The origin of different bending stiffness between double-stranded RNA and DNA revealed by magnetic tweezers and simulations.

The subtle differences in the chemical structures of double-stranded (ds) RNA and DNA lead to significant variations in their biological roles and medical implications, largely due to their distinct biophysical properties, such as bending stiffness. Although it is well known that A-form dsRNA is stiffer than B-form dsDNA under physiological salt conditions, the underlying cause of this difference remains unclear. In this study, we employ high-precision magnetic-tweezer experiments along with molecular dynamics simulations and reveal that the relative bending stiffness between dsRNA and dsDNA is primarily determined by the structure- and salt-concentration-dependent ion distribution around their helical structures. At near-physiological salt conditions, dsRNA shows a sparser ion distribution surrounding its phosphate groups compared to dsDNA, causing its greater stiffness. However, at very high monovalent salt concentrations, phosphate groups in both dsRNA and dsDNA become fully neutralized by excess ions, resulting in a similar intrinsic bending persistence length of approximately 39 nm. This similarity in intrinsic bending stiffness of dsRNA and dsDNA is coupled to the analogous fluctuations in their total groove widths and further coupled to the similar fluctuation of base-pair inclination, despite their distinct A-form and B-form helical structures.

Universality in RNA and DNA deformations induced by salt, temperature change, stretching force, and protein binding.

Nucleic acid deformations play important roles in many biological processes. The physical understanding of nucleic acid deformation by environmental stimuli is limited due to the challenge in the precise measurement of RNA and DNA deformations and the complexity of interactions in RNA and DNA. Magnetic tweezers experiments provide an excellent opportunity to precisely measure DNA and RNA twist changes induced by environmental stimuli. In this work, we applied magnetic tweezers to measure double-stranded RNA twist changes induced by salt and temperature changes. We observed RNA unwinds when lowering salt concentration, or increasing temperature. Our molecular dynamics simulations revealed the mechanism: lowering salt concentration or increasing temperature enlarges RNA major groove width, which causes twist decrease through twist-groove coupling. Combining these results with previous results, we found some universality in RNA and DNA deformations induced by three different stimuli: salt change, temperature, and stretching force. For RNA, these stimuli first modify the major groove width, which is transduced into twist change through twist-groove coupling. For DNA, these stimuli first modify diameter, which is transduced into twist change through twist-diameter coupling. Twist-groove coupling and twist-diameter coupling appear to be utilized by protein binding to reduce DNA and RNA deformation energy cost upon protein binding.

5-Methyl-cytosine stabilizes DNA but hinders DNA hybridization revealed by magnetic tweezers and simulations.

5-Methyl-cytosine (5mC) is one of the most important DNA modifications and plays versatile biological roles. It is well known that 5mC stabilizes DNA duplexes. However, it remains unclear how 5mC affects the kinetics of DNA melting and hybridization. Here, we studied the kinetics of unzipping and rezipping using a 502-bp DNA hairpin by single-molecule magnetic tweezers. Under constant loading rates, 5mC increases the unzipping force but counterintuitively decreases the rezipping force at various salt and temperature conditions. Under constant forces, the non-methylated DNA hops between metastable states during unzipping and rezipping, which implies low energy barriers. Surprisingly, the 5mC DNA can't rezip after fully unzipping unless much lower forces are applied, where it rezips stochastically in a one-step manner, which implies 5mC kinetically hinders DNA hybridization and high energy barriers in DNA hybridization. All-atom molecular dynamics simulations reveal that the 5mC kinetically hinders DNA hybridization due to steric effects rather than electrostatic effects caused by the additional methyl groups of cytosines. Considering the possible high speed of DNA unzipping and zipping during replication and transcription, our findings provide new insights into the biological roles of 5mC.

Predicting 3D structures and stabilities for complex RNA pseudoknots in ion solutions.

