Biology subjects

Granzhan, A.

Publications and source records attributed to Granzhan, A..

3 recordsLinked to original sources

bioRxiv2023

iMab Antibody Binds Single-Stranded Cytosine-Rich Sequences and Unfolds DNA i-Motifs

i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+{middle dot}C base pairs in cytosine-rich DNA sequences, predominantly at pH < 7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, that was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions (pH 5.8 or 6.5), similarly to what has been observed with hnRNP K, well-studied single- stranded DNA binding protein. Taken together, our results suggest that iMab actually binds to blocks of 2-3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.

biophysics↗

bioRxiv2020

Disclosing the actual efficiency of G-quadruplex DNA-disrupting small molecules

The quest for small molecules that avidly bind to G-quadruplex-DNA (G4-DNA, or G4), so called G4-ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to i- screen or design G4-ligands, ii- evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and iii- use them as unique chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts at developing small molecules aimed at destabilizing G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application to neurobiology as many genetic and age-related diseases are caused by an over-representation of G4s, itself caused by a deficiency of G4-resolving enzymes, the G4-helicases. Herein, we report on our double effort to i- develop a reliable in vitro assay to identify molecules able to destabilize G4s, the G4-unfold assay, and ii- fully characterize the first prototype of G4-disrupting small molecule, a phenylpyrrolcytosine (PhpC)-based G-clamp analog.

biophysics↗

bioRxiv2020

Harnessing intrinsic fluorescence for typing of secondary structures of DNA

DNA is polymorphic since, despite its ubiquitous presence as a double-stranded helix, it is able to fold into a plethora of other secondary structures both in vitro and in cells. Despite the considerable advances that have been made in understanding this structural diversity, its high-throughput investigation still faces severe limitations. This mainly stems from the lack of suitable label-free methods, combining a fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of the suitability of this phenomenon for tracking the conformational changes of DNA, we examined the intrinsic steady-state emission spectra of an 89-membered set of synthetic oligonucleotides with reported conformation (G-quadruplexes, i-motifs, single- and double stranded DNA) by means of multivariate analysis. Specifically, principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, albeit without discrimination between single- and double-stranded structures. Linear discriminant analysis of the same training set was exploited for the assessment of new sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labelling agent or dye, avoiding the related intrinsic bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5'-d[(G3T)3G3]-3' (G3T), the most fluorescent G4 structure reported to date. This property is likely to arise from the similar base-stacking geometry in both types of structures.

biophysics↗