STRUCTURE AND FUNCTION OF NUCLEIC ACID THERAPEUTICS

本研究的目的是详细研究核酸类似物的结构和功能。利用x射线晶体学，化学修饰的核酸片段的三维结构将被确定。该项目的第一个重点是利用积累的结构数据和随后的结构-稳定性和结构-活性相关性作为设计下一代具有潜在抗癌，抗病毒和抗炎适应症的反义疗法的指导。基于对反义化合物以下关键特征的结构见解的改进将特别关注：RNA亲和力，核酸酶抗性以及反义-RNA杂合体对RNase h介导的裂解的敏感性。新设计的修饰的功效将与Isis制药公司合作，使用各种体外和基于细胞的分析来测试。核酸类似物除了作为治疗性反义试剂的潜在用途外，也是研究天然DNA和RNA起源和进化的先决条件。与后者相比，它们的特性发生了改变，这使它们成为医学诊断、材料科学、蛋白质-核酸相互作用分析、DNA电子转移等领域的理想工具。第二。本项目的重点是在上述一些领域正在探索的几种核酸类似物的结构确定和结构功能分析。此外，用某些类似物观察到的良好结晶特性将用于超高分辨率下dna -离子相互作用的研究。本研究的具体目的是：1)分析反义核酸类似物RNA亲和力的结构起源。2)反义核酸类似物耐核酸酶的结构起源分析。第三代反义修饰的结构导向设计，利用研究1和2中出现的原则。利用紫外熔融实验、酶分析和细胞分析，评价含有这些修饰的反义寡核苷酸的性质。4)通过对大肠杆菌RNase H处理的RNA与构象限制性类似物杂交体的结构分析，探讨其底物特异性的结构来源。5)人工核酸配对系统结构的x射线晶体学分析，例如带有己糖或苯乙烯帽的dna。对基于结构数据的单个类似物的热力学稳定性、配对性质和特定功能方面的合理化。6)超高分辨率测定DNA晶体结构和化学修饰DNA，分析金属离子与DNA的配位。在此基础上，详细分析了碱金属离子和土碱金属离子在DNA结构和填充中的作用。7)开发核酸晶体的数据收集和结构测定方案，利用选定的碱和土碱金属离子、硫（类似物）和磷的异常散射成分。

The objective of this research is a detailed investigation of the structure and function of nucleic acid analogs. Using X-ray crystallography, the three-dimensional structures of chemically modified nucleic acid fragments will be determined. The first focus of the project is to use the accumulated structural data and subsequent structure-stability and structure-activity correlations as guides for designing the next generation of antisense therapeutics with potential anticancer, antiviral and antiinflammatory indications. Improvements based on the structural insights of the following key features of antisense compounds will be given particular attention: RNA affinity, nuclease resistance and susceptibility of the antisense-RNA hybrid to RNase H-mediated cleavage. The efficacy of the newly designed modifications will be tested in collaboration with Isis Pharmaceuticals Inc., using a variety of in vitro and cell-based assays. In addition to their potential use as therapeutic antisense reagents, nucleic acid analogs are a prerequisite for studying the origin and evolution of natural DNA and RNA. Their altered properties relative to the latter make them ideal tools in medical diagnostics, material science, analysis of protein-nucleic acid interactions, DNA electron transfer etc. The second. focus of this project is the structure determination and structure-function analysis of several nucleic acid analogs that are being explored in some of the above areas. Moreover, the favorable crystallization properties observed with certain analogs will be exploited for studying DNA-ion interactions at ultra-high resolutions. The specific aims of this research are: 1) Analysis of the structural origins of the RNA affinity of antisense nucleic acid analogs. 2) Analysis of the structural origins of the nuclease resistance of antisense nucleic acid analogs. 3) Structure-guided design of third- generation antisense modifications using principles emerging from studies l and 2. Evaluation of the properties of antisense oligonucleotides containing these modifications, using UV melting experiments, enzyme assays and cell-based assays. 4) Investigation of the structural origins of the substrate specificity of E. coli RNase H by way of structure analysis of hybrids between RNA and conformationally restricted analogs that are processed by the enzyme. 5) X-ray crystallographic analysis of the structures of artificial nucleic acid pairing systems, e.g. DNAs with hexose-based sugars or stilbene caps. A rationalization of the thermodynamic stabilities, pairing properties and particular functional aspects of the individual analogs based on structural data. 6) Determination of crystal structures of DNAs and chemically modified DNAs at ultrahigh resolutions to analyze metal ion coordination to DNA. Based on these and their comparisons with reference structures of similar precision, a detailed analysis of the role of alkali and earth alkali metal ions in DNA structure and packing. 7) Development of data collection and structure determination protocols for nucleic acid crystals that exploit the anomalous scattering component of selected alkali and earth alkali metal ions, sulfur (in analogs) and phosphorus.