Universality in RNA and DNA deformations induced by salt, temperature change, stretching force, and protein binding

Significance

DNA and RNA deformations play crucial roles in biological processes, such as DNA packaging and nucleic acid recognition by proteins. The relevant understanding is limited due to the challenge in the precise measurement of nucleic acid deformations and the complexity of nucleic acid interactions. We solve these two issues using experiments, simulations, and theory. Magnetic tweezers experiments provide an excellent opportunity to precisely measure DNA and RNA twist changes induced by salt, temperature change, and stretching. Surprisingly, our simulations and theory find that common deformation pathways drive DNA and RNA deformations induced by different stimuli. Furthermore, the common deformation pathways appear to be utilized by protein binding to reduce the energy cost of DNA and RNA deformations.

Abstract

Nucleic acid deformations play important roles in many biological processes. The physical understanding of nucleic acid deformation by environmental stimuli is limited due to the challenge in the precise measurement of RNA and DNA deformations and the complexity of interactions in RNA and DNA. Magnetic tweezers experiments provide an excellent opportunity to precisely measure DNA and RNA twist changes induced by environmental stimuli. In this work, we applied magnetic tweezers to measure double-stranded RNA twist changes induced by salt and temperature changes. We observed RNA unwinds when lowering salt concentration, or increasing temperature. Our molecular dynamics simulations revealed the mechanism: lowering salt concentration or increasing temperature enlarges RNA major groove width, which causes twist decrease through twist-groove coupling. Combining these results with previous results, we found some universality in RNA and DNA deformations induced by three different stimuli: salt change, temperature, and stretching force. For RNA, these stimuli first modify the major groove width, which is transduced into twist change through twist-groove coupling. For DNA, these stimuli first modify diameter, which is transduced into twist change through twist-diameter coupling. Twist-groove coupling and twist-diameter coupling appear to be utilized by protein binding to reduce DNA and RNA deformation energy cost upon protein binding.

Double-stranded (ds) DNA and dsRNA deformations play crucial roles in many biological processes, such as DNA packaging (1–6), nucleic acid–protein interactions (7–10), and gene expression (11–13). The deformations can be induced by various stimuli, such as salt changes (14–20), temperature changes (21, 22), external forces (23–25), and protein binding (26). DsDNA and dsRNA deformations can exhibit in numerous forms, such as bending, twisting, diameter variation, and groove width variation. For instance, DNA sharp bending occurs in nucleosomes for DNA compaction (27). Twisting dsDNA by topoisomerases leads to supercoiling and facilitates DNA compaction in bacteria (28). Furthermore, undertwisting dsDNA lowers the energetic cost of separating the two strands and hence promotes DNA replication and transcription (29, 30). Conversely, overtwisting dsDNA enhances the separation energy cost and double-helix stability, which has been adopted by bacteria living at high temperatures (31).

Nucleic acid deformations strongly affect nucleic acid–protein binding affinity because proteins often recognize nucleic acids through shape readout (32–36). Upon protein binding, dsRNA and dsDNA usually deform from their standalone conformations (intrinsic shapes) to the conformations that best fit protein shapes and facilitate nucleic acid–protein binding attractions, such as hydrogen bond formation. A recent study demonstrated that a higher deformation energy cost weakens the dsRNA/dsDNA–protein binding affinity because the deformation energy offsets the binding attraction (36). They found that some DNA mismatches can substantially enhance protein–DNA binding affinities because the new DNA shapes with mismatches can better fit the protein–DNA binding geometry and reduce DNA deformation energy cost (36).

An intriguing property of nucleic acid deformations is that many structural parameters of nucleic acids are correlated, which means that the variation of one structural parameter causes the variation of another one. For instance, stretching DNA leads to overtwisting (23, 37), while stretching RNA leads to undertwisting (24). The correlations or couplings of structural parameters are not straightforward and are often counterintuitive. Extensive simulation and theoretical analysis have been carried out to reveal the mechanisms of these couplings (25, 38) as the couplings of nucleic acid structural parameters play essential roles in the responses of nucleic acid structures to environmental changes, such as salt and temperature changes (38–40).

