Hawkins M E, Pfleiderer W, Balis F M, Porter D, Knutson J R
Pediatric Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892-1928, USA.
Anal Biochem. 1997 Jan 1;244(1):86-95. doi: 10.1006/abio.1996.9879.
Eighteen fluorescent pteridine-based nucleoside analogs have been prepared that are suitable for synthesis as phosphormidites and site-specific incorporation into oligonucleotides. Their quantum yields ranged from < or = 0.03 to 0.88. The maximum excitation and emission wavelenghts 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 the Tm of 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.
已经制备了18种基于蝶啶的荧光核苷类似物，它们适合作为亚磷酰胺进行合成，并可位点特异性地掺入寡核苷酸中。它们的量子产率范围为≤0.03至0.88。七种量子产率>0.15的选定探针的最大激发波长和发射波长分别为334至358nm和400至444nm。这七种探针的荧光衰减曲线是双指数的，平均强度加权寿命范围为0.87至6.54ns。将探针4和17（3-甲基异黄蝶呤和6-甲基异黄蝶呤）掺入寡核苷酸中会显著淬灭它们的荧光信号，淬灭程度与寡核苷酸中嘌呤的数量和距离相关。掺入还导致6-甲基异黄蝶呤的吸收、发射和衰减相关光谱发生偏移。两种探针的衰减曲线复杂度增加，平均寿命降低。双链寡核苷酸的形成并没有显著增加淬灭程度，但通常会增加衰减曲线的复杂度并降低平均寿命。在含3-甲基异黄蝶呤的双链寡核苷酸中观察到熔解温度（Tm）降低，相当于单个碱基对错配的情况，而含6-甲基异黄蝶呤的双链寡核苷酸的Tm不受影响，例如与未标记的双链寡核苷酸相当。这类新型荧光团为研究蛋白质/DNA相互作用提供了有前景的探针。
