Biology subjects

Mitra, A.

Publications and source records attributed to Mitra, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Rapid redistribution and extensive binding of NANOG and GATA6 at shared regulatory elements underlie specification of divergent cell fates

Establishment of divergent cell types from a common progenitor requires transcription factors (TFs) to promote lineage-restricted transcriptional programs while suppressing alternative fates. In the mouse blastocyst, cells of the inner cell mass (ICM) coexpress NANOG and GATA6, two TFs that drive the bifurcation of these progenitors into either the epiblast (Epi) or the primitive endoderm (PrE), respectively. Here, using in vitro differentiation, we describe the molecular mechanisms of how GATA6 quickly induces the PrE fate while repressing the Epi lineage. GATA6 functions as a pioneer TF by inducing nucleosome repositioning at regulatory elements controlling PrE genes, making them accessible for deposition of active histone marks and leading to rewiring of chromatin interactions and ultimately transcriptional activation. GATA6 also binds most regulatory elements of Epi genes followed by eviction of the Epispecific TFs NANOG and SOX2, loss of active histone marks, and reduction in chromatin accessibility that culminates in transcriptional repression. Unexpectedly, evicted NANOG and SOX2 transiently bind PrE regulatory elements occupied by GATA6. Our study shows that GATA6 binds and modulate the same regulatory elements as Epi TFs, a phenomenon we also validated in blastocysts. We propose that the ability of PrE and Epi-specific TFs to extensively bind and regulate the same gene networks contributes to ICM plasticity and allows rapid cell lineage specification by coordinating both activation

developmental biology↗

bioRxiv2021

Transcriptional heterogeneity of ventricular zone cells throughout the embryonic mouse forebrain

The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression. However, a detailed characterization of transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the mouse embryonic cortex and ganglionic eminences (GEs). By using a transgenic mouse line to enrich for VZ cells, we detect significant transcriptional heterogeneity within VZ and SVZ progenitors, both between forebrain regions and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of telencephalic VZ cells that will aid our understanding of initial fate decisions in the forebrain.

neuroscience↗

bioRxiv2021

The terminal heme synthetic enzyme, Coproheme Decarboxylase, coordinates heme synthesis and uptake in response to iron in Mycobacteria

Heme is both an essential cofactor and an abundant source of nutritional iron for the human pathogen Mycobacterium tuberculosis (Mtb). While heme is required for Mtb survival and virulence, it is also potentially cytotoxic. Since Mtb has the ability to both make and uptake heme, the de novo synthesis of heme and its acquisition from the host must be balanced in order to mitigate heme toxicity. However, the mechanisms employed by Mtb to regulate heme uptake, synthesis, and bioavailability are poorly understood. By integrating ratiometric heme sensors with mycobacterial genetics, cell biology, and biochemistry, we determined that the terminal heme biosynthetic enzyme, coproheme decarboxylase (ChdC), plays a role in regulating both heme bioavailability and uptake in Mtb. Moreover, we found that Mtb has a preference for scavenging reduced ferrous heme and exhibits a cell surface heme reductase activity that is regulated by ChdC. In Mtb, ChdC expression is down-regulated when iron is limiting, which in-turn increases both heme import and bioavailability. Such a mechanism may serve to protect cells from heme toxicity while trying to meet the nutritional demand for iron. Our results demonstrate that heme synthesis and uptake are tightly integrated in mycobacteria and represent the first example of a heme synthetic enzyme playing a role in controlling heme uptake. Significance StatementHeme is an essential but potentially cytotoxic cofactor and iron source for the pathogen, Mycobacterium tuberculosis (Mtb). To understand how Mtb coordinates heme uptake and synthesis to mitigate heme toxicity, we integrated heme sensors with mycobacterial genetics and biochemical approaches to probe the interplay between heme synthesis and scavenging. We discovered that the terminal heme synthetic enzyme, coproheme decarboxylase (ChdC), negatively regulates heme uptake and utilization in response to iron availability through a mechanism involving control of a ferric heme reductase. During iron limitation, ChdC is downregulated, thereby enhancing exogenous heme reduction, uptake and utilization while simultaneously suppressing heme synthesis, which allows Mtb to avoid heme toxicity. Our results highlight the close coordination between heme synthesis and uptake in mycobacteria. ClassificationBiological sciences : Biochemistry

microbiology↗

bioRxiv2021

Single cell chromatin accessibility reveals regulatory elements and developmental trajectories in the embryonic forebrain

A comprehensive characterization of epigemonic organization in the embryonic mouse forebrain will enhance our understanding of neurodevelopment and provide insight into mechanisms of neurological disease. We collected single-cell chromatin accessibility profiles from four distinct neurogenic regions of the embryonic mouse forebrain using single nuclei ATAC-Seq (snATAC-Seq). We identified thousands of differentially accessible peaks, many restricted to distinct progenitor cell types or brain regions. We integrated snATAC-Seq and single cell transcriptome data to characterize changes of chromatin accessibility at enhancers and promoters with associated transcript abundance. Multi-modal integration of histone modifications (CUT&Tag and CUT&RUN), promoter-enhancer interactions (Capture-C) and high-order chromatin structure (Hi-C) extended these initial observations. This dataset reveals a diverse chromatin landscape with region-specific regulatory mechanisms and genomic interactions in distinct neurogenic regions of the embryonic mouse brain and represents an extensive public resource of a ground truth epigenomic landscape at this critical stage of neurogenesis.

neuroscience↗

bioRxiv2021

Cardiac pathologies in mouse loss of imprinting models are due to misexpression of H19 long noncoding RNA

Maternal loss of imprinting (LOI) at the H19/IGF2 locus results in biallelic IGF2 and reduced H19 expression and is associated with Beckwith Wiedemann syndrome (BWS). We use mouse models for LOI to understand the relative importance of Igf2 and H19 mis-expression in BWS phenotypes. Here we focus on cardiovascular phenotypes and show that neonatal cardiomegaly is exclusively dependent on increased Igf2. Circulating IGF2 binds cardiomyocyte receptors to hyperactivate mTOR signaling, resulting in cellular hyperplasia and hypertrophy. These Igf2-dependent phenotypes are transient: cardiac size returns to normal once Igf2 expression is suppressed postnatally. However, reduced H19 expression is sufficient to cause progressive heart pathologies including fibrosis and reduced ventricular function. In the heart, H19 expression is concentrated predominantly in endothelial cells (ECs) and regulates EC differentiation both, in vivo and in vitro. Finally, we establish novel mouse models to show that cardiac phenotypes depend on H19 lncRNA interactions with let7 microRNA.

developmental biology↗

bioRxiv2021

Occurrences of protonated base triples in RNA are determined by their cooperative binding energies and specific functional requirements

With wide ranging diversity in their geometries, binding strengths and chemical properties, noncanonical base pairs are equipped to intricately regulate and control the structural dynamics of RNA molecules. Protonation of nucleobases adds to the diversity. Compared to the unprotonated scenario, on one hand they open up new alternatives for base pairing interactions (Class I) while on the other, they modulate the geometry and stability of existing base pairing interactions (Class II). In both cases, compensation of the energetic cost associated with nucleobase protonation at physiological pH, can be understood in terms of protonation induced restructuring of charge distribution. This not only leads to modifications in existing base-base interactions but often also leads to additional stabilizing interactions, resulting in the formation of protonated base triples. Here we report our detailed quantum chemical studies, in conjunction with structural bioinformatics based analysis of RNA crystal and NMR structure datasets, probing into the contribution of such protonated triples in the structural dynamics of RNA. Our studies revealed more than 55 varieties of protonated triples in RNA, some of which occur recurrently within conserved structural motifs present in rRNAs, tRNAs and in other synthetic RNAs. Our studies suggest that high occurrence frequencies are associated with protonated triples which satisfy the specific structural requirements of conserved motifs where they occur. For example, protonated triples with flexible geometries are involved in the formation of tertiary contacts between different distant motifs. Stabilization of protonated base pairs, through the induction of additional energetically cooperative interactions, appears to be another factor. These results provide significant insights into the sequence-structure-function relationships in RNA.

biophysics↗

bioRxiv2021

Molecular analysis of prostate cancer: prostate tissue and urine proteomics based approach

