The effects of "molecular crowding" on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop-receptor folding to isolate a single RNA tertiary interaction using timecorrelated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high-molecularweight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG°∼ −2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (K eq ) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in K eq is dominated by a 35-fold increase in tetraloop-receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple "hard-sphere" treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG°∼ −5 kcal/mol free-energy stabilization at excluded volume fractions of 30%.scaled particle theory | fluorescence | PEG

Mg 2þ is essential for the proper folding and function of RNA, though the effect of Mg 2þ concentration on the free energy, enthalpy, and entropy landscapes of RNA folding is unknown. This work exploits temperature-controlled single-molecule FRET methods to address the thermodynamics of RNA folding pathways by probing the intramolecular docking/undocking kinetics of the ubiquitous GAAA tetraloop−receptor tertiary interaction as a function of [Mg 2þ ]. These measurements yield the barrier and standard state enthalpies, entropies, and free energies for an RNA tertiary transition, in particular, revealing the thermodynamic origin of [Mg 2þ ]-facilitated folding. Surprisingly, these studies reveal that increasing [Mg 2þ ] promotes tetraloop-receptor interaction by reducing the entropic barrier (−T ΔS ‡ dock ) and the overall entropic penalty (−T ΔS°d ock ) for docking, with essentially negligible effects on both the activation enthalpy (ΔH ‡ dock ) and overall exothermicity (ΔH°d ock ). These observations contrast with the conventional notion that increasing [Mg 2þ ] facilitates folding by minimizing electrostatic repulsion of opposing RNA helices, which would incorrectly predict a decrease in ΔH ‡ dock and ΔH°d ock with [Mg 2þ ]. Instead we propose that higher [Mg 2þ ] can aid RNA folding by decreasing the entropic penalty of counterion uptake and by reducing disorder of the unfolded conformational ensemble.T he folding of RNA proceeds hierarchically, whereby secondary structure is formed rapidly and subsequent slow helical packing is mediated by tertiary interactions (1, 2). RNA secondary structure prediction from the known thermodynamics is quite reliable (3), though correspondingly accurate prediction of tertiary structure remains a major challenge (1). Static tertiary structure data alone are also not enough to predict RNA functionality, as time-dependent conformational dynamics occur during biochemical processes (4, 5). As a result, one needs the full free energy, enthalpy, and entropy landscapes for folding. A major road block in achieving a predictive understanding of RNA folding landscapes is that they are often "rugged,", i.e., with alternative conformations acting as kinetic traps (6, 7). Moreover, the electrostatic challenge of folding a charged biopolymer highlights the particularly critical role of Mg 2þ and other counterions in the folding process.Characterization of folding transition states-and the role of Mg 2þ in stabilizing transition states-remains a crucial bottleneck for reconciling the kinetics and thermodynamics of RNA folding (8-13). Some insight into the free energy landscapes for RNA folding can be obtained from temperature-dependent stopped-flow kinetic studies, which offer the ability to deconstruct free energy barriers (ΔG ‡ ) into enthalpic (ΔH ‡ ) and entropic (−TΔS ‡ ) components. However, with such methods, only the net rate constant (i.e., k total ¼ k fold þ k unfold ) for approach to equilibrium can be observed, which requires strong assumptions (e.g., that k fold ≫ k unfol...

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k'on = 2.1(1) × 10(6) M(-1) s(-1)) and 6 bp (k'on = 5.0(1) × 10(6) M(-1) s(-1)) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s(-1)) to 6 bp (koff = 14 s(-1)) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, kon increases by >40-fold (kon = 0.10(1) s(-1) to 4.0(4) s(-1) between [NaCl] = 25 mM and 1 M), whereas in contrast, koff decreases by fourfold (0.72(3) s(-1) to 0.17(7) s(-1)) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.

Knowledge of the structure and dynamics of RNA molecules is critical to understand their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be significantly enhanced by methods that enable incorporation of modified or labeled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. We have developed a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labeling. We demonstrate its utility by successfully preparing various isotope- or fluorescently-labeled versions of the 71-nucleotide aptamer domain of an adenine riboswitch1 for nuclear magnetic resonance (NMR) spectroscopy or single molecule Förster resonance-energy transfer (smFRET), respectively. Those RNAs include molecules that were selectively isotope-labeled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently-labeled in and near kissing loops. These selectively labeled RNAs have the same fold as those transcribed using conventional methods, but greatly simplified the interpretation of NMR spectra. The single-position isotope-labeled and fluorescently-labeled RNA samples revealed multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labeling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection2 and disease diagnostics3,4.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 1.2 million deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

The association of biomolecules is the elementary event of communication in biology. Most mechanistic information of how the interactions between binding partners form or break is, however, hidden in the transition paths, the very short parts of the molecular trajectories from the encounter of the two molecules to the formation of a stable complex. Here we use single-molecule spectroscopy to measure the transition path times for the association of two intrinsically disordered proteins that form a folded dimer upon binding. The results reveal the formation of a metastable encounter complex that is electrostatically favored and transits to the final bound state within tens of microseconds. Such measurements thus open a new window into the microscopic events governing biomolecular interactions.

No abstract

Cations have long been associated with formation of native RNA structure and are commonly thought to stabilize the formation of tertiary contacts by favorably interacting with the electrostatic potential of the RNA, giving rise to an “ion atmosphere”. A significant amount of information regarding the thermodynamics of structural transitions in the presence of an ion atmosphere has accumulated and suggests stabilization is dominated by entropic terms. This work provides an analysis of how RNA–cation interactions affect the entropy and enthalpy associated with an RNA tertiary transition. Specifically, temperature-dependent single-molecule fluorescence resonance energy transfer studies have been exploited to determine the free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) of folding for an isolated tetraloop–receptor tertiary interaction as a function of Na+ concentration. Somewhat unexpectedly, increasing the Na+ concentration changes the folding enthalpy from a strongly exothermic process [e.g., ΔH° = −26(2) kcal/mol at 180 mM] to a weakly exothermic process [e.g., ΔH° = −4(1) kcal/mol at 630 mM]. As a direct corollary, it is the strong increase in folding entropy [Δ(ΔS°) > 0] that compensates for this loss of exothermicity for the achievement of more favorable folding [Δ(ΔG°) < 0] at higher Na+ concentrations. In conjunction with corresponding measurements of the thermodynamics of the transition state barrier, these data provide a detailed description of the folding pathway associated with the GAAA tetraloop–receptor interaction as a function of Na+ concentration. The results support a potentially universal mechanism for monovalent facilitated RNA folding, whereby an increasing monovalent concentration stabilizes tertiary structure by reducing the entropic penalty for folding.