RNA pseudoknots are a kind of important tertiary motif, and the structures and stabilities of pseudoknots are generally critical to the biological functions of RNAs with the motifs. In this work, we have carefully refined our previously developed coarse-grained model with salt effect through involving a new coarse-grained force field and a replica-exchange Monte Carlo algorithm, and employed the model to predict structures and stabilities of complex RNA pseudoknots in ion solutions beyond minimal H-type pseudoknots. Compared with available experimental data, the newly refined model can successfully predict 3D structures from sequences for the complex RNA pseudoknots including SARS-CoV-2 programming-1 ribosomal frameshifting element and Zika virus xrRNA, and can reliably predict the thermal stabilities of RNA pseudoknots with various sequences and lengths over broad ranges of monovalent/divalent salts. In addition, for complex pseudoknots including SARS-CoV-2 frameshifting element, our analyses show that their thermally unfolding pathways are mainly dependent on the relative stabilities of unfolded intermediate states, in analogy to those of minimal H-type pseudoknots.

RNA 3D Structure Prediction: Progress and Perspective.

Ribonucleic acid (RNA) molecules play vital roles in numerous important biological functions such as catalysis and gene regulation. The functions of RNAs are strongly coupled to their structures or proper structure changes, and RNA structure prediction has been paid much attention in the last two decades. Some computational models have been developed to predict RNA three-dimensional (3D) structures in silico, and these models are generally composed of predicting RNA 3D structure ensemble, evaluating near-native RNAs from the structure ensemble, and refining the identified RNAs. In this review, we will make a comprehensive overview of the recent advances in RNA 3D structure modeling, including structure ensemble prediction, evaluation, and refinement. Finally, we will emphasize some insights and perspectives in modeling RNA 3D structures.

rsRNASP: A residue-separation-based statistical potential for RNA 3D structure evaluation.

Knowledge-based statistical potentials have been shown to be rather effective in protein 3-dimensional (3D) structure evaluation and prediction. Recently, several statistical potentials have been developed for RNA 3D structure evaluation, while their performances are either still at a low level for the test datasets from structure prediction models or dependent on the "black-box" process through neural networks. In this work, we have developed an all-atom distance-dependent statistical potential based on residue separation for RNA 3D structure evaluation, namely rsRNASP, which is composed of short- and long-ranged potentials distinguished by residue separation. The extensive examinations against available RNA test datasets show that rsRNASP has apparently higher performance than the existing statistical potentials for the realistic test datasets with large RNAs from structure prediction models, including the newly released RNA-Puzzles dataset, and is comparable to the existing top statistical potentials for the test datasets with small RNAs or near-native decoys. In addition, rsRNASP is superior to RNA3DCNN, a recently developed scoring function through 3D convolutional neural networks. rsRNASP and the relevant databases are available to the public.

FebRNA: An automated fragment-ensemble-based model for building RNA 3D structures.

Knowledge of RNA three-dimensional (3D) structures is critical to understanding the important biological functions of RNAs. Although various structure prediction models have been developed, the high-accuracy predictions of RNA 3D structures are still limited to the RNAs with short lengths or with simple topology. In this work, we proposed a new model, namely FebRNA, for building RNA 3D structures through fragment assembly based on coarse-grained (CG) fragment ensembles. Specifically, FebRNA is composed of four processes: establishing the library of different types of non-redundant CG fragment ensembles regardless of the sequences, building CG 3D structure ensemble through fragment assembly, identifying top-scored CG structures through a specific CG scoring function, and rebuilding the all-atom structures from the top-scored CG ones. Extensive examination against different types of RNA structures indicates that FebRNA consistently gives the reliable predictions on RNA 3D structures, including pseudoknots, three-way junctions, four-way and five-way junctions, and RNAs in the RNA-Puzzles. FebRNA is available on the Web site: https://github.com/Tan-group/FebRNA.

Salt effect on thermodynamics and kinetics of a single RNA base pair.

Due to the polyanionic nature of RNAs, the structural folding of RNAs are sensitive to solution salt conditions, while there is still lack of a deep understanding of the salt effect on the thermodynamics and kinetics of RNAs at a single base-pair level. In this work, the thermodynamic and the kinetic parameters for the base-pair AU closing/opening at different salt concentrations were calculated by 3-µsec all-atom molecular dynamics (MD) simulations at different temperatures. It was found that for the base-pair formation, the enthalpy change [Formula: see text] is nearly independent of salt concentration, while the entropy change [Formula: see text] exhibits a linear dependence on the logarithm of salt concentration, verifying the empirical assumption based on thermodynamic experiments. Our analyses revealed that such salt concentration dependence of the entropy change mainly results from the dependence of ion translational entropy change for the base pair closing/opening on salt concentration. Furthermore, the closing rate increases with the increasing of salt concentration, while the opening rate is nearly independent of salt concentration. Additionally, our analyses revealed that the free energy surface for describing the base-pair opening and closing dynamics becomes more rugged with the decrease of salt concentration.