While nucleic acid deformations are biologically important, the relevant understanding is limited because nucleic acid deformations are usually on very short lengths and the precise measurement of deformations is challenging. Some researchers used the DNA structures in the Protein Data Bank (PDB) to analyze DNA deformations induced by protein binding, while these DNA structures were precisely resolved by X-ray diffraction (36). However, the number of DNA structures in the database is limited. Furthermore, it is impossible or difficult to use these DNA structures to analyze the DNA deformations with environmental changes, such as salt and temperature. A high-throughput and precise method is demanded to measure nucleic acid deformations with environmental changes.

In this work, we employed magnetic tweezers (MT) experiments to precisely measure RNA twist changes induced by salt and temperature changes. MT experiments provide an excellent opportunity to precisely measure DNA or RNA twist changes because even small twist changes can accumulate along a long DNA or RNA molecule and cause a large rotation of the DNA or RNA end (Fig. 1A), which has been demonstrated by recent studies using magnetic or optical tweezers (22–24, 37, 39–43). To explain the experimental results of salt- and temperature-induced RNA twist changes, we performed simulations and theoretical analysis. We found that RNA deformations induced by different environmental stimuli were driven by a common deformation pathway. Inspired by this, we carried out a similar analysis of DNA and also elaborated a common deformation pathway in DNA.

Our work aims to reveal the common deformation pathways in eight different phenomena: RNA and DNA deformations induced by salt, temperature change, stretching force, and protein binding. Among these eight phenomena, RNA deformations induced by salt and temperature changes are interesting results from our experiments and simulations. DNA and RNA deformations induced by protein binding are analyses of existing structures of protein–RNA/DNA complexes. RNA deformations by stretching force (24, 25, 38) as well as DNA deformations by stretching force (23, 25, 37, 38), salt (20, 39, 40), and temperature change (22, 40) have been measured by others and us and were remeasured here using additional DNA/RNA sequences.

Results

Salt-Induced RNA Twist Change.

We used MT experiments to measure the RNA twist changes with the salt concentration, c_salt (Fig. 1A) (16). At each c_salt, we rotated a single torsionally constrained RNA and measured the RNA extension simultaneously, which yields the rotation–extension curves (Fig. 1B). We determined the torsionally relaxed point of RNA to be the crossing point of the two linear fits of the plectoneme ranges. Then, using the same RNA molecule, we changed c_salt and measured the torsionally relaxed point again (Fig. 1 C and D). We recorded the shift in the rotation turns N_turn induced by the salt change. Then, we converted N_turn to the twist change Δω_exp per base pair using Δω_exp = (N_turn × 360^o)/N_bp, where N_bp = 13.6 × 10³ is the number of base pairs. For convenience, we treated 1 M as the reference salt concentration, i.e., the RNA twist change Δω_exp = 0 at c_salt = 1 M.

In the above measurement, the change in c_salt slightly modifies the refractive index of the buffer, which slightly affects the measurement of RNA extension. As described above, determining salt-induced twist changes needs just the peak locations of the curves in Fig. 1 C and D but not the peak heights, i.e., the absolution values of RNA extension. The change in the refractive index has no influence on the peak locations and hence no influence on the results of RNA twist changes. For accuracy, the data of extensions in Fig. 1 have considered the effect of the change in the refractive index on RNA extension measurement (SI Appendix, section S2).

Fig. 2A presents the experimental results of RNA twist changes as a function of c_salt at the temperature of T = 295 K. With the decreasing of c_salt, Δω_exp decreases steadily for all five monovalent ions. The salt-induced RNA twist changes are similar for all five ions, suggesting that the twist changes may be mainly caused by electrostatic screening…