Salt-Dependent Self-Association of Trinucleotide Repeat RNA Sequences.

Repeat RNA sequences self-associate to form condensates. Simulations of a coarse-grained single-interaction site model for (CAG)n (n = 30 and 31) show that the salt-dependent free energy gap, ΔGS, between the ground (perfect hairpin) and the excited state (slipped hairpin (SH) with one CAG overhang) of the monomer for (n even) is the primary factor that determines the rates and yield of self-assembly. For odd n, the free energy (GS) of the ground state, which is an SH, is used to predict the self-association kinetics. As the monovalent salt concentration, CS, increases, ΔGS and GS increase, which decreases the rates of dimer formation. In contrast, ΔGS for shuffled sequences, with the same length and sequence composition as (CAG)31, is larger, which suppresses their propensities to aggregate. Although demonstrated explicitly for (CAG) polymers, the finding of inverse correlation between the free energy gap and RNA aggregation is general.

Competition between Stacking and Divalent Cation-Mediated Electrostatic Interactions Determines the Conformations of Short DNA Sequences.

Interplay between divalent cations (Mg2+ and Ca2+) and single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), as well as stacking interactions, is important in nucleosome stability and phase separation in nucleic acids. Quantitative techniques accounting for ion-DNA interactions are needed to obtain insights into these and related problems. Toward this end, we created a sequence-dependent computational TIS-ION model that explicitly accounts for monovalent and divalent ions. Simulations of the rigid 24 base-pair (bp) dsDNA and flexible ssDNA sequences, dT30 and dA30, with varying amounts of the divalent cations show that the calculated excess number of ions around the dsDNA and ssDNA agree quantitatively with ion-counting experiments. Using an ensemble of all-atom structures generated from coarse-grained simulations, we calculated the small-angle X-ray scattering profiles, which are in excellent agreement with experiments. Although ion-counting experiments mask the differences between Mg2+ and Ca2+, we find that Mg2+ binds to the minor grooves and phosphate groups, whereas Ca2+ binds specifically to the minor groove. Both Mg2+ and Ca2+ exhibit a tendency to bind to the minor groove of DNA as opposed to the major groove. The dA30 conformations are dominated by stacking interactions, resulting in structures with considerable helical order. The near cancellation of the favorable stacking and unfavorable electrostatic interactions leads to dT30 populating an ensemble of heterogeneous conformations. The successful applications of the TIS-ION model are poised to confront many problems in DNA biophysics.

Intraperitoneal Administration of p53-armed Oncolytic Adenovirus Inhibits Peritoneal Metastasis of Diffuse-type Gastric Cancer Cells.

BACKGROUND/AIM: Diffuse-type gastric cancer (GC) frequently exhibits peritoneal metastasis, leading to poor prognosis. However, efforts to develop antitumor strategies for preventing the peritoneal metastasis of GC have been unsuccessful. As diffuse-type GC cells often carry a genetic alteration in the tumor suppressor p53 gene, p53 restoration may be a potent strategy for preventing peritoneal metastasis of GC. In this study, we investigated the therapeutic potential of p53-expressing adenoviral vectors against peritoneal metastasis of diffuse-type GC cells. MATERIALS AND METHODS: Three diffuse-type human GC cell types with different p53 statuses (p53-wild type NUGC-4, p53-mutant type GCIY, and p53-null type KATOIII) were used to evaluate the therapeutic potential of p53 activation induced by the p53-expressing, replication-deficient adenovirus Ad-p53 and oncolytic adenovirus OBP-702. Viability, apoptosis, and autophagy of virus-treated GC cells were analyzed under normal and sphere-forming culture conditions using the XTT assay and western blot analysis. The in vivo antitumor effects of OBP-702 and Ad-p53 were assessed using xenograft tumor models involving peritoneal metastasis of NUGC-4 and GCIY cells. RESULTS: Under normal and sphere-forming culture conditions, OBP-702 induced a significantly greater antitumor effect in GC cells compared with Ad-p53 by strongly inducing p53-mediated apoptosis and autophagy and receptor tyrosine kinase suppression. In vivo experiments demonstrated that intraperitoneal administration of OBP-702 significantly suppressed the peritoneal metastasis of NUGC-4 and GCIY cells compared with Ad-p53, leading to prolonged survival of mice. CONCLUSION: Intraperitoneal administration of OBP-702 inhibits the peritoneal metastasis of GC cells by inducing p53-mediated cytopathic activity.

p53-armed oncolytic adenovirus induces autophagy and apoptosis in KRAS and BRAF-mutant colorectal cancer cells.

