2'-mercaptonucleotide interference reveals regions of close packing within folded RNA molecules

The 2'-hydroxyl group makes essential contributions to RNA structure and function. As an approach to assess the ability of a mercapto group to serve as a functional analogue for the 2'-hydroxyl group, we synthesized 2'-mercaptonucleotides for use in nucleotide analogue interference mapping. To correlate the observed interference effects with tertiary structure, we used the independently folding DeltaC209 P4-P6 domain from the Tetrahymena group I intron. We generated populations of DeltaC209 P4-P6 molecules containing 2'-mercaptonucleotides located randomly throughout the domain and separated the folded molecules from the unfolded molecules by nondenaturing gel electrophoresis. Iodine-induced cleavage of the RNA molecules revealed the sites at which 2'-mercaptonucleotides interfere with folding. These interferences cluster in the most densely packed regions of the tertiary structure, occurring only at sites that lack the space and flexibility to accommodate a sulfur atom. Interference mapping with 2'-mercaptonucleotides therefore provides a method by which to identify structurally rigid and densely packed regions within folded RNA molecules.     

Selective stabilization of natively folded RNA structure by DNA constraints

Learning how native RNA conformations can be stabilized relative to unfolded states is an important objective, for both understanding natural RNAs and improving the design of artificial functional RNAs. Here we show that covalently attached double-stranded DNA constraints (ca. 14 base pairs in length) can significantly stabilize the native conformation of an RNA molecule. Using the P4-P6 domain of the Tetrahymena group I intron as the test system, we identified pairs of RNA sites where attaching a DNA duplex is predicted to be structurally compatible with only the folded state of the RNA. The DNA-constrained RNAs were synthesized and shown by nondenaturing polyacrylamide gel electrophoresis (native PAGE) to have substantial decreases in their Mg2+ midpoints ([Mg2+]1/2 values). These changes are equivalent to free energy stabilizations as large as DeltaDeltaGdegrees = -2.5 kcal/mol, which is approximately 14% of the total tertiary folding energy. For comparison, the sole modification of P4-P6 previously reported to stabilize this RNA is a single-nucleotide deletion (DeltaC209) that provides only 1.1 kcal/mol of stabilization. Our findings indicate that nature has not completely optimized P4-P6 RNA folding. Furthermore, the DNA constraints are designed not to interact directly and extensively with the RNA, but rather more indirectly to modulate the relative stabilities of folded and unfolded RNA states. The successful implementation of this strategy to further stabilize a natively folded RNA conformation suggests an important element of modularity in stabilization of RNA structure, with implications for how nature might use other molecules such as proteins to stabilize specific RNA conformations.     

Determining the Mg2+ stoichiometry for folding an RNA metal ion core

The folding and catalytic function of RNA molecules depend on their interactions with divalent metal ions, such as magnesium. As with every molecular process, the most basic knowledge required for understanding the close relationship of an RNA with its metal ions is the stoichiometry of the interaction. Unfortunately, inventories of the numbers of divalent ions associated with unfolded and folded RNA states have been unattainable. A common approach has been to interpret Hill coefficients fit to folding equilibria as the number of metal ions bound upon folding. However, this approach is vitiated by the presence of diffusely associated divalent ions in a dynamic ion atmosphere and by the likelihood of multiple transitions along a folding pathway. We demonstrate that the use of molar concentrations of background monovalent salt can alleviate these complications. These simplifying solution conditions allow a precise determination of the stoichiometry of the magnesium ions involved in folding the metal ion core of the P4-P6 domain of the Tetrahymena group I ribozyme. Hill analysis of hydroxyl radical footprinting data suggests that the P4-P6 RNA core folds cooperatively upon the association of two metal ions. This unexpectedly small stoichiometry is strongly supported by counting magnesium ions associated with the P4-P6 RNA via fluorescence titration and atomic emission spectroscopy. By pinpointing the metal ion stoichiometry, these measurements provide a critical but previously missing step in the thermodynamic dissection of the coupling between metal ion binding and RNA folding.     