Multivalent Cations Reverse the Twist-Stretch Coupling of RNA.

When stretched, both DNA and RNA duplexes change their twist angles through twist-stretch coupling. The coupling is negative for DNA but positive for RNA, which is not yet completely understood. Here, our magnetic tweezers experiments show that the coupling of RNA reverses from positive to negative by multivalent cations. Combining with the previously reported tension-induced negative-to-positive coupling reversal of DNA, we propose a unified mechanism of the couplings of both RNA and DNA based on molecular dynamics simulations. Two deformation pathways are competing when stretched: shrinking the radius causes positive couplings but widening the major groove causes negative couplings. For RNA whose major groove is clamped by multivalent cations and canonical DNA, their radii shrink when stretched, thus exhibiting positive couplings. For elongated DNA whose radius already shrinks to the minimum and canonical RNA, their major grooves are widened when stretched, thus exhibiting negative couplings.

Structure folding of RNA kissing complexes in salt solutions: predicting 3D structure, stability, and folding pathway.

RNA kissing complexes are essential for genomic RNA dimerization and regulation of gene expression, and their structures and stability are critical to their biological functions. In this work, we used our previously developed coarse-grained model with an implicit structure-based electrostatic potential to predict three-dimensional (3D) structures and stability of RNA kissing complexes in salt solutions. For extensive RNA kissing complexes, our model shows great reliability in predicting 3D structures from their sequences, and our additional predictions indicate that the model can capture the dependence of 3D structures of RNA kissing complexes on monovalent/divalent ion concentrations. Moreover, the comparisons with extensive experimental data show that the model can make reliable predictions on the stability for various RNA kissing complexes over wide ranges of monovalent/divalent ion concentrations. Notably, for RNA kissing complexes, our further analyses show the important contribution of coaxial stacking to the 3D structures and stronger stability than the corresponding kissing-interface duplexes at high salts. Furthermore, our comprehensive analyses for RNA kissing complexes reveal that the thermally folding pathway for a complex sequence is mainly determined by the relative stability of two possible folded states of kissing complex and extended duplex, which can be significantly modulated by its sequence.

What is the best reference state for building statistical potentials in RNA 3D structure evaluation?

Knowledge-based statistical potentials have been shown to be efficient in protein structure evaluation/prediction, and the core difference between various statistical potentials is attributed to the choice of reference states. However, for RNA 3D structure evaluation, a comprehensive examination on reference states is still lacking. In this work, we built six statistical potentials based on six reference states widely used in protein structure evaluation, including averaging, quasi-chemical approximation, atom-shuffled, finite-ideal-gas, spherical-noninteracting, and random-walk-chain reference states, and we examined the six reference states against three RNA test sets including six subsets. Our extensive examinations show that, overall, for identifying native structures and ranking decoy structures, the finite-ideal-gas and random-walk-chain reference states are slightly superior to others, while for identifying near-native structures, there is only a slight difference between these reference states. Our further analyses show that the performance of a statistical potential is apparently dependent on the quality of the training set. Furthermore, we found that the performance of a statistical potential is closely related to the origin of test sets, and for the three realistic test subsets, the six statistical potentials have overall unsatisfactory performance. This work presents a comprehensive examination on the existing reference states and statistical potentials for RNA 3D structure evaluation.

Cytosine Methylation Enhances DNA Condensation Revealed by Equilibrium Measurements Using Magnetic Tweezers.

