Substrates for Investigation of DNA Polymerase Function: Synthesis and Properties of 4′-C-Alkylated Oligonucleotides

In addition to their potential as diagnostic and therapeutic agents, modified oligonucleotides have also been shown to be highly valuable tools for examination of complex biological processes. Carefully designed nucleotide analogues have therefore found considerable application in investigations of DNA polymerase function and mechanism. To examine the contribution of primarily steric constraints on DNA polymerase selectivity, we have developed a new functional strategy based on the use of modified nucleotide analogues that differ primarily in their steric demand. Here we report the efficient synthesis of modified thymidine analogues bearing 4′-alkyl groups with varying steric demand, the effects of 4′-alkylation on sugar puckering, and the incorporation of these analogues into oligonucleotides by use of automated solid-phase DNA synthesis. We also studied the pairing properties of 4′-alkylated oligonucleotide duplexes in relation to those of their natural counterparts under a range of buffer conditions. In general, our studies indicate that 4′-alkylation of thymidines has little effect on nucleoside and oligonucleotide conformation. These results have relevance to the previously reported action of 4′-alkylated nucleotides and oligonucleotides as probes of DNA polymerase function and mechanism. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)