Chen F M
Department of Chemistry, Tennessee State University, Nashville 37209-1561, USA.
Biochemistry. 1998 Mar 17;37(11):3955-64. doi: 10.1021/bi972110x.
Actinomycin D (ACTD) binding propensities of DNA with CXG trinucleotide repeats were investigated using oligomers of the form d[AT(CXG)n = 2-4AT] and their corresponding heteroduplexes, where X = A, C, G, or T. These oligonucleotides contain -CXGCXG-, -CXGCXGCXG-, and -CXGCXGCXGCXG- units that can form homoduplexes containing one, two, and three GpC binding sites, respectively, with flanking X/X mismatches. The corresponding heteroduplexes contain these same sites with flanking Watson-Crick base pairs. It was found that oligomers with X = G exhibit weak ACTD affinities whereas those with X not equal to G and n = 3 exhibit unusually strong ACTD binding affinities with binding constants ranging from 2.3 x 10(7) to 3.3 x 10(7) M-1 and binding densities of approximately 1 drug molecule/strand (or 2/duplex). These binding affinities are considerably higher than those of their shorter and longer counterparts and are about 2- and 10-fold stronger than the corresponding CAG.CTG and CGG.CCG heteroduplexes, respectively. The CTG-containing oligomer d[AT(CTG)3AT] stands out as unique in having its ACTD dissociation kinetics being dominated by a strikingly slow process with a characteristic time of 205 min at 20 degrees C, which is 100-fold slower than d[AT(CAG)3AT], nearly 10-fold slower than the corresponding heteroduplex, and considerably slower than d[AT(CTG)2AT] (63 min) and d[AT(CTG)4AT] (16 min). The faster dissociation rate of the n = 4 oligomer compared to its n = 2 counterpart is in apparent contrast with the observed 10-fold stronger ACTD binding affinity of the former. It was also found that d[AT(CCG)3AT] exhibits the slowest dissociation rate of the CGG/CCG series, being more than an order of magnitude slower than that of its heteroduplex (tau slow of 43 vs 2 min). The finding that a homoduplex d[AT-CXG-CXG-CXG-AT]2 can bind two ACTD molecules tightly is significant since it was thought unlikely for two consecutive GpC sites separated by a single T/T mismatch to do so.
使用d[AT(CXG)n = 2 - 4AT]形式的寡聚物及其相应的异源双链体研究了具有CXG三核苷酸重复序列的DNA与放线菌素D（ACTD）的结合倾向，其中X = A、C、G或T。这些寡核苷酸包含 -CXGCXG-、-CXGCXGCXG- 和 -CXGCXGCXGCXG- 单元，它们可以分别形成含有一个、两个和三个GpC结合位点的同型双链体，两侧为X/X错配。相应的异源双链体包含这些相同的位点以及两侧的沃森 - 克里克碱基对。发现X = G的寡聚物表现出较弱的ACTD亲和力，而X不等于G且n = 3的寡聚物表现出异常强的ACTD结合亲和力，结合常数范围为2.3×10⁷至3.3×10⁷ M⁻¹，结合密度约为1个药物分子/链（或2个/双链体）。这些结合亲和力明显高于其较短和较长的对应物，分别比相应的CAG.CTG和CGG.CCG异源双链体强约2倍和10倍。含CTG的寡聚物d[AT(CTG)₃AT]很独特，其ACTD解离动力学主要由一个极其缓慢的过程主导，在20℃下特征时间为205分钟，比d[AT(CAG)₃AT]慢100倍，比相应的异源双链体慢近10倍，比d[AT(CTG)₂AT]（63分钟）和d[AT(CTG)₄AT]（16分钟）慢得多。与n = 2的对应物相比，n = 4的寡聚物解离速率更快，这与观察到的前者ACTD结合亲和力强10倍明显矛盾。还发现d[AT(CCG)₃AT]在CGG/CCG系列中表现出最慢的解离速率，比其异源双链体慢一个多数量级（tau slow为43分钟对2分钟）。同型双链体d[AT - CXG - CXG - CXG - AT]₂能紧密结合两个ACTD分子这一发现很重要，因为人们认为由单个T/T错配分隔的两个连续GpC位点不太可能这样做。
