CONFORMATIONAL VARIABILITY OF DNA

DNA 的构象变异

基本信息

批准号：
2178744
负责人：
JULI FEIGON
金额：
$ 22.15万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-07-01 至 1999-03-31
项目状态：
已结题

项目摘要

The overall goal of the work described in this proposal is aimed at understanding the range of conformations that DNA can adopt, both in vivo and in vitro, and the principles underlying the folding of DNA into these non-B-DNA conformations. To this end, we propose to investigate two classes of folded DNA molecules: (1) DNA triplexes and (2) DNA aptamers. Multidimensional proton and heteronuclear NMR methods will be used to study the conformation and dynamics of the DNA oligonucleotides. DNA triplexes: In order to elucidate the factors governing sequence specific recognition of DNA by third strand binding to form triplexes, we propose to investigate the structures and cation dependence of several different triplexes and related structures. These include DNA triplexes with modified bases, ribose and 2'Omethyl ribose third strands, purine third strands, and crossover triplexes. We also propose to determine optimal loop sequences for intramolecular triplexes. Studies of a novel triplex-guanine 'clamp' are also planned. DNA aptamers: Aptamers are RNA or DNA molecules which have been selected, from a large pool of oligonucleotides containing a region of random nucleotide sequence, for binding to a specific target molecule. In collaboration with the Szostak laboratory, we will determine the structures of a variety of DNA aptamers which have been selected for binding to the cofactors ATP. GTP, biotin, and riboflavin. These will provide insight into the tertiary structure of nucleic acid binding pockets for protein cofactors. Ultimately, we also plan to determine the structures of DNA catalysts which contain cofactor binding sites. Since many of the folded oligonucleotides which we propose to study are too large to obtain well-defined structures using 1H spectroscopy alone, we plan to develop and optimize methods for synthesizing uniformly 15N and/or 13C labeled DNA oligonucleotides. Once the labeled DNA oligonucleotides are obtained, optimal methods for assignment and structure determination using double and triple-resonance NMR experiments will be developed, similar to those already widely used in protein NMR spectroscopy and more recently applied to labeled RNA oligonucleotides. Where possible, complete three-dimensional structures will be refined from starting structures generated by metric matrix distance geometry calculations. An understanding of the three-dimensional non-B-DNA structures that DNA can adopt and the factors that stabilize these conformations is important for a number of reasons. The solution of new DNA structures should lead to a better understanding of the principles of nucleic acid folding, stability, and sequence specific recognition by ligands including other nucleic acids. These studies should also provide a structural basis for potential applications of nucleic acids as pharmaceuticals, biosensors, and diagnostics.

本提案中描述的工作的总体目标是针对了解DNA可以采用的一系列构型，均在体内并在体外，以及将DNA折叠为这些的原理非B-DNA构象。为此，我们建议调查两个折叠DNA分子的类别：（1）DNA三折和（2）DNA适体。多维质子和异核NMR方法将用于研究 DNA寡核苷酸的构象和动力学。 DNA三倍：为了阐明序列的因素通过第三链结合与形成三链的特定识别，我们建议研究几个的结构和阳离子依赖性不同的三环和相关结构。这些包括DNA三倍具有改良的碱，核糖和2'omethyl核糖第三链，嘌呤第三链和跨界三链。我们还建议确定分子内三倍体的最佳循环序列。小说的研究还计划了三曲线弹式“夹具”。 DNA适体：适体是已选择的RNA或DNA分子，从包含随机区域的大量寡核苷酸池中核苷酸序列，以与特定靶标分子结合。在与Szostak实验室的合作，我们将确定结构已选择用于结合的多种DNA适体辅因子ATP。 GTP，生物素和核黄素。这些将提供洞察力进入蛋白质核酸结合口袋的三级结构辅因子。最终，我们还计划确定DNA的结构包含辅因子结合位点的催化剂。由于我们建议研究的许多折叠寡核苷酸也是仅使用1H光谱学获得明确定义的结构，我们计划开发和优化统一合成15N和/或的方法 13C标记的DNA寡核苷酸。一旦标记为DNA寡核苷酸获得了分配和结构确定的最佳方法将开发使用双呼和三谐和NMR实验，类似于已经广泛用于蛋白质NMR光谱的那些和更多最近应用于标记的RNA寡核苷酸。在可能的情况下，完成三维结构将从起始结构中进行完善由度量矩阵距离几何计算产生。了解DNA可以的三维非B-DNA结构采用和稳定这些构型的因素对许多原因。新的DNA结构的解决方案应导致更好地了解核酸折叠，稳定性的原理，配体（包括其他核酸）的序列特异性识别。这些研究还应为潜在的结构基础提供核酸作为药物，生物传感器和诊断。