Structure and Function of Nucleic Acid Therapeutics

核酸疗法的结构和功能

• 批准号: 7654586
• 负责人: MARTIN EGLI
• 金额: $ 30.17万
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7654586
• 关键词: Active Sites; Address; Affinity; Anti-Inflammatory Agents; Anti-inflammatory; Anticodon; Antisense Oligonucleotides; Antiviral Agents; Base Pairing; Behavior; Binding; Biochemical; Biological; Biological Models; Calculi; Catalytic Domain; Chemicals; Cleaved cell; Collaborations; Complex; Crystallography; DNA; DNA Damage; DNA Structure; DNA polymerase A; DNA-Directed DNA Polymerase; Data; Development; Diagnostic; Double-Stranded RNA; Drug Delivery Systems; Environment; Enzymes; Etiology; Exhibits; Future; Generations; Genomics; Glycols; Hybrids; Hydration status; Hydrogen Bonding; Investigation; Kinetics; Laboratories; Left; Lesion; Light; Messenger RNA; Mind; Modification; Molecular Biology; Nature; Neutrons; Nucleic Acid Probes; Nucleic Acids; Nucleotides; Oligonucleotides; Organic Chemistry; Peptide Nucleic Acids; Pharmaceutical Preparations; Pharmacologic Substance; Play; Process; Property; Protein Analysis; Proteins; RNA; RNA Interference; RNA Processing; Relative (related person); Research; Resistance; Resolution; Ribonuclease H; Ribose; Role; Shapes; Small Interfering RNA; Specificity; Structure; Substrate Specificity; System; Techniques; Testing; Therapeutic; Thermodynamics; Thymine; Time; Transfer RNA; Transferase; Validation; Variant; Vertebral column; Water; Work; X-Ray Crystallography; analog; antigene; base; chemical stability; deoxyguanosine triphosphate; design; difluorotoluene; drug discovery; ds-DNA; endonuclease; hydroxyl group; improved; improved functioning; in vivo; inorganic phosphate; insight; member; next generation; novel; nuclease; nucleic acid analog; nucleic acid binding protein; nucleic acid structure; prevent; programs; public health relevance; research and development; ribonuclease H1; three dimensional structure; tool; uptake

DESCRIPTION (provided by applicant): Chemically modified nucleic acids (CNAs) function as potential antigene-, antisense-, or RNA interference (RNAi)-based drugs, as model systems for native DNA and RNA, as chemical probes in diagnostics and in high-throughput genomics and drug target validation, or the analysis of protein-nucleic acid interactions, and as tools for structure determination. This application is a continuation of our research directed at CNAs, with the long-term objectives to optimize their structure and activity for future applications as antisense oligonucleotide (AON) and small interfering RNA (siRNA) therapeutics, to devise an etiology of nucleic acid structure, and to determine the origins of substrate recognition by selected DNA- and RNA-processing enzymes. We propose four aims of broad biological significance in understanding the consequences of chemical modification for nucleic acid structure and stability and, by probing nucleic acid-protein interactions structurally and functionally by way of CNAs, the principles affording substrate recognition and processing by RNase H and A- and Y-class DNA polymerases. Aim (1) focuses on investigations of the conformational features underlying the stability and efficacy of modifications assessed in connection with the discovery and development of the next generation of AON and siRNA therapeutics. This work will be carried out in collaboration with two world leaders in R&D of nucleic acid drugs, Alnylam Pharmaceuticals Inc. and Isis Pharmaceuticals, Inc. In Aim (2) we will scrutinize the pairing and structure of glycol nucleic acid (GNA), the simplest artificial pairing system with a phosphate backbone found to cross-pair with RNA. We will also use neutron macromolecular crystallography (NMC) to delve deeper into aspects of nucleic acid structure that have eluded characterization using standard techniques, such as the orientations of water molecules and ribose 22-hydroxyl groups. Work in Aim (3) is directed at RNase H, an endonuclease that plays a key role in antisense applications by way of destroying the mRNA targeted by certain AONs. By way of 3D structural data for complexes with duplexes that are bound but not cleaved, we will probe features of nucleic acids central to recognition. The conformational range of the strand opposite RNA tolerated by the enzyme will be gauged with 3D structures of complexes with AON/RNA hybrids. In Aim (4) we will address the recent hypothesis that certain DNA polymerases appear to rely more on shape than hydrogen bonding for accurate and efficient replication. Building on our recent structures of CNAs containing 2,4-difluorotoluene (F, an apolar T mimic) and complexes of F-modified templates with a trans-lesion (Y-class) DNA Pol, we will determine structures of ternary Pol-DNA-dNTP complexes containing F or dFTP of a replicative (A-class) DNA Pol, and correlate these data with activity data in the pre-steady- and steady-states. The main tool to be used is X-ray crystallography. Other approaches we will rely on to achieve our objectives are synthetic organic chemistry, biochemical and molecular biology tools as well a thermodynamics, kinetics and single-crystal NMC. PUBLIC HEALTH RELEVANCE: A comprehensive structure-based program to analyze and improve RNA affinity, chemical stability and ultimately efficacy of chemically modified antisense and siRNA oligonucleotides with implications for drug discovery of nucleic acid therapeutics, to gain insight into self-pairing and cross-pairing with DNA and/or RNA of nucleic acid analogs analyzed in the context of an etiology of nucleic acid structure, and, by using chemically modified nucleotides, to investigate and thereby refute or confirm existing hypotheses regarding the chemical and structural bases for substrate recognition by the RNase H endonuclease and nucleotide insertion by A- and Y-class DNA polymerases.

