Synergistic and non-specific nucleic acid production by T7 RNA polymerase and Bsu DNA polymerase catalyzed by single-stranded polynucleotides

Abstract

Point-of-care molecular diagnostic tests show great promise for providing accurate, timely results in low-infrastructure healthcare settings and at home. The design space for these tests is limited by a variety of possible background reactions, which often originate from relatively weak promiscuous activities of the enzymes used for nucleic acid amplification. When this background signal is amplified alongside the signal of the intended biomarker, the dynamic range of the test can be severely compromised. Therefore, a detailed knowledge of potential side reactions arising from enzyme promiscuity can improve rational design of point-of-care molecular diagnostic tests. Towards this end, we report a previously unknown synergistic reaction between T7 RNA polymerase and Bsu DNA polymerase that produces nucleic acid in the presence of single-stranded DNA or RNA. This reaction occurs in the absence of any previously reported substrates for either polymerases and compromises a theoretical microRNA amplification scheme utilizing these polymerases.