Biology subjects

Pratihar, S.

Publications and source records attributed to Pratihar, S..

6 recordsLinked to original sources

bioRxiv2025

RNA 2'-OH modification with stable reagents enabled bynucleophilic catalysis

RNA modification at 2-OH has typically required highly reactive acylating species that exhibit short half-lives in water, challenging purification, and limited shelf lives. Here, we investigate the use of more stable species as electrophilic reagents, employing nucleophilic catalysis to promote reactions. Results show that multiple previously unreported electrophiles can react in high stoichiometric yields with RNA under appropriate catalysis. Most notably, aryl esters can transfer acyl groups to RNA in one hour, but are stable for months even in pure water. The results expand the functional chemotypes of RNA-reactive species, and identify reagent classes with improved stability and selectivity.

biochemistry↗

bioRxiv2025

Sequence-Specific Installation of Aryl Groups in RNA via DNA-Catalyst Conjugates

Installing functional groups at specific sites in existing RNA molecules remains a challenge for modification, labeling, and therapeutic strategies. Here we describe the use of DNA oligonucleotides carrying a catalytic amine group to effect the aqueous SNAr arylation of 2'-OH groups at sequence-complementary sites in RNAs. Chloro-pyrimidine electrophiles are shown to react with amino-DNA conjugates, resulting in a proposed transient ammonium aryl intermediate that can react with RNA near the DNA binding site, delivering the heterocycle to the RNA in high yields. In a test of utility, we construct an aryl electrophile carrying an azide group, and apply this strategy to fluorescently label messenger RNAs locally at the polyA tail. We also employ the approach to direct in vitro arylation in the coding region of a messenger RNA, knocking down protein expression selectively in the presence of another coding RNA. This sequence-directed catalytic strategy enables multiple applications in RNA labeling and modification.

biochemistry↗

bioRxiv2025

Hierarchically ordered multi-timescale structural dynamics of the intrinsically disordered p53 transactivation domain

Intrinsically disordered proteins (IDPs) exhibit pronounced structural dynamics, which is crucial for their functional versatility. Yet their dynamics slower than nanoseconds remain largely elusive. We combined high-power relaxation dispersion nuclear magnetic resonance spectroscopy with molecular dynamics simulations to characterize these kinetics and the underlying structural interconversions of a prototypical IDP, the N-terminal transactivation domain of the tumor suppressor p53 (p53-TAD). We find a complex hierarchy of structural dynamics on timescales covering over seven orders of magnitude, ranging from fast nanoseconds backbone re-orientations, via sub-microsecond helix-formation dynamics involving many structural sub-states and transition times, to transient tertiary structure formation slower than 25 microseconds. These rich structural dynamics of p53-TAD, and likely those of other IDPs, parallel the timescale hierarchy of the conformational dynamics of folded proteins. One-Sentence summaryA hierarchical energy landscape governs kinetics and structural dynamics of the disordered p53 transactivation domain.

biophysics↗

bioRxiv2025

Development of Bioisosteric Iboga-alkaloids as Antinociceptive and Anxiolytic Agents with Neuroprotective Effects

The clinical importance of iboga alkaloids lies in their efficacy in reversing drug addiction and modulating drug tolerance. However, due to safety concerns, their use is restricted to appropriate medical supervision. These alkaloids often cause severe hallucinogenic effects due to differential binding to various brain receptors and cardiotoxicity by blocking the human ether-a-go-go-related gene (hERG) potassium channel. To create safer analogs, our group previously synthesized various benzofuran-containing iboga analogs with good opioid binding selectivity and excellent antinociceptive property. However, the present manuscript disclosed a step-economical and cost-effective synthesis of modified ibogaine/ibogamine analogs (C1, C2, C3 & C4) with bio-isosteric replacement of the indole scaffold with a benzofuran moiety, and comparing their antinociceptive/anxiolytic activity with their natural counterparts. Among the synthesized iboga analogs, the Endo-iboga analogs (C2 & C4, epimers of C1 and C3, respectively) not only exhibited superior anti-inflammatory and oxidative stress-relieving activity, but also effectively improved restricted locomotor activity in a formalin-induced acute pain model in mice. These Endo-iboga analogs significantly elevated the levels of inhibitory neurotransmitters (GABA and dopamine) and brain-derived neurotrophic factor (BDNF) compared to their Exo-counterparts or previously published benzofuran-containing iboga analogs lacking the tetrahydroazepine ring. Amongst, C2 and C4, the latter exhibited superior cardiac safety profile in C2C12 cells (IC50 = 235 {micro}M) and showed no adverse effects on rat hearts during in vivo ECG tests, indicated by no significant QTc prolongation. Overall, the development of bioisosteric iboga analogs, particularly C4, demonstrated significant potential for acute pain management without notable cardiotoxicity, representing a breakthrough in pain therapy innovation.

physiology↗

bioRxiv2024

An A-T Hoogsteen base pair in a naked DNA hairpin motif: A Protein-Recognized Conformation

In duplex DNA, A-T and G-C form Watson-Crick base pairs, and Hoogsteen pairing only dominates upon protein binding or DNA damage. Using NMR, we show that an A-T Hoogsteen base pair previously observed in crystal structures of transposon DNA hairpins bound to TnpA protein forms in solution even in the absence of TnpA. This Hoogsteen base pair, located adjacent to a dinucleotide apical loop, exists in dynamic equilibrium with a minor Watson-Crick conformation (population [~]11% and lifetime [~]55 {micro}s). Extending the apical loop to three residues inverted the equilibrium, making Watson-Crick the dominant state and the Hoogsteen conformation recognized by TnpA a minor state (population [~]14% and lifetime [~]28 {micro}s). The propensity for Hoogsteen pairing depended on apical loop residues, which form contacts directly or indirectly stabilizing the Hoogsteen conformation. A structure survey did not reveal Hoogsteen pairing near RNA apical loops making them unique to DNA. Our results demonstrate that Hoogsteen can be the dominant state even in naked unmodified duplex DNA and identify 5-CTT(T/C)AG-3 as a DNA-specific apical loop motif stabilized by Hoogsteen pairing. Hoogsteen base pairs may be prevalent in DNA hairpins forming during replication and transcription, with broad implications for the genomic landscape.

biochemistry↗

bioRxiv2023

mRNA interactions with disordered regions control protein activity

The cytoplasm is compartmentalized into different translation environments. mRNAs use their 3'UTRs to localize to distinct cytoplasmic compartments, including TIS granules (TGs). Many transcription factors, including MYC, are translated in TGs. It was shown that translation of proteins in TGs enables the formation of protein complexes that cannot be established when these proteins are translated in the cytosol, but the mechanism is poorly understood. Here we show that MYC protein complexes that involve binding to the intrinsically disordered region (IDR) of MYC are only formed when MYC is translated in TGs. TG-dependent protein complexes require TG-enriched mRNAs for assembly. These mRNAs bind to a new and widespread RNA-binding domain in neutral or negatively charged IDRs in several transcription factors, including MYC. RNA-IDR interaction changes the conformational ensemble of the IDR, enabling the formation of MYC protein complexes that act in the nucleus and control functions that cannot be accomplished by cytosolically-translated MYC. We propose that certain mRNAs have IDR chaperone activity as they control IDR conformations. In addition to post-translational modifications, we found a novel mode of protein activity regulation. Since RNA-IDR interactions are prevalent, we suggest that mRNA-dependent control of protein functional states is widespread.

molecular biology↗