| Summary: | <p>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.</p>
<p>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.</p>
<p>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.</p>
|
|---|