Sequence- and structure-dependent DNA base dynamics: synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA.
Sequence- and structure-dependent DNA base dynamics: synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA.
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DOI：
10.1021/bi00450a036
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发表时间：
1989-11
期刊：
影响因子：
2.9
通讯作者：
A. Spaltenstein;B. Robinson;P. B. Hopkins
中科院分区：
文献类型：
--
作者：
A. Spaltenstein;B. Robinson;P. B. Hopkins
氮氧自由基自旋标记的胸腺嘧啶核苷类似物（1a），其中甲基被取代的乙炔系氮氧自由基，作为探针的序列特异性自旋标记的DNA的结构和动力学研究进行了评估。通过使用自动化亚磷酸三酯方法将残基1a掺入合成的脱氧寡核苷酸中。1H NMR、CD和热变性研究表明，1a（T*）不会显著改变天然十二聚体的5 '-d（CGCGAATT*CGCG）的结构。单体，单链和双链DNA的EPR研究表明，1a很容易区分不同刚性的环境。几个小的双链体（12聚体，24聚体）与模拟EPR光谱假设各向同性运动的EPR光谱观察到的一般线形特征的比较表明，探针1A监测全球翻滚的小双链体。增加的DNA寡聚体的长度导致显着偏离各向同性运动，与线形特征类似的计算光谱的对象与各向同性旋转相关时间为20-100 ns。自旋标记的GT错配和长DNA中的T凸起的EPR谱不同于自旋标记的沃森-克里克配对DNA的EPR谱，进一步证明了EPR作为评价大分子中局部动力学和结构特征的工具的价值。
A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. 1H NMR, CD, and thermal denaturation studies indicate that 1a (T*) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules.