Conformational Heterogeneity in a Fully Complementary DNA Three-Way Junction with a GC-Rich Branchpoint
- Anita Toulmin
- ,
- Laura E. Baltierra-Jasso
- ,
- Michael J. Morten
- ,
- Tara Sabir
- ,
- Peter McGlynn
- ,
- Gunnar F. Schröder
- ,
- Brian O. Smith
- , and
- Steven W. Magennis
View Author Information
† The School of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, U.K.
‡ The Photon Science Institute, The University of Manchester, Alan Turing Building, Oxford Road, Manchester M13 9PL, U.K.
§ School of Chemistry, WestCHEM, University of Glasgow, Joseph Black Building, University Avenue, Glasgow G12 8QQ, U.K.
∥ Department of Biology, University of York, Wentworth Way, York YO10 5DD, U.K.
⊥ Institute of Complex Systems (ICS-6), Forschungszentrum Jülich, 52425 Jülich, Germany
# Physics Department, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany
@ Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, U.K.
*E-mail: [email protected]
Copyright © 2017 American Chemical Society. This publication is licensed under CC-BY.
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Abstract
DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the global structure of a fully complementary 3WJ and found that it contained unpaired bases at the branchpoint, which is consistent with previous observations of branch flexibility and branchpoint reactivity. By combining high-resolution single-molecule Förster resonance energy transfer, molecular modeling, time-resolved ensemble fluorescence spectroscopy, and the first 19F nuclear magnetic resonance observations of fully complementary 3WJs, we now show that the 3WJ structure can adopt multiple distinct conformations depending upon the sequence at the branchpoint. A 3WJ with a GC-rich branchpoint adopts an open conformation with unpaired bases at the branch and at least one additional conformation with an increased number of base interactions at the branchpoint. This structural diversity has implications for branch interactions and processing in vivo and for technological applications.
Branched nucleic acid molecules are key intermediates and structural elements in vivo (1, 2) and are important components in structural and dynamic DNA nanoscience. (3) Arguably the simplest branched structure is the three-way junction (3WJ), formed from three single nucleic acid strands. We recently used high-resolution single-molecule Förster resonance energy transfer (SM-FRET (4)) to show that a DNA 3WJ with a branchpoint that is not GC-rich does not adopt a fully paired conformation as expected but instead has a nanoscale cavity at the branchpoint. (5) The structures of fully complementary 3WJs have previously eluded structural characterization at atomic resolution because their dynamic nature has precluded their crystallization and rendered 1H nuclear magnetic resonance (NMR) signals of the junction bases unobservable. Since then, 3WJs have continued to attract attention in a range of contexts, including for controlled charge transport, (6-8) ion sensing, (9) molecular recognition, (10) and as scaffolds for light harvesting. (11, 12)
In this work, we examine whether the expanded branchpoint described previously (5) is a general phenomenon for all fully complementary 3WJs or if it is dependent on sequence. We use SM-FRET and molecular dynamics (MD) simulations to study local and global 3WJ structure. In addition, we report the first use of 19F NMR spectroscopy to study fully complementary 3WJs, by incorporating a 5-fluorocytosine near the branchpoint. We find that a 3WJ with a GC-rich branchpoint can adopt at least two conformations in solution. The major conformation is similar to that previously observed, with unpaired bases at the branch and a Y-shaped, pyramidal structure. There is also at least one minor conformation with an altered branchpoint structure, which we attribute to an increased level of base pairing at the branch. These structures are in exchange on the supramillisecond time scale. The structural heterogeneity observed upon changing the branch sequence will have consequences for the reactivity of fully complementary 3WJs and illustrates the conformational diversity that branched DNA molecules may possess.
Materials and Methods
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Preparation of Three-Way Junctions
Oligonucleotides were synthesized and labeled by Purimex GmbH (Grebenstein, Germany). The NHS esters of Alexa488 (5′/6′ mixed isomer, Invitrogen) or Cy5 (GE Healthcare) were attached via a 5-C6-aminoallyl-deoxythymidine. Annealing of samples for branched DNA was performed in buffer [13 mM Tris and 65 mM NaCl (pH 7.5)]. For all structures, the ratio of donor strand to other strands was 1:3. Samples were heated to 90 °C in a water bath and left to cool slowly overnight. For measurement, all samples were diluted into buffer containing 20 mM Tris, 15 mM NaCl, and 1 mM ascorbic acid. Prior to sample addition, the buffer was stirred with activated charcoal to remove fluorescent impurities. The measurement buffer contained either 0 or 1 mM MgCl2.
Sequences of Oligonucleotides for FRET Studies
The sequences used to construct the 3WJs are shown below. For labeling positions, see Figure 1: 3WJa, 5′ GTC GGA TCC TCT AGA TAT CTC CAT GCT CAC TGG TTA TAG GAG AAT CCG GG, 5′ AAT CTC ACA GCT GAT CAC ATT GCT ACA TGG AGA TAT CTA GAG GAT CCG AC, 5′ CCC GGA TTC TCC TAT AAC CAG TGA GTA GCA ATG TGA TCA GCT GTG AGA TT; 3WJb, 5′ GTC GGA TCC TCT AGA TAT CTC CAC GCG CAC TGG TTA TAG GAG AAT CCG GG, 5′ AAT CTC ACA GCT GAT CAC ATT GCC GCG TGG AGA TAT CTA GAG GAT CCG AC, 5′ CCC GGA TTC TCC TAT AAC CAG TGC GCG GCA ATG TGA TCA GCT GTG AGA TT.
Figure 1
Figure 1. Sequences and dye positions for DNA three-way junctions 3WJa and 3WJb [note that this is a composite of all dye positions; measured structures have only one donor (D) and one acceptor (A) dye]. 3WJa and 3WJb differ only in the bases at the branchpoint (colored red).
Multiparameter Fluorescence Detection (MFD)
MFD is based on burstwise detection of fluorescence as single molecules diffuse through the focus of a confocal microscope. Photon counting detection by multiple detectors allows simultaneous detection of the color, lifetime, polarization, and intensity of fluorescence for each molecule. (13) MFD measurements were performed using a home-built system, which we described previously. (14) The process for calculating the FRET distances from the MFD data was described previously. (5) The FRET distances reported here were reproducible to a ≤2 Å standard deviation.
19F NMR Spectroscopy
Oligonucleotides were synthesized and labeled by Purimex GmbH and were received lyophilized; the sequences are shown below. They were initially dissolved in NMR buffer [20 mM phosphate buffer (pH 7.5), 0.01% sodium azide, and 50 mM NaCl] that was prepared in ultrapure water (Direct Q3, Merck Millipore). Strands NMR_1, NMR_2, and NMR_3 were annealed together to create the 3WJ in a 1:2:2 ratio. NMR_1 was used alone for the single-stranded DNA (ssDNA) measurement, and NMR_1 and NMR_4 formed the double-stranded DNA (dsDNA) structure in a 1:2 ratio. Samples were heated to 90 °C and left to cool overnight. D2O was then added to all samples to a final concentration of 5%, with trimethylsilylpropionic acid (TSP) added to a final concentration of 0.00025%. The 3WJ was also later re-lyophilized and re-dissolved in 100% D2O.
NMR data were recorded on a Bruker AVIII 500 MHz spectrometer, fitted with a BBFO+ probe, where the operating temperatures of the spectrometer were calibrated using a sample of D, 99.8% methanol-d4. The NMR spectra were processed using Topspin, and all chemical shifts are reported relative to the 1H resonance of TSP, using the position of the TSP signal in the corresponding 1H one-dimensional (1D) spectra, recorded across the temperature range for reference. (15)
Sequences of Oligonucleotides for NMR Studies
The sequences for the 3WJ, ssDNA, and dsDNA that were studied using 19F NMR spectroscopy are shown below. The position of the 5-fluorocytosine is underlined: NMR_1, 5′ TAT CTC CAC GCG CAC TGG TT; NMR_2, 5′ AAC CAG TGC GCG GCA ATG TG; NMR_3, 5′ CAC ATT GCC GCG TGG AGA TA; NMR_4, 5′ AAC CAG TGC GCG TGG AGA TA.
Ensemble Optical Spectroscopy
Absorption spectra were recorded on a Cary 50 (Agilent Technologies) spectrometer. Steady-state and time-resolved fluorescence were measured under magic angle conditions using a fluorescence spectrometer (FluoTime 300, Picoquant GmbH) with a hybrid PMT detector (PMA Hybrid 40, Picoquant). The absorbance of the sample was low (<0.05), so that inner-filter effects were negligible. Time-resolved fluorescence spectroscopy was performed using the technique of time-correlated single-photon counting. The excitation source was a super continuum laser (WL-SC-400-4-PP, Fianium) with a tunable wavelength filter and a multimode fiber (Superchrome-Vis-FDS-MM, Fianium) at a wavelength of 488 nm, a bandpass of 10 nm, and a repetition rate of 10 MHz. The instrument response function (IRF) was ∼130 ps full width at half-maximum; the time per channel was 8 ps. Decay curves were analyzed using a standard iterative reconvolution method (FluoFit, Picoquant GmbH), assuming a multiexponential decay function. The quality of the fit was judged on the basis of the reduced χ2 statistic and the randomness of residuals.
Molecular Dynamics (MD) Simulations
The procedure for deriving structures from FRET distance restraints was described previously for 3WJa. (5)
For the starting model, the three DNA arms of the three-way junction were modeled as B-DNA using the program Hyperchem (version 8.0.8 for Windows, Molecular Modeling System, Lightwave Scientific). The program CNS (16) was then used for the simulated annealing molecular dynamics calculation. During the simulation, FRET distance restraints were applied to model the geometry of the DNA. We used the mean dye positions for Alexa488 and Cy5 obtained from a MD simulation that had been published previously. (17)
In brief, the mean positions were fixed relative to the surface of the DNA by distance restraints. The distance restraints were generated by the DEN (Deformable Elastic Network) feature (18) in CNS that selects random atom pairs that are within a specified distance range in the starting structure. It should be noted that we did not actually use deformable restraints but rather used regular (nondeformable) harmonic distance restraints during the refinement.
Results
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GC-Rich 3WJ Structure from Single-Molecule FRET and MD
To test whether the unpairing of bases at the branchpoint of fully complementary 3WJs is a general phenomenon or is specific to the previously studied 3WJ (here named 3WJa), we modified the sequence so that the four nucleotides next to the branchpoint in each arm had the potential to form two G:C Watson–Crick pairs (3WJb). The sequences of 3WJa and 3WJb are shown in Figure 1, together with the positions used for fluorescent dye labeling; the 12 nucleotides closest to the branchpoint are colored red. We postulated that the GC-rich branchpoint of 3WJb might make the formation of a fully paired structure more likely.
We studied 3WJs labeled with one donor dye and one acceptor dye. The six dye positions (Figure 1) allowed seven different combinations of donor and acceptor dyes to be used for single-molecule FRET measurements using MFD. (13) MFD is ideal for measuring accurate dye–dye distances for structural determination. (19) For each FRET pair, 3WJb was measured in a buffer containing 0 or 1 mM Mg2+, to check for potential ion-induced effects. In each case, the data were qualitatively similar to those for 3WJa, showing a donor-only peak and a FRET population (Figure 2a). MFD data for all the 3WJb samples are given in Figures S1 and S2.
Figure 2
Figure 2. Single-molecule FRET shows a GC-rich 3WJ adopts multiple conformations. (a) Two-dimensional burst frequency histograms of FRET efficiency (E) or donor anisotropy (rD) vs donor lifetime [τD(A)]. The overlaid red line is the theoretical FRET relationship E = 1 – [τD(A)/τD], where τD = 4.1 ns. The blue overlaid line is the Perrin equation rD = r0/[1 + τD(A)/ρD], where mean rotational correlation time ρD = 0.35 ns and fundamental anisotropy r0 = 0.375. (b) A sub-ensemble analysis of the whole FRET population can be fitted (black) as two Gaussian distributions (blue). The data in panels a and b are for 3WJb in 0 mM MgCl2 (dye positions D1 and A1) but were recorded on different days.
Although the MFD plots for 3WJa and 3WJb are qualitatively similar, upon closer inspection of the MFD plots it is apparent that the FRET populations for 3WJb (Figures 2a, S1, and S2) are much broader and less symmetric than those of 3WJa and other branched DNA structures that we have examined previously. (5, 14) The three broadest FRET distributions (for the D1–A1, D1–A3, and D3–A1 pairs) could be reliably fitted to double Gaussian distributions (see Table S1). For example, the FRET lifetime distribution in Figure 2a can be fitted with two Gaussians (Figure 2b) with lifetimes of 1.93 (0.1) ns (23%) and 3.24 (0.03) ns (77%). In these cases, the minor population, which ranged from 17 to 34%, has a higher FRET efficiency. In contrast, for the other FRET pairs, the distribution of lifetimes could not be reliably separated and the distribution fits a single Gaussian. These results can be explained by the existence of a minor population, which adopts a conformation in which two arms are closer to each other than in the major species. The D1–A1, D1–A3, and D3–A1 FRET pairs are all associated with the same two arms, which might mean that FRET distances involving the third arm are less affected by a change in conformation. Notably, the entire FRET population lies along the theoretical FRET line, implying that there are at least two conformations that are static or in slow exchange on the millisecond time scale required for diffusion through the confocal volume. (20) A direct comparison between the MFD plots of 3WJa and 3WJb for these three FRET pairs illustrates the very different shapes and breadths of their FRET distributions in 0 and 1 mM Mg2+ (Figures S3 and S4, respectively). In support of the SM-FRET data, bulk lifetime measurements of 3WJb (dye position D1 and A1 in 1 mM Mg2+) using time-correlated single-photon counting (TCSPC) were clearly biexponential (Figure S5), with lifetimes and weights similar to those of the SM-FRET sub-ensemble fits.
