Comparative ability of RNA and DNA to prime DNA synthesis in vitro: role of sequence, sugar, and structure of template-primer.

Abstract

The priming efficiency of oligo(RNA) vs. oligo(DNA) in a homopolymer template-homooligomer primer system was compared using four DNA polymerases. The templates included (dT)n, (dA)n, (dC)n, and (dI)n. Primers were the oligomers of the complementary DNA OR RNA with chain lengths of 6 to 23. The DNA polymerases used were from Micrococcus luteus, avian myeloblastosis virus (AMV), and Escherichia coli (polymerase I and polymerase III). The polymerases demonstrated a preference for the DNA primers with (dC)n, (dA)n, and (dI)n templates. However, when (dT)n was the template, all but the AMV polymerase preferred (rA)11 more than 200-fold better than (dA)12. This preference was due to the physical structure of the initiation complex. The structures of the oligo-polymer complexes were characterized by mixing curves, melting curves, and analytical bouyant density analyses. (rA)11 + (dT)n formed predominatly a duplex structure, whereas (dA)12 + (dT)n formed the three- stranded structure, (dA12-2(dT)n. The Km of the duplex with E. coli Pol III was 2.9 mugM (rA)11. The Ki of the triplex was 2.2 mugM (dA)12, indicating that Pol III could bind to the triplex but would not elongate the (dA)12 primer. The influence of structure on priming also was demonstrated with longer oligomers, (dA)23 and (rA)23, where the (dA)23 formed more duplex-like structures and primed more than the (dA)12.(dT)10 + (dA)n complexes also were shown to form triplex structures that inhibited priming. These results show that template-primer structure has more influence on priming than the sugar moiety or the sequence of the nucleic acid.