MicroRNAs are non-coding RNA oligonucleotide sequences, approximately 22 nucleotides long
that function in post transcriptional gene regulation. They are emerging biomarkers for many
diseases, most notably cancer, and can be found in detectable quantities freely circulating in
biological fluids. Up-regulation of one such microRNA (miR-141) is associated with locally
advanced and metastatic prostate cancer and has potential as a minimally invasive biomarker
for prostate cancer. Sequence specific detection of miRNAs is challenging because of their short
length and high sequence homology between miRNAs. These problems can be tackled using
Peptide Nucleic Acid (PNA) hybridisation probes and Oligonucleotide Templated Reactions
(OTRs).
PNA is a synthetic DNA analogue, in which the sugar phosphate backbone is replaced by
an aminoethylglycine peptide chain. PNAs bind to natural nucleic acids (DNA or RNA) with
high affinity and sequence specificity. Oligonucleotide templated reactions are reactions between
chemically reactive moieties attached to oligonucleotides, catalysed by complementary binding
a target sequence. Most OTRs reported generate an optical readout. The combination of PNA
and OTR has previously been exploited in our lab as a valid approach for detecting microRNA
from biological samples. Herein, we report on a new design of OTR with an electrochemical
readout dubbed Electrochemical Oligonucleotide Templated Reaction (EOTR).
Chapter 2 demonstrates the first example of EOTR between PNA probes complementary
to miR-141. Ratiometric, quantitative and sequence-specific detection of miR-141 in vitro is
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achieved. Chapter 3 uses computational molecular dynamics to simulate EOTRs as a potential
new tool for in silico optimisation of PNA probes for miRNA sensing. Chapter 4 shows progress
towards translating EOTR onto suitable platform via immobilising one of the PNA probes onto
the surface of gold electrodes.
Overall, this work shows the development of a new biosensing strategy that will hopefully
be further translated into a miRNA sensing device for cancer diagnosis.