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Nucleic Acids Research, 1995, Vol. 23, No. 15 2872-2880
© 1995

METHODS

Incorporation of a fluorescent guanosine analog into olignoucleotides and its application to a real time assay for the HIV-1 integrase 3'-Processing reaction

Mary E. Hawkins*, Wolfgang Pfieiderer1, Abhijit Mazumder2, Yves G. Pommier2 and Frank M. Balis

Pediatric Branch, National Cancer Institute Building 10/13N240, 10 Center Drive, MSC 1928, Bethesda, MD 20892–1928, USA 1Universitaät Konstanz, Fakultaät fuür Chemie, Universitaätsstrasse 10 D-78464 Konstanz, Germany 2Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute 9000 Rockville Pike, Bethesda, MD 20892–4255, USA

*To Whom correspondence should be addressed

Received May 4, 1995. Accepted June 30, 1995.

We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-ß-Dribofuranosyl)isoxanthopterin (3-MI) in a dlmethoxytrityl, 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-processlng 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 dlnucleotide 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.


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