UC Riverside

An extended genetic alphabet can provide insight to the development of biological structure and function with respect to the evolution and mechanism of DNA and RNA polymerization. Designing an unnatural base pair (UBP) that is isosteric to the canonical bases would increase their affinity for recognition by polymerases in enzymatic processes. The isosteric metal-mediated base pairs (MMBPs) purine-2,6-dicarboxylate and 3-pyridine (PurDC•3Py) met many of the conditions needed for polymerization due to their Watson-Crick like pairing. The need for a MMBP with a higher affinity and fidelity towards enzymatic processes led to investigating imidazole (Im), 2-methylimidazole (2Me), 4-methyimidazole (4Me), 1,2,3-triazole (3Y), and 1,2,4-triazole (4Y) as potential pyrimidine analogs complimentary to PurDC. Oligonucleotides incorporating Im•PurDC or 4Me•PurDC metallo-base pairs were found to be the most thermally stable. Their stability depended on the presence of Cu2+, and was comparable to the canonical A•T pair.Incorporating a novel set of UBPs requires reliable enzymatic recognition of the UBPs by polymerizes to perform subsequent replication, transcription, or translation. Im•PurDC or 4Me•PurDC in the presence of Cu2+ were characterized and evaluated for enzymatic compatibility via single nucleotide insertion and extension by varying polymerases. Both insertion and extension for PurDC•Im and PurDC•4Me pairs were investigated. Therminator and Sulfolobus polymerases were found to produce full extension for the azole template. The role of Cu2+ was investigated and the Cu2+ threshold required for primer extension was found to be 10 µM. In addition, insertion across an azole template could be stopped with the removal of Cu2+ via a Cu2+ ligand. The same was not true for insertion across a PurDC template. An RNA that can act as its own enzyme in polymerization offers support for an RNA world. A hairpin ribooligonucleotide that acts as both template and primer in non-enzymatic polymerization has been shown to elongate in the presence of C and G residues, albeit over a length of time not ideal in prebiotic conditions. In order to increase the rate of template-directed nonenzymatic oligomerization, four hairpin templates with varying TNA residues were investigated. The presence of 2-AmImpG and 2-AmImpC to create a reactive imidazolium-bridged dinucleotide intermediate was not found to produce extension in under 48 hours.