Internal redox labelling of oligonucleotides

• Main Author: Cabezas-Hayes, Sinead
• Other Authors: Frost, Christopher
• Published: University of Bath 2018
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.767586

The aim of this thesis was to improve the detection method of electrochemical solution-based DNA diagnostics. Electrochemical solution-based detection lacks in sensitivity compared to surface-immobilised techniques or fluorescent probes, however benefits from cheaper equipment and materials, and lower levels of user-input required. Improvements in electrochemical signal upon detection of DNA would allow for improved sensitivity of the probes and therefore more useful diagnostic devices. To achieve this, it was hypothesised that the incorporation of several ferrocene labels onto oligodeoxynucleotides could offer improved electrochemical performance. The synthesis of a library of ferrocene-based nucleotides is discussed in Chapter 2, with a view to increasing the possible increase in electrochemical signal analysed via differential pulse voltammetry that can be achieved via the introduction of multiple ferrocene units on a single strand. This was achieved through the conjugation of ferrocene onto 2'-deoxyuridine utilising the copper-catalysed azide-alkyne cycloaddition, followed by solid phase synthesis of oligonucleotides to allow the introduction of up to five ferrocenes on a single oligonucleotide strand. The digestion of two DNA sequences was studied with S1 nuclease, showing an increase in signal is possible in a single-stranded assay. Chapter 3 expands on this methodology, next synthesising ferrocene labels with increased sensitivity vs. those developed in Chapter 2. This was achieved through the introduction of two ferrocenes onto a single nucleotide, followed by incorporation into a clinically relevant DNA sequence, allowing the synthesis of oligonucleotides containing ten ferrocene units. The probes were again treated with S1 nuclease displaying increased sensitivity measured via differential pulse voltammetry over the traditional 5'-labelling method, as well as the probes developed in the previous Chapter. Chapter 4 details the incorporation of the probes previously developed into diagnostic assays, using both T7 exonuclease and lambda exonuclease to digest the novel probes. The internal labelling strategy was shown to be active towards T7 exonuclease digestion, however this did not offer improved digestion properties compared to traditional methods. The use of multiplex detection system was suitable for the detection of two clinically relevant DNA sequences simultaneously, while also offering insight into the mechanistic properties of T7 exonuclease. The incorporation of internal labels enabled the use of lambda exonuclease, the use of which has been limited by the previous labelling strategy.