To derive a structure from the SM-FRET data for 3WJb comparable to that calculated for 3WJa, (5) we fitted a small region of the peak of the MFD distribution (the region had a width in τ of 0.7 ns and a height in E of 0.07); importantly, the lifetime recovered from a single Gaussian fit of this small region matched the lifetime of the major population measured by a double Gaussian fit to the whole FRET region (see Table S1). The fluorescence lifetime, τD(A), for the major FRET population was extracted from three separate measurements and converted to a dye–dye distance. The average τD(A) from these experiments and the resulting dye–dye distances (r) are listed in Table 1. The global structure was modeled using molecular dynamics simulations (MD) as done previously for 3WJa, (5) treating the arms as duplexes with B-form structure, while using the FRET distances listed in Table 1 as structural restraints. The FRET distances were imposed as the distance ± 0.5 Å using a harmonic square well potential. Four different sets of restraints were applied, differing in the number of bases paired at the junction (termed 0-free, 1-free, 2-free, and 3-free). The distance root-mean-square deviations (DRMSDs) between the model generated and the experimental dye–dye distances are listed in Table S2; these DRMSDs are a measure of how well the structures fit the FRET restraints. As with 3WJa, the model with the lowest DRMSD, and therefore the model that most closely matches the experimentally measured FRET distances, is the 2-free model. In other words, the FRET restraints produce a structure with unpairing at the branchpoint. The five lowest-restraint energy conformations with the 2-free restraint applied are shown in Figure 3.
Table 1. FRET Species Lifetimes [τD(A)] and Calculated Dye–Dye Distances (r) for the Major 3WJb Structurea
| 0 mM Mg2+ | 1 mM Mg2+ | |||||
|---|---|---|---|---|---|---|
| structure | τD(A) (ns) | r (Å) | 3WJa r (Å) | τD(A) (ns) | r (Å) | 3WJa r (Å) |
| D1–A1 | 3.20 | 64 (0.9) | 69 | 3.10 | 63 (<0.1) | 66 |
| D1–A2 | 3.15 | 63 (0.2) | 64 | 2.99 | 61 (<0.1) | 62 |
| D1–A3 | 3.13 | 63 (0.9) | 68 | 3.09 | 62 (0.6) | 67 |
| D2–A1 | 3.41 | 69 (0.7) | 71 | 3.19 | 64 (0.4) | 65 |
| D2–A3 | 3.69 | 76 (2.3) | 81 | 3.49 | 70 (1.7) | 78 |
| D3–A1 | 3.25 | 65 (1.5) | 69 | 3.18 | 64 (0.7) | 67 |
| D3–A2 | 3.28 | 66 (0.3) | 66 | 3.07 | 62 (0.1) | 64 |
a
Distances for 3WJa are also shown for comparison. Lifetimes were recovered from a single Gaussian fit of a sub-ensemble region centered on the FRET maximum of MFD data (the selected region had a width in τ of 0.7 ns and a height in E of 0.07). The standard deviation for r is indicated in parentheses.
Figure 3
Figure 3. Global structure of 3WJb derived from SM-FRET distance restraints for the major FRET population and MD simulations showing that the unpaired branchpoint is present with and without MgCl2 (a) and that the structures for 3WJa (yellow) and the major conformation of 3WJb (orange) are similar (b). The five lowest-restraint energy conformations are superposed using the DNA backbone atoms.
As seen for 3WJa, (5) there is little change in structure upon addition of 1 mM Mg2+ (Figure 3a). Comparison of the 3WJa ensemble and the major conformation of 3WJb (Figure 3b and Figure S6) shows that despite the significant differences in the FRET distances measured between some donor–acceptor pairs (Table 1), 3WJb clearly adopts an extended Y-shaped conformation similar to that of 3WJa (Figure 3b) with no evidence of coaxial stacking of the arms at either 0 or 1 mM Mg2+.
GC-Rich 3WJ Conformation Monitored by 19F NMR Spectroscopy
SM-FRET and TCSPC measurements provided strong evidence that 3WJb can adopt at least two distinct conformations. To corroborate the findings from the fluorescence measurements and characterize the conformations of the bases at the junction site, we sought to apply solution NMR spectroscopy. However, while 1H NMR structures of 3WJs that incorporate bulges in one strand have been determined, (21, 22) previous attempts to characterize perfectly complementary 3WJs reportedly failed. (23) The most likely explanation for this is that the particular dynamic nature of the systems studied resulted in the critical 1H signals being broadened to an extent that they were either undetectable or could not be resolved, likely because of intermediate exchange.
Because the effects of such exchange broadening depend on the difference in frequency between the NMR signals of the exchanging species, we reasoned that the synthesis of oligonucleotides that incorporated an NMR active nucleus with a frequency range much wider than that of 1H could restore the ability to observe signals in the slow exchange regime. 19F has a chemical shift range much wider than that of 1H, is highly sensitive to its chemical environment, and can be stably incorporated into nucleotide analogues, allowing the site specific labeling of synthetic oligonucleotides. (24, 25) Fluorine’s chemical shift range is wider than that of 1H, and because its gyromagnetic ratio is similar, its NMR signals are spread over a much wider range of frequencies at a particular magnetic field strength. We therefore designed a 3WJ made from three 20mers of identical sequence to the central 20 nucleotides used in 3WJb and labeled at the branchpoint with 5-fluoro-dC (Figure 4a). Although the 19F 3WJ is a truncated version of 3WJb, it should still be very stable, as there should be at least 8 bp in each arm.
Figure 4
Figure 4. NMR spectra of a 19F-labeled 3WJ. A 3WJ with a GC-rich branchpoint was modified to include a 5-fluorodeoxycytosine. (a) The schematic shows the sequence of the 3WJ, where the position of the 5F-dC is colored red, and its chemical structure is given in the inset. (b) 1D 19F NMR spectra of the 3WJ at temperatures from 12 to 40 °C. The 3WJ spectra show two major peaks that become broader with a decrease in temperature. (c) 1D 19F NMR spectra of ssDNA (top), dsDNA (middle), and the 3WJ (bottom) at 30 °C. Note that the ssDNA is capable of forming a hairpin, positioning the 5F-dC in a loop environment that results in the more upfield peak. The more downfield peak represents a ssDNA conformation that has no defined secondary structure. The vertical dashed line marks the position of the ssDNA peak.
We also recorded data from samples of the 19F-labeled oligonucleotide alone and incorporated into a fully complementary duplex structure, to establish reference chemical shifts for single- and double-stranded conformations. In 1D 19F spectra measured at 470 MHz (11.4 T, 500 MHz for 1H), our single-stranded 20mer with a 5F-dC at position 11 contains two peaks at 30 °C, indicating that it acts as a bistable oligonucleotide exchanging between a single-stranded conformation and a second conformation that is most likely a 4 bp double-stranded hairpin structure in which the 5F-dC is found at the 5′ position of the intervening four-nucleotide loop (Figure 4c). We can assign the downfield peak to the single-stranded conformation because it becomes more highly populated with an increase in temperature (Figure S5b). In contrast, in the double-stranded state, the 5F-dC gives rise to the expected single, sharp peak at a chemical shift between the single-stranded and loop chemical shifts (Figure 4c), whose position changes little at temperatures from 12 to 40 °C (Figure S7).
A 5F-dC label has previously been used to study DNA secondary structure. (25) This study found that the thermodynamic stability of labeled and unlabeled DNA and RNA molecules was unaffected by labeling. The chemical shift of the 19F can be influenced by a number of factors, including the identity of the bases stacked on either side of the GC base pair, whether the labeled base is at the end of the dsDNA, and whether the nucleic acid is an A- or B-form helix. Similarly, noncanonical structures could result in different magnetic environments. Although the dsDNA chemical shift we observe is not as upfield as previously reported (by ∼0.5 ppm), this is likely because differences in sequence context exist (ACXTT in ref 25 compared to CGXGC) and also because our spectra were recorded in 95% H2O (the 5-19F-dC resonance shifts progressively downfield as H2O is added to a D2O solution, which is attributed to a secondary isotope effect caused by nearby imino hydrogens (25)).
The 1D 19F spectrum of the 5F-dC in the assembled 3WJ is characterized by the presence of two distinct peaks at −164.16 and −164.52 ppm indicating that the 5F-dC is sampling (at least) two distinct conformations (Figure 4b). The relative intensities of these peaks change with temperature, indicating that the states are in exchange. The downfield peak’s chemical shift is very close to the single-stranded chemical shift, which we interpret to mean that the base spends a significant proportion of the time in an unstacked, single-stranded conformation. The small chemical shift difference with ssDNA could be due to the exchange process. Additionally, in unstructured ssDNA, the chemical shift is a product of rapid averaging among many different environments. If the constrained 3WJ context changes the weighting of the populations sampled, that would also change the shift. The upfield peak matches neither the double-stranded chemical shift nor the loop shift seen for the bistable oligonucleotide and is further upfield than either (Figure 4c). There are a number of possible conformations that could give rise to such an upfield shift, including non-B-form double-stranded structures or placement in the last base pair of a double-stranded region. The similar pattern of peaks seen for the 3WJ in 100% D2O (Figure S5) excludes the possibility that the spectral features seen in H2O arise from secondary isotope effects.
Fitting the 3WJ spectrum at 30 °C with Dynamic NMR (26, 27) (TEDDY DNMR) suggests that the true chemical shifts of the exchanging species are very close to the shifts observed and that the rate of exchange between the states is around 15 Hz with a 60/40 population ratio. This agrees with our interpretation of the SM-FRET data as representing two or more conformations that are stable on the time scale of diffusion through the confocal volume (several milliseconds).
Discussion
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Pioneering ensemble studies had previously illustrated the power of FRET to probe the conformation and heterogeneity of branched DNA molecules such as 3WJs. (28, 29) The emergence of SM-FRET as a powerful approach to determining structure (17, 19, 30-32) has subsequently made it possible to unambiguously probe the structure and heterogeneity of branched DNA in unprecedented detail. (5, 14) In an earlier report, SM-FRET and MD were combined with ensemble time-resolved fluorescence of a nucleobase analogue (2-AP) to yield information about both global structure (Y-shaped with pyramidal geometry) and local structure (observation of an unpaired branchpoint) in a fully complementary 3WJ. (5) Although this work reconciled much of the previous literature about this class of branched molecules, it was not known whether this was a general result for all fully complementary 3WJs.
In this work, we have attempted to address this issue by studying a 3WJ with a GC-rich branchpoint, hypothesizing that this could provide the stability required for full Watson–Crick pairing. Although the changes to the overall sequence were small, amounting to only four AT pairs exchanged for GCs, the change was clearly manifested in the heterogeneous SM-FRET data, which was interpreted as arising from at least two different conformations. The combination of FRET restraints from the major conformation of 3WJb and MD produced a global structure that was in excellent agreement with that derived for 3WJa, with an asymmetric Y shape and a shallow pyramidal geometry. This is also in agreement with recent small angle X-ray scattering (SAXS) experiments on a 3WJ, which confirmed that the arms are in a B form and that the global structure is asymmetric. (33)
The lack of an adenine at the branchpoint precluded us from utilizing 2-AP as a probe of local branch structure, as was done for 3WJa. (5) Instead, we used 19F to label a cytosine at the branch, thereby allowing us to report the first NMR spectra of a branchpoint base on a fully complementary 3WJ. The two resonances observed for the 3WJ can be assigned as the cytosine in different environments: one resonance resembles that of the same nucleotide in ssDNA, while the other indicates that the base populates a distinct DNA secondary structure. The latter conformation does not match either dsDNA or the hairpin structure formed for the labeled single strand; therefore, it may be due to a distorted Watson–Crick pair, an underwound helical conformation, or alternative secondary structure.
We did not attempt to model the minor conformation(s) observed via SM-FRET as we did for the major species. Even if we assume that there are only two conformations, we were able to reliably assign different FRET populations, and therefore derive distances, for only the most heterogeneous FRET distributions. The simplest model that is consistent with the SM-FRET and NMR data is for a 3WJ that can adopt two conformations, which interconvert on a time scale of tens of milliseconds (Figure 5). However, we note that we cannot rule out the possibility of multiple secondary conformations.
Figure 5
Figure 5. Simple model of 3WJ conformational heterogeneity assuming a two-state system. The left image represents the major open structure derived from FRET restraints (Figure 3), which can convert via base pairing at the branch to a constrained structure in which there are strengthened interactions at the branchpoint and two of the arms are closer together. The exchange rate is an estimate from DNMR measurements (see the text).
This work demonstrates that SM-FRET, MD, and NMR spectroscopy are a powerful combination of techniques for studying nucleic acid structures. They have been used here to demonstrate the structural complexity in a simple branched nucleic acid molecule, a DNA 3WJ. The observed conformational heterogeneity may have an impact on the accessibility and reactivity of the 3WJ branchpoint, and the manner in which they are recognized and processed by enzymes. It also indicates the potential for subtle control of DNA structure and dynamics at the nanoscale through judicious choice of sequence and environment.
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00677.
