Abstract:
Strand separation by negative supercoiling of B-DNA is known to give rise to noncanonical DNA structures. It is hypothesized that negative supercoiling might give rise to the formation of G-quadruplexes. Current literature involving in vitro studies offer contrasting results both for and against the above hypothesis. Ensemble assays like chemical foot printing, S1 nuclease cleavage and 2D-gel electrophoresis show correlation between negative superhelical stress and G-quadruplex formation in only in certain cases. On the other hand, force-extension measurements using magnetooptical tweezers and single molecule studies using atomic force microscopy (AFM) support the hypothesis. A real time dynamic assay will be useful to determine circumstances in which the hypothesis is true. Intercalation Induced Supercoiling of DNA (ISD) is a dynamic single-molecule technique to generate and visualize supercoiled DNA with the help of fluorescence microscopy. SG4 is a recently developed anti-G-quadruplex nanobody. We aim to use fluorescently labelled SG4 to probe G-quadruplex formation in negatively supercoiled DNA generated by ISD. Negatively supercoiled DNA was successfully generated through ISD assay. The purified SG4 nanobody however did not show G-quadruplex binding in gel mobility shift assay. Work needs to be done in optimizing conditions for the activity of the protein, only then can it be used as a probe.