CpG methylation of DNA is common in mammalian cells. In sperm, the DNA has the highest level of CpG methylation and is condensed into toroidal structures. How CpG methylation affects DNA structures and interactions is important to understand its biological roles but is largely unknown. Using an RNA-DNA-RNA structure, we observed the equilibrium hopping dynamics between the condensed and extended states of DNA in the presence of polyamines or polylysine peptide as a reduced model of histone tails. Combing with the measured DNA elasticities, we report that CpG methylation of each cytosine nucleotide substantially increases DNA-DNA attraction by up to 0.2 kBT. For the DNA with 57% GC content, the relative increase caused by CpG methylation is up to 32% for the spermine-induced DNA-DNA attraction and up to 9% for the polylysine-induced DNA-DNA attraction. These findings help us to evaluate the energetic contributions of CpG methylation in sperm development and chromatin regulation.

Opposite Effects of High-Valent Cations on the Elasticities of DNA and RNA Duplexes Revealed by Magnetic Tweezers.

We report that trivalent cobalt hexammine cations decrease the persistence length, stretching modulus, helical density, and size of plectonemes formed under torque of DNA but increase those of RNA. Divalent magnesium cations, however, decrease the persistence lengths, contour lengths, and sizes of plectonemes while increasing the helical densities of both DNA and RNA. The experimental results are explained by different binding modes of the cations on DNA and RNA in our all-atom molecular dynamics simulations. The significant variations of the helical densities and structures of DNA and RNA duplexes induced by high-valent cations may affect interactions of the duplexes with proteins.

Structural Flexibility of DNA-RNA Hybrid Duplex: Stretching and Twist-Stretch Coupling.

DNA-RNA hybrid (DRH) duplexes play essential roles during the replication of DNA and the reverse transcription of RNA viruses, and their flexibility is important for their biological functions. Recent experiments indicated that A-form RNA and B-form DNA have a strikingly different flexibility in stretching and twist-stretch coupling, and the structural flexibility of DRH duplex is of great interest, especially in stretching and twist-stretch coupling. In this work, we performed microsecond all-atom molecular dynamics simulations with new AMBER force fields to characterize the structural flexibility of DRH duplex in stretching and twist-stretch coupling. We have calculated all the helical parameters, stretch modulus, and twist-stretch coupling parameters for the DRH duplex. First, our analyses on structure suggest that the DRH duplex exhibits an intermediate conformation between A- and B-forms and closer to A-form, which can be attributed to the stronger rigidity of the RNA strand than the DNA strand. Second, our calculations show that the DRH duplex has the stretch modulus of 834 ± 34 pN and a very weak twist-stretch coupling. Our quantitative analyses indicate that, compared with DNA and RNA duplexes, the different flexibility of the DRH duplex in stretching and twist-stretch coupling is mainly attributed to its apparently different basepair inclination in the helical structure.

Predicting 3D structure and stability of RNA pseudoknots in monovalent and divalent ion solutions.

RNA pseudoknots are a kind of minimal RNA tertiary structural motifs, and their three-dimensional (3D) structures and stability play essential roles in a variety of biological functions. Therefore, to predict 3D structures and stability of RNA pseudoknots is essential for understanding their functions. In the work, we employed our previously developed coarse-grained model with implicit salt to make extensive predictions and comprehensive analyses on the 3D structures and stability for RNA pseudoknots in monovalent/divalent ion solutions. The comparisons with available experimental data show that our model can successfully predict the 3D structures of RNA pseudoknots from their sequences, and can also make reliable predictions for the stability of RNA pseudoknots with different lengths and sequences over a wide range of monovalent/divalent ion concentrations. Furthermore, we made comprehensive analyses on the unfolding pathway for various RNA pseudoknots in ion solutions. Our analyses for extensive pseudokonts and the wide range of monovalent/divalent ion concentrations verify that the unfolding pathway of RNA pseudoknots is mainly dependent on the relative stability of unfolded intermediate states, and show that the unfolding pathway of RNA pseudoknots can be significantly modulated by their sequences and solution ion conditions.

Apparent repulsion between equally and oppositely charged spherical polyelectrolytes in symmetrical salt solutions.

Ion-mediated interactions are very important for the properties of colloids and biomacromolecules such as nucleic acids and proteins. In this work, the ion-mediated interactions between equally and oppositely charged spherical polyelectrolytes (SPEs) in symmetrical divalent electrolytes have been investigated by Monte Carlo simulations, and an unexpected apparent repulsion was observed at high divalent salt concentration. Our investigations also show that the effective repulsion becomes more pronounced for SPEs with higher charge densities and for counterions with larger sizes and was found to be tightly accompanied with the over-neutralization to SPEs by condensed counterions and their release upon the approach of SPEs. Such attractive interaction can be reproduced by our proposed modified Poisson-Boltzmann model and is mainly attributed to the increase in the electrostatic repulsion between on charged SPE and the over-neutralized counterions around the other oppositely SPE with the approach of the two SPEs.

