Chen F M, Sha F
Department of Chemistry, Tennessee State University, Nashville 37209-1561, USA.
Biochemistry. 1998 Aug 11;37(32):11143-51. doi: 10.1021/bi980950l.
DNA binding modes of distamycin (DST) were investigated via comparative binding studies with oligomeric duplexes of the form d(GCG-X-GCG).d(CGC-Y-CGC), where Y is complementary to X and X = 4- or 5-base binding site. It was found that 1:1 and 2:1 drug-duplex complexes exhibit distinctly different circular dichroic (CD) spectral characteristics and can, thus, serve as diagnostic tools for binding mode differentiation. CD intensity profiles at 265 or 275 nm as a function of drug to DNA ratios can reveal the extent of binding cooperativity for 2:1 complex formation (i.e., the relative binding affinities of 2:1 vs 1:1) at a 5-base-paired binding site. Comparison of these profiles leads to the following qualitative ranking for the binding cooperativity for the studied sites: AAGTT, ATATA >/= AAACT > AATAA, AAATA, AAAGT > AATAT > TAAAA >/= AAATT >/= AAAAA >/= ATAAA, AAAAT. The plausibility of this ordering is strengthened by its agreement with the ranking established by earlier NMR studies on some of the sequences. The significantly slower DST dissociation kinetics of the 2:1 complexes as compared to those of 1:1 made the kinetic measurements of SDS-induced dissociation by the stopped-flow technique possible. The results indicate that the AAGTT site exhibits the slowest DST dissociation rate, with a characteristic time of 35 s. The rates of dissociation in general correlate reasonably well with the cooperativity order found via equilibrium CD measurements (the higher the binding cooperativity, the slower the rate of dissociation). Base sequence specific binding of DST was also found for the 1:1 complex formation at the 4-base-paired sites, with AAAA, TTTT, ATTT, and AAAT sequences exhibiting the highest binding affinities.
通过与d(GCG-X-GCG).d(CGC-Y-CGC)形式的寡聚双链体进行比较结合研究，对放线菌素（DST）的DNA结合模式进行了研究，其中Y与X互补，X为4或5个碱基的结合位点。研究发现，1:1和2:1的药物-双链体复合物表现出明显不同的圆二色性（CD）光谱特征，因此可作为区分结合模式的诊断工具。在265或275 nm处，CD强度谱随药物与DNA比例的变化，可以揭示在5个碱基对结合位点形成2:1复合物时的结合协同程度（即2:1与1:1的相对结合亲和力）。比较这些谱图可得出所研究位点结合协同性的以下定性排序：AAGTT、ATATA >= AAACT > AATAA、AAATA、AAAGT > AATAT > TAAAA >= AAATT >= AAAAA >= ATAAA、AAAAT。该排序与早期对部分序列进行的核磁共振研究确定的排序一致，从而增强了其合理性。与1:1复合物相比，2:1复合物中DST的解离动力学明显较慢，这使得采用停流技术对SDS诱导的解离进行动力学测量成为可能。结果表明，AAGTT位点的DST解离速率最慢，特征时间为35秒。一般来说，解离速率与通过平衡CD测量得到的协同性顺序具有合理的相关性（结合协同性越高，解离速率越慢）。在4个碱基对位点形成1:1复合物时，也发现了DST的碱基序列特异性结合，其中AAAA、TTTT、ATTT和AAAT序列表现出最高的结合亲和力。