Single-molecule FRET plots for 3WJa and 3WJb; TCSPC data for 3WJb; overlaid molecular models of the global structure of 3WJa and 3WJb in 0 mM Mg2+ buffer; 1D 19F NMR spectra of a GC-rich 3WJ in D2O from 12 to 40 °C, ssDNA in H2O from 21 to 30 °C, and dsDNA in H2O from 12 to 38 °C; sub-ensemble analysis of 3WJb FRET data; and DRMSD from the FRET-based restraints in 3WJb (PDF)
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Author Information
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- Steven W. Magennis - School of Chemistry, WestCHEM, University of Glasgow, Joseph Black Building, University Avenue, Glasgow G12 8QQ, U.K.;http://orcid.org/0000-0002-4327-8366;
- Laura E. Baltierra-Jasso - The School of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, U.K.; The Photon Science Institute, The University of Manchester, Alan Turing Building, Oxford Road, Manchester M13 9PL, U.K.; School of Chemistry, WestCHEM, University of Glasgow, Joseph Black Building, University Avenue, Glasgow G12 8QQ, U.K.;http://orcid.org/0000-0002-4824-5926
This work was supported by EPSRC Grant EP/L027003/1 (M.J.M.), BBSRC Grant BB/G00269X/1 (T.S.), and a CONACyT scholarship (L.E.B.-J.).
References
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This article references 33 other publications.
- 1Lilley, D. M. J. (2000) Structures of helical junctions in nucleic acids Q. Rev. Biophys. 33, 109– 159 DOI: 10.1017/S0033583500003590Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjvVWqtw%253D%253D&md5=40a7d48ff2c61797e7b73170fb92a3d8Structures of helical junctions in nucleic acidsLilley, David M. J.Quarterly Reviews of Biophysics (2000), 33 (2), 109-159CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)A review with 232 refs. Topics discussed include the occurrence of helical junctions in nucleic acids, the structure of the four-way DNA junction, the role of metal ions in the folding of the four-way DNA junction, conformational variation in the four-way junctions, branch migration, three-way DNA junctions, helical junctions in RNA, and the recognition and distortion of four-way DNA junctions by proteins.
- 2Atkinson, J. and McGlynn, P. (2009) Replication fork reversal and the maintenance of genome stability Nucleic Acids Res. 37, 3475– 3492 DOI: 10.1093/nar/gkp244Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnvVyhsbw%253D&md5=94b01aff9aba8b42654f746a7f49a7c3Replication fork reversal and the maintenance of genome stabilityAtkinson, John; McGlynn, PeterNucleic Acids Research (2009), 37 (11), 3475-3492CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)A review. The progress of replication forks is often threatened in vivo, both by DNA damage and by proteins bound to the template. Blocked forks must somehow be restarted, and the original blockage cleared, in order to complete genome duplication, implying that blocked fork processing may be crit. for genome stability. One possible pathway that might allow processing and restart of blocked forks, replication fork reversal, involves the unwinding of blocked forks to form four-stranded structures resembling Holliday junctions. This concept has gained increasing popularity recently based on the ability of such processing to explain many genetic observations, the detection of unwound fork structures in vivo and the identification of enzymes that have the capacity to catalyze fork regression in vitro. Here, the authors discuss the contexts in which fork regression might occur, the factors that may promote such a reaction and the possible roles of replication fork unwinding in normal DNA metab.
- 3Seeman, N. C. (2010) Nanomaterials based on DNA Annu. Rev. Biochem. 79, 65– 87 DOI: 10.1146/annurev-biochem-060308-102244Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXpslShsbo%253D&md5=b842821a6e15581c40329e75e219c0c7Nanomaterials based on DNASeeman, Nadrian C.Annual Review of Biochemistry (2010), 79 (), 65-87CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. The combination of synthetic stable branched DNA and sticky-ended cohesion has led to the development of structural DNA nanotechnol. over the past 30 years. The basis of this enterprise is that it is possible to construct novel DNA-based materials by combining these features in a self-assembly protocol. Thus, simple branched mols. lead directly to the construction of polyhedrons, whose edges consist of double helical DNA and whose vertices correspond to the branch points. Stiffer branched motifs can be used to produce self-assembled 2-dimensional and 3-dimensional periodic lattices of DNA (crystals). DNA was also used to make a variety of nanomech. devices, including mols. that change their shapes and mols. that can walk along a DNA sidewalk. Devices were incorporated into 2-dimensional DNA arrangements; sequence-dependent devices are driven by increases in nucleotide pairing at each step in their machine cycles.
- 4Roy, R., Hohng, S., and Ha, T. (2008) A practical guide to single-molecule FRET Nat. Methods 5, 507– 516 DOI: 10.1038/nmeth.1208Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmsVOjt7Y%253D&md5=d6207e40c17cec6d202f7f46030aa575A practical guide to single-molecule FRETRoy, Rahul; Hohng, Sungchul; Ha, TaekjipNature Methods (2008), 5 (6), 507-516CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Single-mol. fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-mol. techniques. Despite the explosive growth in the application of smFRET to answer biol. questions in the last decade, the technique has been practiced mostly by biophysicists. The authors provide a practical guide to using smFRET, focusing on the study of immobilized mols. that allow measurements of single-mol. reaction trajectories from 1 ms to many minutes. The authors discuss issues a biologist must consider to conduct successful smFRET expts., including exptl. design, sample prepn., single-mol. detection and data anal. The authors also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.
- 5Sabir, T., Toulmin, A., Ma, L., Jones, A. C., McGlynn, P., Schroeder, G. F., and Magennis, S. W. (2012) Branchpoint Expansion in a Fully Complementary Three-Way DNA Junction J. Am. Chem. Soc. 134, 6280– 6285 DOI: 10.1021/ja211802zGoogle Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XitFCjsLs%253D&md5=212a7c044d4c79fb2c3a4e8c14e54550Branchpoint Expansion in a Fully Complementary Three-Way DNA JunctionSabir, Tara; Toulmin, Anita; Ma, Long; Jones, Anita C.; McGlynn, Peter; Schroder, Gunnar F.; Magennis, Steven W.Journal of the American Chemical Society (2012), 134 (14), 6280-6285CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Branched nucleic acid mols. serve as key intermediates in DNA replication, recombination, and repair; architectural elements in RNA; and building blocks and functional components for nanoscience applications. Using a combination of high-resoln. single-mol. FRET, time-resolved spectroscopy, and mol. modeling, we have probed the local and global structure of a DNA three-way junction (3WJ) in soln. We found that it adopts a Y-shaped, pyramidal structure, in which the bases adjacent to the branchpoint are unpaired, despite the full Watson-Crick complementarity of the mol. The unpairing allows a nanoscale cavity to form at the junction center. Our structure accounts for earlier observations made of the structure, flexibility, and reactivity of 3WJs. We anticipate that these results will guide the development of new DNA-based supramol. receptors and nanosystems.
- 6Young, R. M., Singh, A. P. N., Thazhathveetil, A. K., Cho, V. Y., Zhang, Y. Q., Renaud, N., Grozema, F. C., Beratan, D. N., Ratner, M. A., Schatz, G. C., Berlin, Y. A., Lewis, F. D., and Wasielewski, M. R. (2015) Charge Transport across DNA-Based Three-Way Junctions J. Am. Chem. Soc. 137, 5113– 5122 DOI: 10.1021/jacs.5b00931Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlsFGhu7w%253D&md5=16043c16064fbbd6728265b40b102d50Charge Transport across DNA-Based Three-Way JunctionsYoung, Ryan M.; Singh, Arunoday P. N.; Thazhathveetil, Arun; Cho, Vincent Y.; Zhang, Yuqi; Renaud, Nicolas; Grozema, Ferdinand C.; Beratan, David N.; Ratner, Mark A.; Schatz, George C.; Berlin, Yuri A.; Lewis, Frederick D.; Wasielewski, Michael R.Journal of the American Chemical Society (2015), 137 (15), 5113-5122CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)DNA-based mol. electronics will require charges to be transported from one site within a 2D or 3D architecture to another. While this has been shown previously in linear, π-stacked DNA sequences, the dynamics and efficiency of charge transport across DNA three-way junction (3WJ) have yet to be detd. Here, the authors present a study of hole transport and trapping across a DNA-based three-way junction systems by a combination of femtosecond transient absorption spectroscopy and mol. dynamics simulations. Hole transport across the junction is proposed to be gated by conformational fluctuations in the ground state which bring the transiently populated hole carrier nucleobases into better aligned geometries on the nanosecond time scale, thus modulating the π-π electronic coupling along the base pair sequence.
- 7Zhang, Y. Q., Young, R. M., Thazhathveetil, A. K., Singh, A. P. N., Liu, C. R., Berlin, Y. A., Grozema, F. C., Lewis, F. D., Ratner, M. A., Renaud, N., Siriwong, K., Voityuk, A. A., Wasielewski, M. R., and Beratan, D. N. (2015) Conformationally Gated Charge Transfer in DNA Three-Way Junctions J. Phys. Chem. Lett. 6, 2434– 2438 DOI: 10.1021/acs.jpclett.5b00863Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpsFOlt7g%253D&md5=93b935ef85e287c2a6e3139ddddf77dcConformationally Gated Charge Transfer in DNA Three-Way JunctionsZhang, Yuqi; Young, Ryan M.; Thazhathveetil, Arun K.; Singh, Arunoday P. N.; Liu, Chaoren; Berlin, Yuri A.; Grozema, Ferdinand C.; Lewis, Frederick D.; Ratner, Mark A.; Renaud, Nicolas; Siriwong, Khatcharin; Voityuk, Alexander A.; Wasielewski, Michael R.; Beratan, David N.Journal of Physical Chemistry Letters (2015), 6 (13), 2434-2438CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)Mol. structures that direct charge transport in two or three dimensions possess some of the essential functionality of elec. switches and gates. We use theory, modeling, and simulation to explore the conformational dynamics of DNA three-way junctions (TWJs) that may control the flow of charge through these structures. Mol. dynamics simulations and quantum calcns. indicate that DNA TWJs undergo dynamic interconversion among "well stacked" conformations on the time scale of nanoseconds, a feature that makes the junctions very different from linear DNA duplexes. The studies further indicate that this conformational gating would control charge flow through these TWJs, distinguishing them from conventional (larger size scale) gated devices. Simulations also find that structures with polyethylene glycol linking groups ("extenders") lock conformations that favor CT for 25 ns or more. The simulations explain the kinetics obsd. exptl. in TWJs and rationalize their transport properties compared with double-stranded DNA.
- 8Zhang, Y. Q., Zhang, W. B., Liu, C. R., Zhang, P., Balaeff, A., and Beratan, D. N. (2016) DNA charge transport: Moving beyond 1D Surf. Sci. 652, 33– 38 DOI: 10.1016/j.susc.2016.03.011Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xkt1Gjurc%253D&md5=50e28ed5ed174ee81b673cb536f379b0DNA charge transport: Moving beyond 1DZhang, Yuqi; Zhang, William B.; Liu, Chaoren; Zhang, Peng; Balaeff, Alexander; Beratan, David N.Surface Science (2016), 652 (), 33-38CODEN: SUSCAS; ISSN:0039-6028. (Elsevier B.V.)A review. Charge transport across novel DNA junctions has been studied for several decades. From early attempts to move charge across DNA double crossover junctions to recent studies on DNA 3-way junctions and G-quadruplex (G4) motifs, it is becoming clear that efficient cross-junction charge migration requires strong base-to-base electronic coupling at the junction, facilitated by favorable pi-stacking. Here, the authors review recent progress toward the goal of manipulating and controlling charge transport through DNA junctions.
- 9Ma, L., Wu, G. R., Li, Y. F., Qin, P., Meng, L. P., Liu, H. Y., Li, Y. Y., and Diao, A. P. (2015) A reversible metal ion fueled DNA three-way junction molecular device for ″turn-on and -off″ fluorescence detection of mercury ions (II) and biothiols respectively with high selectivity and sensitivity Nanoscale 7, 18044– 18048 DOI: 10.1039/C5NR04688BGoogle ScholarThere is no corresponding record for this reference.
- 10Barros, S. A. and Chenoweth, D. M. (2014) Recognition of Nucleic Acid Junctions Using Triptycene-Based Molecules Angew. Chem., Int. Ed. 53, 13746– 13750 DOI: 10.1002/anie.201407061Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVCit73O&md5=b3d59010e6f130eab638869353164ca2Recognition of Nucleic Acid Junctions Using Triptycene-Based MoleculesBarros, Stephanie A.; Chenoweth, David M.Angewandte Chemie, International Edition (2014), 53 (50), 13746-13750CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The modulation of nucleic acids by small mols. is an essential process across the kingdoms of life. Targeting nucleic acids with small mols. represents a significant challenge at the forefront of chem. biol. Nucleic acid junctions are ubiquitous structural motifs in nature and in designed materials. Herein, we describe a new class of structure-specific nucleic acid junction stabilizers based on a triptycene scaffold. Triptycenes provide significant stabilization of DNA and RNA three-way junctions, providing a new scaffold for the development of nucleic acid junction binders with enhanced recognition properties. Addnl., we report cytotoxicity and cell uptake data in two human ovarian carcinoma cell lines.
- 11Probst, M., Langenegger, S. M., and Haner, R. (2014) A modular LHC built on the DNA three-way junction Chem. Commun. 50, 159– 161 DOI: 10.1039/C3CC47490AGoogle Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVKjtLjF&md5=e7b616c5bbd54019a2f7d679c95b0faeA modular LHC built on the DNA three-way junctionProbst, Markus; Langenegger, Simon M.; Haener, RobertChemical Communications (Cambridge, United Kingdom) (2014), 50 (2), 159-161CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A light-harvesting complex composed of a π-stacked multichromophoric array in a DNA three-way junction is described. The modular design allows for a ready exchange of non-covalently attached energy acceptors.