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

Double-stranded (ds) RNAs play essential roles in many processes of cell metabolism. The knowledge of three-dimensional (3D) structure, stability, and flexibility of dsRNAs in salt solutions is important for understanding their biological functions. In this work, we further developed our previously proposed coarse-grained model to predict 3D structure, stability, and flexibility for dsRNAs in monovalent and divalent ion solutions through involving an implicit structure-based electrostatic potential. The model can make reliable predictions for 3D structures of extensive dsRNAs with/without bulge/internal loops from their sequences, and the involvement of the structure-based electrostatic potential and corresponding ion condition can improve the predictions for 3D structures of dsRNAs in ion solutions. Furthermore, the model can make good predictions for thermal stability for extensive dsRNAs over the wide range of monovalent/divalent ion concentrations, and our analyses show that the thermally unfolding pathway of dsRNA is generally dependent on its length as well as its sequence. In addition, the model was employed to examine the salt-dependent flexibility of a dsRNA helix, and the calculated salt-dependent persistence lengths are in good accordance with experiments.

Competitive Binding of Mg2+ and Na+ Ions to Nucleic Acids: From Helices to Tertiary Structures.

Nucleic acids generally reside in cellular aqueous solutions with mixed divalent/monovalent ions, and the competitive binding of divalent and monovalent ions is critical to the structures of nucleic acids because of their polyanionic nature. In this work, we first proposed a general and effective method for simulating a nucleic acid in mixed divalent/monovalent ion solutions with desired bulk ion concentrations via molecular dynamics (MD) simulations and investigated the competitive binding of Mg2+/Na+ ions to various nucleic acids by all-atom MD simulations. The extensive MD-based examinations show that single MD simulations conducted using the proposed method can yield desired bulk divalent/monovalent ion concentrations for various nucleic acids, including RNA tertiary structures. Our comprehensive analyses show that the global binding of Mg2+/Na+ to a nucleic acid is mainly dependent on its structure compactness, as well as Mg2+/Na+ concentrations, rather than the specific structure of the nucleic acid. Specifically, the relative global binding of Mg2+ over Na+ is stronger for a nucleic acid with higher effective surface charge density and higher relative Mg2+/Na+ concentrations. Furthermore, the local binding of Mg2+/Na+ to a phosphate of a nucleic acid mainly depends on the local phosphate density in addition to Mg2+/Na+ concentrations.

Precise Antibody-Independent m6A Identification via 4SedTTP-Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution.

Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this "RT-silent" modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution.

Divalent Ion-Mediated DNA-DNA Interactions: A Comparative Study of Triplex and Duplex.

Ion-mediated interaction between DNAs is essential for DNA condensation, and it is generally believed that monovalent and nonspecifically binding divalent cations cannot induce the aggregation of double-stranded (ds) DNAs. Interestingly, recent experiments found that alkaline earth metal ions such as Mg2+ can induce the aggregation of triple-stranded (ts) DNAs, although there is still a lack of deep understanding of the surprising findings at the microscopic level. In this work, we employed all-atom dynamic simulations to directly calculate the potentials of mean force (PMFs) between tsDNAs, between dsDNAs, and between tsDNA and dsDNA in Mg2+ solutions. Our calculations show that the PMF between tsDNAs is apparently attractive and becomes more strongly attractive at higher [Mg2+], although the PMF between dsDNAs cannot become apparently attractive even at high [Mg2+]. Our analyses show that Mg2+ internally binds into grooves and externally binds to phosphate groups for both tsDNA and dsDNA, whereas the external binding of Mg2+ is much stronger for tsDNA. Such stronger external binding of Mg2+ for tsDNA favors more apparent ion-bridging between helices than for dsDNA. Furthermore, our analyses illustrate that bridging ions, as a special part of external binding ions, are tightly and positively coupled to ion-mediated attraction between DNAs.