Origin Of High Fidelity In Target-sequence Recognition By Pna Ce(iv)/edta Combinations As Site-selective Dna Cutters

摘要

Double-duplex invasion of pseudocomplementary peptide nucleic acid (pcPNA) is one of the most important strategies for recognizing a specific site in double-stranded DNA (Proc. Natl. Acad. Sci. U.S.A. 1999, 96,11804-11808). This strategy has recently been used to develop artificial restriction DNA cutters (ARCUTs) for site-selective scission of double-stranded DNA, in which a hot spot formed by double-duplex invasion of PNA was hydrolyzed by Ce(IV)/EDTA (Nat. Protoc. 2008, 3, 655-662). The present paper shows how and where the target sequence in double-stranded DNA is recognized by the PNA-Ce(IV)/ EDTA combinations for site-selective scission. The mismatch-recognizing activities in both the invasion process and the whole scission process are evaluated. When both pcPNA additives are completely complementary to each strand of the DNA, site-selective scission is the most efficient, as expected. Upon exchange of one DNA base pair at the invasion site with another base pair, which introduces mismatches between the pcPNAs and the DNA, the site-selective scission by the ARCUT is notably diminished. Mismatches in (or near) the central double-invasion region are especially fatal, showing that Watson-Crick pairings of the DNA bases in this region with the pcPNA strands are essential for precise recognition of the target sequence. Both gel-shift assays and melting temperature measurements on the double-duplex invasion process have confirmed that the fidelity in this process primarily governs the fidelity of the DNA scission. According to these systematic analyses, the typical ARCUT involving two 15-mer pcPNAs precisely recognizes 14 -16 base pairs in substrate DNA. This remarkable fidelity is accomplished at rather high salt concentrations that are similar to the values in cells.