- 12Su, W., Bagshaw, C. R., and Burley, G. A. (2013) Addressable and unidirectional energy transfer along a DNA three-way junction programmed by pyrrole-imidazole polyamides Sci. Rep. 3, 1883 DOI: 10.1038/srep01883Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3snmsFyksg%253D%253D&md5=1ba61151bbb29917c536468d9940d7a8Addressable and unidirectional energy transfer along a DNA three-way junction programmed by pyrrole-imidazole polyamidesSu Wu; Bagshaw Clive R; Burley Glenn AScientific reports (2013), 3 (), 1883 ISSN:.We describe a photonic waveguide where FRET is routed uni-directionally along a double-stranded DNA track. The efficiency of FRET is modulated by the supramolecular control of fluorophores along double-stranded DNA using fluorophore-tethered Pyrrole-Imidazole polyamides (PAs). We show that uni-directional FRET is enhanced by the complete assembly of each of the constituent parts, resulting in the selective routing of light along simple DNA duplexes as well as a three-way junction (3WJ).
- 13Kühnemuth, R. and Seidel, C. A. M. (2001) Principles of single molecule multiparameter fluorescence spectroscopy Single Mol. 2, 251– 254 DOI: 10.1002/1438-5171(200112)2:4<251::AID-SIMO251>3.3.CO;2-KGoogle Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtlCltQ%253D%253D&md5=46bc301eecf524bfa54b8be2b6c46732Principles of single molecule multiparameter fluorescence spectroscopyKuhnemuth, Ralf; Seidel, Claus A. M.Single Molecules (2001), 2 (4), 251-254CODEN: SGMCF7; ISSN:1438-5163. (Wiley-VCH Verlag Berlin GmbH)A review. In this review a short introduction to the concept and exptl. realization of single mol. multiparameter fluorescence spectroscopy is presented. The approach of Multiparameter Fluorescence Detection (MFD) is essentially the time-resolved observation of all five intrinsic properties of a chromophore that can be probed in a fluorescence expt., i.e. spectral properties of absorption and fluorescence, F(λA, λ), fluorescence quantum yield, φF, fluorescence lifetime, τ, and anisotropy, r. This harbors the potential to combine Fluorescence Correlation Spectroscopy (FCS) and Time-Correlated Single Photon Counting (TCSPC) in a single expt. Addnl., species selective anal. of subensembles and even direct studies on single mol. dynamics become accessible. The application of MFD to complex multichromophoric systems is discussed. Examples range from studies of Donor-Acceptor pairs showing intra- or intermol. Fluorescence Resonance Energy Transfer (FRET) for revealing dynamical and statical structural informations of biol. relevant macromols. to the development of vastly improved anal. tools for the accurate identification of target mols. in multicomponent systems as needed in todays and future High-Throughput Screening (HTS) efforts. Many theor. models of interactions and chem. reactions have been described on the mol. level although the primary source for our knowledge on chem. structure and dynamics so far are studies on mol. ensembles. Such studies, however, did not yet provide an answer to the question whether all members of the ensemble have the same properties. Here, expts. on single mols. promise new and unexpected insights, because they eliminate ensemble averaging and provide direct information on heterogeneity and kinetics of the system. That way distributions and spectra of different species in specific states are directly accessible even in heterogeneous systems (Fig.1).
- 14Sabir, T., Schröder, G. F., Toulmin, A., McGlynn, P., and Magennis, S. W. (2011) Global structure of forked DNA in solution revealed by high-resolution single-molecule FRET J. Am. Chem. Soc. 133, 1188– 1191 DOI: 10.1021/ja108626wGoogle Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsF2kurbO&md5=a327b9308801c6b70d4e65bdf4634e27Global Structure of Forked DNA in Solution Revealed by High-Resolution Single-Molecule FRETSabir, Tara; Schrodr, Gunnar F.; Toulmin, Anita; McGlynn, Peter; Magennis, Steven W.Journal of the American Chemical Society (2011), 133 (5), 1188-1191CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Branched DNA structures play crit. roles in DNA replication, repair, and recombination in addn. to being key building blocks for DNA nanotechnol. Here we combine single-mol. multiparameter fluorescence detection and mol. dynamics simulations to give a general approach to global structure detn. of branched DNA in soln. We reveal an open, planar structure of a forked DNA mol. with three duplex arms and demonstrate an ion-induced conformational change. This structure will serve as a benchmark for DNA-protein interaction studies.
- 15Harris, R. K., Becker, E. D., Cabral De Menezes, S. M., Goodfellow, R., and Granger, P. (2001) NMR nomenclature. Nuclear spin properties and conventions for chemical shifts - (IUPAC recommendations 2001) Pure Appl. Chem. 73, 1795– 1818 DOI: 10.1351/pac200173111795Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XitVeltbw%253D&md5=678eae2d74c6c1e4321cd907a3d2129bNMR nomenclature. Nuclear spin properties and conventions for chemical shifts (IUPAC recommendations 2001)Harris, Robin K.; Becker, Edwin D.; Cabral De Menezes, Sonia M.; Goodfellow, Robin; Granger, PierrePure and Applied Chemistry (2001), 73 (11), 1795-1818CODEN: PACHAS; ISSN:0033-4545. (International Union of Pure and Applied Chemistry)A review. A unified scale is recommended for reporting the NMR chem. shifts of all nuclei relative to the 1H resonance of tetramethylsilane (TMS). The unified scale is designed to provide a precise ratio, Ξ, of the resonance frequency of a given nuclide to that of the primary ref., the 1H resonance of TMS in dil. soln. (vol. fraction, φ < 1%) in CHCl3. Referencing procedures are discussed, including matters of practical application of the unified scale. Special attention is paid to recommended ref. samples, and values of Ξ for secondary refs. on the unified scale are listed, many of which are the results of new measurements. Some earlier recommendations relating to the reporting of chem. shifts are endorsed. The chem. shift, δ, is redefined to avoid previous ambiguities but to leave practical usage unchanged. Relations between the unified scale and recently published recommendations for referencing in aq. solns. (for specific use in biochem. work) are discussed, as well as the special effects of working in the solid state with magic-angle spinning. In all, nine new recommendations relating to chem. shifts are made. Standardized nuclear spin data are also presented in tabular form for the stable (and some unstable) isotopes of all elements with nonzero quantum nos. The information given includes quantum nos., isotopic abundances, magnetic moments, magnetogyric ratios and receptivities, together with quadrupole moments and line-width factors where appropriate.
- 16Brunger, A. T. (2007) Version 1.2 of the Crystallography and NMR system Nat. Protoc. 2, 2728– 2733 DOI: 10.1038/nprot.2007.406Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlSlsrzK&md5=92276efc33435ac14906f42613368c9fVersion 1.2 of the Crystallography and NMR systemBrunger, Axel T.Nature Protocols (2007), 2 (11), 2728-2733CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Version 1.2 of the software system, termed Crystallog. and NMR system (CNS), for crystallog. and NMR structure detn. has been released. Since its first release, the goals of CNS have been (i) to create a flexible computational framework for exploration of new approaches to structure detn., (ii) to provide tools for structure soln. of difficult or large structures, (iii) to develop models for analyzing structural and dynamical properties of macromols. and (iv) to integrate all sources of information into all stages of the structure detn. process. Version 1.2 includes an improved model for the treatment of disordered solvent for crystallog. refinement that employs a combined grid search and least-squares optimization of the bulk solvent model parameters. The method is more robust than previous implementations, esp. at lower resoln., generally resulting in lower R values. Other advances include the ability to apply thermal factor sharpening to electron d. maps. Consistent with the modular design of CNS, these addns. and changes were implemented in the high-level computing language of CNS.
- 17Woźniak, A. K., Schröder, G. F., Grubmüller, H., Seidel, C. A. M., and Oesterhelt, F. (2008) Single-molecule FRET measures bends and kinks in DNA Proc. Natl. Acad. Sci. U. S. A. 105, 18337– 18342 DOI: 10.1073/pnas.0800977105Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVyhsrrK&md5=29584669731e101909f635cebd32f2cbSingle-molecule FRET measures bends and kinks in DNAWozniak, Anna K.; Schroder, Gunnar F.; Grubmueller, Helmut; Seidel, Claus A. M.; Oesterhelt, FilippProceedings of the National Academy of Sciences of the United States of America (2008), 105 (47), 18337-18342, S18337/1-S18337/19CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The authors present advances in the use of single-mol. FRET measurements with flexibly linked dyes to derive full 3D structures of DNA constructs based on abs. distances. The resoln. obtained by this single-mol. approach harbors the potential to study in detail also protein- or damage-induced DNA bending. If one is to generate a geometric structural model, distances between fixed positions are needed. These are usually not exptl. accessible because of unknown fluorophore-linker mobility effects that lead to a distribution of FRET efficiencies and distances. To solve this problem, the authors performed studies on DNA double-helixes by systematically varying donor acceptor distances from 2 to 10 nm. Anal. of dye-dye quenching and fluorescence anisotropy measurements reveal slow positional and fast orientational fluorophore dynamics, that results in an isotropic av. of the FRET efficiency. The authors use a nonlinear conversion function based on mol. dynamics simulations that allows the authors to include this effect in the calcn. of abs. FRET distances. To obtain unique structures, the authors performed a quant. statistical anal. for the conformational search in full space based on triangulation, which uses the known helical nucleic acid features. The authors' higher accuracy allowed the detection of sequence-dependent DNA bending by 16°. For DNA with bulged adenosines, the authors also quantified the kink angles introduced by the insertion of 1, 3 and 5 bases to be 32° ± 6°, 56° ±4° and 73±2°, resp. Moreover, the rotation angles and shifts of the helixes were calcd. to describe the relative orientation of the two arms in detail.
- 18Schröder, G. F., Brunger, A. T., and Levitt, M. (2007) Combining efficient conformational sampling with a deformable elastic network model facilitates structure refinement at low resolution Structure 15, 1630– 1641 DOI: 10.1016/j.str.2007.09.021Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2sjjs1Onsw%253D%253D&md5=0f7df5635fb0b44134e8bff03c018a85Combining efficient conformational sampling with a deformable elastic network model facilitates structure refinement at low resolutionSchroder Gunnar F; Brunger Axel T; Levitt MichaelStructure (London, England : 1993) (2007), 15 (12), 1630-41 ISSN:0969-2126.Structural studies of large proteins and protein assemblies are a difficult and pressing challenge in molecular biology. Experiments often yield only low-resolution or sparse data that are not sufficient to fully determine atomistic structures. We have developed a general geometry-based algorithm that efficiently samples conformational space under constraints imposed by low-resolution density maps obtained from electron microscopy or X-ray crystallography experiments. A deformable elastic network (DEN) is used to restrain the sampling to prior knowledge of an approximate structure. The DEN restraints dramatically reduce over-fitting, especially at low resolution. Cross-validation is used to optimally weight the structural information and experimental data. Our algorithm is robust even for noise-added density maps and has a large radius of convergence for our test case. The DEN restraints can also be used to enhance reciprocal space simulated annealing refinement.
- 19Kalinin, S., Peulen, T., Sindbert, S., Rothwell, P. J., Berger, S., Restle, T., Goody, R. S., Gohlke, H., and Seidel, C. A. M. (2012) A toolkit and benchmark study for FRET-restrained high-precision structural modeling Nat. Methods 9, 1218– 1225 DOI: 10.1038/nmeth.2222Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1Ckt7nE&md5=ff95e353c33d3e845da14c480b36c1bbA toolkit and benchmark study for FRET-restrained high-precision structural modelingKalinin, Stanislav; Peulen, Thomas; Sindbert, Simon; Rothwell, Paul J.; Berger, Sylvia; Restle, Tobias; Goody, Roger S.; Gohlke, Holger; Seidel, Claus A. M.Nature Methods (2012), 9 (12), 1218-1225CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We present a comprehensive toolkit for Foerster resonance energy transfer (FRET)-restrained modeling of biomols. and their complexes for quant. applications in structural biol. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calcd. by rigorous error estn. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with an r.m.s. deviation of 0.5 Å. Furthermore, we introduce FRET-guided 'screening' of a large structural ensemble created by mol. dynamics simulations. We used this hybrid approach to det. the formerly unknown configuration of the flexible single-strand template overhang.
- 20Kalinin, S., Valeri, A., Antonik, M., Felekyan, S., and Seidel, C. A. M. (2010) Detection of structural dynamics by FRET: a photon distribution and fluorescence lifetime analysis of systems with multiple states J. Phys. Chem. B 114, 7983– 7995 DOI: 10.1021/jp102156tGoogle Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmt12ksLk%253D&md5=1d5212f1c66259d3b877b212ad88292cDetection of Structural Dynamics by FRET: A Photon Distribution and Fluorescence Lifetime Analysis of Systems with Multiple StatesKalinin, Stanislav; Valeri, Alessandro; Antonik, Matthew; Felekyan, Suren; Seidel, Claus A. M.Journal of Physical Chemistry B (2010), 114 (23), 7983-7995CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)Two complementary methods in confocal single-mol. fluorescence spectroscopy are presented to analyze conformational dynamics by Forster resonance energy transfer (FRET) measurements considering simulated and exptl. data. First, an extension of photon distribution anal. (PDA) is applied to characterize conformational exchange between two or more states via global anal. of the shape of FRET peaks for different time bins. PDA accurately predicts the shape of FRET efficiency histograms in the presence of FRET fluctuations, taking into account shot noise and background contributions. Dynamic-PDA quant. recovers FRET efficiencies of the interconverting states and relaxation times of dynamics on the time scale of the diffusion time td (typically milliseconds), with a dynamic range of the method of about ± 1 order of magnitude with respect to td. Correction procedures are proposed to consider the factors limiting the accuracy of dynamic-PDA, such as brightness variations, shortening of the observation time due to diffusion, and a contribution of multimol. events. Second, an anal. procedure for multiparameter fluorescence detection is presented, where intensity-derived FRET efficiency is correlated with the fluorescence lifetime of the donor quenched by FRET. If a max. likelihood estimator is applied to compute a mean fluorescence lifetime of mixed states, one obtains a fluorescence weighted mean lifetime. Thus a mixed state is detected by a characteristic shift of the fluorescence lifetime, which becomes longer than that expected for a single species with the same intensity-derived FRET efficiency. Anal. tools for direct visual inspection of two-dimensional diagrams of FRET efficiency vs. donor lifetime are presented for the cases of static and dynamic FRET. Finally these new techniques are compared with fluorescence correlation spectroscopy.
