Fluorescent labelling of abasic sites: a novel methodology to detect closely-spaced damage sites in DNA.
OBJECTIVE
To test the ability of a new fluorescent reagent to label abasic sites in DNA and to use fluorescent energy transfer as a measure of closely-spaced abasic sites in DNA.


METHODS
A fluorescein-conjugated hydroxylamine derivative (FARP, 5-(((2-(carbohydrazino)-methyl)thio)acetyl)aminofluorescein, aminooxyacetyl hydrazide) that reacts covalently with open chain aldehydes in DNA has been synthesized. Upon depurination of plasmid DNA and reaction with FARP a stable oxime bond is 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 become fluorescently labelled. The limit of sensitivity with the fluorometer used is approximately 90 femtomole abasic sites, corresponding to 1 abasic site per 17000 base pairs. DNA can be fluorescently labelled over 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 abasic sites labelled with FARP, that undergo fluorescence energy transfer. Treatment of heavily labelled DNA samples with nuclease 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 quantitative measure of the proximity of labelled abasic sites. Furthermore 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 approach 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 energy transfer. The technique also comprises a general and convenient method to fluorescently label nucleic acids without introducing strand breaks as a result of the labelling procedure.
Joint Center for Radiation Therapy and Department of Radiation Oncology, Harvard Medical School, Boston, MA 02215, USA.
G M
GM
Makrigiorgos
S
S
Chakrabarti
A
A
Mahmood
D000447:Aldehydes / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000528:radiation effects*; D004249:DNA Damage / Q000235:genetics*; D004996:Ethidium / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006898:Hydroxylamines / Q000378:metabolism; D015394:Molecular Structure; D010957:Plasmids / Q000378:metabolism; D011687:Purines / Q000528:radiation effects; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000447:Aldehydes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006898:Hydroxylamines; D011687:Purines; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004996:Ethidium