Deoxyribozymes with 2'-5' RNA ligase activity

In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg(2+)-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 degrees C, and they afford 40-50% yield in 1 h at pH 9.0 and 37 degrees C. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUA GGAA or UAUN GGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2'-5' linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg(2+)-dependent folding (DeltaDeltaG degrees ' < 0.2 kcal/mol). This demonstrates that a 2'-5' linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.     

Learning the Fastest RNA Folding Path Based on Reinforcement Learning and Monte Carlo Tree Search

RNA molecules participate in many important biological processes, and they need to fold into well-defined secondary and tertiary structures to realize their functions. Like the well-known protein folding problem, there is also an RNA folding problem. The folding problem includes two aspects: structure prediction and folding mechanism. Although the former has been widely studied, the latter is still not well understood. Here we present a deep reinforcement learning algorithms 2dRNA-Fold to study the fastest folding paths of RNA secondary structure. 2dRNA-Fold uses a neural network combined with Monte Carlo tree search to select residue pairing step by step according to a given RNA sequence until the final secondary structure is formed. We apply 2dRNA-Fold to several short RNA molecules and one longer RNA 1Y26 and find that their fastest folding paths show some interesting features. 2dRNA-Fold is further trained using a set of RNA molecules from the dataset bpRNA and is used to predict RNA secondary structure. Since in 2dRNA-Fold the scoring to determine next step is based on possible base pairings, the learned or predicted fastest folding path may not agree with the actual folding paths determined by free energy according to physical laws.     

Fast folding of RNA pseudoknots initiated by laser temperature-jump

RNA pseudoknots are examples of minimal structural motifs in RNA with tertiary interactions that stabilize the structures of many ribozymes. They also play an essential role in a variety of biological functions that are modulated by their structure, stability, and dynamics. Therefore, understanding the global principles that determine the thermodynamics and folding pathways of RNA pseudoknots is an important problem in biology, both for elucidating the folding mechanisms of larger ribozymes as well as addressing issues of possible kinetic control of the biological functions of pseudoknots. We report on the folding/unfolding kinetics of a hairpin-type pseudoknot obtained with microsecond time-resolution in response to a laser temperature-jump perturbation. The kinetics are monitored using UV absorbance as well as fluorescence of extrinsically attached labels as spectroscopic probes of the transiently populated RNA conformations. We measure folding times of 1-6 ms at 37 °C, which are at least 100-fold faster than previous observations of very slow folding pseudoknots that were trapped in misfolded conformations. The measured relaxation times are remarkably similar to predictions of a computational study by Thirumalai and co-workers (Cho, S. S.; Pincus, D.L.; Thirumalai, D. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 17349-17354). Thus, these studies provide the first observation of a fast-folding pseudoknot and present a benchmark against which computational models can be refined.     

2'-Fluoro substituents can mimic native 2'-hydroxyls within structured RNA

The ability of fluorine in a C-F bond to act as?a hydrogen bond acceptor is controversial. To test such ability in complex RNA macromolecules, we have replaced native 2'-OH groups with 2'-F and 2'-H groups in two related systems, the Tetrahymena group I ribozyme and the ΔC209 P4-P6 RNA domain. In three cases the introduced 2'-F mimics the native 2'-OH group, suggesting that the fluorine atom can accept a hydrogen bond. In each of these cases the native hydroxyl group interacts with a purine exocyclic amine. Our results give insight about the properties of organofluorine and suggest a possible general biochemical signature for tertiary interactions between 2'-hydroxyl groups and exocyclic amino groups within RNA.     

Toward a digital gene response: RNA G-quadruplexes with fewer quartets fold with higher cooperativity

Changes in RNA conformation can alter gene expression. The guanine quadruplex sequence (GQS) is an RNA motif that folds in the presence of K(+) ions. Changes in the conformation of this motif could be especially important in regulating gene expression in plants because intracellular K(+) concentrations often increase during drought stress. Little is known about the folding thermodynamics of RNA GQS. We show here that RNA GQS with tracts containing three G's [e.g., (GGGxx)(4)] have a modest dependence on the K(+) concentration, folding with no or even negative cooperativity (Hill coefficients ≤1), and are associated with populated folding intermediates. In contrast, GQS with tracts containing just two G's [e.g., (GGxx)(4)] have a steep dependence on the K(+) concentration and fold with positive cooperativity (Hill coefficients of 1.7-2.7) without significantly populating intermediate states. We postulate that in plants, the more stable G3 sequences are largely folded even under unstressed conditions, while the less stable G2 sequences fold only at the higher K(+) concentrations associated with cellular stress, wherein they respond sharply to changing K(+) concentrations. Given the binary nature of their folding, G2 sequences may find application in computation with DNA and in engineering of genetic circuits.     