Benign prostatic hyperplasia (BPH) and prostate cancer are the most frequently diagnosed conditions in men above 60 years. BPH manifests as benign enlargement of the prostate gland causing pain and difficulty in micturition, often associated with other lower urinary tract symptoms. On the other hand, prostate cancer might initially set in as a tumor of no clinical significance, but can ultimately develop into an aggressively metastatic cancer at later stages. Due to overlapping symptomatic manifestations with BPH, it might be difficult to diagnose prostate cancer and differentiate it from BPH. Screening for prostate cancer is based on examining the levels of prostate specific antigen (PSA), a clinical biomarker for prostate cancer in blood. However, several reported cases indicate that PSA might lack the sensitivity and specificity required to differentiate between the cancerous and benign conditions of prostate. Therefore, in the absence of non-invasive biomarkers in a diagnostic setup, an intensely invasive surgical removal of a part of the tissue and subsequent histopathologial examination is the only available procedure to confirm the cancer. In this study, we used tissue and urine proteomics platforms to profile respective proteomes in both clinical conditions. We observed that latent transforming growth factor binding protein was significantly under-expressed in the both tissue and urine proteome of prostate cancer. We propose that the down-regulation of the latent transforming growth factor binding protein 4 might be explored in a large set of patients with prostate cancer to develop a non-invasive urine based biomarker for the diagnosis of prostate cancer.

cancer biology↗

bioRxiv2020

Differential expression of proteins in human prostate cancer tissues probed by MALDI imaging mass spectrometry

Histopathology, the gold-standard method for diagnosis of human prostate cancer, is based on the analysis of changes in cellular morphology and tissue architecture in chemically stained tissue sections. Even at the very early stages of cancer with minimal phenotypic changes in cellular morphologies, there might be detectable changes in the expression profiles of proteins. Over the last decade, imaging mass spectrometry has been used to explore the spatial distribution and expression profiles of several molecules with their twodimensional heterogeneity retained across the tissue section. In the present study, using MALDI mass spectrometry based tissue imaging, we report the differential expression of three proteins, vinculin, ribonuclease T2 and 60 kDa heat shock protein across human prostate tissue sections in regions pathologically demarcated as frankly malignant. In an independent analysis, quantitative proteomics revealed that in cancerous prostate tissues, ribonuclease T2 and 60 kDa heat shock protein were significantly overexpressed by 22.26- and 6 folds respectively compared to benign condition. Our results show the utility of this approach to probe differential protein expression in architecturally intact cancer tissues. In addition, we propose that ribonuclease T2 and 60 kDa heat shock protein might be developed as diagnostic biomarkers for prostate cancer in future.

biophysics↗

bioRxiv2020

NMDAR-mediated transcriptional control of geneexpression in the specification of interneuron subtype identity

Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+ and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical and physiological properties. However, the molecular mechanisms regulating their diversity remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates subtype-specific transcriptional regulation of gene expression in MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific conditional Grin1 loss results in a systemic downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders, particularly schizophrenia. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders.

neuroscience↗

bioRxiv2020

Analyzing and interpreting DNA double-strand break sequencing data

DNA double-strand breaks (DSBs), are a major threat to genomic stability and may lead to cancer. Several technologies to accurately detect DSBs genome-wide have been developed recently, but still lacking publicly available tools for analysis of the resulting data. Here, we present a step-by-step iSeq package (http://breakome.utmb.edu/software.html), custom designed for analysis and interpretation of DSB-sequencing data. iSeq performs barcode trimming and read counting, and identifies DSB-enriched regions by statistical test and annotate them to the desired genomic features. Applying this package, users can identify and annotate DSB-enriched regions from base pair (eg. Cas9 cleavage sites) up to megabase (eg. DNA replication stress-induced) resolution, and if possible quantify DSB frequencies per cell genome-wide by combining with qDSB-Seq. iSeq can be used for any sequencing-based DSB detection techniques. The analysis for Steps 1-19 can be performed within ~4 hours.

bioinformatics↗

bioRxiv2020

A Comprehensive Survey on the Nature of Ring:Ring Nucleobase Stacking Interactions in RNA: Occurrence, Structural Variability and Classification of the Associated Contacts

