Thesis Defense: Michael Mohsen, Kool Group

DNA polymerases constitute a class of enzymes that catalyze the synthesis of DNA molecules from their building blocks, the deoxynucleoside triphosphates (dNTPs). These enzymes are responsible for accurately replicating the entire genome of organisms across the domains of life. They have also found widespread utility in nucleic acid amplification techniques such as polymerase chain reaction (PCR). Using chemical strategies, we have sought to expand the functionality of DNA polymerase enzymes with the development of two novel classes of chimeric dinucleotides that can substitute for canonical dNTPs as substrates of DNA polymerase: ATP-releasing nucleotides (ARNs) and dicaptides.

 

ARNs are comprised of four dinucleotide compounds each with a riboadenosine moiety linked via a tetraphosphate bridge to one of the four canonical deoxynucleoside bases found in DNA. We demonstrated that ATP released upon incorporation of ARNs by DNA polymerase could be quantified using a luciferase-based assay, effectively linking DNA synthesis to luminescence signaling. We employed ARNs in this strategy to report on DNA polymerase activity. This led to the development of a new method, polymerase-amplified release of ATP (POLARA) to detect point mutations associated with specific cancers and diseases. In addition to being rapid and sensitive, this method is luminescence-based and isothermal, indicating its potential for diagnostic testing in point-of-care and low-resource environments.

A second class of chimeric dinucleotides known as dicaptides consists of two deoxynucleoside heads bridged by a pentaphosphate linker. Existing as either of two pseudocomplentary pairs, dicaptides replace dNTPs in DNA synthesis and contain the reactivity of two nucleotides at once. Dicaptides offer several benefits including increased thermal stability and decreased production of the inhibitory pyrophosphate byproduct, two issues that hinder the effectiveness of dNTPs. Dicaptides were shown to outperform dNTPs during PCR under extended thermal cycling conditions.