Is there a unique melting temperature for two-state proteins?

Thermal unfolding (or folding) in many proteins occurs in an apparent two-state manner, suggesting that only two states, unfolded and folded, are populated. At the melting temperature, Tm, the two states coexist. Using lattice models with side chains we show that individual residues become structured at temperatures that deviate from Tm, which implies that partially folded conformations make substantial contribution to thermodynamic properties of two-state proteins. We also find that the folding cooperativity for a given residue is linked to its accessible surface area. These results are consistent with the experiments on GCN4-like zipper peptide, which showed that local melting temperatures differ from Tm. Analysis of thermal unfolding of six proteins shows that deltaT/Tm approximately N(-1), where deltaT is the transition width and N is the number of residues. This scaling allows us to conclude that, when corrected for finite size effects, folding cooperativity can be captured using coarse grained models.     

Topology-based models and NMR structures in protein folding simulations

Topology-based interaction potentials are simplified models that use the native contacts in the folded structure of a protein to define an energetically unfrustrated folding funnel. They have been widely used to analyze the folding transition and pathways of different proteins through computer simulations. Obviously, they need a reliable, experimentally determined folded structure to define the model interactions. In structures elucidated through NMR spectroscopy, a complex treatment of the raw experimental data usually provides a series of models, a set of different conformations compatible with the available experimental data. Here, we use an efficient coarse-grained simulation technique to independently consider the contact maps from every different NMR model in a protein whose structure has been resolved by the use of NMR spectroscopy. For lambda-Cro repressor, a homodimeric protein, we have analyzed its folding characteristics with a topology-based model. We have focused on the competition between the folding of the individual chains and their binding to form the final quaternary structure. From 20 different NMR models, we find a predominant three-state folding behavior, in agreement with experimental data on the folding pathway for this protein. Individual NMR models, however, show distinct characteristics, which are analyzed both at the level of the interplay between tertiary/quaternary structure formation and also regarding the thermal stability of the tertiary structure of every individual chain.     

RNA Hairpin Folding in the Crowded Cell

Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin‐structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high‐molecular‐weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.     

The shape-shifting quasispecies of RNA: one sequence, many functional folds

E Unus pluribum, or "Of One, Many", may be at the root of decoding the RNA sequence-structure-function relationship. RNAs embody the large majority of genes in higher eukaryotes and fold in a sequence-directed fashion into three-dimensional structures that perform functions conserved across all cellular life forms, ranging from regulating to executing gene expression. While it is the most important determinant of the RNA structure, the nucleotide sequence is generally not sufficient to specify a unique set of secondary and tertiary interactions due to the highly frustrated nature of RNA folding. This frustration results in folding heterogeneity, a common phenomenon wherein a chemically homogeneous population of RNA molecules folds into multiple stable structures. Often, these alternative conformations constitute misfolds, lacking the biological activity of the natively folded RNA. Intriguingly, a number of RNAs have recently been described as capable of adopting multiple distinct conformations that all perform, or contribute to, the same function. Characteristically, these conformations interconvert slowly on the experimental timescale, suggesting that they should be regarded as distinct native states. We discuss how rugged folding free energy landscapes give rise to multiple native states in the Tetrahymena Group I intron ribozyme, hairpin ribozyme, sarcin-ricin loop, ribosome, and an in vitro selected aptamer. We further describe the varying degrees to which folding heterogeneity impacts function in these RNAs, and compare and contrast this impact with that of heterogeneities found in protein folding. Embracing that one sequence can give rise to multiple native folds, we hypothesize that this phenomenon imparts adaptive advantages on any functionally evolving RNA quasispecies.     

