Biology subjects

Chaires, J.

Publications and source records attributed to Chaires, J..

4 recordsLinked to original sources

bioRxiv2021

POT1 Stability and Binding Measured by Fluorescence Thermal Shift Assays

The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 - DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

biophysics↗

bioRxiv2019

Folding landscape of a parallel G-quadruplex

Circular dichroism and stopped-flow UV spectroscopies were used to investigate the thermodynamic stability and the folding pathway of d(TGAG3TG3TAG3TG3TA2) at 25C in solutions containing 25 mM KCl. Under these conditions the oligonucleotide adopts a thermally stable, all-parallel G-quadruplex topography containing three stacked quartets. K+-induced folding shows three resolved relaxation times, each with distinctive spectral changes. Folding is complete within 200 s. These data indicate a folding pathway that involves at least two populated intermediates, one of which seems to be an antiparallel structure that rearranges to the final all-parallel conformation. Molecular dynamics reveals a stereochemically plausible folding pathway that does not involve complete unfolding of the intermediate. The rate of unfolding was determined using complementary DNA to trap transiently unfolded states to form a stable duplex. As assessed by 1D-1H NMR and fluorescence spectroscopy, unfolding is extremely slow with only one observable rate-limiting relaxation time.

biophysics↗

bioRxiv2017

A high-throughput fluorescent indicator displacement assay and principal component/cluster data analysis for determination of ligand-nucleic acid structural selectivity

We describe a high-throughput fluorescence indicator displacement assay (HT-FID) to evaluate the affinity and the selectivity of compounds binding to different DNA structures. We validated the assay using a library of 30 well-known nucleic acid binders containing a variety chemical scaffolds. We used a combination of principal component analysis and hierarchical clustering analysis to interpret the results obtained. This analysis classified compounds based on selectivity for AT-rich, GC-rich and G4 structures. We used the FID assay as a secondary screen to test the binding selectivity of an additional 20 compounds selected from the NCI diversity set III library that were identified as G4 binders using a thermal shift assay. The results showed G4 binding selectivity for only a few of the 20 compounds. Overall, we show that this HT-FID assay, coupled with PCA and HCA, provides a useful tool for the discovery of ligands selective for particular nucleic acid structures.

biophysics↗

bioRxiv2017

G-quadruplex secondary structure from circular dichroism spectroscopy

A curated library of circular dichroism spectra of 23 G-quadruplexes of known structure was built and analyzed. The goal of this study was to use this reference library to develop an algorithm to derive quantitative estimates of the secondary structure content of quadruplexes from their experimental CD spectra. Principle component analysis and singular value decomposition were used to characterize the reference spectral library. CD spectra were successfully fit to obtain estimates of the amounts of base steps in anti-anti, syn-anti or anti-syn conformations, in diagonal or lateral loops or in other conformations. The results show that CD spectra of nucleic acids can be analyzed to obtain quantitative structural information about secondary structure content in an analogous way to methods used to analyze protein CD spectra.

biophysics↗