Mujeeb A, Reynolds M A, James T L
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Biochemistry. 1997 Mar 4;36(9):2371-9. doi: 10.1021/bi963008n.
A high-resolution proton NMR study has been performed on a hybrid duplex formed by a methylphosphonate (MP) oligodeoxyribonucleotide (MPO) and its target oligoribonucleotide, d(T(MP)CC(MP)-TT(MP)AG(MP)CT(MP)CC(MP)TG) x r(CAGGAGCUAAGGA), where MP corresponds to positions of methylphosphonate linkages in the pure R(p) stereoconfiguration. MP-containing analogs of DNA are reported to be effective antisense agents capable of specifically inhibiting protein synthesis with the R(p) chiral MPOs exhibiting greater affinity for the target mRNA than their S(p) counterparts. Nearly complete proton resonance assignments of the hybrid duplex have been made using two-dimensional nuclear Overhauser effect (2D NOE) spectra, at three different mixing times, and double quantum-filtered COSY (2QF-COSY) spectra. The 2QF-COSY cross-peak patterns which are resolved have been analyzed qualitatively to suggest sugar conformations. Distance restraints have been obtained from the 2D NOE spectra of the duplex in D(2)O. These interproton distance restraints were determined using a complete relaxation matrix method to improve accuracy. Specifically, a new approach termed RANDMARDI has been utilized to calculate these distance restraints, accounting for spectral noise and errors in 2D NOE peak volume integration. The calculated interproton distances and sugar puckers have been analyzed to assess the solution conformation of the hybrid. The hybrid duplex appears to have an overall solution structure which is distinct from standard B- and A-forms, but the RNA strand exhibits features of the A-form. The absence of H1'-H2' cross-peaks in the 2QF-COSY spectrum indicates a C3'-endo type of conformation for ribose sugars in the RNA strand. The deoxyriboses in the antisense DNA strand exhibit a mixed behavior with almost equal scalar coupling constant values for H1'-H2' and H1'-H2" and a strong H3'-H4' 2QF-COSY peak pattern. Variations in calculated values of interproton distances and sixth-root R factor analysis of experimental intensities indicate that the hybrid duplex may have a DNA strand with significant conformational plasticity.
对由甲基膦酸酯（MP）寡脱氧核糖核苷酸（MPO）及其靶寡核糖核苷酸d(T(MP)CC(MP)-TT(MP)AG(MP)CT(MP)CC(MP)TG)×r(CAGGAGCUAAGGA)形成的杂交双链体进行了高分辨率质子核磁共振研究，其中MP对应于纯R(p)立体构型中甲基膦酸酯键的位置。据报道，含MP的DNA类似物是有效的反义剂，能够特异性抑制蛋白质合成，R(p)手性MPO对靶mRNA的亲和力高于其S(p)对应物。利用二维核Overhauser效应（2D NOE）光谱（在三个不同混合时间）和双量子滤波COSY（2QF-COSY）光谱对杂交双链体进行了几乎完整的质子共振归属。对已解析的2QF-COSY交叉峰模式进行了定性分析，以推测糖的构象。从D₂O中双链体的2D NOE光谱获得了距离限制。这些质子间距离限制是使用完整弛豫矩阵方法确定的，以提高准确性。具体而言，一种称为RANDMARDI的新方法已被用于计算这些距离限制，同时考虑了光谱噪声和2D NOE峰体积积分中的误差。对计算出的质子间距离和糖的褶皱进行了分析，以评估杂交体的溶液构象。杂交双链体似乎具有与标准B型和A型不同的整体溶液结构，但RNA链表现出A型的特征。2QF-COSY光谱中不存在H1'-H2'交叉峰表明RNA链中核糖糖的构象为C3'-内型。反义DNA链中的脱氧核糖表现出混合行为，H1'-H2'和H1'-H2"的标量耦合常数几乎相等，并且有很强的H3'-H4' 2QF-COSY峰模式。质子间距离计算值的变化以及实验强度的六次根R因子分析表明，杂交双链体的DNA链可能具有显著的构象可塑性。
