Design synthesis and evaluation of novel nucleotides for use in nanowire-based DNA analysis and diagnostic devices

Sanger DNA sequencing has been the gold standard for over 30 years and was instrumental in the completion of Human Genome Project in 2004. Whilst the method provides highly accurate sequence information, it is relatively expensive and the need for the electrophoretic separation of dideoxynucleotide-terminated DNA fragments limits its use for large-scale parallel sequencing or as a procedure for use outside the laboratory. In contrast, recent procedures such as pyrosequencing and sequencing by synthesis (SBS) dispense with the need for electrophoresis and offer solutions to some drawbacks of Sanger sequencing. SBS identifies each template base sequentially by the addition of the cognate nucleotide bearing a unique and cleavable tag (e.g fluorophore) to the primer strand. Nucleotide incorporation is accompanied by termination of DNA synthesis. The associated tag identifies the incorporated nucleotide and is then removed allowing the next nucleotide to be added and identified. Ideally the cleavable tag should be attached to the 3'-OH of the nucleotide such that it also functions as a blocking group preventing further chain extension. Unfortunately dNTPs with modified 3'-OH groups are typically not well-tolerated by most polymerases. However, progress has been achieved following directed evolution of existing polymerases to produce novel enzymes with an enhanced ability to incorporate a given nucleotide analogue. The most common approach in SBS has been to attach the cleavable tag to the base of the nucleotide which is also modified or blocked at the 3'-position. In a number of different examples the 3'-blocking group was chosen such that its removal (to produce a free 3'-OH for further extension) could be achieved simultaneously to the cleavage of the tag by using the same reagent in a single step. Several different 3'-blocking groups have been reported and include acyl, allyl, methoxymethyl, o-nitrobenzyl, azidomethyl and recently 3'-aminooxy analogues. QuantuMDx (QMDx) have developed biosensors employing nanowires and microfluidic technologies that can be used for the detection of biological analytes including nucleic acids within small handheld units. These devices rely upon the interaction of a charged biomolecule with an immobilised interacting partner attached to a nanowire which produces a small but measurable change in the electrical resistance of the nanowire. These devices have the capacity to extract and amplify DNA from patient samples and the current focus is to develop methodology for these devices which will rely upon DNA sequence analysis using SBS but crucially allow disease diagnoses or genome analysis at the point of care. The basis of the SBS methodology for development within the QMDx devices relies on a DNA primer immobilized via its 5'-terminus to a nanowire and annealed to the DNA template sequence. It is envisaged that nucleotides bearing a suitably charged cleavable tag rather than a fluorophore (commonly employed in SBS) will allow the identification of the inserted nucleotide. The initial stages of the project will focus on a TTP analogue, C5-propargylamino-dUTP to which will we will attach a cleavable linker containing one of the modifications listed above which in turn is appended to a negatively charged tag. This will use established amide coupling chemistry and explore the use of differing length linkers and effective charge on tag on the abilities of the analogues to be incorporated by one of a variety of DNA polymerases. Initially we will examine the incorporation of tagged nucleotides with unblocked 3'-OH groups and progress to explore the use of reversible blocking groups. It is also envisaged that analogues bearing the cleavable tag on the 3'-OH will be investigated. In this aspect of the work the evolution of novel polymerases will be undertaken by QMDx in ongoing studies in this area. Optimisation of the TTP analogue will be followed by synthesis of the A,C and G nucleotides.