SEQUENCE-SPECIFIC SCISSION OF DNA BY THE CHEMICAL NUCLEASE ACTIVITY OF 1,10-PHENANTHROLINE-COPPER(I) TARGETED BY RNA

RNAs modified with the chemical nuclease 1,10-phenanthroline-copper(I) can achieve the sequence-specific scission of single- and double-stra nded DNA targets. The RNAs are prepared in vitro by using 5-(3-aminoal lyl)-UTP as the sole source of UTP and can be readily modified with 1, 10-phenanthroline by using N-succinimidyl 3-(2-pyridyl-dithio)propiona te (SPDP) to cross-link the ligand to the aminoallyl moiety. Single-st randed DNAs are efficiently cleaved at multiple sites because 1,10-phe nanthroline is incorporated at several uridines in the sequence. Seque nce-specific double-stranded scission of duplex DNA can also be accomp lished with 1,10-phenanthroline-derivatized RNA within R loops. These triple-stranded structures form in 70% formamide and involve the displ acement of one strand of DNA by the RNA of identical sequence. R loop- directed scission is the first method for DNA scission applicable to a ny sequence. A unique application of R loop-targeted nucleolytic sciss ion, which relies on its ability to cut DNA at any sequence, is the de termination of the distance between two marker DNA sequences within a target. In this case, 1,10-phenanthroline-linked RNAs are prepared fro m 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 se quences. For example, the distance separating two chicken delta crysta llin (delta1 and delta2) genes has been confirmed as 24 kilobases by t his method.