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

Abstract

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.

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

“…Incorporation of the 3-methyl-8-(2-deoxy-␤-D-ribofuranosyl) isoxanthopterin (3MI) probe into ODN and fluorescence properties of the PTER1 ODN (Fig. 1G) have been described (19,20). Double-stranded ODNs were obtained by mixing equimolar amounts of complementary strands in a 20 mM Tris⅐HCl buffer (pH 7.2) containing 100 mM NaCl.…”

“…The experiments described above with fluorescein-labeled ODNs were repeated with the 21-mer, basemodified ODN, PTER1 (Fig. 1G), in which the guanosine at position 19 (from the 3Ј end) was replaced by the fluorescent guanosine analog, 3MI (19,20). This substitution had no detectable effect on either 3Ј processing or integration reaction (not shown).…”

Fluorescence Fluctuation Analysis for the Study of Interactions between Oligonucleotides and Polycationic Polymers

“…Although the reason is unclear, the presence of the pDMAEMA chains results in a changed environment of the Rh-ONs, which may alter their fluorescence properties. Several groups reported that the fluorescence of dyes such as 5-carboxytetramethylrhodamine could be partially quenched upon binding to an oligonucleotide, the degree of quenching being dependent on the fluorophore's proximity to purines and its position in the oligonucleotide (Hawkins et al, 1995;Vamosi et al, 1996). It is hypothesized that the pDMAEMA chains might lead to a 'dequenching' of the rhodamine molecules bound to the ONs (the increased intensity corresponds to an increased average fluorescence lifetime; (results not shown).…”

Synthesis and fluorescence study of 7-azaindole in DNA oligonucleotides replacing a purine base

“…There are mainly two groups of 'bases' synthesized and incorporated into DNA oligomers: those made by minor modification of the natural bases or the derivatives of cyclic aromatic compounds. While the former includes 2-aminopurine [1][2][3][4][5][6][7][8], inosine [9] and isoinosine [9][10][11], 3-and 7-deazaadenine [12][13][14][15], and 3-and 7-deazaguanine [14,16], the latter includes analogs of pteridine [17][18][19] and indole [20][21][22][23][24], benzene [23][24][25][26], naphthalene and pyrene derivatives [24], The first group of compounds, when replacing a natural base in duplex DNA, can still form hydrogen bonds with the base in the opposite strand, while the second group of compounds cannot form any hydrogen bonds.…”

“…The fluorescence properties of the above base analogs have been utilized for studying DNA structure and duplex stability [1,2,10,14,15,22,26], protein-DNA interactions [4][5][6][7][8]13,18,25], or simply as universal base analogs [20,27]. Some of the unusual 'bases' are used as anti-HIV or anti-cancer drugs [28][29][30].…”