Colorectal cancer (CRC) cells harboring KRAS or BRAF mutations show a more-malignant phenotype than cells with wild-type KRAS and BRAF. KRAS/BRAF-wild-type CRCs are sensitive to epidermal growth factor receptor (EGFR)-targeting agents, whereas KRAS/BRAF-mutant CRCs are resistant due to constitutive activation of the EGFR-downstream KRAS/BRAF signaling pathway. Novel therapeutic strategies to treat KRAS/BRAF mutant CRC cells are thus needed. We recently demonstrated that the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 exhibit therapeutic potential against KRAS-mutant human pancreatic cancer cells. In this study, we evaluated the therapeutic potential of OBP-301 and OBP-702 against human CRC cells with differing KRAS/BRAF status. Human CRC cells with wild-type KRAS/BRAF (SW48, Colo320DM, CACO-2), mutant KRAS (DLD-1, SW620, HCT116), and mutant BRAF (RKO, HT29, COLO205) were used in this study. The antitumor effect of OBP-301 and OBP-702 against CRC cells was analyzed using the XTT assay. Virus-mediated modulation of apoptosis, autophagy, and the EGFR-MEK-ERK and AKT-mTOR signaling pathways was analyzed by Western blotting. Wild-type and KRAS-mutant CRC cells were sensitive to OBP-301 and OBP-702, whereas BRAF-mutant CRC cells were sensitive to OBP-702 but resistant to OBP-301. Western blot analysis demonstrated that OBP-301 induced autophagy and that OBP-702 induced autophagy and apoptosis in human CRC cells. In BRAF-mutant CRC cells, OBP-301 and OBP-702 suppressed the expression of EGFR, MEK, ERK, and AKT proteins, whereas mTOR expression was suppressed only by OBP-702. Our results suggest that p53-armed oncolytic virotherapy is a viable therapeutic option for treating KRAS/BRAF-mutant CRC cells via induction of autophagy and apoptosis.

Watching ion-driven kinetics of ribozyme folding and misfolding caused by energetic and topological frustration one molecule at a time.

Folding of ribozymes into well-defined tertiary structures usually requires divalent cations. How Mg2+ ions direct the folding kinetics has been a long-standing unsolved problem because experiments cannot detect the positions and dynamics of ions. To address this problem, we used molecular simulations to dissect the folding kinetics of the Azoarcus ribozyme by monitoring the path each molecule takes to reach the folded state. We quantitatively establish that Mg2+ binding to specific sites, coupled with counter-ion release of monovalent cations, stimulate the formation of secondary and tertiary structures, leading to diverse pathways that include direct rapid folding and trapping in misfolded structures. In some molecules, key tertiary structural elements form when Mg2+ ions bind to specific RNA sites at the earliest stages of the folding, leading to specific collapse and rapid folding. In others, the formation of non-native base pairs, whose rearrangement is needed to reach the folded state, is the rate-limiting step. Escape from energetic traps, driven by thermal fluctuations, occurs readily. In contrast, the transition to the native state from long-lived topologically trapped native-like metastable states is extremely slow. Specific collapse and formation of energetically or topologically frustrated states occur early in the assembly process.

Odd-even disparity in the population of slipped hairpins in RNA repeat sequences with implications for phase separation.

Low-complexity nucleotide repeat sequences, which are implicated in several neurological disorders, undergo liquid-liquid phase separation (LLPS) provided the number of repeat units, n, exceeds a critical value. Here, we establish a link between the folding landscapes of the monomers of trinucleotide repeats and their propensity to self-associate. Simulations using a coarse-grained Self-Organized Polymer (SOP) model for (CAG)n repeats in monovalent salt solutions reproduce experimentally measured melting temperatures, which are available only for small n. By extending the simulations to large n, we show that the free-energy gap, ΔGS, between the ground state (GS) and slipped hairpin (SH) states is a predictor of aggregation propensity. The GS for even n is a perfect hairpin (PH), whereas it is a SH when n is odd. The value of ΔGS (zero for odd n) is larger for even n than for odd n. As a result, the rate of dimer formation is slower in (CAG)30 relative to (CAG)31, thus linking ΔGS to RNA-RNA association. The yield of the dimer decreases dramatically, compared to the wild type, in mutant sequences in which the population of the SH decreases substantially. Association between RNA chains is preceded by a transition to the SH even if the GS is a PH. The finding that the excitation spectrum-which depends on the exact sequence, n, and ionic conditions-is a predictor of self-association should also hold for other RNAs (mRNA for example) that undergo LLPS.

