Discovery and Modulation of an Unusual Terminal Mismatch Bypass Activity Involving 3′-Phosphorothioated Primers and Commercial Strand Displacement DNA Polymerases

Abstract Phosphorothioated (PST) oligonucleotides are extensively used in RNA therapeutics and biosensing applications. PST-modified linkages remain part of 13 approved oligonucleotide drug backbones (out of 23 total approved drugs). Despite the widespread utilization of PST linkages in oligonucleotide therapeutics, the full spectrum of their interactions with nontarget nucleic acids and DNA polymerases remains inadequately characterized. In this study, we report a novel discovery and the modalities (sequence dependence and length) of an unusual 3′-mismatch bypass activity involving 3′-PST primers, circular DNA (CD) template, and strand displacement DNA polymerases. We demonstrate that this activity persists for at least 24 nt of mismatch, is sequence-independent, and occurs when processed by three major commercially available strand displacement DNA polymerases commonly used in molecular diagnostics (phi29, BST large fragment (LF), BSU large fragment (LF)) with operational conditions spanning a large temperature range and buffer conditions. The 3′-terminal mismatch bypass activity of these three polymerases additionally extended to therapeutically relevant modifications such as locked nucleic acid (LNA) and 2′-O-methyl (2′-OMe). We also found that this mismatch bypass activity could be regulated by oligonucleotides complementary to the mismatch region. The significance of this study lies not only in the discovery and analytical investigation of the novel observation but also in its ramifications for biosensing and the nonspecific interactions of PST oligonucleotides in RNA therapeutics.