Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths (l p ) of ssRNA. We observe valence and ionic strength-dependent differences in l p in a direct comparison between 40-mers of deoxythymidylate (dT 40 ) and uridylate (rU 40 ) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo.single molecule FRET | small angle X-ray scattering | worm-like chain | ion-nucleic acid interactions N ucleic acids in the cell are dynamic and undergo structural changes as they transmit and process genetic information. Dynamic processes related to biological function (e.g., transcription for DNA and recognition and folding for RNA) involve nonbase-paired regions that confer flexibility to the overall structure. For RNAs like riboswitches that exchange between multiple structures in equilibrium (1), conformational disorder is often an intrinsic property of the molecule and important for biological function. Even relatively stable molecules like catalytic introns and transfer RNA must pass through a disordered phase while folding. Thus, progress toward a mechanistic understanding of RNA folding and dynamics will require detailed knowledge of nucleic acid chain flexibility and its dependence on base content, solution conditions, and molecular context.In light of its importance to biology, it is surprising that RNA flexibility has not been studied in as much detail as DNA flexibility. Despite the chemical similarity of the RNA and DNA backbone, there is ample evidence from X-ray crystallography that the identity of the sugar (ribose vs. deoxy-ribose) affects backbone conformations (2). However, researchers have used the properties of DNA to understand RNA folding (3) because corresponding information for RNA was lacking. This difficulty motivates our present efforts to measure and directly compare the flexibilities of single-stranded nucleic acids (ssRNA and ssDNA).In the cell, RNA and DNA interact with cations that screen the negatively charged phosphate backbone. Both diffuse and specifically bound ions are important for RNA folding (4), and divalent ions are almost always required to stabilize RNA tertiary structures (5-8). A full mechanistic description of these ion effects is complicated by the fact that ions can interact with RNA differently during various stages of folding (9, ...

About 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS coronavirus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule–based interventions. All coronaviruses utilize a molecular mechanism called programmed −1 ribosomal frameshift (−1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high −1 PRF rates and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar −1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate −1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

Time-resolved small-angle x-ray scattering was used to measure the radius of gyration of cytochrome c after initiation of folding by a pH jump. Submillisecond time resolution was obtained with a microfabricated diffusional mixer and synchrotron radiation. The results show that the protein first collapses to compact denatured structures before folding very fast to the native state.The stability and folding speed of a protein depend on the structures of the denatured as well as the native state, raising the question: do proteins fold more rapidly from a denatured state of expanded structures or from one of compact structures? Lattice simulations of simplified representations of proteins suggest that slow folding amino acid sequences collapse to compact structures with non-native topologies before folding, while fast folders collapse and fold simultaneously (1-4). We have begun to address this question experimentally with submillisecond small-angle x-ray scattering (SAXS) using synchrotron radiation and a microfabricated diffusional mixer to rapidly initiate folding. SAXS yields the radius of gyration, the most unambiguous measure of compactness. Here we show that, in contrast to the simulations, one of the fastest-folding proteins (cytochrome c: folding ϭ 400 s) first collapses to compact structures before forming the final native state. X-ray scattering by proteins in solution is sensitive to spatial variations in electron density. Scattering at the smallest angles yields the radius of gyration, R g , which in conjunction with the protein molecular weight provides a measure of the compactness of globular proteins. Additional structural information can be obtained from scattering at larger angles, which reflects electron density correlations on length scales shorter than R g . For a compact polymer, such as the native protein or compact denatured structures, I(q)q 2 increases at low q, goes through a maximum, and decreases at large q where I(q)␣q Ϫ4 [I(q) is the scattered intensity; q ϭ 4sin ͞, is the momentum transfer;is the x-ray wavelength, 1.54 Å; and 2 is the scattering angle] (5). In contrast, for a polymer chain undergoing a random walk in space, as can occur for an unfolded protein under strongly denaturing conditions, I(q)q 2 first increases, then plateaus (6), and, at large q, where I(q)␣q