Advances in the determination of nucleic acid conformational ensembles

Abstract

Conformational changes in nucleic acids play a key role in the way genetic information is stored, transferred, and processed in living cells. Here, we describe new approaches that employ a broad range of experimental data, including NMR-derived chemical shifts and residual dipolar couplings, small-angle X-ray scattering, and computational approaches such as molecular dynamics simulations to determine ensembles of DNA and RNA at atomic resolution. We review the complementary information that can be obtained from diverse sets of data and the various methods that have been developed to combine these data with computational methods to construct ensembles and assess their uncertainty. We conclude by surveying RNA and DNA ensembles determined using these methods, highlighting the unique physical and functional insights obtained so far.

Figure 1

Experimental data used in ensemble determination of nucleic acids. (A) SAXS profile for an RNA and the corresponding pair distribution function. Adapted from Wang et al.(18) (B) Au-SAXS measurement of probability distance distributions for two attached gold nanocrystal probes. Adapted from Shi et al.(23) (C) NMR chemical shifts are sensitive to electronic environment and local structure. (D) NMR residual dipolar couplings provide angular information on the orientation of bond relative to the overall molecular alignment frame (A_xx, A_yy, A_zz).

Figure 2

Ensemble determination. (A) Typical behaviour of active and passive data reproduction as a function of the number of conformers in the ensemble refinement approach. Adapted from Schwieters and Clore(15) (B) Flow chart for data guided selection of conformers form a pool. (C) Typical behaviour of active and passive data reproduction as a function of the number of conformers in data guided ensemble selection approach. Adapted from Salmon et al.(105)

Figure 3

Evaluating HIV-1 TAR ensemble using RDCs and CSs. (A, B) Reproduction of RDCs measured in four differentially elongated TAR RNA constructs (indicated with different colors) using (A) MD starting conformational pool and (B) 20 conformer selected ensemble. (C–F). Cross-validation using (C) randomly selected RDCs (D) individual RDC data sets (E) magnetic field induced RDCs (F) and CSs. In E and F, grey and red data represent reproduction from the starting pool and RDC selected ensemble. Adapted from Salmon et al.(105)

Figure 4

Testing the accuracy of an ensemble determination using simulated data. Distribution of angles in HIV-1 TAR ensemble. (A) Euler angles (α_h,β_h,γ_h) defining the inter-helical orientations(131) (B) intra- and inter-base pair angular parameters for the A22-U40 base-pair (buckle, propeller twist and opening and tilt, roll and twist) and (C) sugar torsion angles (ν₀-ν₄) for the A22-U40 base-pair (top) A22, (bottom) U40. Starting pool, target ensemble, and RDC-selected ensembles are shown in grey, blue and red, respectively. Adapted from Salmon et al.(105)

Figure 5

Ensembles of nucleic acids. (A) Ensemble description of the Dickerson DNA dodecamer. Structural representation of the four conformer ensemble and distribution of angular parameters describing the dodecamer base pairs. Angular parameters are plotted on clockfaces where the top corresponds to 0°, the right side to 90°, and the bottom to 180°. The helical rise is plotted on a bar chart with ticks at 1 Å intervals, with the bottom being at 1 Å. The probability increases from blue to green to red. Abbreviations: prop, propeller twist; twist, helical twist. Adapted from Schwieters and Clore(15). (B) Experimentally observed distance distributions between the two gold probes attached to a DNA duplex using Au-SAXS. The gold probes are separated by different numbers of base-pair steps, as indicated by the color-coded numbers. Adapted from Shi et al.(23) (C) SAXS visualization of the ensemble representing the orientation of two helices at various metal concentrations. One helix is depicted in gray while the second is represented using balls that are color-coded according to energetic differences (Red: 0–1 k_BT, orange: 1–2 k_BT, yellow: 2–3 k_BT, blue >3 k_BT). Adapted from Bai et al.(108) (D) Overlay of the 13 free state conformations of SAM-1 riboswitch aptamer domain with helices are represented as cylinders. The bound state is depicted in grey. Adapted from Stoddard et al.(120) (E) Analysis of HIV-1 TAR ensemble. Schematic representation of the three clusters and a proposed ordering for transitioning between conformations with different bend angles. Curved arrows indicate local dynamics. Interactions with helix II are indicated with a dashed line. Structural similarity between (orange) the RDC selected ensemble and (grey) three distinct ligand bound TAR structures. Adapted from Salmon et al.(105)