Quantification of the π-π interactions that govern tertiary structure in donor-acceptor [2]pseudorotaxanes

Flexibility in pseudorotaxanes and interlocked molecules that rely on interactions between π-donor-acceptor subunits provides access to folded structures reminiscent of the tertiary structure of proteins. While they have been described before, only now have we been able to quantify one such tertiary structure by making use of pseudorotaxanes designed for the purpose. Here, the enhanced stability of a pseudorotaxane inside a folded structure is measured to be ΔG = ca. 0.5 kcal mol(-1). The tertiary structure is stabilized by a charge-transfer interaction between a tetrathiafulvalene-based π-donor that can situate alongside a π-accepting paraquat-based macrocycle by folding of a flexible linker. At room temperature, it was estimated that 70% of the pseudorotaxanes examined here exist in their folded state. This quantitative information is critical for the creation of interlocked molecular machines that have predictable energetics and structures and for revealing a complexity approaching biological molecules.     

Biomimetic Transmembrane Channels with High Stability and Transporting Efficiency from Helically Folded Macromolecules

Membrane channels span the cellular lipid bilayers to transport ions and molecules into cells with sophisticated properties including high efficiency and selectivity. It is of particular biological importance in developing biomimetic transmembrane channels with unique functions by means of chemically synthetic strategies. An artificial unimolecular transmembrane channel using pore‐containing helical macromolecules is reported. The self‐folding, shape‐persistent, pore‐containing helical macromolecules are able to span the lipid bilayer, and thus result in extraordinary channel stability and high transporting efficiency for protons and cations. The lifetime of this artificial unimolecular channel in the lipid bilayer membrane is impressively long, rivaling those of natural protein channels. Natural channel mimics designed by helically folded polymeric scaffolds will display robust and versatile transport‐related properties at single‐molecule level.     

Tryptophan-BODIPY: a versatile donor-acceptor pair for probing generic changes of intraprotein distances

We demonstrate that Tryptophan (Trp) and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide (BODIPY) is a suitable donor-acceptor (D-A) pair for intraprotein distance measurements, applicable to the study of protein folding. The suitability of the Trp-BODIPY electronic energy transfer is exemplified on the extensively-characterised two-state protein, S6, from Thermus thermophilus. This protein has proved to be useful for the elucidation of folding cooperativity and nucleation, as well as the changes upon induction of structural transitions. For a comprehensive structural coverage, BODIPY molecules were anchored by Cys insertions at four different positions on the S6 surface. Trp residues at position 33 or 62 acted as donors of electronic energy to the BODIPY groups. None of the D-A pairs show any detectable difference in the folding kinetics (or protein stability), which supports the notion that the two-state transition of S6 is a highly concerted process. Similar results are obtained for mutants affecting the N- and C-terminus. The kinetic analyses indicate that changes of the transition state occur through local unfolding of the native state, rather than by a decrease of the folding cooperativity. The distances obtained from the analysis of the time-resolved fluorescence experiments in the native state were compared to those calculated from X-ray structure. As an additional measure, molecular dynamics simulations of the different protein constructs were performed to account for variability in the BODIPY location on the protein surface. The agreement between fluorescence and X-ray data is quite convincing, and shows that energy transfer measurements between Trp and BODIPY can probe distances between ca. 17 to 34 A, with an error better than 10%.     

Ubiquitin: a small protein folding paradigm

For the past twenty years, the small, 76-residue protein ubiquitin has been used as a model system to study protein structure, stability, folding and dynamics. In this time, ubiquitin has become a paradigm for both the experimental and computational folding communities. The folding energy landscape is now uniquely characterised with a plethora of information available on not only the native and denatured states, but partially structured states, alternatively folded states and locally unfolded states, in addition to the transition state ensemble. This Perspective focuses on the experimental characterisation of ubiquitin using a comprehensive range of biophysical techniques.     

