Lee S P, Porter D, Chirikjian J G, Knutson J R, Han M K
Department of Biochemistry, Georgetown University Medical Center, Washington, DC 20007.
Anal Biochem. 1994 Aug 1;220(2):377-83. doi: 10.1006/abio.1994.1353.
Fluorescently labeled oligonucleotides and DNA fragments have promise in nucleic acid research with applications that include DNA hybridization, automated DNA sequencing, fluorescence anisotropy, and resonance energy transfer studies. Past concerns with fluorescent-labeled DNA arose from interactions between fluorophores and DNA that result in quenched fluorescence. This quenching phenomenon is most problematic in fluorescence resonance energy transfer studies because quenching of the donor fluorescence could result from either resonance energy transfer or nontransfer effects. In the present study, relief of nontransfer quenching of a 14-mer fluorescein 5-isothiocyanate (FITC)-labeled oligonucleotide containing the BamHI restriction site was characterized with both steady-state and time-resolved fluorescence techniques. The FITC-labeled single strand was best fit by a triexponential decay with lifetimes of 0.5, 2.7, and 4.2 ns. The 4.2-ns component was found to contribute more than 80% of the total steady-state intensity. Upon annealing with an unmodified complementary strand, the contribution from the 4.2-ns component was significantly decreased, resulting in twofold quenching of total fluorescence. We reasoned that this quenching phenomenon should be a reversible process and could be employed to study strand separation processes in molecular biology. Hence, cleavage of the fluorescently labeled substrate was examined using DNAase I and BamHI restriction endonuclease. Our results show that the quenched fluorescence is totally recovered upon cleavage (compared to that of the single strand). The extent of cleavage measured by fluorescence was confirmed by nondenaturing polyacrylamide gel electrophoresis analysis. We believe this fluorescence "dequenching" technique may be used to quantify the kinetics of other DNA strand separation and cleavage processes in molecular biology.
荧光标记的寡核苷酸和DNA片段在核酸研究中具有广阔前景，其应用包括DNA杂交、自动化DNA测序、荧光各向异性以及共振能量转移研究。过去对荧光标记DNA的担忧源于荧光团与DNA之间的相互作用，这种相互作用会导致荧光猝灭。在荧光共振能量转移研究中，这种猝灭现象最为棘手，因为供体荧光的猝灭可能是由共振能量转移或非转移效应引起的。在本研究中，我们使用稳态和时间分辨荧光技术对含有BamHI限制性位点的14聚体异硫氰酸荧光素（FITC）标记的寡核苷酸的非转移猝灭缓解情况进行了表征。FITC标记的单链最适合用三指数衰减来拟合，其寿命分别为0.5、2.7和4.2纳秒。发现4.2纳秒的成分对总稳态强度的贡献超过80%。与未修饰的互补链退火后，4.2纳秒成分的贡献显著降低，导致总荧光猝灭两倍。我们推测这种猝灭现象应该是一个可逆过程，可以用来研究分子生物学中的链分离过程。因此，我们使用DNA酶I和BamHI限制性内切酶检测了荧光标记底物的切割情况。我们的结果表明，切割后猝灭的荧光完全恢复（与单链相比）。通过荧光测量的切割程度通过非变性聚丙烯酰胺凝胶电泳分析得到了证实。我们相信这种荧光“去猝灭”技术可用于量化分子生物学中其他DNA链分离和切割过程的动力学。
