Enzymatic synthesis of chemically modified DNA for analytical and diagnostic applications

Functionalised nucleotides have found widespread adoption in applications ranging from genome labelling to disease diagnostics. A popular means of introducing these modifications into nucleic acids is via the copper-catalysed azide-alkyne cycloaddition reaction. However, the toxicity of copper limits its use in vivo. Therefore, the copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (IEDDA) reactions were evaluated as a means of directly and indirectly labelling DNA and RNA. A variety of cyclooctyne, trans-cyclooctene and azide functionalised deoxyuridine triphosphates were synthesised and shown to be incorporated by primer extension, polymerase chain reaction (PCR) and reverse transcription. Bulky cyclooctyne and trans-cyclooctene triphosphates demonstrated moderate incorporation efficiency, while azide nucleotides with smaller functional groups were better substrates. PCR products of ca. 500 base pairs with 100% sequence fidelity can be formed, where all thymidine sites are replaced with azidohexanamidopropargyl deoxyuridine. These modified duplex products were efficiently labelled with fluorophores using the SPAAC and IEDDA reactions, and can be converted to fluorescent single-stranded probes through exonuclease digestion and streptavidin magnetic bead separation of the duplex followed by fluorophore-labelling. To expand the utility of this methodology, a new and simple single-tube dual labelling strategy was demonstrated to give a range of mixed dual coloured probes. Nucleosides bearing azide, trans-cylcooctene modifications and a fluorescent base analogue were evaluated for direct cellular DNA labelling and detection. When cultured adenocarcinoma A549 cells were treated with these nucleosides, then chemically fixed and fluorescently labelled by the SPAAC or IEDDA reaction, cytoplasmic staining without nuclear DNA labelling was observed. To improve the efficiency of fluorescent labelling of genomic DNA in cultured cells, phenyl isopropyl alanine-protected nucleoside monophosphates were prepared in an attempt to directly deliver 5' phosphorylated nucleosides into the cells. Preliminary fluorescence studies indicate bright foci in addition to cytoplasmic staining, the origins of which require further study.