Biology subjects

DasGupta, S.

Publications and source records attributed to DasGupta, S..

4 recordsLinked to original sources

bioRxiv2023

Molecular crowding facilitates ribozyme-catalyzed RNA assembly

Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg2+ to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg2+ such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis. Further, prebiotic cell-like compartments bounded by fatty acid membranes are destabilized in the presence of Mg2+, making ribozyme function inside prebiotically relevant protocells a significant challenge. Therefore, we sought to identify conditions that would enable ribozymes to retain activity in low concentrations of Mg2+. Inspired by the ability of ribozymes to function inside crowded cellular environments with <1 mM free Mg2+, we tested molecular crowding as a potential mechanism to lower the Mg2+ concentration required for ribozyme-catalyzed RNA assembly. Here, we show that the ribozyme-catalyzed ligation of phosphorimidazolide RNA substrates is significantly enhanced in the presence of the artificial crowding agent polyethylene glycol. We also found that molecular crowding preserves ligase activity under denaturing conditions such as alkaline pH and the presence of urea. We also show that crowding-induced stimulation of RNA-catalyzed RNA assembly is not limited to phosphorimidazolide ligation but extends to the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzed RNA ligation is also stimulated by the presence of prebiotically relevant small molecules such as ethylene glycol, ribose, and amino acids, consistent with a role for molecular crowding in primordial ribozyme function and more generally, in the emergence of RNA-based cellular life. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/538884v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@17d282borg.highwire.dtl.DTLVardef@1d8531aorg.highwire.dtl.DTLVardef@1380f7borg.highwire.dtl.DTLVardef@180708f_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISMolecular crowding enables ribozyme-catalyzed RNA assembly under low Mg2+ conditions, and consequently may have played an essential role in the origin and evolution of RNA-based primordial life.

biochemistry↗

bioRxiv2023

RNA-catalyzed RNA Ligation within Prebiotically Plausible Model Protocells

Demonstrating RNA catalysis within prebiotically relevant models of primordial cells (protocells) remains a challenge in Origins of life research. Fatty acid vesicles encapsulating genomic and catalytic RNAs (ribozymes) are attractive models for protocells; however, RNA catalysis has largely been incompatible with fatty acid vesicles due to their instability in the presence of Mg2+ at concentrations required for ribozyme function. Here, we report a ribozyme that catalyzes template-directed RNA ligation at low Mg2+ concentrations and thus remains active within stable vesicles. Ribose and adenine, both prebiotically relevant molecules, were found to greatly reduce Mg2+-induced RNA leakage from vesicles. When we co-encapsulated the ribozyme, substrate, and template within fatty acid vesicles, we observed efficient RNA-catalyzed RNA ligation upon subsequent addition of Mg2+. Our work shows that RNA-catalyzed RNA assembly can occur efficiently within prebiotically plausible fatty acid vesicles and represents a step toward the replication of primordial genomes within self-replicating protocells.

biochemistry↗

bioRxiv2022

REVERSE: A user-friendly web server for analyzing next-generation sequencing data from in vitro selection/evolution experiments

Next-generation sequencing (NGS) enables the identification of functional nucleic acid sequences from in vitro selection/evolution experiments and illuminates the evolutionary process at single nucleotide resolution. However, analyzing the vast output from NGS can be daunting, especially with limited programming skills. No single platform exists that performs all the steps necessary to generate publishable results starting with raw sequence data. We developed REVERSE (Rapid EValuation of Experimental RNA Selection/Evolution) (https://www.reverseserver.org/), a web server that incorporates an integrated computational pipeline through a graphical user interface, which performs both pre-processing and sequence level analyses within minutes. FASTQ files from multiple rounds are quality filtered, dereplicated, and trimmed before being analyzed by two pipelines. The first pipeline counts, sorts, and tracks enrichment of unique sequences and tracks the enrichment of sequence motifs. It also identifies mutational intermediates present in the sequence data that connect two input sequences. The second pipeline sorts similar sequences into clusters and tracks enrichment of peak sequences. It also performs nucleotide conservation analysis on the cluster of choice. Both pipelines generate downloadable high-resolution figures. Collectively, REVERSE is a one stop-solution for the rapid analysis of NGS data obtained from in vitro selection/evolution experiments that obviates the need for computational expertise.

bioinformatics↗

bioRxiv2021

Design, Fabrication, and Theoretical Investigation of a Cost-Effective Laser Printing Based Colorimetric Paper Sensor for Non-Invasive Glucose and Ketone Detection

Diabetes, a chronic condition, is one of the prevalent afflictions of the 21st century, and if left unchecked, this ailment could lead to severe life-threatening complications. A widely accepted methodology for monitoring diabetes is the estimation of the glucose and ketone contents in the body-fluids, viz. blood, urine, etc. Additionally, certain conditions such as starvation, and following a protein rich diet (e.g., keto-diet) could also lead to significant changes in the ketone content, thereby resulting in false-positive diagnosis. Hence, a precise, portable, and on-demand procedure for the rapid and combined estimation of glucose and ketone in the bodily-fluids is of utmost importance. To that end, paper-based analytical devices (PADs) are promising tools, owing to their multitudinous advantages, and compatibility with biofluids. Although, numerous researchers have contributed substantially in the fundamental investigation, design, and fabrication of PADs for various applications, a combined platform capable of rapid, accurate and on-demand glucose and ketone detection, that is easy to fabricate, is still relatively unexplored. Moreover, the flow dynamics of an analyte, in combination with enzyme-catalysed (for glucose) and uncatalyzed reactions (for ketone), within a porous paper matrix is also vaguely understood. Herein, we present a facile laser-printing based fabrication of colorimetric sensors on a filter paper, for rapid, and non-invasive estimation of glucose and ketone contents in urine. The urine sample, upon being deposited in a particular expanse, is wicked through the paper matrix, and reacts with specific reagents in the designated zone(s), giving rise to a final color, concomitant with the glucose or ketone content in the sample. The device design enables the liquid to be wicked into the porous matrix in a way that would concentrate the colored product in a dedicated detection zone, thereby augmenting the feasibility for accurate colorimetric detection. Furthermore, we present for the first time, a detailed dynamic model of the flow-field in a variable cross-section paper device using the Richards equation, while also considering the species transport and reaction kinetics within the porous media. The results of the numerical simulation agree well with those observed experimentally, thereby validating the present model. Finally, we also developed a web and desktop-based application that would enable the user to upload the images of the colored zones to provide an accurate estimate of the glucose and ketone content in the sample. We believe that our model, in combination with the proposed fabrication methodology, and the in-house developed app., would enable rapid and reliable fabrication of PADs for various fundamental investigations, and applications pertaining to affordable health-care monitoring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/461386v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@49e824org.highwire.dtl.DTLVardef@27a512org.highwire.dtl.DTLVardef@c65242org.highwire.dtl.DTLVardef@429896_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