Interstrand cross-link formation in duplex and triplex DNA by modified pyrimidines.
Interstrand cross-link formation in duplex and triplex DNA by modified pyrimidines.
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DOI：
10.1021/ja802177u
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发表时间：
2008-08-06
影响因子：
15
通讯作者：
Greenberg MM
中科院分区：
文献类型：
--
作者：
Peng X;Hong IS;Li H;Seidman MM;Greenberg MM
DNA链间交联具有重要的生物学后果，并且是有用的生物技术工具。当被NaIO 4氧化时，胸腺嘧啶核苷（1）和5-甲基-2 ′-脱氧胞苷（5）的苯硒基取代衍生物在双链DNA中产生链间交联。该机制涉及[2，3]-sigmatropic重排相应的硒氧化物相应的甲基化物型中间体，最终产生链间交联。硒氧化物重排的速率常数的测定表明，交联的速率决定步骤是甲基化物形成后。双链体DNA中的胸苷衍生物的交联显示出适度的动力学偏好时，两侧的嘧啶，而不是嘌呤。相比之下，双链体DNA中5个相对dG形成交联的速率常数强烈依赖于侧翼序列，并且通常比在其他方面相同的序列中1个dG形成交联的速率常数慢至少一个数量级。在5侧翼的碱基对处引入错配或相对dG被dI取代显著增加了交联的速率常数和产率，表明与dA和衍生自1的相应亲电体相比，衍生自它和dG的甲基化物之间的氢键更强，通过增加旋转成所需顺式构象的屏障来限制反应。在利用Hoogsteen碱基配对的三链体形成寡核苷酸（TFO）中掺入1或5也产生链间交联。当掺入TFO的5′-末端时，dC衍生物产生ICL的速度比胸苷衍生物快约10倍，当掺入内部位点时，产量更高。由1产生的较慢、效率较低的ICL形成归因于N1-dA下的反应，这需要双链体的局部熔融。相反，5通过与N7-dG反应产生交联。1和5的交联反应说明了这些分子作为生物技术的机械探针和工具的多功能性和实用性。
DNA interstrand cross-links have important biological consequences and are useful biotechnology tools. Phenylselenyl substituted derivatives of thymidine (1) and 5-methyl-2′-deoxycytidine (5) produce interstrand cross-links in duplex DNA when oxidized by NaIO4. The mechanism involves a [2,3]-sigmatropic rearrangement of the respective selenoxides to the corresponding methide type intermediates, which ultimately produce the interstrand cross-links. Determination of the rate constants for the selenoxide rearrangements indicates that the rate-determining step for cross-linking is after methide formation. Cross-linking by the thymidine derivative in duplex DNA shows a modest kinetic preference when flanked by pyrimidines as opposed to purines. In contrast, the rate constant for cross-link formation from 5 opposite dG in duplex DNA is strongly dependent upon the flanking sequence and, in general, is at least an order of magnitude slower than that for 1 in an otherwise identical sequence. Introduction of mispairs at the base pairs flanking 5 or substitution of the opposing dG by dI significantly increases the rate constant and yield for cross-linking, indicating that stronger hydrogen bonding between the methide derived from it and dG compared to dA and the respective electrophile derived from 1 limits reaction by increasing the barrier to rotation into the required syn-conformation. Incorporation of 1 or 5 in triplex forming oligonucleotides (TFOs) that utilize Hoogsteen base pairing also yields interstrand cross-links. The dC derivative produces ICLs approximately 10× faster than the thymidine derivative when incorporated at the 5′-termini of the TFOs and higher yields when incorporated at internal sites. The slower, less efficient ICL formation emanating from 1 is attributed to reaction at N1-dA, which requires local melting of the duplex. In contrast, 5 produces cross-links by reacting with N7-dG. The cross-linking reactions of 1 and 5 illustrate the versatility and utility of these molecules as mechanistic probes and tools for biotechnology.
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