Tuning RNA Flexibility with Helix Length and Junction Sequence

Abstract

The increasing awareness of RNA's central role in biology calls for a new understanding of how RNAs, like proteins, recognize biological partners. Because RNA is inherently flexible, it assumes a variety of conformations. This conformational flexibility can be a critical aspect of how RNA attracts and binds molecular partners. Structurally, RNA consists of rigid basepaired duplexes, separated by flexible non-basepaired regions. Here, using an RNA system consisting of two short helices, connected by a single-stranded (non-basepaired) junction, we explore the role of helix length and junction sequence in determining the range of conformations available to a model RNA. Single-molecule Förster resonance energy transfer reports on the RNA conformation as a function of either mono- or divalent ion concentration. Electrostatic repulsion between helices dominates at low salt concentration, whereas junction sequence effects determine the conformations at high salt concentration. Near physiological salt concentrations, RNA conformation is sensitive to both helix length and junction sequence, suggesting a means for sensitively tuning RNA conformations.

Conformations of an RNA Helix-Junction-Helix Construct Revealed by SAXS Refinement of MD Simulations

“…1. We use the phosphorus atom of C12 as the new origin and the direction of central line of H1 as þz at x ¼ x 0 > 0, (22). The error bars are the standard deviations of at leaset two independent measurements.…”

“…Naively, one might expect a continuous change in the conformation of this model system as a function of increasing [KCl] because both the screening length of the ionic cloud around the helices and the linker stiffness decrease with added salt (21). Surprisingly, previous studies of the HJH system by single-molecule fluorescence resonance energy transfer (smFRET) showed a nonmonotonic energy transfer (E FRET ) with increasing KCl concentrations (22), suggesting that the dyes attached to the two helices do not report a continuous molecular collapse as the salt concentration increases (23). From FRET studies carried out with different constructs, we arrived at the following qualitative explanation of the measured salt dependence: repulsion between helices determines conformations at low salt, junction sequence determines conformation at high salt, and these effects compete at moderate (close to physiological) salt.…”

“…The molecule is constructed by pairing a 29-nucleotide backbone with two complementary 12-nucleotide oligonucleotides at the ends. The long backbone has the same mixed sequence as in (22), CCC UAU ACU CCC UUU UUC CUC CUA AUC GC.…”

Modeling Structure, Stability, and Flexibility of Double-Stranded RNAs in Salt Solutions

“…8, A and B. This indicates the stronger bending conformations and the higher bending fluctuation for the dsRNA helix at higher ion concentrations, which are attributed to the stronger ion neutralization on P bead charges and consequently the reduced electrostatic repulsion due to bending (86)(87)(88)(89)(90)(91)(92)(93)(94). The RMSD variance of the dsRNA helix at different [Na þ ] values calculated based on the conformation-averaged reference structure also indicates that the dsRNA helix would become more flexible with the increase of [Na þ ]; see Fig.…”

“…DsRNAs generally are rather flexible in ion solutions because of their polymeric nature, and the flexibility is extremely important for their biological functions. Additionally, dsRNA flexibility is highly dependent on solution ion conditions (85)(86)(87)(88)(89)(90)(91)(92)(93). In this section, we further employed our model to examine the flexibility of a 40-bp RNA helix in ion solutions.…”