Probing DNA polymerase activity with stereoisomeric 2′-fluoro-β-D-arabinose (2′F-araNTPs) and 2′-fluoro-β-D-ribose (2′F-rNTPs) nucleoside 5-triphosphates

2'-Deoxy-2'-fluoro-beta-D-ribonucleosides (2'F-rN) and 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides (2'F-araN) differ solely in the stereochemistry at the 2'-carbon of the furanose sugar ring. 2'F-rN 5'-triphosphates (2'F-rNTPs) are among the most commonly used sugar-modified nucleoside 5'-triphosphates (NTPs) for in vitro selection; however, the epimeric 2'F-araN 5'-triphosphates (2,F-araNTPs) have only recently been applied to polymerase-directed biosynthesis [C.G. Peng and M.J. Damha. J. Am. Chem. Sec. 129, 5310 (2007) The present study describes primer extension assays that compare, for the first time, the incorporation efficiency of the two isomeric NTPs, namely, 2'F-araNTPs or 2'F-rNTPs. by four DNA polymerases [Deep Vent (exo-), 9 degrees N-m, HIV-1 RT, and MMLV-RT]. Under the conditions used. incorporation of 2'F-araTTP proceeded more efficiently relative to 2'F-rUTP, while the incorporation of 2'F-araCTP is comparable or slightly less efficient than that observed with 2'F-rCTP. Interestingly, these preferences were observed for all four of the DNA polymerases tested. Unexpected differences in NTP incorporation were observed for 2'F-rCTP vs. rCTP. Despite their seemingly similar conformation, they behaved striking differently in the in vitro polymerization assays. 2'F-rCTP is a much better substrate than the native counterpart (rCTP), an observation first made with human DNA polymerases [F.C. Richardson, R.D. Kuchta, A. Mazurkiewicz, K.A. Richardson. Biochem. Pharmacol. 59, 1045 (2000)]. In contrast, 2'F-rUTP behaved like rUTP, providing poor yield of full-length products. Taken to-ether. this indicates that 2'F-rCTP is very unusual with regard to enzyme/substrate recognition; an observation that can be exploited for the production of DNA oligomers enriched with both ribose and arabinose modifications. These findings are timely given the significant interest and growing need to develop chemically modified oligonucleotides for therapeutic and diagnostic research. By examining the structure-activity relationship (SAR) of the ribose and arabinose sugar, this Study furthers Our understanding of how the nature of the 2' substituent (e.g., alpha vs. beta; F vs. OH) and the heterocyclic base affect NTP selection (specificity) by DNA polymerases.