描述（由申请人提供）：化学修饰的核酸 (CNA) 可以作为潜在的基于反基因、反义或 RNA 干扰 (RNAi) 的药物，作为天然 DNA 和 RNA 的模型系统，作为诊断和高通量测序中的化学探针。 -通量基因组学和药物靶点验证，或蛋白质-核酸相互作用的分析，以及作为结构测定的工具。该应用是我们针对 CNAs 研究的延续，长期目标是优化其结构和活性，以供未来作为反义寡核苷酸 (AON) 和小干扰 RNA (siRNA) 治疗剂的应用，设计核酸结构的病因学，并确定选定的 DNA 和 RNA 加工酶识别底物的起源。我们提出了四个具有广泛生物学意义的目标，以了解化学修饰对核酸结构和稳定性的影响，并通过 CNA 探索核酸-蛋白质结构和功能上的相互作用，提供 RNase H 和 A 的底物识别和加工原理- 和 Y 类 DNA 聚合酶。目标 (1) 重点研究与下一代 AON 和 siRNA 疗法的发现和开发相关的修饰稳定性和功效的构象特征。这项工作将与核酸药物研发领域的两家世界领先企业 Alnylam Pharmaceuticals Inc. 和 Isis Pharmaceuticals, Inc. 合作进行。在 Aim (2) 中，我们将仔细研究乙二醇核酸 (GNA) 的配对和结构，最简单的人工配对系统，具有与 RNA 交叉配对的磷酸盐主链。我们还将使用中子大分子晶体学 (NMC) 更深入地研究使用标准技术无法表征的核酸结构方面，例如水分子和核糖 22-羟基的方向。 Aim (3) 中的工作针对 RNase H，这是一种核酸内切酶，通过破坏某些 AON 靶向的 mRNA，在反义应用中发挥关键作用。通过具有结合但未切割的双链体的复合物的 3D 结构数据，我们将探测对于识别至关重要的核酸特征。酶耐受的 RNA 相对链的构象范围将通过 AON/RNA 杂合体复合物的 3D 结构来测量。在目标 (4) 中，我们将解决最近的假设，即某些 DNA 聚合酶似乎更多地依赖于形状而不是氢键来实现准确和有效的复制。基于我们最近含有 2,4-二氟甲苯（F，非极性 T 模拟物）的 CNA 结构以及 F 修饰模板与跨损伤（Y 类）DNA Pol 的复合物，我们将确定三元 Pol-DNA 的结构-dNTP 复合物包含复制型（A 类）DNA Pol 的 F 或 dFTP，并将这些数据与稳态前和稳态的活动数据相关联。使用的主要工具是X射线晶体学。我们实现目标所依赖的其他方法包括合成有机化学、生物化学和分子生物学工具以及热力学、动力学和单晶 NMC。公共卫生相关性：一个基于结构的综合计划，用于分析和提高化学修饰反义和 siRNA 寡核苷酸的 RNA 亲和力、化学稳定性和最终功效，对核酸疗法的药物发现具有影响，以深入了解自配对和交叉配对在核酸结构病因学的背景下分析核酸类似物的DNA和/或RNA，并通过使用化学修饰的核苷酸来研究并从而反驳或证实有关核酸的现有假设RNase H 核酸内切酶底物识别和 A 类和 Y 类 DNA 聚合酶核苷酸插入的化学和结构基础。