- 21Leontis, N. B., Hills, M. T., Piotto, M., Malhotra, A., Nussbaum, J., and Gorenstein, D. G. (1993) A model for the solution structure of a branched, 3-strand DNA complex J. Biomol. Struct. Dyn. 11, 215– 223 DOI: 10.1080/07391102.1993.10508722Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXpvFKmtg%253D%253D&md5=b5ef4910e26ec0722f0a9c5018931cc1A model for the solution structure of a branched, three-strand DNA complexLeontis, Neocles B.; Hills, Michael T.; Piotto, Martial; Malhotra, Arun; Nussbaum, Jean; Gorenstein, David G.Journal of Biomolecular Structure & Dynamics (1993), 11 (2), 215-23, 1 plateCODEN: JBSDD6; ISSN:0739-1102.The soln. structure of a DNA three-way junction (TWJ) contg. two unpaired thymidines was elucidated using two- and three-dimensional 1H NMR spectroscopy. TWJs with unpaired nucleotides are ubiquitous structural motifs of complex single-stranded nucleic acids. In the presence of Mg2+, the TWJ complex adopts a unique conformation in which the bases of one of the oligonucleotides ("strand 1") are continuously stacked across the junction. Guanosine 8 of strand 3 (S3-G8), which pairs with S1-C5, stacks on S2-G5, which is paired to S1-C6. The unpaired thymidine bases (S3-T6 and S3-T7) are exposed to the solvent, whereas the sugar of S3-G8 is largely buried. S3-T6 also interacts with the sugar residue of S3-G11. All three stems conform to B-type DNA.
- 22Wu, B., Girard, F., van Buuren, B., Schleucher, J., Tessari, M., and Wijmenga, S. (2004) Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold Nucleic Acids Res. 32, 3228– 3239 DOI: 10.1093/nar/gkh645Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXltVWltrs%253D&md5=07720f0443df0c3edd23046450ed9493Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H foldWu, Bin; Girard, Frederic; Van Buuren, Bernd; Schleucher, Juergen; Tessari, Marco; Wijmenga, SybrenNucleic Acids Research (2004), 32 (10), 3228-3239CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Three-way junctions (3H) are the simplest and most commonly occurring branched nucleic acids. They consist of three double helical arms (A to C), connected at the junction point, with or without a no. of unpaired bases in one or more of the three different strands. Three-way junctions with two unpaired bases in one strand (3HS2) have a high tendency to adopt either of two alternative stacked conformations in which two of the three arms A, B and C are coaxially stacked, i.e. A/B-stacked or A/C-stacked. Empirical stacking rules, which successfully predict for DNA 3HS2 A/B-stacking preference from sequence, have been extended to A/C-stacked conformations. Three novel DNA 3HS2 sequences were designed to test the validity of these extended stacking rules and their conformational behavior was studied by soln. NMR. All three show the predicted A/C-stacking preference even in the absence of multivalent cations. The stacking preference for both classes of DNA 3HS2 can thus be predicted from sequence. The high-resoln. NMR soln. structure for one of the stacked 3HS2 is also reported. It shows a well-defined local and global structure defined by an extensive set of classical NMR restraints and residual dipolar couplings. Anal. of its global conformation and that of other representatives of the 3H family, shows that the relative orientations of the stacked and non-stacked arms, are restricted to narrow regions of conformational space, which can be understood from geometric considerations. Together, these findings open up the possibility of full prediction of 3HS2 conformation (stacking and global fold) directly from sequence.
- 23Ouporov, I. V. and Leontis, N. B. (1995) Refinement of the solution structure of a branched DNA 3-way junction Biophys. J. 68, 266– 274 DOI: 10.1016/S0006-3495(95)80183-9Google ScholarThere is no corresponding record for this reference.
- 24Seela, F. and Xu, K. (2008) DNA with stable fluorinated dA and dG substitutes: syntheses, base pairing and F-19-NMR spectra of 7-fluoro-7-deaza-2 ′-deoxyadenosine and 7-fluoro-7-deaza-2 ′-deoxyguanosine Org. Biomol. Chem. 6, 3552– 3560 DOI: 10.1039/b806145aGoogle ScholarThere is no corresponding record for this reference.
- 25Puffer, B., Kreutz, C., Rieder, U., Ebert, M.-O., Konrat, R., and Micura, R. (2009) 5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D F-19 NMR spectroscopy Nucleic Acids Res. 37, 7728– 7740 DOI: 10.1093/nar/gkp862Google ScholarThere is no corresponding record for this reference.
- 26Rohonczy, J. (1992) Total lineshape analysis of DNMR spectra by IBM personal computer Kem. Kozl. 74, 161– 200Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXjtVarsw%253D%253D&md5=e36eb30e3f517ed4bbd160327411dcebTotal lineshape analysis of dynamic NMR spectra by IBM personal computerRohonczy, JanosKemiai Kozlemenyek (1992), 74 (1-2), 161-200CODEN: KEKOAS; ISSN:0022-9814.A DNMR spectrum simulation program TEDDY was developed, that applies spin group-, kinetic parameter- and mole fraction-factorization and solves Liouville-von Neumann equations. A heuristic iteration procedure dets. the best spectroscopic and kinetic parameters of the spin systems. Activation enthalpy and entropy calcn. options are available. Examples: 13C DNMR of 1,2-dimethyl-cyclohexane ring inversion, 1H DNMR of correlated rotation of the iso-Pr-groups of N,N-diisopropylcarbamic acid trimethylsilyl ester. A review with 25 refs.
- 27Rohonczy, J. (2007) DNMR Lineshape Analysis Software Manual, version 1.1, revision 071103, Bruker BioSpin GmbH, Rheinstetten, Germany.Google ScholarThere is no corresponding record for this reference.
- 28Yang, M. S. and Millar, D. P. (1996) Conformational flexibility of three-way DNA junctions containing unpaired nucleotides Biochemistry 35, 7959– 7967 DOI: 10.1021/bi952892zGoogle ScholarThere is no corresponding record for this reference.
- 29Stühmeier, F., Welch, J. B., Murchie, A. I. H., Lilley, D. M. J., and Clegg, R. M. (1997) Global structure of three-way DNA junctions with and without additional unpaired bases: A fluorescence resonance energy transfer analysis Biochemistry 36, 13530– 13538 DOI: 10.1021/bi9702445Google ScholarThere is no corresponding record for this reference.
- 30Sisamakis, E., Valeri, A., Kalinin, S., Rothwell, P. J., and Seidel, C. A. M. (2010) Accurate single-molecule FRET studies using multiparameter fluorescence detection Methods Enzymol. 475, 455– 514 DOI: 10.1016/S0076-6879(10)75018-7Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVyltb7L&md5=20d586eb83ef1fc641b7a75154e39a2aAccurate single-molecule FRET studies using multiparameter fluorescence detectionSisamakis, Evangelos; Valeri, Alessandro; Kalinin, Stanislav; Rothwell, Paul J.; Seidel, Claus A. M.Methods in Enzymology (2010), 475 (Single Molecule Tools, Part B), 455-514CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)A review. In the recent decade, single-mol. (sm) spectroscopy has come of age and is providing important insight into how biol. mols. function. So far our view of protein function is formed, to a significant extent, by traditional structure detn. showing many beautiful static protein structures. Recent expts. by single-mol. and other techniques have questioned the idea that proteins and other biomols. are static structures. In particular, Foerster resonance energy transfer (FRET) studies of single mols. have shown that biomols. may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophys. behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amts. of contaminants. We demonstrate that the simultaneous acquisition of a max. of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and anal. of individual species present in a population of mols. After a short introduction, the data anal. procedure is described in detail together with some exptl. considerations. The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure detn. based on FRET. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when detg. FRET-related distances with high accuracy. Furthermore, the broad time resoln. (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomol. of interest explores its structural energy landscape.
- 31Muschielok, A., Andrecka, J., Jawhari, A., Brueckner, F., Cramer, P., and Michaelis, J. (2008) A nano-positioning system for macromolecular structural analysis Nat. Methods 5, 965– 971 DOI: 10.1038/nmeth.1259Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlSktrfK&md5=bacd89b1f60852fc59260932486929afA nano-positioning system for macromolecular structural analysisMuschielok, Adam; Andrecka, Joanna; Jawhari, Anass; Brueckner, Florian; Cramer, Patrick; Michaelis, JensNature Methods (2008), 5 (11), 965-971CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Very often, the positions of flexible domains within macromols. as well as within macromol. complexes cannot be detd. by std. structural biol. methods. To overcome this problem, we developed a method that uses probabilistic data anal. to combine single-mol. measurements with X-ray crystallog. data. The method dets. not only the most likely position of a fluorescent dye mol. attached to the domain but also the complete three-dimensional probability distribution depicting the exptl. uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quant. tool for investigating the position and dynamics of flexible domains within macromol. complexes. We applied this method to find the position of the 5' end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.
- 32Brunger, A. T., Strop, P., Vrljic, M., Chu, S., and Weninger, K. R. (2011) Three-dimensional molecular modeling with single molecule FRET J. Struct. Biol. 173, 497– 505 DOI: 10.1016/j.jsb.2010.09.004Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvFWntb4%253D&md5=c8c4224ee322b2b82ee22b9969be9ff4Three-dimensional molecular modeling with single molecule FRETBrunger, Axel T.; Strop, Pavel; Vrljic, Marija; Chu, Steven; Weninger, Keith R.Journal of Structural Biology (2011), 173 (3), 497-505CODEN: JSBIEM; ISSN:1047-8477. (Elsevier B.V.)A review. Single mol. fluorescence energy transfer expts. enable investigations of macromol. conformation and folding by the introduction of fluorescent dyes at specific sites in the macromol. Multiple such expts. can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple mols. Distances that are derived from such expts. can be used for detn. of the fluorophore positions by triangulation. When combined with a known structure of the macromol.(s) to which the fluorophores are attached, a three-dimensional model of the system can be detd. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the exptl. and computational methods used for three-dimensional modeling based on single mol. fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromol. complexes.
- 33Im, K., Jeong, D., Hur, J., Kim, S. J., Hwang, S., Jin, K. S., Park, N., and Kim, K. (2013) Robust analysis of synthetic label-free DNA junctions in solution by X-ray scattering and molecular simulation Sci. Rep. 3, 3226 DOI: 10.1038/srep03226Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2c7mvFSqsg%253D%253D&md5=2f999b5de9c6ad39c0bf0fb5de36c26bRobust analysis of synthetic label-free DNA junctions in solution by X-ray scattering and molecular simulationIm Kyuhyun; Jeong Daun; Hur Jaehyun; Kim Sung-Jin; Hwang Sungwoo; Jin Kyeong Sik; Park Nokyoung; Kim KinamScientific reports (2013), 3 (), 3226 ISSN:.Structural analysis of branched DNA molecules (BDM) is important as model systems for DNA junctions and also as building units for DNA assembly. Although there have been efforts to study the structures of BDM, label-free solution structures have not been well determined yet. Here, we used a combination of synchrotron-based experimental tools and computational simulation to study the global structures of label-free BDM in solution. Overall structures of 3-arm and 4-arm BDM were revealed as an asymmetric T(or Y)-shape and a distorted X-shape, respectively. The internal structures of the DNA double helix were shown to have a canonical B-form for both the BDM. We also reconstructed the thermal denaturation process of BDM by determining the transient global structures over a wide range of temperatures. The proposed high-resolution structures of BDM are expected to provide fundamental information for studies of the biological function of junction DNAs and DNA assembly.
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Abstract
Figure 1
Figure 1. Sequences and dye positions for DNA three-way junctions 3WJa and 3WJb [note that this is a composite of all dye positions; measured structures have only one donor (D) and one acceptor (A) dye]. 3WJa and 3WJb differ only in the bases at the branchpoint (colored red).
Figure 2
Figure 2. Single-molecule FRET shows a GC-rich 3WJ adopts multiple conformations. (a) Two-dimensional burst frequency histograms of FRET efficiency (E) or donor anisotropy (rD) vs donor lifetime [τD(A)]. The overlaid red line is the theoretical FRET relationship E = 1 – [τD(A)/τD], where τD = 4.1 ns. The blue overlaid line is the Perrin equation rD = r0/[1 + τD(A)/ρD], where mean rotational correlation time ρD = 0.35 ns and fundamental anisotropy r0 = 0.375. (b) A sub-ensemble analysis of the whole FRET population can be fitted (black) as two Gaussian distributions (blue). The data in panels a and b are for 3WJb in 0 mM MgCl2 (dye positions D1 and A1) but were recorded on different days.
