, Cong; Wang, Jie; Tang, XinjingNucleic Acids Research (2013), 41 (1), 677-686CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)We synthesized three 20mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10mer oligodeoxynucleotides (ODNs). With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by RNase H were readily photomodulated. RNA cleavage rates were upregulated ∼43-, 25- and 15-fold for R20, R20B2 and R20B4, resp., upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amt. of RNA digestion was still obsd. The loss of binding ability indicated promising gene photoregulation through a non-enzymic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2'-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both RNase H and non-enzyme involved antisense strategies.
- 34Alouane, A.; Labruère, R.; Le Saux, T.; Schmidt, F.; Jullien, L. Self-Immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications. Angew. Chem., Int. Ed. 2015, 54 (26), 7492– 7509, DOI: 10.1002/anie.20150008834https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXps1eguro%253D&md5=fe98dd1aff7066a9a58073ee3c57f688Self-immolative spacers: kinetic aspects, structure-property relationships, and applicationsAlouane, Ahmed; Labruere, Raphael; Le Saux, Thomas; Schmidt, Frederic; Jullien, LudovicAngewandte Chemie, International Edition (2015), 54 (26), 7492-7509CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Self-immolative spacers are covalent assemblies tailored to correlate the cleavage of two chem. bonds after activation of a protective part in a precursor: Upon stimulation, the protective moiety is removed, which generates a cascade of disassembling reactions leading to the temporally sequential release of smaller mols. Originally introduced to overcome limitations for drug delivery, self-immolative spacers have gained wide interest in medicinal chem., anal. chem., and material science. For most applications, the kinetics of the disassembly of the activated self-immolative spacer governs functional properties. This Review addresses kinetic aspects of self-immolation. It provides information for selecting a particular self-immolative motif for a specific demand. Moreover, it should help researchers design kinetic expts. and fully exploit the rich perspectives of self-immolative spacers.
- 35Yazawa, R.; Hirono, I.; Aoki, T. Characterization of promoter activities of four different Japanese flounder promoters in transgenic zebrafish. Mar. Biotechnol. (NY) 2005, 7 (6), 625– 33, DOI: 10.1007/s10126-005-0011-135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2MnjtVGjtA%253D%253D&md5=7d0ab93150e29fc93c052363f4fe91dcCharacterization of promoter activities of four different Japanese flounder promoters in transgenic zebrafishYazawa Ryosuke; Hirono Ikuo; Aoki TakashiMarine biotechnology (New York, N.Y.) (2005), 7 (6), 625-33 ISSN:1436-2228.An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5' flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish.
- 36Udvadia, A. J.; Linney, E. Windows into development: historic, current, and future perspectives on transgenic zebrafish. Dev. Biol. 2003, 256 (1), 1– 17, DOI: 10.1016/S0012-1606(02)00083-036https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXitlWhurc%253D&md5=0c78aa9cd90ab01349053d9bd929ae96Windows into development: historic, current, and future perspectives on transgenic zebrafishUdvadia, Ava J.; Linney, ElwoodDevelopmental Biology (Orlando, FL, United States) (2003), 256 (1), 1-17CODEN: DEBIAO; ISSN:0012-1606. (Elsevier Science)A review. The recent explosion of transgenic zebrafish lines in the literature demonstrates the value of this model system for detailed in vivo anal. of gene regulation and morphogenetic movements. The optical clarity and rapid early development of zebrafish provides the ability to follow these events as they occur in live, developing embryos. This article will review the development of transgenic technol. in zebrafish as well as the current and future uses of transgenic zebrafish to explore the dynamic environment of the developing vertebrate embryo.
