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。使用X射线晶体学，将确定化学修饰的核酸片段的三维结构。该项目的第一个重点是使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指导，用于设计具有潜在抗癌，抗病毒和抗肿瘤适应症的下一代反义治疗药物。基于反义化合物的以下关键特征的结构见解的改进将被给予特别关注：RNA亲和力、核酸酶抗性和反义RNA杂交体对RNA酶H介导的切割的敏感性。新设计的修改的有效性将与Isis制药公司合作进行测试，使用多种体外和基于细胞的测定。除了作为治疗性反义试剂的潜在用途之外，核酸类似物是研究天然DNA和RNA的起源和进化的先决条件。它们相对于后者的改变的性质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电子转移等的理想工具。该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测定和结构-功能分析。此外，某些类似物观察到的有利结晶特性将被用于研究超高分辨率的DNA-离子相互作用。本研究的具体目的是：1）分析反义核酸类似物的RNA亲和力的结构来源。2)分析反义核酸类似物的核酸酶抗性的结构起源。3)第三代反义修饰的结构指导设计，使用研究1和2中出现的原理。使用UV熔解实验、酶测定和基于细胞的测定来评价含有这些修饰的反义寡核苷酸的性质。4)探讨了E. coli RNase H，通过对RNA和由该酶加工的构象限制性类似物之间的杂合体进行结构分析。5)人工核酸配对系统结构的X射线晶体学分析，例如具有己糖基糖或芪帽的DNA。基于结构数据的热力学稳定性，配对性质和个别类似物的特定功能方面的合理化。6)以100倍分辨率测定DNA和化学修饰的DNA的晶体结构，以分析金属离子与DNA的配位。在此基础上，并与具有相似精度的参考结构进行比较，详细分析了碱金属和碱土金属离子在DNA结构和包装中的作用。7)开发用于核酸晶体的数据收集和结构确定方案，其利用所选碱金属和碱土金属离子、硫（类似物）和磷的异常散射组分。

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.