The astonishing diversity in folding patterns of RNA 3D structures is crafted by myriads of noncovalent contacts, of which base pairing and stacking are the most prominent. Although the classification scheme proposed by Leontis and Westhof (RNA (2001), 7, 499) has been widely accepted for the annotation of RNA base pairs, the absence of an unambiguous classification system for base stacks appears to be a roadblock for exploring the stacking diversity in RNA. Here we provide an unambiguous and structurally-intuitive scheme for a geometry cum topology based classification of base stacking, where a stack is essentially classified in terms of the topology of the interacting nucleobase faces and the geometry described by the relative orientation of the glycosidic bonds. For heterodimeric stacks, this generates eight basic stacking geometric families, whereas for homodimeric stacks, this generates six of those. Further annotation in terms of the identity of the bases and the region of involvement of purines (5-membered, 6-membered or both rings), leads to the enumeration of 384 distinct RNA base stacks. Based on our classification scheme, we also present an algorithm for automated identification of stacks in RNA crystal structures. Overall, the work described here is expected to greatly facilitate structure-based RNA research.

bioinformatics↗

bioRxiv2020

Correlated motions of conserved polar motifs lay out a plausible mechanism of G protein-coupled receptor activation.

Recent advancements in the field of experimental structural biology have provided high-resolution structures of active and inactive state G protein-coupled receptors (GPCRs), a highly important pharmaceutical target family, but the process of transition between these states is poorly understood. According to the current theory, GPCRs exist in structurally distinct, dynamically interconverting functional states of which populations are shifted upon binding of ligands and intracellular signaling proteins. However, explanation of the activation mechanism on an entirely structural basis gets complicated when multiple activation pathways and active receptor states are considered. Our unbiased, atomistic molecular dynamics simulations of the mu-opioid receptor in a physiological environment revealed that external stimulus is propagated to the intracellular surface of the receptor through subtle, concerted movements of highly conserved polar amino acid side chains along the 7th transmembrane helix. To amend the widely accepted theory we suggest that the initiation event of GPCR activation is the shift of macroscopic polarization between the ortho- and allosteric binding pockets and the intracellular G protein-binding interface. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/920769v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@102f33org.highwire.dtl.DTLVardef@8af6bforg.highwire.dtl.DTLVardef@14d9192org.highwire.dtl.DTLVardef@1903819_HPS_FORMAT_FIGEXP M_FIG Table of Contents Graphic C_FIG

molecular biology↗

bioRxiv2020

Regulation of human development by ubiquitin chain editing of chromatin remodelers

Embryonic development occurs through commitment of pluripotent stem cells to differentiation programs that require highly coordinated changes in gene expression. Chromatin remodeling of gene regulatory elements is a critical component of how such changes are achieved. While many factors controlling chromatin dynamics are known, mechanisms of how different chromatin regulators are orchestrated during development are not well understood. Here, we describe LINKED (LINKage-specific-deubiquitylation-deficiency-induced Embryonic Defects) syndrome, a novel multiple congenital anomaly disorder caused by hypomorphic hemizygous missense variants in the deubiquitylase OTUD5/DUBA. Studying LINKED mutations in vitro, in mouse, and in models of neuroectodermal differentiation of human pluripotent stem cells, we uncover a novel regulatory circuit that coordinates chromatin remodeling pathways during early differentiation. We show that the K48-linkage-specific deubiquitylation activity of OTUD5 is essential for murine and human development and, if reduced, leads to aberrant cell-fate specification. OTUD5 controls differentiation through preventing the degradation of multiple chromatin regulators including ARID1A/B and HDAC2, mutation of which underlie developmental syndromes that exhibit phenotypic overlap with LINKED patients. Accordingly, loss of OTUD5 during early differentiation leads to less accessible chromatin at neural and neural crest enhancers and thus aberrant rewiring of gene expression networks. Our work identifies a novel mechanistic link between phenotypically related developmental disorders and an essential function for linkagespecific ubiquitin editing of substrate groups (i.e. chromatin remodeling complexes) during early cellfate decisions - a regulatory concept, we predict to be a general feature of embryonic development.

genetics↗

bioRxiv2020

Non-essential function of KRAB zinc finger gene clusters in retrotransposon suppression