- 37Yang, L.; Eberwine, J. H.; Dmochowski, I. J. Caspase-Activated Oligonucleotide Probe. Bioconjugate Chem. 2020, 31 (9), 2172– 2178, DOI: 10.1021/acs.bioconjchem.0c0036237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGju7zL&md5=adc5d603692003b1dedd34ca19d0b2c1Caspase-Activated Oligonucleotide ProbeYang, Linlin; Eberwine, James H.; Dmochowski, Ivan J.Bioconjugate Chemistry (2020), 31 (9), 2172-2178CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Light-activated ("caged") oligonucleotides provide a strategy for modulating the activity of antisense oligos, siRNA, miRNA, aptamers, DNAzymes, and mRNA-capturing probes with high spatiotemporal resoln. However, the near-UV and visible wavelengths that promote these bond-breaking reactions poorly penetrate living tissue, which limits some biol. applications. To address this issue, we describe the first example of a protease-activated oligonucleotide probe, capable of reporting on caspase-3 during cellular apoptosis. The 2'-F RNA-peptide substrate-peptide nucleic acid (PNA) hairpin structure was generated in 30% yield in a single bioconjugation step.
- 38Bonomo, R. A. β-Lactamases: A Focus on Current Challenges. Cold Spring Harbor Perspect. Med. 2017, 7 (1), a025239, DOI: 10.1101/cshperspect.a02523938https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFKms7rK&md5=ecbc5d8ec1265dab4c924a9724cee6b7β-lactamases: a focus on current challengesBonomo, Robert A.Cold Spring Harbor Perspectives in Medicine (2017), 7 (1), a025239/1-a025239/16CODEN: CSHPFV; ISSN:2157-1422. (Cold Spring Harbor Laboratory Press)β-Lactamases, the enzymes that hydrolyze β-lactam antibiotics, remain the greatest threat to the usage of these agents. In this review, the mechanism of hydrolysis is discussed for both those enzymes that use serine at the active site and those that require divalent zinc ions for hydrolysis. The β-lactamases now include >2000 unique, naturally occurring amino acid sequences. Some of the clin. most important of these are the class A penicillinases, the extended-spectrum β-lactamases (ESBLs), the AmpC cephalosporinases, and the carbapenem-hydrolyzing enzymes in both the serine and metalloenzyme groups. Because of the versatility of these enzymes to evolve as ne β-lactams are used therapeutically, new approaches to antimicrobial therapy may be required.
- 39Tooke, C. L.; Hinchliffe, P.; Bragginton, E. C.; Colenso, C. K.; Hirvonen, V. H. A.; Takebayashi, Y.; Spencer, J. beta-Lactamases and beta-Lactamase Inhibitors in the 21st Century. J. Mol. Biol. 2019, 431 (18), 3472– 3500, DOI: 10.1016/j.jmb.2019.04.00239https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnt1Cmur8%253D&md5=abb0775c85a735c83a96dabac95a7ca5β-Lactamases and β-Lactamase Inhibitors in the 21st CenturyTooke, Catherine L.; Hinchliffe, Philip; Bragginton, Eilis C.; Colenso, Charlotte K.; Hirvonen, Viivi H. A.; Takebayashi, Yuiko; Spencer, JamesJournal of Molecular Biology (2019), 431 (18), 3472-3500CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)A review. The β-lactams retain a central place in the antibacterial armamentarium. In Gram-neg. bacteria, β-lactamase enzymes that hydrolyze the amide bond of the four-membered β-lactam ring are the primary resistance mechanism, with multiple enzymes disseminating on mobile genetic elements across opportunistic pathogens such as Enterobacteriaceae (e.g., Escherichia coli) and non-fermenting organisms (e.g., Pseudomonas aeruginosa). β-Lactamases divide into four classes; the active-site serine β-lactamases (classes A, C and D) and the zinc-dependent or metallo-β-lactamases (MBLs; class B). Here we review recent advances in mechanistic understanding of each class, focusing upon how growing nos. of crystal structures, in particular for β-lactam complexes, and methods such as neutron diffraction and mol. simulations, have improved understanding of the biochem. of β-lactam breakdown. A second focus is β-lactamase interactions with carbapenems, as carbapenem-resistant bacteria are of grave clin. concern and carbapenem-hydrolyzing enzymes such as KPC (class A) NDM (class B) and OXA-48 (class D) are proliferating worldwide. An overview is provided of the changing landscape of β-lactamase inhibitors, exemplified by the introduction to the clinic of combinations of β-lactams with diazabicyclooctanone and cyclic boronate serine β-lactamase inhibitors, and of progress and strategies toward clin. useful MBL inhibitors. Despite the long history of β-lactamase research, we contend that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallog.; the need for new inhibitors, particularly for MBLs; the likely impact of new β-lactam:inhibitor combinations and the continuing clin. importance of β-lactams mean that this remains a rewarding research area.
