Chen F M, Sha F
Department of Chemistry, Tennessee State University, Nashville, Tennessee 37209-1561, USA.
Biochemistry. 2001 May 1;40(17):5218-25. doi: 10.1021/bi010047c.
Despite the absence of the GpC sequence and complete self-complementarity, d(CGTCGTCG) has recently been shown to bind strongly to actinomycin D (ACTD) with a binding density of about one drug molecule per strand. To further elucidate the nature of such a binding, studies are herein made with single-base G --> A and C --> T replacements in d(CGTCGTCG) to identify the DNA bases that play important roles in the strong ACTD binding of this oligomer. On the basis of these results, the octamer d(TGTCATTG) has been identified as a potentially strong ACTD binder. Indeed, binding titration confirms such an expectation and reveals an ACTD binding constant of about 1 x 10(7) M(-1) and a binding density of roughly 0.8 drug molecule per DNA strand for this strong binding mode. Similar binding studies with single-base substitutions on d(TGTCATTG) further reveal the relative importance of the C and G bases on its ACTD binding, with the 3'-terminus G appearing to be the most crucial base. Further base substitutions lead to the conclusion that these C and G bases act in concert rather than individually in the ACTD binding of d(TGTCATTG). Spectral comparisons with the apparently single-stranded GpC-containing d(TGCTTTG) led to the proposal of a speculated monomeric hairpin binding model to account for the experimental observations. This model makes use of the notion that ACTD prefers to have the 3'-sides of both G bases stacking on the opposite faces of its planar phenoxazone chromophore, a principle akin to its classic preference for the GpC sequence in duplex form. The finding that ACTD can bind strongly to single-stranded DNA of special sequence motifs may have important implications.
尽管缺乏GpC序列且不存在完全的自我互补性，但最近研究表明，d(CGTCGTCG)能与放线菌素D（ACTD）强烈结合，结合密度约为每条链一个药物分子。为进一步阐明这种结合的本质，本文对d(CGTCGTCG)进行单碱基G→A和C→T替换研究，以确定在该寡聚物与ACTD的强结合中起重要作用的DNA碱基。基于这些结果，八聚体d(TGTCATTG)被确定为一种潜在的强ACTD结合剂。事实上，结合滴定证实了这一预期，并揭示出这种强结合模式下ACTD的结合常数约为1×10⁷ M⁻¹，每条DNA链的结合密度约为0.8个药物分子。对d(TGTCATTG)进行单碱基替换的类似结合研究进一步揭示了C和G碱基在其与ACTD结合中的相对重要性，其中3'-末端的G似乎是最关键的碱基。进一步的碱基替换得出结论，在d(TGTCATTG)与ACTD的结合中，这些C和G碱基协同作用而非单独起作用。与明显为单链的含GpC的d(TGCTTTG)进行光谱比较后，提出了一种推测的单体发夹结合模型来解释实验观察结果。该模型利用了这样一种概念，即ACTD倾向于使两个G碱基的3'-侧堆叠在其平面吩恶嗪发色团的相对面上，这一原理类似于其对双链形式的GpC序列的经典偏好。ACTD能与特殊序列基序的单链DNA强烈结合这一发现可能具有重要意义。
