Fluorescence Properties of Pteridine Nucleoside Analogs as Monomers and Incorporated into Oligonucleotides

Autor: Wolfgang Pfleiderer, Frank M. Balis, Mary E. Hawkins, Jay R. Knutson, Denise Porter
Rok vydání: 1997
Předmět:
Zdroj: Analytical Biochemistry. 244:86-95
ISSN: 0003-2697
DOI: 10.1006/abio.1996.9879
Popis: Eighteen fluorescent pteridine-based nucleoside analogs have been prepared that are suitable for synthesis as phosphoramidites and site-specific incorporation into oligonucleotides. Their quantum yields ranged from ≤0.03 to 0.88. The maximum excitation and emission wavelengths of seven selected probes with quantum yields >0.15 ranged from 334 to 358 and 400 to 444 nm, respectively. Fluorescence decay curves of the seven probes were biexponential, and the mean intensity-weighted lifetimes ranged from 0.87 to 6.54 ns. Incorporation of probes 4 and 17 (3-methylisoxanthopterin and 6-methylisoxanthopterin) into oligonucleotides significantly quenched their fluorescence signal, and the degree of quench correlated with the number and proximity of purines in the oligonucleotide. Incorporation also resulted in a shift in absorbance-, emission-, and decay-associated spectra for 6-methylisoxanthopterin. An increase in the complexity of the decay curve and 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 curves and decreased the mean lifetimes. Melting temperature,Tm, depression equivalent to that of a single base pair mismatch was observed in 3-methylisoxanthopterin-containing double-stranded oligonucleotides, while theTmof 6-methylisoxanthopterin-containing double-stranded oligonucleotides were unperturbed, e.g., equivalent to unlabeled double-stranded oligonucleotides. This new class of fluorophore yields promising probes for the study of protein/DNA interactions.
Databáze: OpenAIRE
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