Biology subjects

Hori, N.

Publications and source records attributed to Hori, N..

12 recordsLinked to original sources

bioRxiv2024

Shaving black fur uncovers hidden issues in p16-3MR mice

The p16-3MR mouse model has previously been reported to effectively visualize and eliminate senescent cells in vivo. However, we now report significant issues with this model. Noninvasive bioluminescence imaging without shaving the black fur detected almost no luminescence signals, but they became visible after shaving. However, these signals were very faint and attributed to the auto-luminescence of coelenterazine-h, rather than reporter gene expression. Further experiments showed no significant bioluminescent changes with aging, doxorubicin treatment, or during wound healing, contradicting earlier reports. Comparative analysis between p16-3MR mice from different sources revealed no differences, suggesting functional issues with Renilla luciferase (Rluc) and herpes simplex virus 1 thymidine kinase (HSV-TK) expression in this mouse model. Our findings underscore the necessity of using white-furred mice or ensuring proper fur shaving for accurate in vivo bioluminescence imaging. We recommend the reevaluation of previously published studies that used p16-3MR mice without proper controls to ensure data accuracy. These results provide critical insights for researchers using p16-3MR mice in senescence studies.

molecular biology↗

bioRxiv2023

Salt-dependent self-association of trinucleotide repeat RNA sequences

Low complexity repeat RNA sequences self-associate by homotypic interactions to form condensates. Using simulations of a coarse grained Single-Interaction Site model for (CAG)n (n = 30 and 31), we show that the salt-dependent free energy gap, {triangleup}GS, between the ground (perfect hairpin) and the excited state (slipped hairpin (SH) with one CAG overhang) of monomer (n even) is the primary factor that determines the rates and yield of self-assembly. For odd n, the SH ground state free energy (GS) is used to predict self-association kinetics. As the monovalent salt concentration, CS, increases {triangleup}GS and GS increases, which in turn decreases the self-association rates. In contrast, {triangleup}GS for scrambled sequences, with the same length and sequence composition as (CAG)31 but with higher complexity, is larger which greatly suppresses the propensities to aggregate. Although demonstrated explicitly for (CAG)30 and (CAG)31 polymers, the finding that there is an inverse correlation between CS-dependent {triangleup}GS or GS and RNA aggregation is general. Our predictions are amenable to experimental tests.

biophysics↗

bioRxiv2023

Competition between stacking and divalent cation mediated electrostatic interactions determines the conformations of short DNA sequences

Interactions between divalent cations (Mg2+ and Ca2+) and highly charged single stranded DNA (ssDNA) and double stranded DNA (dsDNA), as well as stacking interactions, are important in a variety of problems, including 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. Towards this end, we created a computational model that explicitly takes into account monovalent and divalent ions, within the framework of the sequence-dependent coarse-grained Three Interaction Site (TIS) model for DNA. Molecular simulations of the rigid 24 base-pair (bp) dsDNA and flexible ssDNA sequences, dT30 and dA30, in a buffer containing Na+ and Cl-, with varying amounts of the divalent cations, are used to 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 (SAXS) profiles, which are also in excellent agreement with experiments. Strikingly, recapitulation of all the experimental findings was achieved without adjusting any of the parameters in the energy function to fit the data. At a molecular level, we find that Mg2+ and Ca2+ sense the differences between the major and minor grooves in dsDNA even though they are masked in ion-counting and SAXS experiments. The smaller Mg2+ binds predominantly to the minor grooves and phosphate groups whereas Ca2+ binds specifically only to the minor groove. The dA30 conformations are dominated by stacking interactions, resulting in structures with considerable helical order. In contrast, the near cancellation of the favorable stacking and unfavorable electrostatic interactions leads to dT30 populating an ensemble of heterogeneous conformations.

biophysics↗

bioRxiv2023

Watching ion-driven folding kinetics of a ribozyme 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/527349v3_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1a69418org.highwire.dtl.DTLVardef@144e1a3org.highwire.dtl.DTLVardef@139b8e2org.highwire.dtl.DTLVardef@993ca2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

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, {Delta}GS, 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 {Delta}GS (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 {Delta}GS 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 is 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 spectra, 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.

biophysics↗

bioRxiv2021

Condensates in RNA Repeat Sequences are Heterogeneously Organized and Exhibit Reptation-like Dynamics

Although it is known that RNA undergoes liquid-liquid phase separation (LLPS), the interplay between the molecular driving forces and the emergent features of the condensates, such as their morphologies and dynamical 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 such as Huntington disease and amyotrophic lateral sclerosis. After establishing that the simulations reproduce key experimental findings (length and concentration dependence of the phase transition in (CAG)n repeats), 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.