Figure 3
Figure 3. Global structure of 3WJb derived from SM-FRET distance restraints for the major FRET population and MD simulations showing that the unpaired branchpoint is present with and without MgCl2 (a) and that the structures for 3WJa (yellow) and the major conformation of 3WJb (orange) are similar (b). The five lowest-restraint energy conformations are superposed using the DNA backbone atoms.
Figure 4
Figure 4. NMR spectra of a 19F-labeled 3WJ. A 3WJ with a GC-rich branchpoint was modified to include a 5-fluorodeoxycytosine. (a) The schematic shows the sequence of the 3WJ, where the position of the 5F-dC is colored red, and its chemical structure is given in the inset. (b) 1D 19F NMR spectra of the 3WJ at temperatures from 12 to 40 °C. The 3WJ spectra show two major peaks that become broader with a decrease in temperature. (c) 1D 19F NMR spectra of ssDNA (top), dsDNA (middle), and the 3WJ (bottom) at 30 °C. Note that the ssDNA is capable of forming a hairpin, positioning the 5F-dC in a loop environment that results in the more upfield peak. The more downfield peak represents a ssDNA conformation that has no defined secondary structure. The vertical dashed line marks the position of the ssDNA peak.
Figure 5
Figure 5. Simple model of 3WJ conformational heterogeneity assuming a two-state system. The left image represents the major open structure derived from FRET restraints (Figure 3), which can convert via base pairing at the branch to a constrained structure in which there are strengthened interactions at the branchpoint and two of the arms are closer together. The exchange rate is an estimate from DNMR measurements (see the text).
References
ARTICLE SECTIONSThis article references 33 other publications.
- 1Lilley, D. M. J. (2000) Structures of helical junctions in nucleic acids Q. Rev. Biophys. 33, 109– 159 DOI: 10.1017/S00335835000035901https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjvVWqtw%253D%253D&md5=40a7d48ff2c61797e7b73170fb92a3d8Structures of helical junctions in nucleic acidsLilley, David M. J.Quarterly Reviews of Biophysics (2000), 33 (2), 109-159CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)A review with 232 refs. Topics discussed include the occurrence of helical junctions in nucleic acids, the structure of the four-way DNA junction, the role of metal ions in the folding of the four-way DNA junction, conformational variation in the four-way junctions, branch migration, three-way DNA junctions, helical junctions in RNA, and the recognition and distortion of four-way DNA junctions by proteins.
- 2Atkinson, J. and McGlynn, P. (2009) Replication fork reversal and the maintenance of genome stability Nucleic Acids Res. 37, 3475– 3492 DOI: 10.1093/nar/gkp2442https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnvVyhsbw%253D&md5=94b01aff9aba8b42654f746a7f49a7c3Replication fork reversal and the maintenance of genome stabilityAtkinson, John; McGlynn, PeterNucleic Acids Research (2009), 37 (11), 3475-3492CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)A review. The progress of replication forks is often threatened in vivo, both by DNA damage and by proteins bound to the template. Blocked forks must somehow be restarted, and the original blockage cleared, in order to complete genome duplication, implying that blocked fork processing may be crit. for genome stability. One possible pathway that might allow processing and restart of blocked forks, replication fork reversal, involves the unwinding of blocked forks to form four-stranded structures resembling Holliday junctions. This concept has gained increasing popularity recently based on the ability of such processing to explain many genetic observations, the detection of unwound fork structures in vivo and the identification of enzymes that have the capacity to catalyze fork regression in vitro. Here, the authors discuss the contexts in which fork regression might occur, the factors that may promote such a reaction and the possible roles of replication fork unwinding in normal DNA metab.
- 3Seeman, N. C. (2010) Nanomaterials based on DNA Annu. Rev. Biochem. 79, 65– 87 DOI: 10.1146/annurev-biochem-060308-1022443https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXpslShsbo%253D&md5=b842821a6e15581c40329e75e219c0c7Nanomaterials based on DNASeeman, Nadrian C.Annual Review of Biochemistry (2010), 79 (), 65-87CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. The combination of synthetic stable branched DNA and sticky-ended cohesion has led to the development of structural DNA nanotechnol. over the past 30 years. The basis of this enterprise is that it is possible to construct novel DNA-based materials by combining these features in a self-assembly protocol. Thus, simple branched mols. lead directly to the construction of polyhedrons, whose edges consist of double helical DNA and whose vertices correspond to the branch points. Stiffer branched motifs can be used to produce self-assembled 2-dimensional and 3-dimensional periodic lattices of DNA (crystals). DNA was also used to make a variety of nanomech. devices, including mols. that change their shapes and mols. that can walk along a DNA sidewalk. Devices were incorporated into 2-dimensional DNA arrangements; sequence-dependent devices are driven by increases in nucleotide pairing at each step in their machine cycles.
- 4Roy, R., Hohng, S., and Ha, T. (2008) A practical guide to single-molecule FRET Nat. Methods 5, 507– 516 DOI: 10.1038/nmeth.12084https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmsVOjt7Y%253D&md5=d6207e40c17cec6d202f7f46030aa575A practical guide to single-molecule FRETRoy, Rahul; Hohng, Sungchul; Ha, TaekjipNature Methods (2008), 5 (6), 507-516CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Single-mol. fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-mol. techniques. Despite the explosive growth in the application of smFRET to answer biol. questions in the last decade, the technique has been practiced mostly by biophysicists. The authors provide a practical guide to using smFRET, focusing on the study of immobilized mols. that allow measurements of single-mol. reaction trajectories from 1 ms to many minutes. The authors discuss issues a biologist must consider to conduct successful smFRET expts., including exptl. design, sample prepn., single-mol. detection and data anal. The authors also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.
- 5Sabir, T., Toulmin, A., Ma, L., Jones, A. C., McGlynn, P., Schroeder, G. F., and Magennis, S. W. (2012) Branchpoint Expansion in a Fully Complementary Three-Way DNA Junction J. Am. Chem. Soc. 134, 6280– 6285 DOI: 10.1021/ja211802z5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XitFCjsLs%253D&md5=212a7c044d4c79fb2c3a4e8c14e54550Branchpoint Expansion in a Fully Complementary Three-Way DNA JunctionSabir, Tara; Toulmin, Anita; Ma, Long; Jones, Anita C.; McGlynn, Peter; Schroder, Gunnar F.; Magennis, Steven W.Journal of the American Chemical Society (2012), 134 (14), 6280-6285CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Branched nucleic acid mols. serve as key intermediates in DNA replication, recombination, and repair; architectural elements in RNA; and building blocks and functional components for nanoscience applications. Using a combination of high-resoln. single-mol. FRET, time-resolved spectroscopy, and mol. modeling, we have probed the local and global structure of a DNA three-way junction (3WJ) in soln. We found that it adopts a Y-shaped, pyramidal structure, in which the bases adjacent to the branchpoint are unpaired, despite the full Watson-Crick complementarity of the mol. The unpairing allows a nanoscale cavity to form at the junction center. Our structure accounts for earlier observations made of the structure, flexibility, and reactivity of 3WJs. We anticipate that these results will guide the development of new DNA-based supramol. receptors and nanosystems.
- 6Young, R. M., Singh, A. P. N., Thazhathveetil, A. K., Cho, V. Y., Zhang, Y. Q., Renaud, N., Grozema, F. C., Beratan, D. N., Ratner, M. A., Schatz, G. C., Berlin, Y. A., Lewis, F. D., and Wasielewski, M. R. (2015) Charge Transport across DNA-Based Three-Way Junctions J. Am. Chem. Soc. 137, 5113– 5122 DOI: 10.1021/jacs.5b009316https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlsFGhu7w%253D&md5=16043c16064fbbd6728265b40b102d50Charge Transport across DNA-Based Three-Way JunctionsYoung, Ryan M.; Singh, Arunoday P. N.; Thazhathveetil, Arun; Cho, Vincent Y.; Zhang, Yuqi; Renaud, Nicolas; Grozema, Ferdinand C.; Beratan, David N.; Ratner, Mark A.; Schatz, George C.; Berlin, Yuri A.; Lewis, Frederick D.; Wasielewski, Michael R.Journal of the American Chemical Society (2015), 137 (15), 5113-5122CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)DNA-based mol. electronics will require charges to be transported from one site within a 2D or 3D architecture to another. While this has been shown previously in linear, π-stacked DNA sequences, the dynamics and efficiency of charge transport across DNA three-way junction (3WJ) have yet to be detd. Here, the authors present a study of hole transport and trapping across a DNA-based three-way junction systems by a combination of femtosecond transient absorption spectroscopy and mol. dynamics simulations. Hole transport across the junction is proposed to be gated by conformational fluctuations in the ground state which bring the transiently populated hole carrier nucleobases into better aligned geometries on the nanosecond time scale, thus modulating the π-π electronic coupling along the base pair sequence.
- 7Zhang, Y. Q., Young, R. M., Thazhathveetil, A. K., Singh, A. P. N., Liu, C. R., Berlin, Y. A., Grozema, F. C., Lewis, F. D., Ratner, M. A., Renaud, N., Siriwong, K., Voityuk, A. A., Wasielewski, M. R., and Beratan, D. N. (2015) Conformationally Gated Charge Transfer in DNA Three-Way Junctions J. Phys. Chem. Lett. 6, 2434– 2438 DOI: 10.1021/acs.jpclett.5b008637https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpsFOlt7g%253D&md5=93b935ef85e287c2a6e3139ddddf77dcConformationally Gated Charge Transfer in DNA Three-Way JunctionsZhang, Yuqi; Young, Ryan M.; Thazhathveetil, Arun K.; Singh, Arunoday P. N.; Liu, Chaoren; Berlin, Yuri A.; Grozema, Ferdinand C.; Lewis, Frederick D.; Ratner, Mark A.; Renaud, Nicolas; Siriwong, Khatcharin; Voityuk, Alexander A.; Wasielewski, Michael R.; Beratan, David N.Journal of Physical Chemistry Letters (2015), 6 (13), 2434-2438CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)Mol. structures that direct charge transport in two or three dimensions possess some of the essential functionality of elec. switches and gates. We use theory, modeling, and simulation to explore the conformational dynamics of DNA three-way junctions (TWJs) that may control the flow of charge through these structures. Mol. dynamics simulations and quantum calcns. indicate that DNA TWJs undergo dynamic interconversion among "well stacked" conformations on the time scale of nanoseconds, a feature that makes the junctions very different from linear DNA duplexes. The studies further indicate that this conformational gating would control charge flow through these TWJs, distinguishing them from conventional (larger size scale) gated devices. Simulations also find that structures with polyethylene glycol linking groups ("extenders") lock conformations that favor CT for 25 ns or more. The simulations explain the kinetics obsd. exptl. in TWJs and rationalize their transport properties compared with double-stranded DNA.
- 8Zhang, Y. Q., Zhang, W. B., Liu, C. R., Zhang, P., Balaeff, A., and Beratan, D. N. (2016) DNA charge transport: Moving beyond 1D Surf. Sci. 652, 33– 38 DOI: 10.1016/j.susc.2016.03.0118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xkt1Gjurc%253D&md5=50e28ed5ed174ee81b673cb536f379b0DNA charge transport: Moving beyond 1DZhang, Yuqi; Zhang, William B.; Liu, Chaoren; Zhang, Peng; Balaeff, Alexander; Beratan, David N.Surface Science (2016), 652 (), 33-38CODEN: SUSCAS; ISSN:0039-6028. (Elsevier B.V.)A review. Charge transport across novel DNA junctions has been studied for several decades. From early attempts to move charge across DNA double crossover junctions to recent studies on DNA 3-way junctions and G-quadruplex (G4) motifs, it is becoming clear that efficient cross-junction charge migration requires strong base-to-base electronic coupling at the junction, facilitated by favorable pi-stacking. Here, the authors review recent progress toward the goal of manipulating and controlling charge transport through DNA junctions.
- 9Ma, L., Wu, G. R., Li, Y. F., Qin, P., Meng, L. P., Liu, H. Y., Li, Y. Y., and Diao, A. P. (2015) A reversible metal ion fueled DNA three-way junction molecular device for ″turn-on and -off″ fluorescence detection of mercury ions (II) and biothiols respectively with high selectivity and sensitivity Nanoscale 7, 18044– 18048 DOI: 10.1039/C5NR04688BThere is no corresponding record for this reference.
- 10Barros, S. A. and Chenoweth, D. M. (2014) Recognition of Nucleic Acid Junctions Using Triptycene-Based Molecules Angew. Chem., Int. Ed. 53, 13746– 13750 DOI: 10.1002/anie.20140706110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVCit73O&md5=b3d59010e6f130eab638869353164ca2Recognition of Nucleic Acid Junctions Using Triptycene-Based MoleculesBarros, Stephanie A.; Chenoweth, David M.Angewandte Chemie, International Edition (2014), 53 (50), 13746-13750CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The modulation of nucleic acids by small mols. is an essential process across the kingdoms of life. Targeting nucleic acids with small mols. represents a significant challenge at the forefront of chem. biol. Nucleic acid junctions are ubiquitous structural motifs in nature and in designed materials. Herein, we describe a new class of structure-specific nucleic acid junction stabilizers based on a triptycene scaffold. Triptycenes provide significant stabilization of DNA and RNA three-way junctions, providing a new scaffold for the development of nucleic acid junction binders with enhanced recognition properties. Addnl., we report cytotoxicity and cell uptake data in two human ovarian carcinoma cell lines.
