Synthesis and polymerase activity of a fluorescent cytidine TNA triphosphate analogue

Threose nucleic acid (TNA) is an artificial genetic polymer capable of undergoing Darwinian evolution to produce aptamers with affinity to specific targets. This property, coupled with a backbone structure that is refractory to nuclease digestion, makes TNA an attractive biopolymer system for diagnostic and therapeutic applications. Expanding the chemical diversity of TNA beyond the natural bases would enable the development of functional TNA molecules with enhanced physiochemical properties. Here, we describe the synthesis and polymerase activity of a fluorescent cytidine TNA triphosphate analogue (1,3-diaza-2-oxo-phenothiazine, tC(f)TP) that maintains Watson-Crick base pairing with guanine. Polymerase-mediated primer-extension assays reveal that tC(f)TP is efficiently added to the growing end of a TNA primer. Detailed kinetic assays indicate that tC(f)TP and tCTP have comparable rates for the first nucleotide incorporation step (k(obs1)). However, addition of the second nucleotide (k(obs2)) is 700-fold faster for tC(f)TP than tCTP due the increased effects of base stacking. Last, we found that TNA replication using tC(f)TP in place of tCTP exhibits 98.4% overall fidelity for the combined process of TNA transcription and reverse transcription. Together, these results expand the chemical diversity of enzymatically generated TNA molecules to include a hydrophobic base analogue with strong fluorescent properties that is compatible with in vitro selection.