- 40Bagshawe, K. D. Antibody-directed enzyme prodrug therapy (ADEPT) for cancer. Expert Rev. Anticancer Ther 2006, 6 (10), 1421– 31, DOI: 10.1586/14737140.6.10.142140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFeru7zL&md5=a937f4361e244e2d34f63f605891bbcaAntibody-directed enzyme prodrug therapy (ADEPT) for cancerBagshawe, Kenneth D.Expert Review of Anticancer Therapy (2006), 6 (10), 1421-1431CODEN: ERATBJ; ISSN:1473-7140. (Future Drugs Ltd.)A review. Antibody-directed enzyme prodrug therapy was conceived as a means of restricting the action of cytotoxic drugs to tumor sites. Since antigenic targets were a central component of the approach, colonic cancer, with its virtually universal expression of carcinoembryonic antigen at the cellular level, presented an obvious starting point. The principle of antibody-directed enzyme prodrug therapy is to use an antibody directed at a tumor-assocd. antigen to vector an enzyme to tumor sites. The enzyme should be retained at tumor sites after it has cleared from blood and normal tissues. A nontoxic prodrug, a substrate for the enzyme, is then given and, by cleaving an inactivating component from the prodrug, a potent cytotoxic agent is generated. One of the potential advantages of such a system is that a small cytotoxic agent, generated within a tumor site, is much more diffusible than a large antibody mol. Moreover, failure to express the target antigen by cancer cells does not protect them from the bystander action of the cytotoxic agent. This review will primarily consider the studies of the London group since this is the only group that has so far reported clin. trials and it is only through clin. trials that the requirements of a successful antibody-directed enzyme prodrug therapy system can be identified.
- 41Zhang, J.; Kale, V.; Chen, M. Gene-directed enzyme prodrug therapy. AAPS J. 2015, 17 (1), 102– 10, DOI: 10.1208/s12248-014-9675-741https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVequ73L&md5=5d088566e4305bb80be167d2bc8656d7Gene-Directed Enzyme Prodrug TherapyZhang, Jin; Kale, Vijay; Chen, MingnanAAPS Journal (2015), 17 (1), 102-110CODEN: AJAOB6; ISSN:1550-7416. (Springer)A review. As one targeting strategy of prodrug delivery, gene-directed enzyme prodrug therapy (GDEPT) promises to realize the targeting through its three key features in cancer therapy-cell-specific gene delivery and expression, controlled conversion of prodrugs to drugs in target cells, and expanded toxicity to the target cells' neighbors through bystander effects. After over 20 years of development, multiple GDEPT systems have advanced into clin. trials. However, no GDEPT product is currently marketed as a drug, suggesting that there are still barriers to overcome before GDEPT becomes a std. therapy. In this review, we first provide a general introduction of this prodrug targeting strategy. Then, we utilize the four most thoroughly studied systems to illustrate components, mechanisms, preclin. and clin. results, and further development directions of GDEPT. These four systems are herpes simplex virus thymidine kinase/ganciclovir, cytosine deaminase/5-fluorocytosine, cytochrome P 450/oxazaphosphorines, and nitroreductase/CB1954 system. Later, we focus our discussion on bystander effects including local and distant bystander effects. Lastly, we discuss carriers that are used to deliver genes for GDEPT including virus carriers and non-virus carriers. Among these carriers, the stem cell-based gene delivery system represents one of the newest carriers under development, and may brought about a breakthrough to the gene delivery issue of GDEPT.
