Scission of DNA at a preselected sequence using a single-strand-specific chemical nuclease

We were interested in developing a protocol for cleaving large DNAs specifically. Previous attempts to develop such methods have failed to work because of high levels of nonspecific background scission.

R-loop formation was chosen for sequence-specific targeting, a method of hybridization whereby an RNA displaces a DNA strand of identical sequence in 70% formamide using Watson-Crick base-pairing, leading to a three-stranded structure. R-loops are stabilized in aqueous solution by modifying the bases with chemical reagents. The R-loop was cleaved using a novel nuclease prepared from the Thr48-->Cys mutant of the single-strand-specific M-13 gene V protein (GVP), which was alkylated with 5-(iodoacetamido-beta-alanyl)1,10-phenanthroline. The cleavage products of the pGEM plasmid were cloned in to the pCR 2.1-TOPO vector. Adenovirus 2 DNA (35.8 kb; tenfold larger than the pGEM plasmid) was also cleaved quantitatively at a preselected sequence.

A new method for cleaving duplex DNA at any preselected sequence was developed. The cleavage method relies on the chemical conversion of M-13 GVP into a nuclease, reflecting GVP's specificity for single-stranded DNA. The GVP chimera is the first example of a semisynthetic secondary structure specific nuclease. The chemical nuclease activity of 1,10-phenanthroline-copper is uniquely suited to this technique because it oxidizes the deoxyribose moiety without generating diffusible intermediates, providing clonable DNA fragments. The protocol could be useful in generating large DNA fragments for mapping the contiguity of probes or defining the exon-intron structure of transcription units.