González C, Stec W, Kobylanska A, Hogrefe R I, Reynolds M, James T L
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Biochemistry. 1994 Sep 20;33(37):11062-72. doi: 10.1021/bi00203a002.
The solution structure of the thiophosphate-modified DNA.RNA hybrid duplex d(GCTATAApsTGG).r(CCAUUAUAGC) has been studied by NMR. Two samples with pure stereochemistry in the modified phosphate have been investigated. Two-dimensional NMR (2D NMR) methods have been applied to assign nearly all the resonances in both duplexes. Scalar coupling constants have been determined by comparing quantitative simulations with experimental double-quantum filtered COSY (DQF-COSY) cross-peaks. More than 300 distance constraints have been obtained from two-dimensional nuclear Overhauser spectroscopy (2D NOE) spectra recorded in D2O and H2O by using a complete relaxation matrix analysis as implemented in the program MARDIGRAS. This hybrid duplex presents a heteronomous structure. Riboses in the RNA strand are found in a N-type conformation typical of the A-form family as shown by the lack of H1'-H2' cross-peaks in DQF-COSY spectra and confirmed by the measured interproton distances. In contrast, the DNA strand adopts a different conformation with sugar puckers partially in the S-type domain, which is not in agreement with the A-family of structures. Coupling constants in deoxyriboses are not consistent with any single sugar conformation. Therefore, sugar pucker pseudorotation parameters are calculated according to a two-state dynamic equilibrium between N- and S-type conformers. In general, the population of major S conformer is lower than in double-stranded DNA duplexes, indicating that hybrid duplexes may be more flexible than pure DNA or RNA. The only differences observed in the spectra between the two stereoisomers studied originate from resonances of protons located near the modified phosphate. No significant differences in interproton distance have been detected, and only a slight difference of sugar pucker in the 5' neighbor has been found. The sulfur atom appears to be well-accommodated without further changes in the structure of the hybrid.
通过核磁共振研究了硫代磷酸酯修饰的DNA.RNA杂交双链体d(GCTATAApsTGG).r(CCAUUAUAGC)的溶液结构。研究了修饰磷酸盐中具有纯立体化学的两个样品。二维核磁共振（2D NMR）方法已用于确定两个双链体中几乎所有的共振峰。通过将定量模拟与实验双量子滤波COSY（DQF-COSY）交叉峰进行比较来确定标量耦合常数。使用程序MARDIGRAS中实现的完整弛豫矩阵分析，从在D2O和H2O中记录的二维核Overhauser光谱（2D NOE）中获得了300多个距离约束。这种杂交双链体呈现出异源结构。如DQF-COSY光谱中缺乏H1'-H2'交叉峰所示，并经测量的质子间距离证实，RNA链中的核糖呈A族典型的N型构象。相比之下，DNA链采用不同的构象，糖环部分处于S型区域，这与A族结构不一致。脱氧核糖中的耦合常数与任何单一的糖构象都不一致。因此，根据N型和S型构象体之间的双态动态平衡计算糖环假旋转参数。一般来说，主要S型构象体的比例低于双链DNA双链体，这表明杂交双链体可能比纯DNA或RNA更具柔韧性。在所研究的两种立体异构体的光谱中观察到的唯一差异源于靠近修饰磷酸盐的质子的共振峰。未检测到质子间距离的显著差异，仅在5'邻位发现糖环略有差异。硫原子似乎很好地容纳在其中，而杂交体的结构没有进一步变化。
