Biology subjects

Gyawali, P.

Publications and source records attributed to Gyawali, P..

5 recordsLinked to original sources

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↗

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↗

bioRxiv2021

A Single Molecule Investigation of I-Motif: Stability, Folding Kinetics, and Potential as an In-situ pH Sensor

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for physiologically low pH levels (pH<6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Forster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the kinetics of i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor establish the time scale for the pH drop in a commonly used oxygen scavenging system.

biophysics↗