biophysics↗

bioRxiv2020

Molecular Simulations of Mg2+-induced 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. In order 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 0-30 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. O_TEXTBOXSignificance StatementRibosomes are complexes between ribosomal RNA (rRNA) and a number of proteins. Because ribosome assembly begins with rRNA folding, we simulated the molecular details of Mg2+-driven folding of the central domain of the bacterial rRNA. Good agreement with experiments on the folding of the three-way junction in the center of the rRNA validates the model. Coupling of rRNA folding and Mg2+ binding shows that ions interact with rRNA segments in a coordinated manner. The shape of rRNA changes from a sphere in the unfolded state to a prolate ellipsoid at high Mg2+ concentration, which is the opposite of what transpires when a globular protein folds. Our study pro-vides the needed framework for undertaking ion-driven folding of large RNA molecules. C_TEXTBOX

biophysics↗

bioRxiv2019

Folding RNA in mixtures of monovalent and divalent cations: Theory and simulations

RNA molecules cannot fold in the absence of counter ions. 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 in 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 accurately predict 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 ribosomal RNA, 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.\n\nSignificance StatementRNA molecules require ions to fold. The problem of how ions of differing sizes and valences drive the folding of RNA molecules is unsolved. Here, we take a major step in its solution by creating a method, based on the theory of polyatomic liquids, to calculate the potential between divalent ions and the phosphate groups. The resulting model, accounting for inner and outer sphere coordination of Mg2+ and Ca2+ to phosphates, when used in coarse-grained molecular simulations predicts folding free energies for a number of RNA molecules in the presence of both divalent and monovalent ions that are in excellent agreement with experiments. The work sets the stage for probing sequence and ion effects on DNA and synthetic polyelectrolytes.

biophysics↗

bioRxiv2019

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 often Mg2+ ions are needed for catalytic activity. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that the most of the ions are territorially and non-specifically bound to the RNA, as predicted by the counterion condensation (CIC) theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained Three Site Interaction (TIS) 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 at very low 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 non-monotonic shape fluctuations in the ribozyme as it folds with increasing Mg2+ or Ca2+ concentration.

biophysics↗

bioRxiv2018

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 (kFs) from more than 100 folding trajectories for each value of the solvent viscosity ({eta}) at a fixed salt concentration of 200 mM. Using the theoretical estimate ([Formula] where N is number of nucleotides) for folding free energy barrier, kF data for both the RNAs are quantitatively fit using one dimensional Kramers theory with two parameters specifying the curvatures in the unfolded basin and the barrier top. In the high-friction regime ({eta} {gtrsim} 10-5 Pa{middle dot}s), for both HP and PK, kFs decrease as 1/{eta} whereas in the low friction regime kFs increase as{eta} increases, leading to a maximum folding rate at a moderate viscosity (~ 10-6 Pa{middle dot}s), which is the Kramers turnover. From the fits, we find that the speed limit to RNA folding at water viscosity is between (1 - 4)s, 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 proteins.

biophysics↗

bioRxiv2018

Sequence-dependent Three Interaction Site (TIS) 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 reproduces 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 with one hundred and thirty base pairs, 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. 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.

biophysics↗

bioRxiv2017

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 experiments and simulations to examine the folding/unfolding pathways of the VPK pseudoknot, 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, and laser temperature-jump approaches serve as local probes allowing us to monitor the order of assembly of VPK with two helices with different intrinsic stabilities. The experiments and molecular simulations show that at 50 mM KCl the dominant folding pathway populates only the more stable partially folded hairpin. As the salt concentration is increased a parallel folding pathway emerges, involving the less stable hairpin structure as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. The 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. Our study not only unambiguously demonstrates that PK folds by parallel pathways, but also establishes that quantitative description of RNA self-assembly requires a synergistic combination of experiments and simulations.\n\nSIGNIFICANCEThe assembly mechanism of RNA, vital to describing their functions, depends on both the sequence and the metal ion concentration. How the latter influences the folding trajectories remains an important unsolved problem. Here, we examine the folding pathways of an RNA pseudoknot (PK) with key functional roles in transcription and translation, using a combination of experiments and simulations. We demonstrate that the PK, consisting of two helices with differing stabilities, folds by parallel pathways. Surprisingly, the flux between them is modulated by monovalent salt concentration. Our work shows that the order of assembly of PKs is determined by the relative stability of the helices, implying that the folding landscape can be controlled by sequence and ion concentration.

biophysics↗