Sterile protection and transmission blockade by a multistage anti-malarial vaccine in the pre-clinical study.

The Malaria Vaccine Technology Roadmap 2013 (World Health Organization) aims to develop safe and effective vaccines by 2030 that will offer at least 75% protective efficacy against clinical malaria and reduce parasite transmission. Here, we demonstrate a highly effective multistage vaccine against both the pre-erythrocytic and sexual stages of Plasmodium falciparum that protects and reduces transmission in a murine model. The vaccine is based on a viral-vectored vaccine platform, comprising a highly-attenuated vaccinia virus strain, LC16m8Δ (m8Δ), a genetically stable variant of a licensed and highly effective Japanese smallpox vaccine LC16m8, and an adeno-associated virus (AAV), a viral vector for human gene therapy. The genes encoding P. falciparum circumsporozoite protein (PfCSP) and the ookinete protein P25 (Pfs25) are expressed as a Pfs25-PfCSP fusion protein, and the heterologous m8Δ-prime/AAV-boost immunization regimen in mice provided both 100% protection against PfCSP-transgenic P. berghei sporozoites and up to 100% transmission blocking efficacy, as determined by a direct membrane feeding assay using parasites from P. falciparum-positive, naturally-infected donors from endemic settings. Remarkably, the persistence of vaccine-induced immune responses were over 7 months and additionally provided complete protection against repeated parasite challenge in a murine model. We propose that application of the m8Δ/AAV malaria multistage vaccine platform has the potential to contribute to the landmark goals of the malaria vaccine technology roadmap, to achieve life-long sterile protection and high-level transmission blocking efficacy.

Condensates in RNA repeat sequences are heterogeneously organized and exhibit reptation dynamics.

Although it is known that RNA undergoes liquid-liquid phase separation, the interplay between the molecular driving forces and the emergent features of the condensates, such as their morphologies and dynamic properties, is not well understood. We introduce a coarse-grained model to simulate phase separation of trinucleotide repeat RNAs, which are implicated in neurological disorders. After establishing that the simulations reproduce key experimental findings, we show that once recruited inside the liquid droplets, the monomers transition from hairpin-like structures to extended states. Interactions between the monomers in the condensates result in the formation of an intricate and dense intermolecular network, which severely restrains the fluctuations and mobilities of the RNAs inside large droplets. In the largest densely packed high-viscosity droplets, the mobility of RNA chains is best characterized by reptation, reminiscent of the dynamics in polymer melts. Our work provides a microscopic framework for understanding liquid-liquid phase separation in RNA, which is not easily discernible in current experiments.

VGLL3 increases the dependency of cancer cells on de novo nucleotide synthesis through GART expression.

Vestigial-like family member 3 (VGLL3) is a member of the VGLL family that serves as cofactors for TEA-domain transcription factors. Although VGLL3 is involved in the proliferation of cancer cells, the molecular mechanisms underlying VGLL3-mediated cell proliferation remain largely unknown. In this study, we found that stable expression of VGLL3 in human lung cancer A549 cells affects glutamine metabolism and increases their dependency on de novo nucleotide synthesis for proliferation. Mechanistically, VGLL3 was found to induce the expression of GART, which encodes a trifunctional enzyme that catalyzes de novo purine synthesis from glutamine. GART knockdown and the glycinamide ribonucleotide synthase, aminoimidazole ribonucleotide synthase, and glycinamide ribonucleotide formyltransferase trifunctional protein (GART) inhibitor lometrexol repressed the proliferation and survival of A549 cells stably expressing VGLL3. Mesenchymal breast cancer BT549 cells and MDA-MB-231 cells showed high expression of VGLL3, and VGLL3 knockdown was found to reduce GART expression. Lometrexol also repressed the proliferation of these breast cancer cells, whereas addition of inosine monophosphate, an important metabolite downstream of GART, rescued this repression. Taken together, these results suggest that VGLL3 induces GART expression and thereby confers de novo nucleotide-dependent cell proliferation in cancer cells.