- 42Schellmann, N.; Deckert, P. M.; Bachran, D.; Fuchs, H.; Bachran, C. Targeted enzyme prodrug therapies. Mini-Rev. Med. Chem. 2010, 10 (10), 887– 904, DOI: 10.2174/13895571079200719642https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFensLjN&md5=c147e612acd4569b5388ae8b08eafe53Targeted enzyme prodrug therapiesSchellmann, N.; Deckert, P. M.; Bachran, D.; Fuchs, H.; Bachran, C.Mini-Reviews in Medicinal Chemistry (2010), 10 (10), 887-904CODEN: MMCIAE; ISSN:1389-5575. (Bentham Science Publishers Ltd.)A review. The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a no. of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A no. of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview of the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.
- 43Pereira, M. P.; Shi, J.; Kelley, S. O. Peptide Targeting of an Antibiotic Prodrug toward Phagosome-Entrapped Mycobacteria. ACS Infect Dis 2015, 1 (12), 586– 92, DOI: 10.1021/acsinfecdis.5b0009943https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1OhurjJ&md5=d664a0d74e81e7152516326435669291Peptide targeting of an antibiotic prodrug toward phagosome-entrapped mycobacteriaPereira, Mark P.; Shi, Julie; Kelley, Shana O.ACS Infectious Diseases (2015), 1 (12), 586-592CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)Mycobacterial infections are difficult to treat due to the bacterium's slow growth, ability to reside in intracellular compartments within macrophages, and resistance mechanisms that limit the effectiveness of conventional antibiotics. Developing antibiotics that overcome these challenges is therefore crit. to providing a pipeline of effective antimicrobial agents. Here, the authors describe the synthesis and testing of a unique peptide-drug conjugate that exhibits high levels of antimicrobial activity against M. smegmatis and M. tuberculosis as well as clearance of intracellular mycobacteria from cultured macrophages. Using an engineered peptide sequence, they deliver a potent DHFR inhibitor and target the intracellular phagosomes where mycobacteria reside and also incorporate a β-lactamase-cleavable cephalosporin linker to enhance the targeting of quiescent intracellular β-lactam-resistant mycobacteria. By using this type of prodrug approach to target intracellular mycobacterial infections, the emergence of antibacterial resistance mechanisms could be minimized.
- 44Liu, R.; Miller, P. A.; Vakulenko, S. B.; Stewart, N. K.; Boggess, W. C.; Miller, M. J. A Synthetic Dual Drug Sideromycin Induces Gram-Negative Bacteria To Commit Suicide with a Gram-Positive Antibiotic. J. Med. Chem. 2018, 61 (9), 3845– 3854, DOI: 10.1021/acs.jmedchem.8b0021844https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltV2rsbk%253D&md5=0c3db140a8cd4e7c9f533120a2407cf6A Synthetic Dual Drug Sideromycin Induces Gram-Negative Bacteria To Commit Suicide with a Gram-Positive AntibioticLiu, Rui; Miller, Patricia A.; Vakulenko, Sergei B.; Stewart, Nichole K.; Boggess, William C.; Miller, Marvin J.Journal of Medicinal Chemistry (2018), 61 (9), 3845-3854CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Many antibiotics lack activity against Gram-neg. bacteria because they cannot permeate the outer membrane or suffer from efflux and, in the case of β-lactams, are degraded by β-lactamases. Herein, we describe the synthesis and studies of a dual drug conjugate (1) of a siderophore linked to a cephalosporin with an attached oxazolidinone. The cephalosporin component of 1 is rapidly hydrolyzed by purified ADC-1 β-lactamase to release the oxazolidinone. Conjugate 1 is active against clin. isolates of Acinetobacter baumannii as well as strains producing large amts. of ADC-1 β-lactamase. Overall, the results are consistent with siderophore-mediated active uptake, inherent activity of the delivered dual drug, and in the presence of β-lactamases, intracellular release of the oxazolidinone upon cleavage of the cephalosporin to allow the freed oxazolidinone to inactivate its target. The ultimate result demonstrates that Gram-pos. oxazolidinone antibiotics can be made to be effective against Gram-neg. bacteria by β-lactamase triggered release.
