SITE SPECIFIC HYDROLYSIS OF DNA AND RNA

The goal of this project is to develop the chemistry of metal-peroxo complexes for the "hydrolytic" cleavage of DNA and RNA. The basis for this project is our recent observation that the H2O2 adduct of the dinuclear complex Fe2(HPTB)OH(NO3)4 (HPTB = N,N,N',N'-tetrakis(2- benzimidazolylmethyl)- 1,3-diamino-2-hydroxypropane) cleaves supercoiled pBR322 upon mixing at room temperature. The same result is obtained when H2O2 is replaced with O2 and a reductant. This cleavage chemistry is unique in several respects: it is double stranded; it occurs at a specific site; and it affords 3'-OH and 5'-OPO3 ends implicating a hydrolytic, rather than an oxidative mechanism. We propose to carry out experiments to understand the mechanism of cleavage and the underlying basis for the specificity of this relatively simple reagent. The cleavage is hypothesized to be the nucleophilic attack of a metal-activated peroxide on a susceptible phosphate diester moiety. The role of H2O2 or O2 will be ascertained in order to determine whether the phosphate product is labeled when H218O2 or 18O2 is used. These labeling experiments will be carried out with small molecules as well as pBR322 modified with a polylinker incorporated near the cleavage site to afford small enough fragments for high resolution mass spectral analysis. The dinucleating ligand will be varied systematically to uncover features that confer specificity of cleavage. pBR322 will be modified in a number of ways to determine if there is a pattern to the cleavage specificity that is related to sequence or topology. These variations include altering the sequence at the cleavage site, moving the cleavage site to another section of the plasmid, and deleting the cleavage site or residues near the cleavage site from the plasmid. pBR322 will be resolved into species as a function of linkage number to study how the extent of supercoiling affects the cleavage specificity. Related and unrelated DNA molecules will be treated with the cleavage reagent and their cleavage specificities determined. RNA molecules will also be examined to determine whether the cleavage reagent may be useful for probing the rich topology of RNAs. Lastly, complexes of other Lewis acidic (but non-redox active) metal ions will be studied to improve the efficiency of the "hydrolytic" cleavage by eliminating the oxidative side reactions engendered by the diiron complexes.