Biology subjects

Kabir, M. L.

Publications and source records attributed to Kabir, M. L..

6 recordsLinked to original sources

bioRxiv2026

Cap-Independent Translation Regulation of VEGF Using CRISPR-dCas13d

Vascular endothelial growth factor (VEGF) is a central driver of pathological angiogenesis in cancer and retinal disease. A G-quadruplex (GQ) structure that forms in an Internal Ribosome Entry Site (IRES) of VEGF was proposed to recruit the 40S ribosome and initiate cap-independent translation under hypoxia. We demonstrate that blocking this GQ with dCas13d represses VEGF translation by up to 5-fold, without impacting mRNA abundance, under hypoxia. Similar levels of repression were attained in HeLa and human umbilical vein endothelial cells (HUVECs) using immunofluorescence and western blot assays, as well as a bicistronic dual-luciferase experiment. Single molecule Forster resonance energy transfer (smFRET) and fluorescence enhancement assays demonstrated destabilization of the GQ by CRISPR-dCas13d. We also tested the functional consequences of this regulation using tube formation assay in bovine aortic endothelial cells (BAOECs), which demonstrated consistent results with VEGF expression levels. Our study demonstrates the feasibility of using CRISPR-dCas13d as a sequence-specific and transient translation regulator for a large group of genes that contain translationally-active secondary structures.

biophysics↗

bioRxiv2025

Accessibility of Telomeric Overhangs to Stabilizing Small-Molecule Ligands

Human chromosomes terminate in 50-300 nucleotide (nt) long single-stranded telomeric overhangs composed of repeating d(TTAGGG) sequences, which can fold into tandem G-quadruplex (GQ) structures that protect chromosome ends. Stabilization of GQs by small-molecule ligands inhibits telomerase activity, motivating extensive efforts to develop GQ-targeting anti-cancer therapeutics. However, how interactions between successive GQs and bound ligands influence small-molecule accessibility remains poorly understood. Here, we employ single-molecule fluorescence microscopy and stepwise photobleaching analysis to quantify the binding stoichiometry of a fluorescently-labeled oxazole telomestatin derivative (L1Cy5-7OTD) to telomeric overhangs capable of forming 1-6 GQs (30-162 nt long), spanning much of the physiologically relevant range. We find that longer overhangs accommodate more ligands on average but exhibit consistently lower binding stoichiometry than the theoretical maximum, saturating at six molecules even in constructs with twelve binding sites. This trend was further supported by experiments showing increased L1Cy5-7OTD binding when the inter-GQ spacer was extended from 3-nt to 9-nt. This effect was independently confirmed by ensemble fluorescence enhancement experiments utilizing N-methyl mesoporphyrin IX (NMM) as a ligand. Complementary modeling with a one-dimensional lattice model describing equilibrium ligand binding to partially ordered telomeric overhangs revealed positive cooperativity between folding of successive GQs, negative cooperativity between ligands bound opposing (top and bottom) faces of successive GQs, and reduced binding affinity to GQs located at the junction of double stranded and single stranded telomeres. Together, these findings demonstrate how telomeric overhang architecture governs ligand accessibility and provide mechanistic insight to guide the rational design of GQ-targeting anticancer agents.

biophysics↗

bioRxiv2024

Combining the CRISPR Activation and Interference Capabilities Using dCas9 and G-Quadruplex Structures

We demonstrate that both CRISPR interference and CRISPR activation can be achieved at RNA and protein levels by targeting the vicinity of a putative G-quadruplex forming sequence (PQS) in the c-Myc promoter with nuclease-dead Cas9 (dCas9). The achieved suppression and activation in Burkitts Lymphoma cell line and in in vitro studies are at or beyond those reported with alternative approaches. When the template strand (contains the PQS) was targeted with CRISPR-dCas9, the G-quadruplex was destabilized and c-Myc mRNA and protein levels increased by 2.1-fold and 1.6-fold, respectively, compared to controls in the absence of CRISPR-dCas9. Targeting individual sites in the non-template strand with CRISPR-dCas9 reduced both the c-Myc mRNA and protein levels (by 1.8-fold and 2.5-fold, respectively), while targeting two sites simultaneously further suppressed both the mRNA (by 3.6-fold) and protein (by 9.8-fold) levels. These were consistent with cell viability assays when single or dual sites in the non-template strand were targeted (1.7-fold and 4.7-fold reduction in viability, respectively). We also report extensive in vitro biophysical studies which are in quantitative agreement with these cellular studies and provide important mechanistic details about how the transcription is modulated via the interactions of RNA polymerase, CRISPR-dCas9, and the G-quadruplex.