The Kruppel-associated box zinc finger protein (KRAB-ZFP) family amplified and diversified in mammals by segmental duplications, but the function of the majority of this gene family remains largely unexplored due to the inaccessibility of the gene clusters to conventional gene targeting. We determined the genomic binding sites of 61 murine KRAB-ZFPs and genetically deleted in mouse embryonic stem (ES) cells five large KRAB-ZFP gene clusters encoding nearly one tenth of the more than 700 mouse KRAB-ZFPs. We demonstrate that clustered KRAB-ZFPs directly bind and silence retrotransposons and block retrotransposon-borne enhancers from gene activation in ES cells. Homozygous knockout mice generated from ES cells deleted in one of two KRAB-ZFP clusters were born at sub-mendelian frequencies in some matings, but heterozygous intercrosses could also yield knockout progeny with no overt phenotype. We further developed a retrotransposon capture-sequencing approach to assess mobility of the MMETn family of endogenous retrovirus like elements, which are transcriptionally activated in KRAB-ZFP cluster KOs, in a pedigree of KRAB-ZFP cluster KO and WT mice. We identified numerous somatic and several germ-line MMETn insertions, and found a modest increase in activity in mutant animals, but these events were detected in both wild-type and KO mice in stochastic and highly variable patterns. Our data suggests that the majority of young KRAB-ZFPs play a non-essential role in transposon silencing, likely due to the large redundancy with other KRAB-ZFPs and other transposon restriction pathways in mice. One Sentence SummaryMegabase-scale deletions of KRAB-ZFP gene clusters in mice leads to retrotransposon activation.

genetics↗

bioRxiv2019

Dynamic evolution of cervical cancer mutations during chemoradiation using novel sampling approach

ObjectiveThe aim of this study was to validate a whole exome sequencing approach to longitudinally characterize the tumor mutational profile of cervical cancer patients undergoing chemoradiation (CRT). Experimental DesignCervical cancer tumor specimens from twenty-seven patients undergoing chemoradiation were collected before and throughout CRT and whole exome sequencing (WES) was performed to characterize individual mutations and alterations in unique genes. WES data were analyzed from cervical cancer patients in The Cancer Genome Atlas (TCGA) as a comparison group. ResultsOver 93% of mutated genes detected at baseline were present in TCGA. Tumor purity from collected swabs correlated with MRI tumor volumes during the course of treatment (R2=0.969). CDK4/CDK6/cyclin D1-related gene mutations involved in the ERK1/2, p16INK4, and p53 pathway and G1/S checkpoint most commonly persisted at the end of CRT. ConclusionThis non-invasive swab technique to serially sample tumor during CRT will allow new discoveries of dynamic tumor mutational profile changes during chemoradiation for mucosal tumors. Mutations that survived or increased during the initial weeks of radiation treatment are potential drivers of radiation resistance including the CDL4/CDK6/cyclin D1-related pathway. Statement of Translational RelevanceThere are no established biomarkers to predict chemoradiation (CRT) response for cervical cancer patients. Serial biopsies cannot be performed due to risks of bleeding and fistula. We used a novel non-invasive swab-based biopsy technique to obtain serial samples from a cohort of twenty-seven patients through the course of treatment, and validated this approach to obtain whole exome sequencing data. We analyzed dynamic tumor mutation profiles during CRT. Results from this study show that mutations in CDK4/CDK6/cyclin D1-related genes increased at the end of CRT, suggesting this pathway as a potential driver of radiation resistance.

cancer biology↗

bioRxiv2019

The kinesin-14, Ncd, drives a right-handed, helical motion of antiparallel microtubules around each other

Within the mitotic spindle, several kinesin motors crosslink and slide microtubules. While some of them (e.g. kinesin-5, kinesin-8 and kinesin-14) have been shown to exhibit sideways components in their step cycles, the impact of the resulting off-axis power strokes on motility and force generation in the spindle has not been investigated so far. Here, we develop and utilize a novel three-dimensional in vitro motility assay to explore the kinesin-14, Ncd, driven sliding of crosslinked, fluorescently-labeled microtubules. We find that free microtubules, sliding in an antiparallel orientation on microtubules suspended between nanofabricated ridges, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. In contrast, microtubules crosslinked in parallel orientation are static with neither longitudinal nor helical motion. Further, our technique allows us to measure the in situ spatial extension of the motors between the crosslinked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.

