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.

当被掺入DNA时，结构偏向的核苷和核苷酸单体通常会传递这些偏好，从而影响寡聚体的整体拓扑结构。我们以前已经研究了核苷呋喃糖环上的氟取代的结构效应，以及这如何相对于酶结合影响生物活性。正如在上一份年度报告中提到的，我们将其扩展到邻近的2‘.3’-二氟核苷，现在已经完成了对三个二氟尿苷衍生物的结构的核磁共振、从头计算和X射线光谱的全面研究。我们发现，只有当尿苷碱基上的H6质子不存在时，F-F高契效应才是可操作的。这证明了以前的一种断言，即存在一个很强的C3“F-H6引力，作用于带有C3‘-内基的氟原子的嘧啶类化合物。少数研究小组目前正在利用构象“锁定”的核苷构建块来构建具有明显螺旋折叠的寡核苷酸。正如Marquez博士在项目Z01 BC 06174-15LMC中所概述的那样，他的团队一直在改进程序，以制备构象“锁定”的[3.1.0]双环系统，作为2‘-endo(类BDNA)和2’-exo-(ADNA/RNA样)-折叠核苷酸同系物的模板。我们已经在战略位置将这些单体加入到寡核苷酸中，以研究具有定义的糖折叠的碱基对对DNA双链的整体结构的影响。特别是，我们将A类单体掺入典型的B类DNA链中，希望以特定的方式破坏结构。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。我们已经证明了这些双链的熔融温度受序列中A类单体的掺入的影响很大。对双链稳定性的重新评估表明，在某些结构中可能存在平衡的双链和发夹结构的混合。圆二色谱(CD)还表明，大多数链在25oC形成双链，但它们的CD特征与野生型序列不同。核磁共振谱显示，修饰的核苷酸碱基对中包含的质子与其为单体时具有相似的耦合特征，证明了修饰的碱基对在双链中的刚性。我们目前正在通过核磁共振光谱学完成六个寡核苷酸的指定，并正在进行实验，使我们能够测量修改后的双链中任何程度的弯曲。