- 11Probst, M., Langenegger, S. M., and Haner, R. (2014) A modular LHC built on the DNA three-way junction Chem. Commun. 50, 159– 161 DOI: 10.1039/C3CC47490A11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVKjtLjF&md5=e7b616c5bbd54019a2f7d679c95b0faeA modular LHC built on the DNA three-way junctionProbst, Markus; Langenegger, Simon M.; Haener, RobertChemical Communications (Cambridge, United Kingdom) (2014), 50 (2), 159-161CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A light-harvesting complex composed of a π-stacked multichromophoric array in a DNA three-way junction is described. The modular design allows for a ready exchange of non-covalently attached energy acceptors.
- 12Su, W., Bagshaw, C. R., and Burley, G. A. (2013) Addressable and unidirectional energy transfer along a DNA three-way junction programmed by pyrrole-imidazole polyamides Sci. Rep. 3, 1883 DOI: 10.1038/srep0188312https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3snmsFyksg%253D%253D&md5=1ba61151bbb29917c536468d9940d7a8Addressable and unidirectional energy transfer along a DNA three-way junction programmed by pyrrole-imidazole polyamidesSu Wu; Bagshaw Clive R; Burley Glenn AScientific reports (2013), 3 (), 1883 ISSN:.We describe a photonic waveguide where FRET is routed uni-directionally along a double-stranded DNA track. The efficiency of FRET is modulated by the supramolecular control of fluorophores along double-stranded DNA using fluorophore-tethered Pyrrole-Imidazole polyamides (PAs). We show that uni-directional FRET is enhanced by the complete assembly of each of the constituent parts, resulting in the selective routing of light along simple DNA duplexes as well as a three-way junction (3WJ).
- 13Kühnemuth, R. and Seidel, C. A. M. (2001) Principles of single molecule multiparameter fluorescence spectroscopy Single Mol. 2, 251– 254 DOI: 10.1002/1438-5171(200112)2:4<251::AID-SIMO251>3.3.CO;2-K13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtlCltQ%253D%253D&md5=46bc301eecf524bfa54b8be2b6c46732Principles of single molecule multiparameter fluorescence spectroscopyKuhnemuth, Ralf; Seidel, Claus A. M.Single Molecules (2001), 2 (4), 251-254CODEN: SGMCF7; ISSN:1438-5163. (Wiley-VCH Verlag Berlin GmbH)A review. In this review a short introduction to the concept and exptl. realization of single mol. multiparameter fluorescence spectroscopy is presented. The approach of Multiparameter Fluorescence Detection (MFD) is essentially the time-resolved observation of all five intrinsic properties of a chromophore that can be probed in a fluorescence expt., i.e. spectral properties of absorption and fluorescence, F(λA, λ), fluorescence quantum yield, φF, fluorescence lifetime, τ, and anisotropy, r. This harbors the potential to combine Fluorescence Correlation Spectroscopy (FCS) and Time-Correlated Single Photon Counting (TCSPC) in a single expt. Addnl., species selective anal. of subensembles and even direct studies on single mol. dynamics become accessible. The application of MFD to complex multichromophoric systems is discussed. Examples range from studies of Donor-Acceptor pairs showing intra- or intermol. Fluorescence Resonance Energy Transfer (FRET) for revealing dynamical and statical structural informations of biol. relevant macromols. to the development of vastly improved anal. tools for the accurate identification of target mols. in multicomponent systems as needed in todays and future High-Throughput Screening (HTS) efforts. Many theor. models of interactions and chem. reactions have been described on the mol. level although the primary source for our knowledge on chem. structure and dynamics so far are studies on mol. ensembles. Such studies, however, did not yet provide an answer to the question whether all members of the ensemble have the same properties. Here, expts. on single mols. promise new and unexpected insights, because they eliminate ensemble averaging and provide direct information on heterogeneity and kinetics of the system. That way distributions and spectra of different species in specific states are directly accessible even in heterogeneous systems (Fig.1).
- 14Sabir, T., Schröder, G. F., Toulmin, A., McGlynn, P., and Magennis, S. W. (2011) Global structure of forked DNA in solution revealed by high-resolution single-molecule FRET J. Am. Chem. Soc. 133, 1188– 1191 DOI: 10.1021/ja108626w14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsF2kurbO&md5=a327b9308801c6b70d4e65bdf4634e27Global Structure of Forked DNA in Solution Revealed by High-Resolution Single-Molecule FRETSabir, Tara; Schrodr, Gunnar F.; Toulmin, Anita; McGlynn, Peter; Magennis, Steven W.Journal of the American Chemical Society (2011), 133 (5), 1188-1191CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Branched DNA structures play crit. roles in DNA replication, repair, and recombination in addn. to being key building blocks for DNA nanotechnol. Here we combine single-mol. multiparameter fluorescence detection and mol. dynamics simulations to give a general approach to global structure detn. of branched DNA in soln. We reveal an open, planar structure of a forked DNA mol. with three duplex arms and demonstrate an ion-induced conformational change. This structure will serve as a benchmark for DNA-protein interaction studies.
- 15Harris, R. K., Becker, E. D., Cabral De Menezes, S. M., Goodfellow, R., and Granger, P. (2001) NMR nomenclature. Nuclear spin properties and conventions for chemical shifts - (IUPAC recommendations 2001) Pure Appl. Chem. 73, 1795– 1818 DOI: 10.1351/pac20017311179515https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XitVeltbw%253D&md5=678eae2d74c6c1e4321cd907a3d2129bNMR nomenclature. Nuclear spin properties and conventions for chemical shifts (IUPAC recommendations 2001)Harris, Robin K.; Becker, Edwin D.; Cabral De Menezes, Sonia M.; Goodfellow, Robin; Granger, PierrePure and Applied Chemistry (2001), 73 (11), 1795-1818CODEN: PACHAS; ISSN:0033-4545. (International Union of Pure and Applied Chemistry)A review. A unified scale is recommended for reporting the NMR chem. shifts of all nuclei relative to the 1H resonance of tetramethylsilane (TMS). The unified scale is designed to provide a precise ratio, Ξ, of the resonance frequency of a given nuclide to that of the primary ref., the 1H resonance of TMS in dil. soln. (vol. fraction, φ < 1%) in CHCl3. Referencing procedures are discussed, including matters of practical application of the unified scale. Special attention is paid to recommended ref. samples, and values of Ξ for secondary refs. on the unified scale are listed, many of which are the results of new measurements. Some earlier recommendations relating to the reporting of chem. shifts are endorsed. The chem. shift, δ, is redefined to avoid previous ambiguities but to leave practical usage unchanged. Relations between the unified scale and recently published recommendations for referencing in aq. solns. (for specific use in biochem. work) are discussed, as well as the special effects of working in the solid state with magic-angle spinning. In all, nine new recommendations relating to chem. shifts are made. Standardized nuclear spin data are also presented in tabular form for the stable (and some unstable) isotopes of all elements with nonzero quantum nos. The information given includes quantum nos., isotopic abundances, magnetic moments, magnetogyric ratios and receptivities, together with quadrupole moments and line-width factors where appropriate.
- 16Brunger, A. T. (2007) Version 1.2 of the Crystallography and NMR system Nat. Protoc. 2, 2728– 2733 DOI: 10.1038/nprot.2007.40616https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlSlsrzK&md5=92276efc33435ac14906f42613368c9fVersion 1.2 of the Crystallography and NMR systemBrunger, Axel T.Nature Protocols (2007), 2 (11), 2728-2733CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Version 1.2 of the software system, termed Crystallog. and NMR system (CNS), for crystallog. and NMR structure detn. has been released. Since its first release, the goals of CNS have been (i) to create a flexible computational framework for exploration of new approaches to structure detn., (ii) to provide tools for structure soln. of difficult or large structures, (iii) to develop models for analyzing structural and dynamical properties of macromols. and (iv) to integrate all sources of information into all stages of the structure detn. process. Version 1.2 includes an improved model for the treatment of disordered solvent for crystallog. refinement that employs a combined grid search and least-squares optimization of the bulk solvent model parameters. The method is more robust than previous implementations, esp. at lower resoln., generally resulting in lower R values. Other advances include the ability to apply thermal factor sharpening to electron d. maps. Consistent with the modular design of CNS, these addns. and changes were implemented in the high-level computing language of CNS.
- 17Woźniak, A. K., Schröder, G. F., Grubmüller, H., Seidel, C. A. M., and Oesterhelt, F. (2008) Single-molecule FRET measures bends and kinks in DNA Proc. Natl. Acad. Sci. U. S. A. 105, 18337– 18342 DOI: 10.1073/pnas.080097710517https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVyhsrrK&md5=29584669731e101909f635cebd32f2cbSingle-molecule FRET measures bends and kinks in DNAWozniak, Anna K.; Schroder, Gunnar F.; Grubmueller, Helmut; Seidel, Claus A. M.; Oesterhelt, FilippProceedings of the National Academy of Sciences of the United States of America (2008), 105 (47), 18337-18342, S18337/1-S18337/19CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The authors present advances in the use of single-mol. FRET measurements with flexibly linked dyes to derive full 3D structures of DNA constructs based on abs. distances. The resoln. obtained by this single-mol. approach harbors the potential to study in detail also protein- or damage-induced DNA bending. If one is to generate a geometric structural model, distances between fixed positions are needed. These are usually not exptl. accessible because of unknown fluorophore-linker mobility effects that lead to a distribution of FRET efficiencies and distances. To solve this problem, the authors performed studies on DNA double-helixes by systematically varying donor acceptor distances from 2 to 10 nm. Anal. of dye-dye quenching and fluorescence anisotropy measurements reveal slow positional and fast orientational fluorophore dynamics, that results in an isotropic av. of the FRET efficiency. The authors use a nonlinear conversion function based on mol. dynamics simulations that allows the authors to include this effect in the calcn. of abs. FRET distances. To obtain unique structures, the authors performed a quant. statistical anal. for the conformational search in full space based on triangulation, which uses the known helical nucleic acid features. The authors' higher accuracy allowed the detection of sequence-dependent DNA bending by 16°. For DNA with bulged adenosines, the authors also quantified the kink angles introduced by the insertion of 1, 3 and 5 bases to be 32° ± 6°, 56° ±4° and 73±2°, resp. Moreover, the rotation angles and shifts of the helixes were calcd. to describe the relative orientation of the two arms in detail.
- 18Schröder, G. F., Brunger, A. T., and Levitt, M. (2007) Combining efficient conformational sampling with a deformable elastic network model facilitates structure refinement at low resolution Structure 15, 1630– 1641 DOI: 10.1016/j.str.2007.09.02118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2sjjs1Onsw%253D%253D&md5=0f7df5635fb0b44134e8bff03c018a85Combining efficient conformational sampling with a deformable elastic network model facilitates structure refinement at low resolutionSchroder Gunnar F; Brunger Axel T; Levitt MichaelStructure (London, England : 1993) (2007), 15 (12), 1630-41 ISSN:0969-2126.Structural studies of large proteins and protein assemblies are a difficult and pressing challenge in molecular biology. Experiments often yield only low-resolution or sparse data that are not sufficient to fully determine atomistic structures. We have developed a general geometry-based algorithm that efficiently samples conformational space under constraints imposed by low-resolution density maps obtained from electron microscopy or X-ray crystallography experiments. A deformable elastic network (DEN) is used to restrain the sampling to prior knowledge of an approximate structure. The DEN restraints dramatically reduce over-fitting, especially at low resolution. Cross-validation is used to optimally weight the structural information and experimental data. Our algorithm is robust even for noise-added density maps and has a large radius of convergence for our test case. The DEN restraints can also be used to enhance reciprocal space simulated annealing refinement.
- 19Kalinin, S., Peulen, T., Sindbert, S., Rothwell, P. J., Berger, S., Restle, T., Goody, R. S., Gohlke, H., and Seidel, C. A. M. (2012) A toolkit and benchmark study for FRET-restrained high-precision structural modeling Nat. Methods 9, 1218– 1225 DOI: 10.1038/nmeth.222219https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1Ckt7nE&md5=ff95e353c33d3e845da14c480b36c1bbA toolkit and benchmark study for FRET-restrained high-precision structural modelingKalinin, Stanislav; Peulen, Thomas; Sindbert, Simon; Rothwell, Paul J.; Berger, Sylvia; Restle, Tobias; Goody, Roger S.; Gohlke, Holger; Seidel, Claus A. M.Nature Methods (2012), 9 (12), 1218-1225CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We present a comprehensive toolkit for Foerster resonance energy transfer (FRET)-restrained modeling of biomols. and their complexes for quant. applications in structural biol. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calcd. by rigorous error estn. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with an r.m.s. deviation of 0.5 Å. Furthermore, we introduce FRET-guided 'screening' of a large structural ensemble created by mol. dynamics simulations. We used this hybrid approach to det. the formerly unknown configuration of the flexible single-strand template overhang.
