Development of single-molecule DNA hybridisation tools for high-content analysis of protein-DNA interactions and gene expression

Thesis

This thesis presents results in the development of two single-molecule methods which use DNA hybridisation, in vitro and in vivo, to investigate protein-DNA interactions and processes.

Single-molecule DNA sequencing methods have made important contributions to genomics, diagnostics and functional analysis of genetic processes. Despite the fact that there are a large variety of single-molecule sequencing methods, there is currently no systematic way to connect the functional properties, with regards to protein interactions, of a single DNA molecule (single-molecule phenotype) with DNA sequence. In this thesis, I describe proof-of-principle experiments for ‘Gap-Seq’, a short-read single-molecule sequencing method that interrogates DNA sequence through the transient binding of short fluorescent DNA strands to surface-immobilised gapped-DNA substrates. A way to identify a base can be the dwell times of the short DNAs on the immobilised substrates: the correct base shows comparatively long dwells of the short DNAs on the substrates, whereas, non-complementary bases show no binding or comparatively shorter dwells on the substrates. I also show how single base DNA modifications affect protein-DNA interactions and a method for how Gap-Seq can be implemented to evaluate the DNA sequence at the protein binding site. Once developed further, Gap-Seq will fill a significant void in single-molecule DNA/RNA sequencing and will be important for both mechanistic work and screening libraries of nucleic acids against targets of biomedical importance.

I have also used DNA hybridisation to demonstrate the proof-of-principle for a new type of fluorescence in situ hybridisation (FISH) assay in live bacterial cells. Specifically, my work introduces ‘in vivo single-molecule FISH’ (in vivo smFISH), where short doubly labelled single-stranded DNAs are electroporated into cells and subsequently bind transiently or stably to target RNA. I establish that the electroporated probes exhibit site-specific binding and produce bright fluorescence signals that should lead to direct measurements of the kinetics of gene transcription on the chromosome, and advance the understanding of many fundamental processes involving RNA.

Authors: Andrews, R

• DOI: 10.5287/ora-xqnqeydzm
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English