Roongta V A, Jones C R, Gorenstein D G
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Biochemistry. 1990 Jun 5;29(22):5245-58. doi: 10.1021/bi00474a005.
We have previously suggested that variations in the 31P chemical shifts of individual phosphates in duplex oligonucleotides are attributable to torsional angle changes in the deoxyribose phosphate backbone. This hypothesis is not directly supported by analysis of the 1H/31P two-dimensional J-resolved spectra of a number of mismatch dodecamer oligonucleotide duplexes including the following sequences: d-(CGTGAATTCGCG), d(CGUGAATTCGCG), d(CGGGAATTCGCG), d(CGAGAATTCGCG), and d(CGCGAATTCACG). The 31P NMR signals of the dodecamer mismatch duplexes were assigned by 2D 1H/31P pure absorption phase constant time (PAC) heteronuclear correlation spectra. From the assigned H3' and H4' signals, the 31P signals of the base-pair mismatch dodecamers were identified. JH3'-P coupling constants for each of the phosphates of the dodecamers were obtained from 1H/31P J-resolved selective proton flip 2D spectra. By use of a modified Karplus relationship, the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. JH3'-P coupling constants were measured for many of the oligonucleotides as a function of temperature. There exists a good linear correlation between 31P chemical shifts and the epsilon torsional angle. This correlation can be further extended to the C3'-O3'-P-O5' torsional angle (zeta) by using a linear relationship between epsilon and zeta obtained from crystal structure studies. The 31P chemical shifts follow the general observation that the more internally the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. In addition, 31P chemical shifts show sequence- and site-specific variations. Analysis of the backbone torsional angle variations from the coupling constant analysis has provided additional information regarding the origin of these variations in 31P chemical shifts.
我们之前曾提出，双链寡核苷酸中单个磷酸基团的³¹P化学位移变化可归因于脱氧核糖磷酸主链的扭转角变化。对包括以下序列在内的多个错配十二聚体寡核苷酸双链体的¹H/³¹P二维J分辨谱进行分析，并未直接支持这一假设：d-(CGTGAATTCGCG)、d(CGUGAATTCGCG)、d(CGGGAATTCGCG)、d(CGAGAATTCGCG)和d(CGCGAATTCACG)。十二聚体错配双链体的³¹P NMR信号通过二维¹H/³¹P纯吸收相位恒定时间（PAC）异核相关谱进行归属。根据归属的H3'和H4'信号，确定了碱基对错配十二聚体的³¹P信号。通过¹H/³¹P J分辨选择性质子翻转二维谱获得了十二聚体各磷酸基团的JH3'-P耦合常数。利用修正的Karplus关系，得到了C4'-C3'-O3'-P扭转角（ε）。测量了许多寡核苷酸的JH3'-P耦合常数随温度的变化。³¹P化学位移与ε扭转角之间存在良好的线性相关性。通过利用晶体结构研究得到的ε与ζ之间的线性关系，这种相关性可以进一步扩展到C3'-O3'-P-O5'扭转角（ζ）。³¹P化学位移符合一般观察结果，即磷酸基团在寡核苷酸序列中位置越靠内，³¹P共振出现的位置越靠上。此外，³¹P化学位移表现出序列和位点特异性变化。通过耦合常数分析对主链扭转角变化进行分析，为³¹P化学位移这些变化的起源提供了额外信息。
