Synthesis and fluorescence characterization of pteridine adenosine nucleoside analogs for DNA incorporation

Two fluorescent adenosine analogs, 4-amino-6-methyl-8-(2-deoxy-beta -D-ribo furanosyl)-7(8H)-pteridone (6MAP) and 4-amino-2,6-dimethyl-8-(2'-deoxy-beta -D-ribofuranosyl)-7(8H)-pteridone (DMAP), have been synthesized as phospho ramidites. These probes are site-selectively incorporated into oligonucleot ides using automated DNA synthesis. Relative quantum yields are 0.39 for 6M AP and 0.48 for DMAP as monomers and range from >0.01 to 0.11 in oligonucle otides. Excitation maxima are 310 (6MAP) and 330 nm (DMAP) and the emission maximum for each is 430 nm. Fluorescence decay curves of each are monoexpo nential exhibiting lifetimes of 3.8 and 4.8 ns for 6MAP and DMAP, respectiv ely. When these probes are incorporated into oligonucleotides they display quenching of fluorescence intensity, increases in the complexity of decay c urves, and decreases in mean lifetimes. Because these changes are apparentl y mediated by interactions with neighboring bases, spectral changes that oc cur as probe-containing oligonucleotides meet and react with other molecule s provide a means of monitoring these interactions in real time. These prob es are minimally disruptive to DNA structure as evidenced by melting temper atures of probe-containing oligonucleotides that are very similar to those of controls. Digestion of probe-containing oligonucleotides with P1 nucleas e confirms probe stability as fluorescence levels are restored to those exp ected for each monomer. These adenosine analog probes are capable of provid ing information on DNA structure as it responds to binding or catalysis thr ough interaction with other molecules. (C) 2001 Academic Press.