Incorporation of a fluorescent guanosine analog into oligonucleotides and its application to a real time assay for the HIV-1 integrase 3'-processing reaction.
We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions.
Hawkins
M E
ME
Pediatric Branch, National Cancer Institute, Bethesda, MD 20892-1928, USA.
Pfleiderer
W
W
Mazumder
A
A
Pommier
Y G
YG
Balis
F M
FM
D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D005456:Fluorescent Dyes; D016679:Genome, Viral; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D019426:Integrases; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D014976:Xanthopterin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism
C095370:3-methyl-8-(2-deoxyribofuranosyl)isoxanthopterin; D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D006151:Guanosine; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D003849:Deoxyguanosine; D014976:Xanthopterin