Insertion and assembly of membrane proteins via simulation

Interactions of lipids are central to the folding and stability of membrane proteins. Coarse-grained molecular dynamics simulations have been used to reveal the mechanisms of self-assembly of protein/membrane and protein/detergent complexes for representatives of two classes of membrane protein, namely, glycophorin (a simple alpha-helical bundle) and OmpA (a beta-barrel). The accuracy of the coarse-grained simulations is established via comparison with the equivalent atomistic simulations of self-assembly of protein/detergent micelles. The simulation of OmpA/bilayer self-assembly reveals how a folded outer membrane protein can be inserted in a bilayer. The glycophorin/bilayer simulation supports the two-state model of membrane folding, in which transmembrane helix insertion precedes dimer self-assembly within a bilayer. The simulations also suggest that a dynamic equilibrium exists between the glycophorin helix monomer and dimer within a bilayer. The simulated glycophorin helix dimer is remarkably close in structure to that revealed by NMR. Thus, coarse-grained methods may help to define mechanisms of membrane protein (re)folding and will prove suitable for simulation of larger scale dynamic rearrangements of biological membranes.     

Time-resolved RNA SHAPE chemistry

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry yields quantitative RNA secondary and tertiary structure information at single nucleotide resolution. SHAPE takes advantage of the discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl group is modulated by local nucleotide flexibility in the RNA backbone. Flexible nucleotides are reactive toward hydroxyl-selective electrophiles, whereas constrained nucleotides are unreactive. Initial versions of SHAPE chemistry, which employ isatoic anhydride derivatives that react on the minute time scale, are emerging as the ideal technology for monitoring equilibrium structures of RNA in a wide variety of biological environments. Here, we extend SHAPE chemistry to a benzoyl cyanide scaffold to make possible facile time-resolved kinetic studies of RNA in approximately 1 s snapshots. We then use SHAPE chemistry to follow the time-dependent folding of an RNase P specificity domain RNA. Tertiary interactions form in two distinct steps with local tertiary contacts forming an order of magnitude faster than long-range interactions. Rate-determining tertiary folding requires minutes despite that no non-native interactions must be disrupted to form the native structure. Instead, overall folding is limited by simultaneous formation of interactions approximately 55 A distant in the RNA. Time-resolved SHAPE holds broad potential for understanding structural biogenesis and the conformational interconversions essential to the functions of complex RNA molecules at single nucleotide resolution.     

Downhill protein folding modules as scaffolds for broad-range ultrafast biosensors

Conformational switches are macromolecules that toggle between two states (active/inactive or folded/unfolded) upon specific binding to a target molecule. These molecular devices provide an excellent scaffold for developing real-time biosensors. Here we take this concept one step beyond to build high-performance conformational rheostat sensors. The rationale is to develop sensors with expanded dynamic range and faster response time by coupling a given signal to the continuous (rather than binary) unfolding process of one-state downhill folding protein modules. As proof of concept we investigate the pH and ionic-strength sensing capabilities of the small α-helical protein BBL. Our results reveal that such a pH/ionic-strength sensor exhibits a linear response over 4 orders of magnitude in analyte concentration, compared to the 2 orders of magnitude for switches, and nearly concentration-independent microsecond response times.     

MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures

One major challenge in nucleic acids analysis by hybridization probes is a compromise between the probe's tight binding and sequence‐selective recognition of nucleic acid targets folded into stable secondary structures. We have been developing a four‐way junction (4WJ)‐based sensor that consists of a universal stem‐loop (USL) probe immobilized on an electrode surface and two adaptor strands (M and F). The sensor was shown to be highly selective towards single base mismatches at room temperature, able to detect multiple targets using the same USL probe, and have improved ability to detect folded nucleic acids. However, some nucleic acid targets, including natural RNA, are folded into very stable secondary and tertiary structures, which may represent a challenge even for the 4WJ sensors. This work describes a new sensor, named MVF since it uses three probe stands M, V and F, which further improves the performance of 4WJ sensors with folded targets. The MVF sensor interrogating a 16S rRNA NASBA amplicon with calculated folding energy of ?32.82 kcal/mol has demonstrated 2.5‐fold improvement in a signal‐to‐background ratio in comparison with a 4WJ sensor lacking strand V. The proposed design can be used as a general strategy in the analysis of folded nucleic acids including natural RNA.