Vestigial-like family member 3 stimulates cell motility by inducing high-mobility group AT-hook 2 expression in cancer cells.

Vestigial-like family member 3 (VGLL3) is a cofactor for TEA domain transcription factors (TEADs). Although VGLL3 is known to be highly expressed and stimulate cell proliferation in mesenchymal cancer cells, its involvement in mesenchymal phenotypes is largely unknown. In this study, we found that VGLL3 promotes epithelial-to-mesenchymal transition (EMT)-like phenotypic changes. We found that A549 human lung cancer cells stably expressing VGLL3 exhibit spindle-like morphological changes, reduction in the epithelial marker E-cadherin and induction of the mesenchymal marker Snail. Notably, VGLL3-expressing cells exhibited enhanced motility. The DNA-binding protein high-mobility group AT-hook 2 (HMGA2) was found to be a target of the VGLL3-TEAD4 complex, and HMGA2 knockdown repressed EMT-like phenotypic changes in VGLL3-expressing cells. VGLL3-dependent phenotypic changes are involved in transforming growth factor-ß (TGF-ß)-induced EMT progression. VGLL3 or HMGA2 knockdown repressed the motility of the mesenchymal breast cancer MDA-MB-231 cells. Importantly, high levels of VGLL3 expression were shown to have a positive correlation with poor prognosis in various human cancers, such as breast, colon, ovarian, head and neck, pancreatic, renal, gastric and cervical cancers. These results suggest that VGLL3 promotes EMT-like cell motility by inducing HMGA2 expression and accelerates cancer progression.

VGLL3 activates inflammatory responses by inducing interleukin-1α secretion.

Vestigial-like family member 3 (VGLL3), a member of the vestigial-like family, is a cofactor of the TEA-domain-containing transcription factor (TEAD). Although elevation in VGLL3 expression is associated with inflammatory diseases, such as inflammatory sarcomas and autoimmune diseases, the molecular mechanisms underlying VGLL3-mediated inflammation remain largely unknown. In this study, we analyzed the relationship between elevated VGLL3 expression and the levels of NF-κB, a transcription factor that plays a pivotal role in inflammation. NF-κB was found to be activated in a cell line stably expressing VGLL3. Mechanistically, VGLL3 was shown to promote the expression and secretion of the potent NF-κB-activating cytokine interleukin (IL)-1α, probably through its association with TEADs. As VGLL3 is a target of transforming growth factor ß (TGF-ß) signaling, we analyzed IL-1α induction upon TGF-ß stimulation. TGF-ß stimulation was observed to induce IL-1α secretion and NF-κB activation, and VGLL3 was associated with this phenomenon. The TGF-ß transcription factors Smad3 and Smad4 were shown to be necessary for inducing VGLL3 and IL-1α expression. Lastly, we found that VGLL3-dependent IL-1α secretion is involved in constitutive NF-κB activation in highly malignant breast cancer cells. Collectively, the findings suggested that VGLL3 expression and TGF-ß stimulation activate the inflammatory response by inducing IL-1α secretion.

Investigation of Factors Causing Nonuniformity in Luminescence Lifetime of Fast-Responding Pressure-Sensitive Paints.

Factors that cause nonuniformity in the luminescence lifetime of pressure-sensitive paints (PSPs) were investigated. The lifetime imaging method of PSP does not theoretically require wind-off reference images. Therefore, it can improve measurement accuracy because it can eliminate errors caused by the deformation or movement of the model during the measurement. However, it is reported that the luminescence lifetime of PSP is not uniform on the model, even under uniform conditions of pressure and temperature. Therefore, reference images are used to compensate for the nonuniformity of the luminescence lifetime, which significantly diminishes the advantages of the lifetime imaging method. In particular, fast-responding PSPs show considerable variation in luminescence lifetime compared to conventional polymer-based PSPs. Therefore, this study investigated and discussed the factors causing the nonuniformity of the luminescence lifetime, such as the luminophore solvent, luminophore concentrations, binder thickness, and spraying conditions. The results obtained suggest that the nonuniformity of the luminophore distribution in the binder caused by the various factors mentioned above during the coating process is closely related to the nonuniformity of the luminescence lifetime. For example, when the thickness of the binder became thinner than 8 µm, the fast-responding PSPs showed a tendency to vary significantly in the luminescence lifetime. In addition, it was found that the luminescence lifetime of fast-responding PSP could be changed in the depth direction of the binder depending on the coating conditions. Therefore, it is important to distribute the luminophore uniformly in the binder layer to create PSPs with a more uniform luminescence lifetime distribution.

