Biology subjects

Miaro, M.

Publications and source records attributed to Miaro, M..

5 recordsLinked to original sources

bioRxiv2026

Nearest Neighbor Parameters for Estimating RNA Folding Stability with In Vivo-like Conditions

RNAs regulate gene expression and cellular processes, often relying on specific conformations for function. RNA folding is hierarchical and sequence-dependent, with nearest-neighbor thermodynamic models commonly used to predict secondary structure. Current models were developed using optical melting experiments in 1 M NaCl, which does not represent the cellular environment. To address this, we developed a new model in Advanced Dulbecco's Modified Eagle Medium (Adv. DMEM), which mimics mammalian extracellular ionic composition. This in vivo-like model provides RNA folding parameters for helical base stacks and loop motifs. Optical melting experiments revealed helical stacks, particularly tandem G-U pairs, are less stabilizing in Adv. DMEM. Loop parameters were generally destabilizing but highly dependent on both sequence and loop type, with internal loops displaying idiosyncratic behavior. Structure prediction benchmarking revealed minimal differences overall, except for tRNAs, which showed improved prediction reliability and enhanced cloverleaf stability. Notably, tRNAs lack internal loops, suggesting further studies in Adv. DMEM could refine secondary structure predictions. This in vivo-like parameter set is included in the RNAstructure software package. Grounding these parameters in a physiologically relevant environment, we improve the biological relevance of RNA secondary structure predictions and establish a foundation for studying RNA folding under in vivo conditions.

biochemistry↗

bioRxiv2026

Nearest Neighbor Parameters for Estimating the Folding Stability of RNA Including Pseudouridine

Nearest neighbor parameters are widely used in software for estimating the conformational stability of an RNA sequence folding into a specific structure. Folding stability for RNA with canonical nucleotides A, C, G, and U has been widely studied, but the same is not true for most modified nucleotides. In this work, we present a comprehensive set of nearest neighbor parameters for estimating the folding stability of RNA including pseudouridine in helical or loop contexts. These parameters are derived from 210 optical melting experiments involving helices with pseudouridine-A and pseudouridine-G pairs and with pseudouridine in loop motifs. The experiments include sequences with pseudouridine and U in the same strand, including U-A and U-G pairs, allowing us to consider the folding stability of sequences with both U and pseudouridine. On average, pseudouridine stabilizes RNA folding compared to U in an analogous motif, although this effect is sequence-context dependent. These parameters improve the modeling of folding stability for RNA secondary structures containing pseudouridine. We demonstrate that these parameters successfully model the secondary structure change for Saccharomyces cerevisiae U2 snRNA when two additional inducible pseudouridines are present. These parameters are freely available and incorporated into the RNAstructure software package. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/725682v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@e1167aorg.highwire.dtl.DTLVardef@18ac7f0org.highwire.dtl.DTLVardef@4c909eorg.highwire.dtl.DTLVardef@aa8bca_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

RNA Folding Nearest Neighbor Parameters Including the Modification 1-Methyl-Pseudouridine

Nearest neighbor analysis is commonly used to estimate RNA folding stabilities. In this contribution, we report a set of RNA folding nearest neighbor parameters for estimating free energy change for RNA sequences including 1-methyl-pseudouridine. Development of mRNA vaccines has identified 1-methyl-pseudouridine as a key nucleobase modification for suppressing innate immune responses. However, the contributions of these modifications to RNA folding stability were unclear. Our new parameters provide helical terms for 1-methyl-pseudouridine-adenine and 1-methyl-pseudouridine-guanine base pairs. The parameters also estimate loop stabilities for loops with 1-methyl-pseudouridine or a combination of 1-methyl-pseudouridine and uridine. These parameters are derived using 208 optical melting experiments and tested against an additional 16 optical melting experiments. On average, we find that substitution of uridine with 1-methyl-pseudouridine stabilizes RNA folding, with the extent of stabilization depending on adjacent sequence. The estimation of tRNA folding ensembles for tRNA sequences with 1-methyl-pseudouridine was significantly improved using the new nearest neighbor parameters. The new nearest neighbor parameters are provided as part of the RNAstructure software package. With these parameters, the secondary structures of natural sequences with 1-methyl-pseudouridine and mRNA therapeutics fully substituted with 1-methyl-pseudouridine can be modeled.

bioinformatics↗

bioRxiv2025

An oligo walk to identify siRNAs against the circular Tau 12->7 RNA

Circular RNAs are associated with numerous diseases and recent evidence shows that they can be translated into proteins after undergoing RNA modification. Circular RNAs differ from their linear mRNA counterparts in their backsplice site, allowing selective targeting using RNA interference, which however limits the options to place the siRNA. We tested all possible siRNAs against the backsplice site of the circTau 12->7 RNA after it was subjected to adenosine to inosine RNA editing, a modification that promotes translation of the circRNA. Most siRNAs reduced the circRNA and protein abundance, which however did not correlate. We identified an siRNA with an IC50 of 750 pmol efficacy on protein expression. This circRNA fulfilled six of the eight criteria for siRNAs targeting mRNAs. Thus, modified circRNAs expressing protein can be targeted with siRNAs, but their optimal sequence needs to be determined empirically.

molecular biology↗

bioRxiv2020

Mutation of a conserved Gln residue does not abolish desensitization of acid-sensing ion channel 1

Desensitization is a common feature of ligand-gated ion channels although the molecular cause varies widely between channel types. Mutations that substantially reduce or abolish desensitization have been described for many ligand-gated ion channels including glutamate, GABA, glycine and nicotinic receptors but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1 the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes essentially like wild type when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular dynamics simulations indicate that the Gln277 side chain participates in a hydrogen bond network that might stabilize the desensitized conformation. Consistent with this, destabilizing this network with the Q277N or Q277L mutations largely mimics the Q277G phenotype. In human ASIC1a, Q276G does not substantially reduce desensitization but surprisingly slows entry to and exit from the desensitized state, thus requiring longer agonist applications to reach equilibrium. Our data reveal that while the Q/G mutation does not substantially impair desensitization as previously reported, it does point to unexpected differences between chicken and human ASICs and the need for careful scrutiny before using this mutation in future studies.

biophysics↗