Sequence-specific scission of DNA by the chemical nuclease activity of 1,10-phenanthroline-copper(I) targeted by RNA.

Abstract

RNAs modified with the chemical nuclease 1,10-phenanthroline-copper(I) can achieve the sequence-specific scission of single- and double-stranded DNA targets. The RNAs are prepared in vitro by using 5-(3-aminoallyl)-UTP as the sole source of UTP and can be readily modified with 1,10-phenanthroline by using N-succinimidyl 3-(2-pyridyl-dithio)propionate (SPDP) to cross-link the ligand to the aminoallyl moiety. Single-stranded DNAs are efficiently cleaved at multiple sites because 1,10-phenanthroline is incorporated at several uridines in the sequence. Sequence-specific double-stranded scission of duplex DNA can also be accomplished with 1,10-phenanthroline-derivatized RNA within R loops. These triple-stranded structures form in 70% formamide and involve the displacement of one strand of DNA by the RNA of identical sequence. R loop-directed scission is the first method for DNA scission applicable to any sequence. A unique application of R loop-targeted nucleolytic scission, which relies on its ability to cut DNA at any sequence, is the determination of the distance between two marker DNA sequences within a target. In this case, 1,10-phenanthroline-linked RNAs are prepared from the two distinct sequences and used to cut the DNA fragment after R-loop formation. The size of the fragment liberated by these methods is a direct measure in base pairs of the distance between the two DNA sequences. For example, the distance separating two chicken delta crystallin (delta 1 and delta 2) genes has been confirmed as 24 kilobases by this method.