- 45Raz, E.; Zlokarnik, G.; Tsien, R. Y.; Driever, W. beta-lactamase as a marker for gene expression in live zebrafish embryos. Dev. Biol. 1998, 203 (2), 290– 4, DOI: 10.1006/dbio.1998.899945https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXnslyqtr8%253D&md5=229fe38f5712323c662d951118fda525β-Lactamase as a marker for gene expression in live zebrafish embryosRaz, Erez; Zlokarnik, Gregor; Tsien, Roger Y.; Driever, WolfgangDevelopmental Biology (1998), 203 (2), 290-294CODEN: DEBIAO; ISSN:0012-1606. (Academic Press)In this report the authors describe the development of a sensitive assay for gene expression in zebrafish embryos using β-lactamase as a reporter gene. The authors show that injection of a green fluorescent substrate for β-lactamase allows the detection of reporter gene expression in live embryos. The β-lactamase enzyme catalyzes the hydrolysis of the substrate, thereby disrupting fluorescence resonance energy transfer from the donor to the acceptor dye in the mol. As a result, a blue fluorescent product is produced and retained specifically in cells within which the enzyme is expressed. β-Lactamase is therefore suitable for monitoring spatially restricted patterns of gene expression in the early embryo. The authors suggest that this new reporter system provides a major advancement in sensitivity over the existing methods for monitoring gene expression in vivo during early embryogenesis. (c) 1998 Academic Press.
- 46Zlokarnik, G.; Negulescu, P. A.; Knapp, T. E.; Mere, L.; Burres, N.; Feng, L.; Whitney, M.; Roemer, K.; Tsien, R. Y. Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 1998, 279 (5347), 84– 8, DOI: 10.1126/science.279.5347.8446https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjtVSjsg%253D%253D&md5=25d3beee86614403847e2ec7b26aed18Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporterZlokarnik, Gregor; Negulescu, Paul A.; Knapp, Thomas E.; Mere, Lora; Burres, Neal; Feng, Luxin; Whitney, Michael; Roemer, Klaus; Tsien, Roger Y.Science (Washington, D. C.) (1998), 279 (5347), 84-88CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Gene expression was visualized in single living mammalian cells with β-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each enzyme mol. changed the fluorescence of many substrate mols. from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual cells contg. less than 100 β-lactamase mols. The robust change in emission ratio reveals quant. heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.
- 47Boyd, D. B. Electronic structures of cephalosporins and penicillins. 15. Inductive effect of the 3-position side chain in cephalosporins. J. Med. Chem. 1984, 27 (1), 63– 6, DOI: 10.1021/jm00367a01247https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXitVSruw%253D%253D&md5=10aa695ae86dd0b1d9c5f3fbca297555Electronic structures of cephalosporins and penicillins. 15. Inductive effect of the 3-position side chain in cephalosporinsBoyd, Donald B.Journal of Medicinal Chemistry (1984), 27 (1), 63-6CODEN: JMCMAR; ISSN:0022-2623.A discussion with 39 refs. Induction appears to be the primary means by which the side chain at position 3 of the cephem nucleus influences the chem. reactivity of the β-lactam ring. In vitro antibacterial activity data suggest that when the cephalosporin is in the active site of the target bacterial enzymes, the presence of a leaving group in the side chain can promote inhibition.