Shape changes and cooperativity in the folding of the central domain of the 16S ribosomal RNA.

Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg2+ ions or by interaction with ribosomal proteins. To provide a quantitative description of ion-induced folding of the ∼350-nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg2+ concentration between 0 and 30 mM. The Mg2+ dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg2+ concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg2+ ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg2+-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg2+-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome.

Vestigial-like family member 3 (VGLL3), a cofactor for TEAD transcription factors, promotes cancer cell proliferation by activating the Hippo pathway.

Vestigial-like 3 (VGLL3) is a member of the VGLL family, whose members serve as cofactors for TEA domain-containing transcription factors (TEADs). TEADs promote tissue and tumor development together with the cofactors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Although VGLL3 is involved in tumor cell proliferation, its relationship with TEADs and YAP/TAZ remains largely unknown. To close this research gap, here we established tumor cells stably expressing VGLL3 and found that they exhibit enhanced proliferation. Notably, YAP and TAZ were inactivated in the VGLL3-expressing cells, coinciding with activation of the Hippo pathway, which suppresses YAP/TAZ activities. VGLL3 in combination with TEADs promoted expression of the Hippo pathway components large tumor suppressor kinase (LATS2) and angiomotin-like 2 (AMOTL2). VGLL3 was highly expressed in malignant breast tumor cells and osteosarcoma cells, and VGLL3 knockdown increased nuclear localization of YAP and TAZ. Knockdown of LATS2 or AMOTL2, as well as VGLL3 knockdown, repressed proliferation of breast tumor cells. Together, these results suggest that VGLL3 together with TEADs promotes cell proliferation by activating the Hippo pathway through LATS2 and AMOTL2, leading to YAP/TAZ inactivation.

Theory and simulations for RNA folding in mixtures of monovalent and divalent cations.

RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion-phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties.

Ion Condensation onto Ribozyme Is Site Specific and Fold Dependent.

The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state, although Mg2+ ions are needed for catalytic activities. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that most of the ions are territorially and nonspecifically bound to the RNA, as predicted by the counterion condensation theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained three-site interaction model with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible even at very low Mg2+ concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods, in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic nonmonotonic shape fluctuations in the ribozyme as it folds with increasing Mg2+ or Ca2+ concentration.

Molecular Simulations of Ion Effects on the Thermodynamics of RNA Folding.

How ions affect RNA folding thermodynamics and kinetics is an important but a vexing problem that remains unsolved. Experiments have shown that the free-energy change, Δ G( c), of RNA upon folding varies with the salt concentration ( c) as, Δ G( c) = k c ln c + const, where the coefficient k c is proportional to the difference in the ion preferential coefficient, ΔΓ. We performed simulations of a coarse-grained model, by modeling electrostatic interactions implicitly and with explicit representation of ions, to elucidate the molecular underpinnings of the relationship between Δ G and ΔΓ. The simulations quantitatively reproduce the heat capacity for a pseudoknot, thus validating the model. We show that Δ G( c), calculated directly from ΔΓ, varies linearly with ln c ( c < 0.2 M), for a hairpin and the pseudoknot, demonstrating a molecular link between the two quantities. Explicit ion simulations also show the linear dependence of Δ G( c) on ln c at all c with k c = 2 kB T, except that Δ G( c) values are shifted by ∼2 kcal/mol higher than experiments. The discrepancy is due to an underestimation of Γ for both the folded and unfolded states while giving accurate values for ΔΓ. The predictions for the salt dependence of ΔΓ are amenable to test using single-molecule pulling experiments. The framework provided here can be used to obtain accurate thermodynamics for other RNA molecules as well.

Frictional Effects on RNA Folding: Speed Limit and Kramers Turnover.

