Choudhury Jayati Roy, Bierbach Ulrich
Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109-7486, USA.
Nucleic Acids Res. 2005 Sep 28;33(17):5622-32. doi: 10.1093/nar/gki869. Print 2005.
The DNA interactions of PT-BIS(ACRAMTU) (Pt(en)(ACRAMTU)24; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, en = ethylenediamine), a bifunctional platinum-acridine conjugate, have been studied in native and synthetic double-stranded DNAs and model duplexes using various biophysical techniques. These include ethidium-DNA fluorescence quenching and thermal melting experiments, circular dichroism (CD) spectroscopy and plasmid unwinding assays. In addition, the binding mode was studied in a short octamer by NMR spectroscopy in conjunction with molecular modeling. In alternating copolymers, PT-BIS(ACRAMTU) shows a distinct preference for poly(dA-dT)2, which is approximately 3-fold higher than that of ACRAMTU. In the ligand-oligomer complex, d(GCTATAGC)2.PT-BIS(ACRAMTU) (complex I*), PT-BIS(ACRAMTU) increases the thermal stability of the B-form host duplex by DeltaT(m) > 30 K (CD and UV melting experiments). The agent unwinds pSP73 plasmid DNA by 44(+/-2) degrees per bound molecule, indicating bisintercalative binding. A 2-D NMR study unequivocally demonstrates that PT-BIS(ACRAMTU)'s chromophores deeply bisintercalate into the 5'-TA/TA base pair steps in I*, while the platinum linker lies in the minor groove. An AMBER model reflecting the NMR results shows that bracketing of the central AT base pairs in a classical nearest neighbor excluded fashion is feasible. PT-BIS(ACRAMTU) inhibits DNA hydrolysis by BstZ17 I at the enzyme's restriction site, GTA downward arrowTAC. Possible consequences for other relevant DNA-protein interactions, such as those involved in TATA-box-mediated transcription initiation and the utility of the platinum-intercalator technology for the design of sequence-specific agents are discussed.
已使用多种生物物理技术，在天然和合成双链DNA及模型双链体中研究了双功能铂 - 吖啶共轭物PT - BIS(ACRAMTU)（Pt(en)(ACRAMTU)24；ACRAMTU = 1 - [2 - (吖啶 - 9 - 基氨基)乙基] - 1,3 - 二甲基硫脲，en = 乙二胺）与DNA的相互作用。这些技术包括溴化乙锭 - DNA荧光猝灭和热变性实验、圆二色性（CD）光谱以及质粒解旋测定。此外，结合分子建模，通过核磁共振光谱在一个短八聚体中研究了其结合模式。在交替共聚物中，PT - BIS(ACRAMTU)对聚(dA - dT)2表现出明显的偏好，其偏好程度比ACRAMTU高约3倍。在配体 - 寡聚物复合物d(GCTATAGC)2.PT - BIS(ACRAMTU)（复合物I*）中，PT - BIS(ACRAMTU)使B型宿主双链体的热稳定性提高了ΔT(m) > 30 K（CD和紫外热变性实验）。该试剂使pSP73质粒DNA每结合一个分子解旋44(±2)度，表明其为双插入结合。二维核磁共振研究明确表明，PT - BIS(ACRAMTU)的发色团深深双插入复合物I*中的5'-TA/TA碱基对步序中，而铂连接体位于小沟中。反映核磁共振结果的AMBER模型表明，以经典的最近邻排斥方式包围中心AT碱基对是可行的。PT - BIS(ACRAMTU)在酶的识别位点GTA↓TAC处抑制BstZ17 I对DNA的水解。还讨论了对其他相关DNA - 蛋白质相互作用（如参与TATA盒介导的转录起始的相互作用）可能产生的影响，以及铂 - 嵌入剂技术在序列特异性试剂设计中的应用。