biophysics↗

bioRxiv2019

Tmod2 is a regulator of cocaine responses through control of striatal and cortical excitability, and drug-induced plasticity

Drugs of abuse induce neuroadaptations, including synaptic plasticity, that are critical for transition to addiction, and genes and pathways that regulate these neuroadaptations are potential therapeutic targets. Tropomodulin 2 (Tmod2) is an actin-regulating gene that plays an important role in synapse maturation and dendritic arborization and has been implicated in substance-abuse and intellectual disability in humans. Here we mine the KOMP2 data and find that Tmod2 knockout mice show emotionality phenotypes that are predictive of addiction vulnerability. Detailed addiction phenotyping showed that Tmod2 deletion does not affect the acute locomotor response to cocaine administration. However, sensitized locomotor responses are highly attenuated in these knockouts, indicating perturbed drug-induced plasticity. In addition, Tmod2 mutant animals do not self-administer cocaine indicating lack of hedonic responses to cocaine. Whole brain MR imaging shows differences in brain volume across multiple regions although transcriptomic experiments did not reveal perturbations in gene co-expression networks. Detailed electrophysiological characterization of Tmod2 KO neurons, showed increased spontaneous firing rate of early postnatal and adult cortical and striatal neurons. Cocaine-induced synaptic plasticity that is critical for sensitization is either missing or reciprocal in Tmod2 KO nucleus accumbens shell medium spiny neurons, providing a mechanistic explanation of the cocaine response phenotypes. Combined, these data provide compelling evidence that Tmod2 is a major regulator of plasticity in the mesolimbic system and regulates the reinforcing and addictive properties of cocaine.\n\nSignificance statementWe identify, characterize, and establish tropomodulin 2 (Tmod2), an actin-regulating gene exclusively expressed in neurons, as an important regulator of addiction-related phenotypes. We show that Tmod2, knockout mice (Tmod2 KO) exhibit phenotypes that are predictive of addiction. In detailed addiction phenotyping, we find the Tmod2 regulates cocaine sensitization and self-administration. We explore anatomical, transcriptional, electrophysiological mechanisms of this regulation. Combined these studies provide compelling evidence that Tmod2 is critical for synaptic plasticity necessary for transition to addiction.

neuroscience↗

bioRxiv2019

Pairing interactions between nucleobases and ligands in aptamer:ligand complexes of riboswitches: Crystal structure analysis, classification, optimal structures and accurate interaction energies

In the present work, sixty-seven crystal structures of the aptamer domains of RNA riboswitches, are chosen for analysis of the structure and strength of hydrogen bonding (pairing) interactions between nucleobases constituting the aptamer binding pockets and the bound ligands. A total of eighty unique base:ligand hydrogen-bonded pairs containing at least two hydrogen bonds were identified through visual inspection. Classification of these contacts in terms of the interacting edge of the aptamer nucleobase revealed that interactions involving the Watson-Crick edge are the most common, followed by the sugar edge of purines and the Hoogsteen edge of uracil. Alternatively, classification in terms of the chemical constitution of the ligand yields five unique classes of base:ligand pairs: base:base, base:amino acid, base:sugar, base:phosphate and base:other. Further, quantum mechanical (QM) geometry optimizations revealed that sixty seven out of eighty pairs exhibit stable geometries and optimal deviations from their macromolecular crystal occurrences. This indicates that these contacts are well-defined RNA aptamer:ligand interaction motifs. QM calculated interaction energies of base:ligand pairs reveal rich hydrogen bonding landscape, ranging from weak interactions (base:other, -3 kcal/mol) to strong (base:phosphate, -48 kcal/mol) contacts. The analysis was further extended to study the biological importance of base:ligand interactions in the binding pocket of the tetrahydrofolate riboswitch and thiamine pyrophosphate riboswitch. Overall, our study helps in understanding the structural and energetic features of base:ligand pairs in riboswitches, which could aid in developing meaningful hypotheses in context of RNA:ligand recognition. This can, in turn contribute towards current efforts to develop antimicrobials that target RNAs.

biophysics↗