Chen F M
Department of Chemistry, Tennessee State University, Nashville 37209-1561.
Biochemistry. 1991 May 7;30(18):4472-9. doi: 10.1021/bi00232a014.
Six octadecamers with hairpin motifs have been synthesized and investigated for possible intramolecular triplex formation. Electrophoretic, hypochromic, and CD evidence suggest that d(CCCCTTTGGGGTTTGGGG) and d(GGGGTTTGGGGTTTCCCC) can form G.G.C intramolecular triplexes via double hairpin formation in neutral solutions, presumably with the terminal G tract folding back along the groove of the hairpin duplex. In contrast, d(GGGGTTTCCCCTTTGGGG) and the three corresponding 18-mers containing one G and two C tracts each forms a single hairpin duplex with a dangling single strand. The design of the sequences has led to the conclusion that the two G tracts are antiparallel to each other in such a triplex. Magnesium chloride titrations indicate that Mg2+ is not essential for such an intramolecular triplex formation. The main advantage of our constructs when compared to the intermolecular triplex formation is that the shorter triplex stem can be formed in a much lower DNA concentration. The merit of G.G.C triplex, in contrast to that of C+.G.C, lies in the fact that acidic condition is not required in its formation and will, thus, greatly expand our repertoire in the triplex strategy for the recognition and cleavage of duplex DNA. Spectral binding studies with actinomycin D (ACTD) and chromomycin A3 (CHR) as well as fluorescence lifetime measurements with ethidium bromide (EB) suggest that although hairpin duplexes bind these drugs quite well, the intramolecular triplexes bind poorly. Interestingly, the binding densities for the strong-binding hairpins obtained from Scatchard plots are about one ACTD molecule per oligomeric strand, whereas more than two drug molecules are found in the case of CHR, in agreement with the recent NMR studies indicating that CHR binds to DNA in the form of a dimer.
已合成了六个带有发夹基序的十八聚体，并对其形成分子内三链体的可能性进行了研究。电泳、减色和圆二色性证据表明，d(CCCCTTTGGGGTTTGGGG) 和 d(GGGGTTTGGGGTTTCCCC) 可以在中性溶液中通过形成双发夹结构形成G.G.C分子内三链体，推测其末端的G链沿着发夹双链体的凹槽向后折叠。相比之下，d(GGGGTTTCCCCTTTGGGG) 以及另外三个各自含有一个G链和两个C链的18聚体形成了带有单链悬垂的单发卡双链体。序列设计得出的结论是，在这样的三链体中，两条G链彼此反平行。氯化镁滴定表明，Mg2+ 对于这种分子内三链体的形成并非必不可少。与分子间三链体形成相比，我们构建体的主要优势在于，在低得多的DNA浓度下就能形成较短的三链体茎。与C+.G.C三链体相比，G.G.C三链体的优点在于其形成过程不需要酸性条件，因此将极大地扩展我们在双链DNA识别和切割的三链体策略中的应用范围。用放线菌素D（ACTD）和嗜铬菌素A3（CHR）进行的光谱结合研究以及用溴化乙锭（EB）进行的荧光寿命测量表明，虽然发夹双链体与这些药物结合良好，但分子内三链体结合较差。有趣的是，从Scatchard图获得的强结合发夹的结合密度约为每条寡聚链一个ACTD分子，而在CHR的情况下发现有两个以上的药物分子，这与最近的核磁共振研究结果一致，即CHR以二聚体形式与DNA结合。
