Wojtuszewski Poulin Kristi, Smirnov Aleksandr V, Hawkins Mary E, Balis Frank M, Knutson Jay R
Optical Spectroscopy Section, Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry. 2009 Sep 22;48(37):8861-8. doi: 10.1021/bi9003414.
Two different microenvironments in the DNA sequence 5'-act aGa gat ccc tca gac cct ttt agt cag tGt gga-3' (in both single- and double-stranded forms) are explored using two similar fluorescent nucleoside analogues, 3MI and 6MI. Each probe was evaluated in two environments, one strand with the probe flanked by thymines (PTRT) and the other by adenines (PTRA) with positions indicated by G's in the sequence. Both time-resolved anisotropies and lifetimes of the probes depend upon local interactions, and these are altered by duplex formation. Integrals of lifetime curves compared with quantum yields reveal that each probe displays a "dark" component (below detection limits, with a lifetime of <70 ps). For 6MI in PTRA, this QSSQ "quasi-static self-quenching" or "dark" component represents approximately half the molecules, whether in single- or double-stranded form. In PTRT, 6MI displays an unusual increase in the quantum yield upon formation of the double strand (from 0.107 to 0.189) apparently the result of escape from QSSQ which simultaneously declines from 66 to 33%. This is also accompanied by doubling of steady-state anisotropy. Only 6MI in the PTRT duplex displays a rotational correlation time of >7 ns. In other words, the DS 6MI PTRA environment fails to constrain local motion and QSSQ remains the same as in the single strand; in contrast, the flanking T duplex environment restricts local motion and halves QSSQ. We collected both steady-state and time-resolved fluorescence quenching titrations of 3MI and 6MI in solution with the mononucleotides AMP, CMP, GMP, and TMP. The dynamic quenching rank of the free probes (quenching constant, kq: T > A > G > C) is totally different from that of incorporated probes. We hypothesize the production of weak 3MI.C or 6MI.C complexes that are somehow rendered less subject to dynamic quenching by collision with subsequent C molecules.
利用两种相似的荧光核苷类似物3MI和6MI，对DNA序列5'-act aGa gat ccc tca gac cct ttt agt cag tGt gga-3'（单链和双链形式）中的两种不同微环境进行了研究。每个探针在两种环境中进行评估，一种是探针两侧为胸腺嘧啶的链（PTRT），另一种是两侧为腺嘌呤的链（PTRA），序列中的位置由G表示。探针的时间分辨各向异性和寿命均取决于局部相互作用，并且这些会因双链形成而改变。将寿命曲线的积分与量子产率进行比较，结果表明每个探针都显示出一个“暗”成分（低于检测限，寿命<70 ps）。对于PTRA中的6MI，无论处于单链还是双链形式，这种QSSQ“准静态自猝灭”或“暗”成分约占分子总数的一半。在PTRT中，6MI在双链形成时量子产率出现异常增加（从0.107增至0.189），这显然是从QSSQ中逃脱的结果，同时QSSQ从66%降至33%。这还伴随着稳态各向异性的加倍。只有PTRT双链中的6MI显示出大于7 ns的旋转相关时间。换句话说，DS 6MI PTRA环境无法限制局部运动，QSSQ与单链中的情况相同；相反，侧翼为T的双链环境限制了局部运动，并使QSSQ减半。我们收集了3MI和6MI在溶液中与单核苷酸AMP、CMP、GMP和TMP的稳态和时间分辨荧光猝灭滴定数据。游离探针的动态猝灭等级（猝灭常数，kq：T > A > G > C）与掺入探针的完全不同。我们推测会产生弱的3MI.C或6MI.C复合物，这些复合物因某种原因在与后续C分子碰撞时较不易受到动态猝灭。
