Javadekar, SM and Nilavar, NM and Paranjape, A and Das, K and Raghavan, SC (2020) Characterization of G-quadruplex antibody reveals differential specificity for G4 DNA forms. In: DNA Research, 27 (5).
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Abstract
Accumulating evidence suggests that human genome can fold into non-B DNA structures, when appropriate sequence and favourable conditions are present. Among these, G-quadruplexes (G4-DNA) are associated with gene regulation, chromosome fragility and telomere maintenance. Although several techniques are used in detecting such structures in vitro, understanding their intracellular existence has been challenging. Recently, an antibody, BG4, was described to study G4 structures within cells. Here, we characterize BG4 for its affinity towards G4-DNA, using several biochemical and biophysical tools. BG4 bound to G-rich DNA derived from multiple genes that form G-quadruplexes, unlike complementary C-rich or random sequences. BLI studies revealed robust binding affinity (Kd = 17.4 nM). Gel shift assays show BG4 binds to inter- and intramolecular G4-DNA, when it is in parallel orientation. Mere presence of G4-motif in duplex DNA is insufficient for antibody recognition. Importantly, BG4 can bind to G4-DNA within telomere sequence in a supercoiled plasmid. Finally, we show that BG4 binds to form efficient foci in four cell lines, irrespective of their lineage, demonstrating presence of G4-DNA in genome. Importantly, number of BG4 foci within the cells can be modulated, upon knockdown of G4-resolvase, WRN. Thus, we establish specificity of BG4 towards G4-DNA and discuss its potential applications. © 2020 The Author(s).
| Item Type: | Journal Article |
|---|---|
| Publication: | DNA Research |
| Publisher: | Oxford University Press |
| Additional Information: | The copyright for this article belongs to the Author(s). |
| Keywords: | double stranded DNA; guanine quadruplex; antibody; DNA; guanine quadruplex, Article; binding affinity; controlled study; DNA binding; gene knockdown; human; human cell; molecular recognition; telomere; cell line; HEK293 cell line; HeLa cell line; human genome; immunology, Antibodies; Cell Line; DNA; G-Quadruplexes; Genome, Human; HEK293 Cells; HeLa Cells; Humans |
| Department/Centre: | Division of Biological Sciences > Biochemistry |
| Date Deposited: | 11 Jan 2023 09:31 |
| Last Modified: | 11 Jan 2023 09:31 |
| URI: | https://eprints.iisc.ac.in/id/eprint/79049 |
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