Borer P N, Kan L S, Ts'o P O
Biochemistry. 1975 Nov 4;14(22):4847-63. doi: 10.1021/bi00693a012.
1H nuclear magnetic resonance (NMR) spectra of a self-complementary ribosyl hexanucleotide, A2GCU2, are investigated as a function of temperature and ionic strength in D2O. Seventeen nonexchangeable base and ribose-H1' resonances are resolved, and unequivocally assigned by a systematic comparison with the spectra of a series of oligonucleotide fragments of the A2GCU2 sequence varying in chain length from 2 to 5. Changes in the chemical shifts of the 17 protons from the hexamer as well as the six H1'-H2' coupling constants are followed throughout a thermally induced helix-coil transition. These sigma vs. T and J vs. T (degrees C) profiles indicate that the transition is not totally cooperative and that substantial populations of partially bonded structures must exist at intermediate temperatures, with the central G-C region being most stable. Transitions in chemical shift for protons in the same base pair exhibit considerable differences in their Tm values as the data reflect both thermodynamic and local magnetic field effects in the structural transition, which are not readily separable. However, an average of the Tm values agrees well with the value predicted from studies of the thermally induced transition made by optical methods. The values of J1'-2' for all six residues become very small (less than 1.5 Hz) at low temperatures indicating that C3'-endo is the most heavily populated furanose conformation in the helix. The sigma values of protons in the duplex were compared with those calculated from the ring current magnetic anisotropies of nearest and next-nearest neighboring bases using the geometrical parameters of the A'-RNA and B-DNA models. The sigma values of the base protons in the duplex calculated assuming the A'-RNA geometry agree (+/- approximately 0.1 ppm) with the observed values much more accurately than those calculated on the basis of B-DNA geometry. The measured sigma values of the H1' are not accurately predicted from either model. The synthesis of 35 mg of A2GCU2 using primer-dependent polynucleotide phosphorylase is described in detail with extensive discussion in the microfilm edition.
在D2O中，研究了自互补核糖基六核苷酸A2GCU2的1H核磁共振（NMR）谱随温度和离子强度的变化。分辨出17个不可交换的碱基和核糖-H1'共振峰，并通过与一系列链长从2到5不等的A2GCU2序列寡核苷酸片段的谱图进行系统比较，明确地进行了归属。在热诱导的螺旋-线圈转变过程中，跟踪六聚体中17个质子的化学位移变化以及六个H1'-H2'耦合常数。这些化学位移与温度（σ对T）和耦合常数与温度（J对T，单位为℃）的曲线表明，该转变并非完全协同，在中间温度下必然存在大量部分键合结构的群体，其中央G-C区域最稳定。同一碱基对中质子的化学位移转变在其熔点（Tm）值上表现出相当大的差异，因为数据反映了结构转变中的热力学和局部磁场效应，而这两者不易区分。然而，Tm值的平均值与通过光学方法研究热诱导转变所预测的值吻合得很好。在低温下，所有六个残基的J1'-2'值变得非常小（小于1.5 Hz），表明C3'-内向型是螺旋中最常见的呋喃糖构象。将双链体中质子的σ值与使用A'-RNA和B-DNA模型的几何参数，根据最近和次近邻碱基的环电流磁各向异性计算得到的值进行了比较。假设A'-RNA几何结构计算得到的双链体中碱基质子的σ值（±约0.1 ppm）与观测值的吻合程度比基于B-DNA几何结构计算得到的值准确得多。两种模型都无法准确预测测量得到的H1'的σ值。详细描述了使用引物依赖性多核苷酸磷酸化酶合成35 mg A2GCU2的过程，并在缩微胶片版中进行了广泛讨论。