- 48Faraci, W. S.; Pratt, R. F. Elimination of a good leaving group from the 3′-position of a cephalosporin need not be concerted with β-lactam ring opening: TEM-2 β-lactamase-catalyzed hydrolysis of pyridine-2-azo-4′-(N′,N′-dimethylaniline) cephalosporin (PADAC) and of cephaloridine. J. Am. Chem. Soc. 1984, 106 (5), 1489– 1490, DOI: 10.1021/ja00317a05348https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXpvFShtw%253D%253D&md5=343a8f7fde7a9cc798ca696e793f07a0Elimination of a good leaving group from the 3'-position of a cephalosporin need not be concerted with β-lactam ring opening: TEM-2 β-lactamase-catalyzed hydrolysis of pyridine-2-azo-4'-(N',N'-dimethylaniline) cephalosporin (PADAC) and of cephaloridineFaraci, W. Stephen; Pratt, R. F.Journal of the American Chemical Society (1984), 106 (5), 1489-90CODEN: JACSAT; ISSN:0002-7863.The hydrolysis of the chromogenic cephalosporin PADAC and of cephaloridine in the presence of TEM-2 β-lactamase occurs in 2 steps, the 1st enzyme-catalyzed and the 2nd not. It is proposed that the enzyme-catalyzed reaction involves β-lactam ring opening, whereas the 2nd step involves spontaneous elimination of the 3-substituent from the cephalosporate thereby formed. Thus, release of good leaving groups from the 3'-position of cephalosporins need not be concerted with β-lactam ring opening. Other enzymes, e.g., Bacillus β-lactamase II, may catalyze elimination as well as β-lactam hydrolysis, either in a concerted or nonconcerted reaction at the enzyme surface.
- 49Patterson, L. D.; Miller, M. J. Enzymatic deprotection of the cephalosporin 3′-acetoxy group using Candida antarctica lipase B. J. Org. Chem. 2010, 75 (4), 1289– 92, DOI: 10.1021/jo902406b49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVSitrk%253D&md5=21f4867040cea21848961d8c27477339Enzymatic Deprotection of the Cephalosporin 3'-Acetoxy Group Using Candida antarctica Lipase BPatterson, Leslie D.; Miller, Marvin J.Journal of Organic Chemistry (2010), 75 (4), 1289-1292CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)Cephalosporins remain one of the most important classes of antibiotics. A useful site for derivatization involves generation of and chem. at the 3'-hydroxymethyl position. While 3'-acetoxymethyl-substituted cephalosporins are readily available, deacetylation to access the free 3'-hydroxymethyl group is problematic when the carboxylic acid is protected as an ester. Herein we report that this important transformation has been efficiently accomplished using Candida antarctica lipase B. Although this transformation is difficult to carry out using chem. methods, the enzymic deacetylation has been successful on gram scale, when the cephalosporin is protected as either the benzhydryl or tert-Bu esters and on the corresponding sulfoxide and sulfone of the tert-Bu ester.
- 50Sarkar, K.; Madras, G.; Chatterjee, K. Dendron conjugation to graphene oxide using click chemistry for efficient gene delivery. RSC Adv. 2015, 5 (62), 50196– 50211, DOI: 10.1039/C5RA07004J50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpt1Ojtbc%253D&md5=e3ee0d99f8c8880373d492ffe3d42a16Dendron conjugation to graphene oxide using click chemistry for efficient gene deliverySarkar, Kishor; Madras, Giridhar; Chatterjee, KaushikRSC Advances (2015), 5 (62), 50196-50211CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)Owing to its large surface area and rapid cellular uptake, graphene oxide (GO) is emerging as an attractive candidate material for delivery of drugs and genes. The inherent sp2 π-π interaction of GO helps to carry drugs and single stranded RNA (ssRNA) but there is no such interaction with double stranded DNA (dsDNA). In this work, a polyamidoamine (PAMAM) dendron was conjugated with nano GO (nGO) through "click" chem. to improve the DNA complexation capability of GO as well as its transfection efficiency. The DNA complexation capability of GO was significantly enhanced after dendronization of GO yielding spherical nanosized (250-350 nm) particles of the dendronized GO (DGO)/pDNA complex with a pos. zeta potential. The transfection efficiency of GO dramatically increased after conjugation of the PAMAM dendron. Transfection efficiency of 51% in HeLa cells with cell viability of 80% was obsd. The transfection efficiency was significantly higher than that of polyethyleneimine 25 kDa (27% efficiency) and also surpassed that of lipofectamine 2000 (47% efficiency). The uptake of the DGO/pDNA complex by the caveolae mediated endocytosis pathway may significantly contribute to the high transfection efficiency. Thus, dendronized GO is shown to be an efficient gene carrier with minimal toxicity and is a promising candidate for use as a nonviral carrier for gene therapy.
