On-demand production of deoxynucleotides from genomic DNA

Abstract

There is a burgeoning of use of nucleic acid amplification testing (NAAT) diagnostics and a need for their associated reagents, especially in less resourced environments. NAATs are highly sensitive and specific assays, applicable in the detection of infectious and many noncommunicable diseases, and thus are crucial for disease management globally. The high cost of this technology, relative to traditional immunoassays and culture-based methods, has slowed the adoption of NAATs in low- and middle-income countries. This thesis explores a novel synthesis method to produce a key NAAT reagent, namely the 2'-deoxynucleoside 5'-triphosphate (dNTPs) building blocks of DNA, in a manner that can be integrated into a NAAT workflow. A unique top-down approach to produce dNTP monomers from a genomic DNA polymer is investigated, contrary to traditional bottom-up synthesis methods, which rely on inaccessible precursor molecules. Readily available and renewable bacterial genomic DNA provides a crude starting material to generate monophosphorylated deoxynucleotides (dNMPs) by the digestion of DNA with nuclease enzymes. The dNMPs are then phosphorylated to dNTPs by a set of in-house recombinantly produced kinase enzymes, and the produced dNTPs directly applied in NAAT, without the need for additional purification steps. In-house synthesised dNTPs were able to amplify PCR fragments up to 7.5 kb in length and showed successful specific detection of Plasmodium malariae and Plasmodium ovale malaria target genes, demonstrating its potential utility for diagnostics applications. Biocatalysis offers many advantages over chemical synthesis, but most importantly these biocatalysts can easily be regenerated in low-resource settings, building on the previous work for local enzyme production by the Cambridge Analytical Biotechnology Lab. Furthermore, the biocatalysts can be immobilised to silica particles and implemented in a reusable bioreactor device. The silica immobilised kinase enzymes showed reusability up to twelve times over two months, when stored at 4⁰C. Overall, this work has demonstrated a pathway to generate deoxynucleotides from genomic DNA, and investigated an immobilised enzyme milli-bioreactor as a proof-of-principle for how the technology can be translated into practice and integrated with NAAT in low-resource research and diagnostics labs.