Structure and Function of Nucleic Acid Therapeutics

核酸疗法的结构和功能

• 批准号: 7454959
• 负责人: MARTIN EGLI
• 金额: $ 25.55万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7454959
• 关键词: Affect; Affinity; Alkali Metals; Alkalies; Anti-Inflammatory Agents; Anti-inflammatory; Antisense Oligonucleotides; Antisense RNA; Antiviral Agents; Attention; Base Sequence; Binding; Binding Sites; Biochemistry; Biological; Catalytic RNA; Cations; Charge; Chemicals; Class; Cleaved cell; Collaborations; Complex; DNA; DNA Binding; DNA Structure; Data; Data Collection; Development; Dissection; Double-Stranded RNA; Drug Delivery Systems; Enzymes; Escherichia coli; Etiology; Exhibits; Gene Expression; Generations; Glucose; Homo; Hybrids; Hydrolysis; In Vitro; Investigation; Laboratories; Major Groove; Maps; Measures; Mediating; Messenger RNA; Metal Ion Binding; Metals; Methods; Minor; Modification; Molecular Conformation; Neutron Diffraction; Neutrons; Nucleic Acid Conformation; Nucleic Acids; Oligonucleotides; Pathway interactions; Pharmacologic Substance; Photons; Play; Process; Property; Proteins; RNA; RNA Degradation; RNA Interference; Relative (related person); Research; Research Personnel; Resistance; Resolution; Ribonuclease H; Roentgen Rays; Role; Shapes; Signal Pathway; Small Interfering RNA; Solvents; Source; Structure; Substrate Specificity; System; Therapeutic; Therapeutic Agents; Therapeutic Uses; Thermodynamics; Time; Universities; Validation; Vertebral column; Water; Width; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; analog; antigene; base; chemical stability; chemical synthesis; design; ds-DNA; electron density; improved; in vivo; inorganic phosphate; insight; knock-down; methylphosphonate; next generation; novel; nuclease; nucleic acid analog; nucleic acid structure; programs; three dimensional structure; tool

DESCRIPTION (provided by applicant): Our research has three main long-term objectives: (i) Detailed analyses of the structure/function of nucleic acid analogs, for the design of the next generation of antisense oligonucleotide (AON) and RNAi therapeutics with potential anticancer, antiviral and anti-inflammatory indications; (ii) Determination of crystal structures of nucleic acid analogs studied in the context of etiology (origin of nucleic acid structure); (iii) Structural investigation of DNA fine structure, for assessing the relative influence of sequence and cations on conformation. The main tool we will use for determining the three dimensional structures is X-ray crystallography. In the first project, two key features of antisense compounds will be given particular attention: RNA affinity and the origins of antisense-RNA substrate binding and recognition by E.coli RNase H. The crystal structures of selected 2nd and 3rd generation AON and 1st generation siRNA modifications will be determined. The structural data will be correlated with their thermodynamic stabilities, nuclease resistances, susceptility to E. coli RNase H degradation (AONs) and overall efficacy, in collaboration with Isis Pharmaceuticals Inc. (AONs) and Alnylam Pharmaceuticals (siRNAs). In addition, we will seek to answer the question why RNase H binds double-stranded RNA (dsRNA), but does not cleave RNA strands paired to RNA (unlike those paired to DNA), by solving the crystal structures of DNA:RNA-RNase H and dsRNA-RNase H complexes. In the second project, we will analyze the crystal structures of alternatives to the natural nucleic acids with shorter backbones; i.e., threofuranosyl and lyxopyranosyl nucleic acid (TNA and LPNA, resp.), using either partially or all-modified oligonucleotides. The third project is an extension of our previous efforts to maximize the resolution of DNA crystal structures, to gain insight into the relative influence of sequence and metal cations on DNA duplex conformation. We will use neutron scattering, to analyze the structure of a representive oligodeoxynucleotide, and to visualize fully and partially occupied alkali metal ion binding sites. The specific aims of this research are: (1) Crystallographic analyses of 2nd and 3rd generation AON modifications, and correlation of the structures with existing in vitro and in vivo functional data; (2) Crystallographic analyses of siRNA modifications that are tolerated by the RNAi pathway, to improve RNA affinity and nuclease resistance of siRNA; (3) Structural studies of AONs (arabino nucleic acids) that, in complex with RNA, are substrates of RNase H; (4) Crystal structures of DNA:RNARNase H and dsRNA-RNase H complexes; (5) Crystallographic analyses of artificial nucleic acid pairing systems; and (6) Neutron diffraction studies of a DNA duplex and high-resolution X-ray structures of DNAs carrying methylphosphonates.

描述（由申请人提供）：我们的研究有三个主要的长期目标：（i）详细分析核酸类似物的结构/功能，用于设计具有潜在抗癌、抗病毒和抗炎适应症的下一代反义寡核苷酸（AON）和RNAi疗法； (ii) 确定在病因学背景下研究的核酸类似物的晶体结构（核酸结构的起源）； (iii) DN​​A精细结构的结构研究，用于评估序列和阳离子对构象的相对影响。我们用于确定三维结构的主要工具是 X 射线晶体学。在第一个项目中，将特别关注反义化合物的两个关键特征：RNA亲和力以及反义RNA底物结合和被大肠杆菌RNase H识别的起源。将确定选定的第二代和第三代AON以及第一代siRNA修饰的晶体结构。与 Isis Pharmaceuticals Inc. (AONs) 和 Alnylam Pharmaceuticals (siRNAs) 合作，结构数据将与其热力学稳定性、核酸酶抗性、对大肠杆菌 RNase H 降解 (AONs) 的敏感性和总体功效相关联。此外，我们将通过解析 DNA:RNA-RNase H 和 dsRNA-RNase H 复合物的晶体结构，寻求回答为什么 RNase H 结合双链 RNA (dsRNA)，但不切割与 RNA 配对的 RNA 链（与与 DNA 配对的 RNA 链不同）的问题。在第二个项目中，我们将分析具有较短主链的天然核酸替代品的晶体结构；即，使用部分或全部修饰的寡核苷酸的苏呋喃糖基和吡喃来糖基核酸（TNA和LPNA，分别）。第三个项目是我们之前努力的延伸，旨在最大限度地提高 DNA 晶体结构的分辨率，以深入了解序列和金属阳离子对 DNA 双链体构象的相对影响。我们将使用中子散射来分析代表性寡脱氧核苷酸的结构，并可视化完全和部分占据的碱金属离子结合位点。本研究的具体目的是：（1）第二代和第三代AON修饰的晶体学分析，以及结构与现有体外和体内功能数据的相关性； (2) 对RNAi途径耐受的siRNA修饰进行晶体学分析，以提高siRNA的RNA亲和力和核酸酶抗性； (3) AON（阿拉伯核酸）的结构研究，AON 与 RNA 复合，是 RNase H 的底物； (4) DNA:RNARNase H和dsRNA-RNase H复合物的晶体结构； (5)人工核酸配对系统的晶体学分析； (6) DNA 双链体的中子衍射研究和携带甲基膦酸盐的 DNA 的高分辨率 X 射线结构。