NMR Group Project: Structural Analysis of Conformational

NMR 小组项目：构象的结构分析

• 批准号: 6763822
• 负责人: Joseph John Barchi
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6763822
• 关键词: adenosine deaminase; bioimaging /biomedical imaging; biomedical facility; computer simulation; conformation; fluorine; hydrogen ions; molecular dynamics; monomer; nuclear magnetic resonance spectroscopy; nucleic acid structure; nucleosides; nucleotide analog; phosphorylation; polynucleotides; protein structure

When incorporated into DNA, structurally biased nucleoside and nucleotide monomers usually transmit these preferences to influence the overall topology of the oligomer. We have previously studied the structural effects of fluorine substitution in the furanose ring of a nucleoside and how this may affect biological activity, vis-a-vis enzyme binding. As mentioned in the last annual report, we extended this to vicinal 2'.3'- difluorinated nucleosides and have now completed a comprehensive study of the structure of three difluorinated uridine derivatives by NMR, ab initio calculations and X-ray spectroscopy. We found that the F-F gauche effect is operable only when the H6 proton of the uridine base is absent. This proved a previous assertion that there is a strong C3"F-H6 attractive force operating pyrimidines with a C3'-endo disposed fluorine atom. A handful of groups are currently exploiting the use of conformationally "locked" nucleoside building blocks in the construction of oligonucleotides with distinct helical folds. As outlined in project Z01 BC 06174-15 LMC by Dr. Marquez, his group has been refining procedures for the preparation of conformationally "locked" [3.1.0] bicyclic systems as templates for both 2'-endo (B DNA-like) and 2'-exo-(A DNA/RNA-like)-puckered nucleotide congeners. We have incorporated these monomers into oligonucleotides at strategic positions to study the effect that base pairs with defined sugar puckers have on the overall structure of a DNA duplex. In particular, we have incorporated A-like monomers into a typical B-like strand of DNA in hopes of disrupting the structure in defined ways. Currently we are studying six different oligomers, based on the Dickerson-Drew dodecamer, by NMR spectroscopy. We have shown that the melt temperatures for these duplexes are highly affected by the incorporation of A-like monomers into the sequence. A reassessment of the duplex stabilities showed that there may be a mixture of duplex and hairpin structures in equilibrium in some constructs. Circular dichroism (CD) spectroscopy has also shown that most of the strands form duplexes at 25oC, however their CD signatures are different than those from the wild type sequences. NMR spectroscopy has shown that protons contained within modified nucleotide base pairs have similar coupling signatures as when they are monomeric, proving the rigidity of the modified base pairs within the duplex. We are currently completing the assignments of six oligonucleotides by NMR spectroscopy and are implementing experiments that will allow us to measure any degree of bending in the modified duplexes.

当掺入 DNA 时，结构偏向的核苷和核苷酸单体通常会传递这些偏好，从而影响寡聚物的整体拓扑结构。我们之前研究了核苷呋喃糖环中氟取代的结构效应，以及这如何影响与酶结合相关的生物活性。正如上一份年度报告中提到的，我们将其扩展到邻位2'.3'-二氟化核苷，现已通过NMR、从头计算和X射线光谱法完成了对三种二氟化尿苷衍生物结构的全面研究。我们发现只有当尿苷碱基的H6质子不存在时，F-F高斯效应才起作用。这证明了之前的断言，即存在强大的 C3"F-H6 吸引力来操作具有 C3'-内配置氟原子的嘧啶。目前，少数研究小组正在利用构象“锁定”核苷构建块来构建正如 Marquez 博士在 Z01 BC 06174-15 LMC 项目中概述的那样，他的团队一直在改进制备寡核苷酸的程序。构象“锁定”[3.1.0]双环系统作为 2'-endo（B DNA 样）和 2'-exo-（A DNA/RNA 样）褶皱核苷酸同源物的模板。寡核苷酸处于战略位置，以研究具有确定糖褶皱的碱基对对 DNA 双链体整体结构的影响。特别是，我们希望将 A 类单体整合到典型的 B 类 DNA 链中。目前，我们正在通过核磁共振波谱研究基于 Dickerson-Drew 十二聚体的六种不同的低聚物。我们已经表明，这些双链体的熔解温度很大程度上受到 A 类单体掺入序列的影响。对双链体稳定性的重新评估表明，在一些构建体中可能存在平衡的双链体和发夹结构的混合物。圆二色性 (CD) 光谱还表明，大多数链在 25oC 时形成双链体，但它们的 CD 特征与野生型序列的不同。核磁共振波谱表明，修饰的核苷酸碱基对中包含的质子具有与单体时相似的偶联特征，证明了双链体中修饰的碱基对的刚性。我们目前正在通过核磁共振波谱完成六种寡核苷酸的分配，并正在进行实验，使我们能够测量修饰双链体中任何程度的弯曲。