molecular biology↗

bioRxiv2024

Reusable Microfluidic Chambers for Single-Molecule Microscopy

Maintaining a consistent environment in single molecule microfluidic chambers containing surface-bound molecules requires laborious cleaning and surface passivation procedures. Despite such efforts, variations in non-specific binding and background signals commonly occur across different chambers. Being able to reuse the chambers without degrading the surface promises significant practical and fundamental advantages; however, this necessitates removing the molecules attached to the surface, such as DNA, proteins, lipids or nanoparticles. Biotin-streptavidin attachment is widely used for such attachments as biotin can be readily incorporated to these molecules. In this study, we present single-molecule fluorescence experiments that demonstrate effective resetting and recycling of the chambers at least 10 times using photocleavable biotin (PC-biotin) and UV light exposure. This method differs from alternatives as it does not utilize any harsh chemical treatment of the surface. We show that all bound molecules (utilizing various PC-biotin attachment chemistries) can be removed from the surface by a 5-min UV exposure of a specific wavelength. Non-optimal wavelengths and light sources showed varying degrees of effectiveness. Our approach does not result in any detectable degradation of surface quality, as assessed by non-specific binding of fluorescently labeled DNA and protein samples, and recovery of DNA secondary structure and protein activity. The speed and efficiency of the resetting process, the cost-effectiveness of the procedure, and the widespread use of biotin-streptavidin attachment make this approach adaptable for a wide range of single-molecule applications.

biophysics↗

bioRxiv2024

Stalling of Transcription by Putative G-quadruplex Sequences and CRISPR-dCas9

Putative G-quadruplex forming sequences (PQS) have been identified in promoter sequences of prominent genes that are implicated among others in cancer and neurological disorders. We explored mechanistic aspects of CRISPR-dCas9-mediated gene expression regulation, which is transient and sequence specific unlike alternative approaches that lack such specificity or create permanent mutations, using the PQS in tyrosine hydroxylase (TH) and c-Myc promoters as model systems. We performed in vitro ensemble and single molecule investigations to study whether G-quadruplex (GQ) structures or dCas9 impede T7 RNA polymerase (RNAP) elongation process and whether orientation of these factors is significant. Our results demonstrate that dCas9 is more likely to block RNAP progression when the non-template strand is targeted. While the GQ in TH promoter was effectively destabilized when the dCas9 target site partially overlapped with the PQS, the c-Myc GQ remained folded and stalled RNAP elongation. We also determined that a minimum separation between the transcription start site and the dCas9 target site is required for effective stalling of RNAP by dCas9. Our study provides significant insights about the factors that impact dCas9-mediated transcription regulation when dCas9 targets the vicinity of sequences that form secondary structures and provides practical guidelines for designing guide RNA sequences.

molecular biology↗

bioRxiv2023

A computational investigation on Rho-related GTP-binding protein RhoB through molecular modeling and molecular dynamics simulation study

BackgorundRhoB is a key member of the Rho family of isoprenylated small GTPases which modulate the cellular cytoskeletal organization. It has a crucial role in the neoplastic apoptotic mechanism after DNA damage. Due to the unavailability of 3D structure in the protein data bank database, in this study, we evaluated the structure of a protein, Rho-related GTP-binding protein RhoB. ResultsRhoB has a predicted pI of 5.10, indicating that it is acidic. The GMQE value was used to compute the target-template alignment, and 6hxu.1.A from Homo sapiens was chosen as the template structure, with the model construction task completed using swiss-model. The structural compactibility and stability were revealed after a 100ns molecular dynamics simulation using GROMACA employing the OPLS-AA force field. PCA analysis found residues that are relevant based on their fluctuation acitivity while their location is between 100-110 and 140-150. ConclusionThis study will benefit future investigations addressing the association between gene mutation and abnormalities generated by protein Rho-related GTP-binding protein RhoB in apoptotic events by offering insight into the biophysical phenomenon of Rho-related GTP-binding protein RhoB inhibitors.

bioinformatics↗