- 51Halpern, M. E.; Ho, R. K.; Walker, C.; Kimmel, C. B. Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation. Cell 1993, 75 (1), 99– 111, DOI: 10.1016/S0092-8674(05)80087-X51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2c%252Fgs1Gmsg%253D%253D&md5=1858c7da3d2edc4ecd63ea7e91d22680Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutationHalpern M E; Ho R K; Walker C; Kimmel C BCell (1993), 75 (1), 99-111 ISSN:0092-8674.Dorsal mesoderm is thought to provide important signals for axis formation and neural differentiation in vertebrate embryos. We have examined induction and patterning in a zebrafish mutant, no tail, that lacks a derivative of dorsal mesoderm, the notochord. Despite the absence of a differentiated notochord, development of the central nervous system including floor plate appears normal, likely owing to the presence of notochord precursor cells. In contrast, somites are misshapen, and muscle pioneer cells are absent. Wild-type cells transplanted into mutant hosts can autonomously differentiate into notochord and thereby rescue somitic defects, suggesting that interactions between notochord and paraxial mesoderm are necessary for proper somite patterning. Thus, cells derived from dorsal mesoderm may have multiple signaling functions during zebrafish embryogenesis.
- 52Schulte-Merker, S.; van Eeden, F. J. M.; Halpern, M. E.; Kimmel, C. B.; Nüsslein-Volhard, C. no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene. Development 1994, 120 (4), 1009– 15, DOI: 10.1242/dev.120.4.100952https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmsleltr0%253D&md5=322293a463aa41ce475b68f2efd9fc8ano tail (ntl) is the zebrafish homolog of the mouse T (Brachyury) geneSchulte-Merker, S.; van Eeden, F. J. M.; Halpern, M. E.; Kimmel, C. B.; Nusslein-Volhard, C.Development (Cambridge, United Kingdom) (1994), 120 (4), 1009-15CODEN: DEVPED; ISSN:0950-1991.The mouse T (Brachyury) gene is required for normal mesoderm development and the extension of the body axis. Recently, two mutant alleles of a zebrafish gene, no tail (ntl), have been isolated (Halpern, M. E., Ho., R. K., Walker, C. and Kimmel, C. B. (1993) Cell 75, 99-111). Ntl mutant embryos resemble mouse T/T mutant embryos in that they lack a differentiated notochord and the caudal region of their bodies. We report here that this phenotype is caused by mutation of the zebrafish homolog of the T gene. While ntl embryos express mutant mRNA, they show no nuclear protein product. Later, expression of mRNA in mutants, but not in wild types, is greatly reduced along the dorsal midline where the notochord normally forms. This suggests that the protein is required for maintaining transcription of its own gene.
- 53Shabanpoor, F.; Gait, M. J. Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry. Chem. Commun. 2013, 49 (87), 10260– 2, DOI: 10.1039/C3CC46067C53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFynsLzI&md5=fd9873978cb407be3fe54c4ab8ecaa7cDevelopment of a general methodology for labeling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistryShabanpoor, Fazel; Gait, Michael J.Chemical Communications (Cambridge, United Kingdom) (2013), 49 (87), 10260-10262CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)We describe a general methodol. for fluorescent labeling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labeling with a fluorescent compd. (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labeled PMOs was seen in a biol. assay.
- 54O’Callaghan, C. H.; Morris, A.; Kirby, S. M.; Shingler, A. H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1972, 1 (4), 283– 8, DOI: 10.1128/AAC.1.4.28354https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaE2c7ns1agsg%253D%253D&md5=dc646a87f692cf4413e81d22218da743Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrateO'Callaghan C H; Morris A; Kirby S M; Shingler A HAntimicrobial agents and chemotherapy (1972), 1 (4), 283-8 ISSN:0066-4804.A new cephalosporin with a highly reactive beta-lactam ring was found to give an immediate color change in the presence of beta-lactamases from many bacteria, including staphylococci, Bacillus species, Enterobacteriaceae, and Pseudomonas. The reaction is confined to organisms producing beta-lactamases, but it is sufficiently sensitive to indicate the presence of this enzyme is small amounts in strains previously considered not to produce it. The compound has an unusual ultraviolet spectrum, and the color change can be followed quantitatively by measuring changes in absorption which occur in the 380- to 500-nm region, where cephalosporins normally have no absorption. The development of color is thought to be a consequence of the beta-lactam ring being unusually highly conjugated with the 3-substituent. Although in the bacteria only beta-lactamases produce this color change, it was found that serum and tissues from experimental animals also rapidly produced the colored breakdown product, which was then excreted in the urine. The mechanism of the mammalian breakdown was considered to be different from that found in bacteria.
