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The ability to create stimuli-responsive DNA nanostructures has played a prominent role in dynamic DNA nanotechnology. Primary among these is the process of toehold-based strand displacement, where a nucleic acid molecule can act as a trigger to cause conformational changes in custom-designed DNA nanostructures. Here, we add another layer of control to strand displacement reactions through a 'toehold clipping' process. By designing DNA complexes with a photocleavable linker-containing toehold or an RNA toehold, we show that we can use light (UV) or enzyme (ribonuclease) to eliminate the toehold, thus preventing strand displacement reactions. We use molecular dynamics simulations to analyze the structural effects of incorporating a photocleavable linker in DNA complexes. Beyond simple DNA duplexes, we also demonstrate the toehold clipping process in a model DNA nanostructure, by designing a toehold containing double-bundle DNA tetrahedron that disassembles when an invading strand is added, but stays intact after the toehold clipping process even in the presence of the invading strand. This work is an example of combining multiple physical or molecular stimuli to provide additional remote control over DNA nanostructure reconfiguration, advances that hold potential use in biosensing, drug delivery or molecular computation.

 

RNA is critical to a broad spectrum of biological and viral processes. This functional diversity is a result of their dynamic nature; the variety of three-dimensional structures that they can fold into; and a host of post-transcriptional chemical modifications. While there are many experimental techniques to study the structural dynamics of biomolecules, molecular dynamics simulations (MDS) play a significant role in complementing experimental data and providing mechanistic insights. The accuracy of the results obtained from MDS is determined by the underlying physical models i.e., the force-fields, that steer the simulations. Though RNA force-fields have received a lot of attention in the last decade, they still lag compared to their protein counterparts. The chemical diversity imparted by the RNA modifications adds another layer of complexity to an already challenging problem. Insight into the effect of RNA modifications upon RNA folding and dynamics is lacking due to the insufficiency or absence of relevant experimental data. This review provides an overview of the state of MDS of modified RNA, focusing on the challenges in parameterization of RNA modifications as well as insights into relevant reference experiments necessary for their calibration. 

RNA folds cotranscriptionally to traverse out-of-equilibrium intermediate structures that are important for RNA function in the context of gene regulation. To investigate this process, here we study the structure and function of the Bacillus subtilis yxjA purine riboswitch, a transcriptional riboswitch that downregulates a nucleoside transporter in response to binding guanine. Although the aptamer and expression platform domain sequences of the yxjA riboswitch do not completely overlap, we hypothesized that a strand exchange process triggers its structural switching in response to ligand binding. In vivo fluorescence assays, structural chemical probing data and experimentally informed secondary structure modeling suggest the presence of a nascent intermediate central helix. The formation of this central helix in the absence of ligand appears to compete with both the aptamer’s P1 helix and the expression platform’s transcriptional terminator. All-atom molecular dynamics simulations support the hypothesis that ligand binding stabilizes the aptamer P1 helix against central helix strand invasion, thus allowing the terminator to form. These results present a potential model mechanism to explain how ligand binding can induce downstream conformational changes by influencing local strand displacement processes of intermediate folds that could be at play in multiple riboswitch classes.

 

Nucleic acid nanostructures with different chemical compositions have shown utility in biological applications as they provide additional assembly parameters and enhanced stability. The naturally occurring 2′-5′ linkage in RNA is thought to be a prebiotic analogue and has potential use in antisense therapeutics. Here, we report the first instance of DNA/RNA motifs containing 2′-5′ linkages. We synthesized and incorporated RNA strands with 2′-5′ linkages into different DNA motifs with varying number of branch points (a duplex, four arm junction, double crossover motif and tensegrity triangle motif). Using experimental characterization and molecular dynamics simulations, we show that hybrid DNA/RNA nanostructures can accommodate interspersed 2′-5′ linkages with relatively minor effect on the formation of these structures. Further, the modified nanostructures showed improved resistance to ribonuclease cleavage, indicating their potential use in the construction of robust drug delivery vehicles with prolonged stability in physiological conditions. 

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the ‘seed’ region of the miRNA and its counterpart mRNA1. Here we use R1ρ relaxation-dispersion nuclear magnetic resonance2 and molecular simulations3 to reveal a dynamic switch—based on the rearrangement of a single base pair in the miRNA–mRNA duplex—that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago4,5. Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA–mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial ‘screening’ state to an ‘active’ state, and unveil the role of the RNA duplex beyond the seed in Ago2.