Toward a Sensor for DNA Supercoiling

Abstract

DNA in the cell takes on a double helical structure that is then formed into larger loops, compacting into a supercoil. These supercoils are the result of mechanical torsional forces caused by over or underwound DNA. Supercoiling plays a strong role in chromatin compaction and gene expression; and the work of topoisomerases, which wind and unwind the DNA, are able to change topology of the DNA to facilitate transcription or replication. Despite the importance of supercoiling in DNA topology and DNA’s cellular function as a whole, there is not currently a single molecule sensor that can measure the degree of DNA supercoiling in vivo in real time. This thesis proposes a design, synthesis protocol, and incorporation protocol for a sensor that reports twist in DNA strands via fluorescence lifetime imaging (FLIM). The protocol proposed here suggests a synthesis derived from the commercially available FlipperTR molecule, resulting in a sensor that can be attached to alkyne-painted DNA strands via a bioorthogonal Click Chemistry reaction. Additionally, this thesis performs testing in vitro and in vivo in Escherichia coli of the proposed supercoiling sensor in order to establish its potential as an high-resolution in vivo reporter for twist in the genome. Data from this testing indicate that the sensor can be incorporated into the genome as proposed and does respond to torsional state in DNA via FLIM.