FLUORESCENT LABELING OF ABASIC SITES - A NOVEL METHODOLOGY TO DETECT CLOSELY-SPACED DAMAGE SITES IN DNA

Purpose: To test the ability of a new fluorescent reagent to label aba sic sites in DNA and to use fluorescent energy transfer as a measure o f closely-spaced abasic sites in DNA. Materials and methods: A fluores cein-conjugated hydroxylamine derivative (FARP, bohydrazino)-methyl)th io)acetyl)-aminofluorescein, aminooxyacetyl hydrazide) that reacts cov alently with open chain aldehydes in DNA has been synthesized. Upon de purination of plasmid DNA and reaction with FARP a stable oxime bond i s formed between FARP and the generated open chain aldehydes. Results: By independently quantitating the generated abasic sites, it is shown that most of the generated abasic sites under acidic conditions becom e fluorescently labelled. The limit of sensitivity with the fluoromete r used is approximately 90 femtomole abasic sites, corresponding to 1 abasic site per 17000 base pairs. DNA can be fluorescently labelled ov er a wide range of FARP:base pair ratios following different extent of depurination, and fluorescent loadings of 1 FARP:20000 base pairs up to 1 FARP:10 base pairs are demonstrated. The heavily labelled samples display significant fluorescence quenching due to the proximity of ab asic sites labelled with FARP, that undergo fluorescence energy transf er. Treatment of heavily labelled DNA samples with nude ase P1 results in an increase in fluorescence due to the release of the fluorescent labels in the solution. The relative increase in fluorescence is a qua ntitative measure of the proximity of labelled abasic sites. Furthermo re it is shown that if only 1% of DNA contains abasic sites generated in close proximity (within 10-20 base pairs or less of each other) the resulting quenching is significant enough to detect, even if the rest of the DNA contains isolated abasic sites. Conclusions: The present a pproach constitutes a novel fluorescence-based method to detect abasic sites in nucleic acids and demonstrates the feasibility of detecting the presence of closely-spaced damage sites in DNA via fluorescence en ergy transfer. The technique also comprises a general and convenient m ethod to fluorescently label nucleic acids without introducing strand breaks as a result of the labelling procedure.