We investigated frictional effects on the folding rates of a human telomerase hairpin (hTR HP) and H-type pseudoknot from the Beet Western Yellow Virus (BWYV PK) using simulations of the Three Interaction Site (TIS) model for RNA. The heat capacity from TIS model simulations, calculated using temperature replica exchange simulations, reproduces nearly quantitatively the available experimental data for the hTR HP. The corresponding results for BWYV PK serve as predictions. We calculated the folding rates ( kF) from more than 100 folding trajectories for each value of the solvent viscosity (η) at a fixed salt concentration of 200 mM. By using the theoretical estimate (∝ √N where N is the number of nucleotides) for folding free energy barrier, kF data for both the RNAs are quantitatively fit using one-dimensional Kramers's theory with two parameters specifying the curvatures in the unfolded basin and the barrier top. In the high-friction regime (η ≳ 10-5 Pa·s), for both HP and PK, kF values decrease as 1/η, whereas in the low friction regime, kF values increase as η increases, leading to a maximum folding rate at a moderate viscosity (∼10-6 Pa·s), which is the Kramers turnover. From the fits, we find that the speed limit to RNA folding at water viscosity is between 1 and 4 µs, which is in accord with our previous theoretical prediction as well as results from several single molecule experiments. Both the RNA constructs fold by parallel pathways. Surprisingly, we find that the flux through the pathways could be altered by changing solvent viscosity, a prediction that is more easily testable in RNA than in proteins.

Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot.

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes and ribonucleoprotein complexes, are determined by their structure, stability, and dynamics. Therefore, it is important to elucidate the general principles governing their thermodynamics/folding mechanisms. Here, we combine laser temperature-jump experiments and coarse-grained simulations to determine the folding/unfolding pathways of VPK, a variant of the mouse mammary tumor virus (MMTV) PK involved in ribosomal frameshifting. Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at different stem/loop positions in the PK serve as local probes allowing us to monitor the order of assembly of VPK that has two constituent hairpins with different intrinsic stabilities. We show that at 50 mM KCl, the dominant folding pathway populates only the more stable hairpin intermediate; as the salt concentration is increased, a parallel folding pathway emerges involving the less stable hairpin as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. Our findings support the principle that the order of PK structure formation is determined by the relative stabilities of the hairpins, which can be altered by sequence variations or salt concentrations. The experimental results of salt effects on the partitioning between the two folding pathways are in remarkable agreement with simulations that were performed with no adjustable parameters. Our study not only unambiguously demonstrates that VPK folds by parallel pathways but also showcases the power of combining experiments and simulations for a more enriched description of RNA self-assembly.

Sequence-Dependent Three Interaction Site Model for Single- and Double-Stranded DNA.

We develop a robust coarse-grained model for single- and double-stranded DNA by representing each nucleotide by three interaction sites (TIS) located at the centers of mass of sugar, phosphate, and base. The resulting TIS model includes base-stacking, hydrogen bond, and electrostatic interactions as well as bond-stretching and bond angle potentials that account for the polymeric nature of DNA. The choices of force constants for stretching and the bending potentials were guided by a Boltzmann inversion procedure using a large representative set of DNA structures extracted from the Protein Data Bank. Some of the parameters in the stacking interactions were calculated using a learning procedure, which ensured that the experimentally measured melting temperatures of dimers are faithfully reproduced. Without any further adjustments, the calculations based on the TIS model reproduce the experimentally measured salt and sequence-dependence of the size of single-stranded DNA (ssDNA), as well as the persistence lengths of poly(dA) and poly(dT) chains. Interestingly, upon application of mechanical force, the extension of poly(dA) exhibits a plateau, which we trace to the formation of stacked helical domains. In contrast, the force-extension curve (FEC) of poly(dT) is entropic in origin and could be described by a standard polymer model. We also show that the persistence length of double-stranded DNA, formed from two complementary ssDNAs, is consistent with the prediction based on the worm-like chain. The persistence length, which decreases with increasing salt concentration, is in accord with the Odijk-Skolnick-Fixman theory intended for stiff polyelectrolyte chains near the rod limit. Our model predicts the melting temperatures of DNA hairpins with excellent accuracy, and we are able to recover the experimentally known sequence-specific trends. The range of applications, which did not require adjusting any parameter after the initial construction based solely on PDB structures and melting profiles of dimers, attests to the transferability and robustness of the TIS model for ssDNA and dsDNA.