- 20Kalinin, S., Valeri, A., Antonik, M., Felekyan, S., and Seidel, C. A. M. (2010) Detection of structural dynamics by FRET: a photon distribution and fluorescence lifetime analysis of systems with multiple states J. Phys. Chem. B 114, 7983– 7995 DOI: 10.1021/jp102156t20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmt12ksLk%253D&md5=1d5212f1c66259d3b877b212ad88292cDetection of Structural Dynamics by FRET: A Photon Distribution and Fluorescence Lifetime Analysis of Systems with Multiple StatesKalinin, Stanislav; Valeri, Alessandro; Antonik, Matthew; Felekyan, Suren; Seidel, Claus A. M.Journal of Physical Chemistry B (2010), 114 (23), 7983-7995CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)Two complementary methods in confocal single-mol. fluorescence spectroscopy are presented to analyze conformational dynamics by Forster resonance energy transfer (FRET) measurements considering simulated and exptl. data. First, an extension of photon distribution anal. (PDA) is applied to characterize conformational exchange between two or more states via global anal. of the shape of FRET peaks for different time bins. PDA accurately predicts the shape of FRET efficiency histograms in the presence of FRET fluctuations, taking into account shot noise and background contributions. Dynamic-PDA quant. recovers FRET efficiencies of the interconverting states and relaxation times of dynamics on the time scale of the diffusion time td (typically milliseconds), with a dynamic range of the method of about ± 1 order of magnitude with respect to td. Correction procedures are proposed to consider the factors limiting the accuracy of dynamic-PDA, such as brightness variations, shortening of the observation time due to diffusion, and a contribution of multimol. events. Second, an anal. procedure for multiparameter fluorescence detection is presented, where intensity-derived FRET efficiency is correlated with the fluorescence lifetime of the donor quenched by FRET. If a max. likelihood estimator is applied to compute a mean fluorescence lifetime of mixed states, one obtains a fluorescence weighted mean lifetime. Thus a mixed state is detected by a characteristic shift of the fluorescence lifetime, which becomes longer than that expected for a single species with the same intensity-derived FRET efficiency. Anal. tools for direct visual inspection of two-dimensional diagrams of FRET efficiency vs. donor lifetime are presented for the cases of static and dynamic FRET. Finally these new techniques are compared with fluorescence correlation spectroscopy.
- 21Leontis, N. B., Hills, M. T., Piotto, M., Malhotra, A., Nussbaum, J., and Gorenstein, D. G. (1993) A model for the solution structure of a branched, 3-strand DNA complex J. Biomol. Struct. Dyn. 11, 215– 223 DOI: 10.1080/07391102.1993.1050872221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXpvFKmtg%253D%253D&md5=b5ef4910e26ec0722f0a9c5018931cc1A model for the solution structure of a branched, three-strand DNA complexLeontis, Neocles B.; Hills, Michael T.; Piotto, Martial; Malhotra, Arun; Nussbaum, Jean; Gorenstein, David G.Journal of Biomolecular Structure & Dynamics (1993), 11 (2), 215-23, 1 plateCODEN: JBSDD6; ISSN:0739-1102.The soln. structure of a DNA three-way junction (TWJ) contg. two unpaired thymidines was elucidated using two- and three-dimensional 1H NMR spectroscopy. TWJs with unpaired nucleotides are ubiquitous structural motifs of complex single-stranded nucleic acids. In the presence of Mg2+, the TWJ complex adopts a unique conformation in which the bases of one of the oligonucleotides ("strand 1") are continuously stacked across the junction. Guanosine 8 of strand 3 (S3-G8), which pairs with S1-C5, stacks on S2-G5, which is paired to S1-C6. The unpaired thymidine bases (S3-T6 and S3-T7) are exposed to the solvent, whereas the sugar of S3-G8 is largely buried. S3-T6 also interacts with the sugar residue of S3-G11. All three stems conform to B-type DNA.
- 22Wu, B., Girard, F., van Buuren, B., Schleucher, J., Tessari, M., and Wijmenga, S. (2004) Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold Nucleic Acids Res. 32, 3228– 3239 DOI: 10.1093/nar/gkh64522https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXltVWltrs%253D&md5=07720f0443df0c3edd23046450ed9493Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H foldWu, Bin; Girard, Frederic; Van Buuren, Bernd; Schleucher, Juergen; Tessari, Marco; Wijmenga, SybrenNucleic Acids Research (2004), 32 (10), 3228-3239CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Three-way junctions (3H) are the simplest and most commonly occurring branched nucleic acids. They consist of three double helical arms (A to C), connected at the junction point, with or without a no. of unpaired bases in one or more of the three different strands. Three-way junctions with two unpaired bases in one strand (3HS2) have a high tendency to adopt either of two alternative stacked conformations in which two of the three arms A, B and C are coaxially stacked, i.e. A/B-stacked or A/C-stacked. Empirical stacking rules, which successfully predict for DNA 3HS2 A/B-stacking preference from sequence, have been extended to A/C-stacked conformations. Three novel DNA 3HS2 sequences were designed to test the validity of these extended stacking rules and their conformational behavior was studied by soln. NMR. All three show the predicted A/C-stacking preference even in the absence of multivalent cations. The stacking preference for both classes of DNA 3HS2 can thus be predicted from sequence. The high-resoln. NMR soln. structure for one of the stacked 3HS2 is also reported. It shows a well-defined local and global structure defined by an extensive set of classical NMR restraints and residual dipolar couplings. Anal. of its global conformation and that of other representatives of the 3H family, shows that the relative orientations of the stacked and non-stacked arms, are restricted to narrow regions of conformational space, which can be understood from geometric considerations. Together, these findings open up the possibility of full prediction of 3HS2 conformation (stacking and global fold) directly from sequence.
- 23Ouporov, I. V. and Leontis, N. B. (1995) Refinement of the solution structure of a branched DNA 3-way junction Biophys. J. 68, 266– 274 DOI: 10.1016/S0006-3495(95)80183-9There is no corresponding record for this reference.
- 24Seela, F. and Xu, K. (2008) DNA with stable fluorinated dA and dG substitutes: syntheses, base pairing and F-19-NMR spectra of 7-fluoro-7-deaza-2 ′-deoxyadenosine and 7-fluoro-7-deaza-2 ′-deoxyguanosine Org. Biomol. Chem. 6, 3552– 3560 DOI: 10.1039/b806145aThere is no corresponding record for this reference.
- 25Puffer, B., Kreutz, C., Rieder, U., Ebert, M.-O., Konrat, R., and Micura, R. (2009) 5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D F-19 NMR spectroscopy Nucleic Acids Res. 37, 7728– 7740 DOI: 10.1093/nar/gkp862There is no corresponding record for this reference.
- 26Rohonczy, J. (1992) Total lineshape analysis of DNMR spectra by IBM personal computer Kem. Kozl. 74, 161– 20026https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXjtVarsw%253D%253D&md5=e36eb30e3f517ed4bbd160327411dcebTotal lineshape analysis of dynamic NMR spectra by IBM personal computerRohonczy, JanosKemiai Kozlemenyek (1992), 74 (1-2), 161-200CODEN: KEKOAS; ISSN:0022-9814.A DNMR spectrum simulation program TEDDY was developed, that applies spin group-, kinetic parameter- and mole fraction-factorization and solves Liouville-von Neumann equations. A heuristic iteration procedure dets. the best spectroscopic and kinetic parameters of the spin systems. Activation enthalpy and entropy calcn. options are available. Examples: 13C DNMR of 1,2-dimethyl-cyclohexane ring inversion, 1H DNMR of correlated rotation of the iso-Pr-groups of N,N-diisopropylcarbamic acid trimethylsilyl ester. A review with 25 refs.
- 27Rohonczy, J. (2007) DNMR Lineshape Analysis Software Manual, version 1.1, revision 071103, Bruker BioSpin GmbH, Rheinstetten, Germany.There is no corresponding record for this reference.
- 28Yang, M. S. and Millar, D. P. (1996) Conformational flexibility of three-way DNA junctions containing unpaired nucleotides Biochemistry 35, 7959– 7967 DOI: 10.1021/bi952892zThere is no corresponding record for this reference.
- 29Stühmeier, F., Welch, J. B., Murchie, A. I. H., Lilley, D. M. J., and Clegg, R. M. (1997) Global structure of three-way DNA junctions with and without additional unpaired bases: A fluorescence resonance energy transfer analysis Biochemistry 36, 13530– 13538 DOI: 10.1021/bi9702445There is no corresponding record for this reference.
- 30Sisamakis, E., Valeri, A., Kalinin, S., Rothwell, P. J., and Seidel, C. A. M. (2010) Accurate single-molecule FRET studies using multiparameter fluorescence detection Methods Enzymol. 475, 455– 514 DOI: 10.1016/S0076-6879(10)75018-730https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVyltb7L&md5=20d586eb83ef1fc641b7a75154e39a2aAccurate single-molecule FRET studies using multiparameter fluorescence detectionSisamakis, Evangelos; Valeri, Alessandro; Kalinin, Stanislav; Rothwell, Paul J.; Seidel, Claus A. M.Methods in Enzymology (2010), 475 (Single Molecule Tools, Part B), 455-514CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)A review. In the recent decade, single-mol. (sm) spectroscopy has come of age and is providing important insight into how biol. mols. function. So far our view of protein function is formed, to a significant extent, by traditional structure detn. showing many beautiful static protein structures. Recent expts. by single-mol. and other techniques have questioned the idea that proteins and other biomols. are static structures. In particular, Foerster resonance energy transfer (FRET) studies of single mols. have shown that biomols. may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophys. behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amts. of contaminants. We demonstrate that the simultaneous acquisition of a max. of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and anal. of individual species present in a population of mols. After a short introduction, the data anal. procedure is described in detail together with some exptl. considerations. The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure detn. based on FRET. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when detg. FRET-related distances with high accuracy. Furthermore, the broad time resoln. (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomol. of interest explores its structural energy landscape.
- 31Muschielok, A., Andrecka, J., Jawhari, A., Brueckner, F., Cramer, P., and Michaelis, J. (2008) A nano-positioning system for macromolecular structural analysis Nat. Methods 5, 965– 971 DOI: 10.1038/nmeth.125931https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlSktrfK&md5=bacd89b1f60852fc59260932486929afA nano-positioning system for macromolecular structural analysisMuschielok, Adam; Andrecka, Joanna; Jawhari, Anass; Brueckner, Florian; Cramer, Patrick; Michaelis, JensNature Methods (2008), 5 (11), 965-971CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Very often, the positions of flexible domains within macromols. as well as within macromol. complexes cannot be detd. by std. structural biol. methods. To overcome this problem, we developed a method that uses probabilistic data anal. to combine single-mol. measurements with X-ray crystallog. data. The method dets. not only the most likely position of a fluorescent dye mol. attached to the domain but also the complete three-dimensional probability distribution depicting the exptl. uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quant. tool for investigating the position and dynamics of flexible domains within macromol. complexes. We applied this method to find the position of the 5' end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.
- 32Brunger, A. T., Strop, P., Vrljic, M., Chu, S., and Weninger, K. R. (2011) Three-dimensional molecular modeling with single molecule FRET J. Struct. Biol. 173, 497– 505 DOI: 10.1016/j.jsb.2010.09.00432https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvFWntb4%253D&md5=c8c4224ee322b2b82ee22b9969be9ff4Three-dimensional molecular modeling with single molecule FRETBrunger, Axel T.; Strop, Pavel; Vrljic, Marija; Chu, Steven; Weninger, Keith R.Journal of Structural Biology (2011), 173 (3), 497-505CODEN: JSBIEM; ISSN:1047-8477. (Elsevier B.V.)A review. Single mol. fluorescence energy transfer expts. enable investigations of macromol. conformation and folding by the introduction of fluorescent dyes at specific sites in the macromol. Multiple such expts. can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple mols. Distances that are derived from such expts. can be used for detn. of the fluorophore positions by triangulation. When combined with a known structure of the macromol.(s) to which the fluorophores are attached, a three-dimensional model of the system can be detd. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the exptl. and computational methods used for three-dimensional modeling based on single mol. fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromol. complexes.
- 33Im, K., Jeong, D., Hur, J., Kim, S. J., Hwang, S., Jin, K. S., Park, N., and Kim, K. (2013) Robust analysis of synthetic label-free DNA junctions in solution by X-ray scattering and molecular simulation Sci. Rep. 3, 3226 DOI: 10.1038/srep0322633https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2c7mvFSqsg%253D%253D&md5=2f999b5de9c6ad39c0bf0fb5de36c26bRobust analysis of synthetic label-free DNA junctions in solution by X-ray scattering and molecular simulationIm Kyuhyun; Jeong Daun; Hur Jaehyun; Kim Sung-Jin; Hwang Sungwoo; Jin Kyeong Sik; Park Nokyoung; Kim KinamScientific reports (2013), 3 (), 3226 ISSN:.Structural analysis of branched DNA molecules (BDM) is important as model systems for DNA junctions and also as building units for DNA assembly. Although there have been efforts to study the structures of BDM, label-free solution structures have not been well determined yet. Here, we used a combination of synchrotron-based experimental tools and computational simulation to study the global structures of label-free BDM in solution. Overall structures of 3-arm and 4-arm BDM were revealed as an asymmetric T(or Y)-shape and a distorted X-shape, respectively. The internal structures of the DNA double helix were shown to have a canonical B-form for both the BDM. We also reconstructed the thermal denaturation process of BDM by determining the transient global structures over a wide range of temperatures. The proposed high-resolution structures of BDM are expected to provide fundamental information for studies of the biological function of junction DNAs and DNA assembly.
Supporting Information
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ARTICLE SECTIONSThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00677.
Single-molecule FRET plots for 3WJa and 3WJb; TCSPC data for 3WJb; overlaid molecular models of the global structure of 3WJa and 3WJb in 0 mM Mg2+ buffer; 1D 19F NMR spectra of a GC-rich 3WJ in D2O from 12 to 40 °C, ssDNA in H2O from 21 to 30 °C, and dsDNA in H2O from 12 to 38 °C; sub-ensemble analysis of 3WJb FRET data; and DRMSD from the FRET-based restraints in 3WJb (PDF)
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