FLUORESCENCE PROPERTIES OF PTERIDINE NUCLEOSIDE ANALOGS AS MONOMERS AND INCORPORATED INTO OLIGONUCLEOTIDES

Eighteen fluorescent pteridine-based nucleoside analogs have been prep ared that are suitable for synthesis as phosphoramidites and site-spec ific incorporation into oligonucleotides. Their quantum yields ranged from less than or equal to 0.03 to 0.88. The maximum excitation and em ission wavelengths of seven selected probes with quantum yields >0.15 ranged from 334 to 358 and 400 to 444 nm,respectively. Fluorescence de cay curves of the seven probes were biexponential, and the mean intens ity-weighted lifetimes ranged from 0.87 to 6.54 ns. Incorporation of p robes 4 and 17 (3-methylisoxanthopterin and 6-methylisoxanthopterin) i nto oligonucleotides significantly quenched their fluorescence signal, and the degree of quench correlated with the number and proximity of purines in the of oligonucleotide. Incorporation also resulted in a sh ift in absorbance-, emission-, and decay-associated spectra for 6-meth ylisoxanthopterin. An increase in the complexity of the decay curve an d a decrease in the mean lifetime occurred for both probes. Formation of double-stranded oligonucleotides did not substantially increase the degree of quenching but generally increased the complexity of decay c urves and decreased the mean lifetimes. Melting temperature, T-m, depr ession equivalent to that of a single base pair mismatch was observed in 3-methylisoxanthopterin-containing double-stranded oligonucleotides , while the T-m of 6-methylisoxanthopterin-containing double-stranded oligonucleotides were unperturbed, e.g., equivalent to unlabeled doubl e-stranded oligonucleotides. This new class of fluorophore yields prom ising probes for the study of protein/DNA interactions. (C) 1997 Acade mic Press, Inc.