Biology subjects

Kodikara, S. G.

Publications and source records attributed to Kodikara, S. G..

8 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↗

bioRxiv2025

Liquid Crystalline Ordering of Banana-Shaped Gapped DNA Duplexes

In-phase adenine-tracts (A-tracts) introduce intrinsic bending to double stranded DNA resulting in banana-shaped macromolecules. In this study, we investigate how such sequence-dependent bending influences DNA-based liquid crystalline (LC) phases formed by gapped DNA (GDNA) constructs. By incorporating three in-phase A-tracts into each duplex arm, we created a GDNA construct with bent duplexes and examined the LC phases they form using temperature-resolved synchrotron small-angle X-ray scattering and polarizing optical microscopy. Like the analogous constructs containing straight (rod-like) duplexes, the bent constructs exhibit a transition from bi-layer smectic-B phase to a monolayer smectic-A phase, although at ~30 {degrees}C lower temperatures. By comparing the monolayer spacing between the bent and straight constructs, we estimate a bending angle of ~11{degrees} per A-tract at room temperature and at physiologically relevant salt and cDNA. The bending angle decreases with increasing DNA concentration and temperature. Moreover, we demonstrate that divalent cations enhance the stability of the smectic-B phase up to ~30 mM Mg2+ but reduce it beyond that. The reduced thermal stabilities of the bilayer and in-layer ordering of bent duplexes imply reduced propensity for DNA condensation and heterochromatin formation under physiological conditions.

biophysics↗

bioRxiv2025

Locked Nucleic Acid Stabilized Liquid Crystalline Phases

Liquid crystalline (LC) phases formed by gapped DNA (GDNA) constructs, where two rigid duplexes are connected with a flexible single stranded linker, offer a versatile platform to investigate interactions between DNA molecules. Base pairs containing a locked nucleic acid (LNA-DNA or LNA-LNA pairs) are generally more stable compared to DNA-DNA pairs due to enhanced hydrogen bonding and/or attractive base stacking interactions. In concentrated solutions of GDNA constructs, the stability of terminal base pairs and the base stacking interactions between neighboring duplexes are critical for the formation of a bilayer smectic phase. By using temperature-resolved synchrotron small-angle X-ray scattering (SAXS) measurements, we quantified the impact of single LNA modification of terminal base pairs on the thermal stability of smectic LC phases. We observe that LNA-DNA terminal AT base pairing (A+T) increases the stability of the bilayer smectic phase by [~]9-18 {degrees}C relative to DNA-DNA pairing at the same duplex concentrations. While relatively large, this increase is still significantly less than the up to [~]30 {degrees}C increase observed when AT DNA-DNA pairs are replaced by GC pairs, suggesting the stacking interactions between A+T LNA-DNA base pairs are significantly weaker than those between unmodified GC base pairs. Our study illustrates the sensitivity of LC ordering in dense DNA solutions to a single nucleotide modification and demonstrates that LNA modifications can provide a new mechanism for tuning the stability of nucleic acid-based materials.

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↗

bioRxiv2023

Liquid Crystalline Layering and Divalent Cations Cooperatively Enhance DNA Condensation

The layered liquid crystalline (LC) phases formed by DNA molecules which include rigid and flexible segments ( gapped DNA) enable the study of both end-to-end stacking and side-to-side lateral interactions that drive the condensation of DNA molecules. The resulting layer structure exhibits long-range inter-layer and intra-layer positional correlations. Using synchrotron small-angle x-ray scattering (SAXS) measurements, we investigate the impact of divalent Mg2+ cations on the stability of the inter- and intra-layer DNA ordering as a function of temperature between 5-65 {degrees}C and for different terminal base pairings at the blunt ends of the gapped DNA constructs, which mediate the strength of the attractive end-to-end interaction. We demonstrate that the stabilities at a fixed DNA concentration of both inter-layer and intra-layer order are significantly enhanced even at a few mM Mg2+ concentration. The stability continues to increase up to [~]30 mM Mg2+ concentration, but at higher ([~]100 mM) Mg2+ content repulsion between positive ions counteracts and reverses the increase. On the other hand, sufficiently strong base-stacking interactions promote intra-layer order even in the absence of multivalent cations, which demonstrates the impact of liquid crystal layering on the DNA condensation process. We discuss the implications of these results in terms cation-mediated DNA-DNA attraction.

biophysics↗

bioRxiv2023

Effect of selective end-to-end base stacking interactions on the stability of smectic liquid crystal ordering in concentrated gapped DNA solutions

Positionally ordered bilayer liquid crystalline nanostructures formed by gapped DNA (GDNA) constructs provide a practical window into DNA-DNA interactions at physiologically relevant DNA concentrations; concentrations several orders of magnitude greater than those in commonly used biophysical assays. The bilayer structure of these states of matter is stabilized by end-to-end base stacking interactions; moreover, such interactions also promote in-plane positional ordering of duplexes that are separated from each other by less than twice the duplex diameter. The end-to-end stacked, as well as in plane ordered duplexes exhibit distinct signatures when studied via small angle x-ray scattering (SAXS). This enables analysis of the thermal stability of both the end-to-end and side-by-side interactions. We performed synchrotron SAXS experiments over a temperature range of 5-65 {degrees}C on GDNA constructs that differ only by the terminal base-pairs at the blunt duplex ends, resulting in identical side-by-side interactions while end-to-end base stacking interactions are varied. Our key finding is that bilayers formed by constructs with GC termination transition into the monolayer state at temperatures as much as 30 {degrees}C higher than for those with AT termination, while mixed (AT/GC) terminations have intermediate stability. By modeling the bilayer melting in terms of a temperature-dependent reduction in the average fraction of end-to-end paired duplexes, we estimate the stacking free energies in DNA solutions of physiologically relevant concentrations. The free-energies thereby determined are generally smaller than those reported in single molecule studies, which might reflect the elevated DNA concentrations in our studies. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/525591v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1ba488borg.highwire.dtl.DTLVardef@f0cbb2org.highwire.dtl.DTLVardef@cd3eaforg.highwire.dtl.DTLVardef@ce59b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Impact of Shelterin Complex on Telomere Accessibility

Shelterin plays critical roles in maintaining and protecting telomeres by regulating access of various physiological agents to telomeric DNA. We present single molecule measurements investigating the impact of the POT1 and a four-component shelterin complex on the accessibility of human telomeric DNA overhangs with physiologically relevant lengths (28-150 nt), which to our knowledge is the first direct approach to measure this effect on such telomeric constructs. To quantify telomere accessibility, we monitored transient binding events of a short peptide nucleic acid (PNA) probe that is complementary to telomeric overhangs using FRET-PAINT. Although POT1 has a mild G-quadruplex unfolding activity, it reduced accessibility of the PNA probe by [~]2.5 fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin reduced the accessibility of telomeric overhangs by [~]5-fold. This enhanced protection suggests shelterin restructures the region between single and double stranded telomere, which is otherwise the most accessible part of the overhang, by a synergistic cooperation of shelterin components located on single and double stranded telomere.

biophysics↗