- 55D’Astolfo, D. S.; Pagliero, R. J.; Pras, A.; Karthaus, W. R.; Clevers, H.; Prasad, V.; Lebbink, R. J.; Rehmann, H.; Geijsen, N. Efficient intracellular delivery of native proteins. Cell 2015, 161 (3), 674– 690, DOI: 10.1016/j.cell.2015.03.02855https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXnt1emtr0%253D&md5=6d1ec33e3e73be57383e1d964e4235b2Efficient intracellular delivery of native proteinsD'Astolfo, Diego S.; Pagliero, Romina J.; Pras, Anita; Karthaus, Wouter R.; Clevers, Hans; Prasad, Vikram; Lebbink, Robert Jan; Rehmann, Holger; Geijsen, NielsCell (Cambridge, MA, United States) (2015), 161 (3), 674-690CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compd. (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner.
- 56Yuan, S.; Sun, Z. Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. J. Visualized Exp. 2009, 27, e1113, DOI: 10.3791/1113There is no corresponding record for this reference.
- 57Rosen, J. N.; Sweeney, M. F.; Mably, J. D. Microinjection of zebrafish embryos to analyze gene function. J. Visualized Exp. 2009, 25, 1115, DOI: 10.3791/1115There is no corresponding record for this reference.
- 58Kimmel, C. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.; Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 1995, 203 (3), 253– 310, DOI: 10.1002/aja.100203030258https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK287nt12qsw%253D%253D&md5=629ae03ffdeb5b0a9f0e5ea52fb3133cStages of embryonic development of the zebrafishKimmel C B; Ballard W W; Kimmel S R; Ullmann B; Schilling T FDevelopmental dynamics : an official publication of the American Association of Anatomists (1995), 203 (3), 253-310 ISSN:1058-8388.We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis--the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts.
- 59Kimmel, C. B.; Law, R. D. Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells. Dev. Biol. 1985, 108 (1), 78– 85, DOI: 10.1016/0012-1606(85)90010-759https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL2M7isl2ksQ%253D%253D&md5=b8b4fa9b5be9054a0e388421ce93f6b9Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cellsKimmel C B; Law R DDevelopmental biology (1985), 108 (1), 78-85 ISSN:0012-1606.Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomere's lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cell's sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.
- 60Hoo, J. Y.; Kumari, Y.; Shaikh, M. F.; Hue, S. M.; Goh, B. H. Zebrafish: A Versatile Animal Model for Fertility Research. Biomed Res. Int. 2016, 2016, 9732780, DOI: 10.1155/2016/973278060https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2szjvVCnsQ%253D%253D&md5=5353875da64e8611a62b4df23b952d9dZebrafish: A Versatile Animal Model for Fertility ResearchHoo Jing Ying; Kumari Yatinesh; Shaikh Mohd Farooq; Hue Seow Mun; Goh Bey HingBioMed research international (2016), 2016 (), 9732780 ISSN:.The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research.
- 61Curado, S.; Stainier, D. Y.; Anderson, R. M. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat. Protoc 2008, 3 (6), 948– 54, DOI: 10.1038/nprot.2008.5861https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmvVemtrg%253D&md5=57fd17f5871cd47ca3443a83f203f7b1Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studiesCurado, Silvia; Stainier, Didier Y. R.; Anderson, Ryan M.Nature Protocols (2008), 3 (6), 948-954CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chem. and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, the authors provide a step-by-step protocol for a hybrid chem.-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the redn. of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR+ cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.
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