NMR Group Project: Structural Analysis of Conformational

当整合到DNA中时，结构上偏倚的核苷和核苷酸单体通常传递这些偏好，从而影响低聚物的整体拓扑结构。我们之前已经研究了核苷呋喃糖环中氟取代的结构效应，以及这可能如何影响生物活性，相对于酶结合。正如在上一份年度报告中提到的，我们将此扩展到2'区。3'-二氟化核苷，现已通过核磁共振、从头计算和x射线光谱学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，只有当尿苷碱的H6质子缺失时，F-F扭扭效应才会发生。这证明了先前的断言，有一个强大的C3'- F-H6引力作用于具有C3'-末端氟原子的嘧啶。目前，一些研究小组正在利用构象“锁定”核苷构建块来构建具有不同螺旋折叠的寡核苷酸。正如Marquez博士在项目Z01 BC 06174-15 LMC中概述的那样，他的团队一直在完善构象“锁定”[3.1.0]双环系统的制备程序，作为2'-endo （B DNA样）和2'-exo-（A DNA/ rna样）折叠核苷酸同源物的模板。我们已将这些单体纳入寡核苷酸的战略位置，以研究具有明确糖袋的碱基对对DNA双链整体结构的影响。特别是，我们将类a单体结合到典型的类b DNA链中，希望以特定的方式破坏其结构。目前，我们正在研究六种不同的低聚物，基于迪克森-德鲁十二聚体，通过核磁共振波谱。我们已经证明，这些双相化合物的熔体温度受到a类单体掺入序列的高度影响。对双相稳定性的重新评估表明，在某些结构中可能存在双相和发夹结构的混合平衡。圆二色性（CD）光谱也表明，大多数链在25℃时形成双链，但它们的CD特征与野生型序列不同。核磁共振波谱显示，在修饰的核苷酸碱基对中含有的质子与它们是单体时具有相似的偶联特征，证明了在双工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个寡核苷酸的分配，并正在实施实验，这将使我们能够测量修饰的双链中的任何程度的弯曲。

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.