Selective deuteration of an RNA:RNA complex for structural analysis using small-angle scattering

The structures of RNA:RNA complexes regulate many biological processes. Despite their importance, protein-free RNA:RNA complexes represent a tiny fraction of experimentally-determined structures. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to structurally interrogate conformational changes in a model RNA:RNA complex. Using SAXS, we measured the solution structures of the individual RNAs in their free state and of the overall RNA:RNA complex. With SANS, we demonstrate, as a proof-of-principle, that isotope labeling and contrast matching (CM) can be combined to probe the bound state structure of an RNA within a selectively deuterated RNA:RNA complex. Furthermore, we show that experimental scattering data can validate and improve predicted AlphaFold 3 RNA:RNA complex structures to reflect its solution structure. Our work demonstrates that in silico modeling, SAXS, and CM-SANS can be used in concert to directly analyze conformational changes within RNAs when in complex, enhancing our understanding of RNA structure in functional assemblies.

(c) Software employed for SAXS/SANS data reduction, analysis, and interpretation.

SAXS SANS
Data reduction I(q) vs q and solvent subtraction using BioXTAS RAW 2.2.1(89)I(q) vs q using drt-SANS, solvent subtraction using BioXTAS RAW 2.
15.12 ± 0.02 27.5 ± 0.1 36.5 ± 0.1 16. *Due the low signal-to-noise ratio in the low-q regime, Guinier fits for the SANS data did not provide reliable coefficient of correlations.

Figure S6 .
Figure S6.Simulated SANS profiles of DIS-C at different contrast conditions show differences in the scattering data.At low neutron contrast and smaller scattering vectors, the forward intensity increases, providing more information about the size and shape of the RNA.In contrast, at high neutron contrast and larger scattering vectors, the data reveals more details about the internal structure.

Figure S7 .
Figure S7.P(r) functions of the selectively deuterated DIS-C:DIS-Gk complexes show successful contrast matching.Comparison of normalized pair distance distribution functions of (A) DIS-C and (B) DIS-Gk to the DIS-C:DIS-Gk complex indicates successful contrast matching.Matched out components are indicated in gray in the legend.

Figure S12 .
Figure S12.Modeling of the DIS RNAs with kissing loops.(A) The RNAMasonry DIS SAS models [DIS-C (top) and DIS-Gk (bottom)] reported in Figures 2 and 4 with the correct secondary structures.(B) AF3 model 4 of the DIS-C:DIS-Gk kissing complex, with the correct KL geometry but incorrect secondary structures.(C) Input structures for RNAMasonry generated by appending the stems of (A) and the apical loops of (B).(D) RNAMasonry DIS + KL SAS models reported in Figure5where the input structures in (C) were further refined to their respective SAS data, keeping the KL residues frozen.

Figure S16 .
Figure S16.SAS-guided DIS-C:DIS-Gk kissing complexes fit to experimental SAXS data.(A) All combinations of the DIS-C:DIS-Gk kissing complexes composed of the individual DIS-C and DIS-Gk RNAs refined to their respective SAS data while keeping the stacking geometry of the KL residues predicted by AF3.The KL residues of the individual SAS-guided DIS RNAs were aligned to their respective RNAs KL residues in AF3 model 4. (B) Theoretical scattering profiles (colored lines) of the possible DIS-C:DIS-Gk complex models in (A) fit to the experimental DIS-C:DIS-Gk SAXS data (black dots, top) and corresponding residuals (bottom).

Figure S17 .
Figure S17.Estimated flexibility of the DIS-C + KL (SANS, 65% D2O):DIS-Gk + KL (SAXS) kissing complex.(A) Alignment of the DIS-C + KL (SANS, 65% D2O):DIS-Gk + KL (SAXS) kissing complex from Figure 5 to its best flexible conformer as estimated by SREFLEX (cyan), with an overall RMSD of 4.1 Å. (B) Structure of the best conformer optimized to the DIS-C:DIS-Gk SAXS data using the SAS-guided complex structure from Figure 5 as the input for SREFLEX.(C) Theoretical scattering of (B) fit to the DIS-C:DIS-Gk SAXS data.

Table S1 . SAXS and SANS parameters for DIS RNA samples according to publication guidelines.(88) (a) Sample details.
(b) SAXS/SANS data collection parameters.

Table S2 . DNA oligonucleotides used in this work.
a m denotes 2′-O-Methyl modification of the nucleotide.bUnderlined nucleotides indicate the T7 promoter sequence.