Benevides J M, Tsuboi M, Bamford J K, Thomas G J
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 64110-2499, USA.
Biophys J. 1997 Jun;72(6):2748-62. doi: 10.1016/S0006-3495(97)78917-3.
Raman tensors for localized vibrations of base (A, U, G, and C), ribose and phosphate groups of double-stranded RNA have been determined from polarized Raman measurements on oriented fibers of the genomic RNA of bacteriophage phi6. Polarized Raman intensities for which electric vectors of both the incident and scattered light are polarized either perpendicular (I[bb]) or parallel (I[cc]) to the RNA fiber axis have been obtained by Raman microspectroscopy using 514.5-nm excitation. Similarly, the polarized Raman components, I(bc) and I(cb), for which incident and scattered vectors are mutually perpendicular, have been obtained. Spectra collected from fibers maintained at constant relative humidity in both H2O and D2O environments indicate the effects of hydrogen-isotopic shifts on the Raman polarizations and tensors. Novel findings are the following: 1) the intense Raman band at 813 cm(-1), which is assigned to phosphodiester (OPO) symmetrical stretching and represents the key marker of the A conformation of double-stranded RNA, is characterized by a moderately anisotropic Raman tensor; 2) the prominent RNA band at 1101 cm(-1), which is assigned to phosphodioxy (PO2-) symmetrical stretching, also exhibits a moderately anisotropic Raman tensor. Comparison with results obtained previously on A, B, and Z DNA suggests that tensors for localized vibrations of backbone phosphodiester and phosphodioxy groups are sensitive to helix secondary structure and local phosphate group environment; and 3) highly anisotropic Raman tensors have been found for prominent and well-resolved Raman markers of all four bases of the RNA duplex. These enable the use of polarized Raman spectroscopy for the determination of purine and pyrimidine base residue orientations in ribonucleoprotein assemblies. The present determination of Raman tensors for dsRNA is comprehensive and accurate. Unambiguous tensors have been deduced for virtually all local vibrational modes of the 300-1800 cm(-1) spectral interval. The results provide a reliable basis for future evaluations of the effects of base pairing, base stacking, and sequence context on the polarized Raman spectra of nucleic acids.
通过对噬菌体phi6基因组RNA定向纤维进行偏振拉曼测量，已确定了双链RNA中碱基（A、U、G和C）、核糖和磷酸基团局部振动的拉曼张量。使用514.5 nm激发的拉曼显微光谱法获得了入射光和散射光的电矢量均垂直（I[bb]）或平行（I[cc]）于RNA纤维轴的偏振拉曼强度。同样，也获得了入射和散射矢量相互垂直的偏振拉曼分量I(bc)和I(cb)。在H2O和D2O环境中保持恒定相对湿度的纤维上收集的光谱表明了氢同位素位移对拉曼偏振和张量的影响。新发现如下：1）813 cm(-1)处的强拉曼带，归属于磷酸二酯（OPO）对称伸缩振动，是双链RNA A构象的关键标记，其特征在于具有适度各向异性的拉曼张量；2）1101 cm(-1)处突出的RNA带，归属于磷酸二氧基（PO2-）对称伸缩振动，也表现出适度各向异性的拉曼张量。与先前在A、B和Z DNA上获得的结果比较表明，主链磷酸二酯和磷酸二氧基基团局部振动的张量对螺旋二级结构和局部磷酸基团环境敏感；3）已发现RNA双链体所有四个碱基的突出且分辨率良好的拉曼标记具有高度各向异性的拉曼张量。这些使得能够利用偏振拉曼光谱法测定核糖核蛋白组装体中嘌呤和嘧啶碱基残基的取向。目前对双链RNA拉曼张量的测定是全面且准确的。对于300 - 1800 cm(-1)光谱区间内几乎所有的局部振动模式，都已推导出明确的张量。这些结果为未来评估碱基配对、碱基堆积和序列背景对核酸偏振拉曼光谱的影响提供了可靠的基础。
