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）充当潜在的抗基因，反义或RNA干扰（RNAI）的药物，作为天然DNA和RNA的模型系统，作为诊断和高通量基因组学和药物靶标验证的诊断中的化学探针，以及对蛋白质 - 核 - 核酸相互作用或分析的工具，或分析的工具，并确定蛋白质 - 和分析。这项应用是我们针对CNA的研究的延续，其长期目标是优化其未来应用的结构和活动，作为反义寡核苷酸（AON）和小型干扰RNA（siRNA）治疗学，以设计核酸结构的病因，以确定选定的DNA-和RNNA-prosess的源代理，以确定源自识别的原始识别。我们提出了四个在理解化学修饰对核酸结构和稳定性的后果方面具有广泛生物学意义的目标，并通过通过CNA在结构和功能上探测核酸 - 蛋白质相互作用，从而可以通过RNase H和A-和A-和Y-Class DNA聚合酶来提供底物识别和处理的原理。 AIM（1）重点是研究与下一代AON和SIRNA Therapeutics的发现和开发评估的修改稳定性和效力的构象特征。这项工作将与两位世界领导人合作进行核酸药物R＆D，Alnylam Pharmaceuticals Inc.和ISIS Pharmaceuticals，Inc。Inc.的AIM（2），我们将仔细检查乙二醇核酸（GNA）的配对和结构，这是最简单的人造配对系统与磷酸盐旁观者与Crossbone to Crossbone to Cross to Cross to rna rna。我们还将使用中子大分子晶体学（NMC）深入研究核酸结构的各个方面，这些方面使用标准技术（例如水分子的方向和核糖22-羟基的方向）进行了表征。 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AONS靶向的mRNA在反义应用中起关键作用。通过3D结构数据，用于具有结合但不裂解的复合物的复合物，我们将探测核酸的特征，中心是识别的中心。酶相反的RNA相对的链的构象范围将用带有AON/RNA杂交的复合物的3D结构进行测量。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，而不是氢键以进行准确有效的复制。 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稳态。要使用的主要工具是X射线晶体学。我们将依靠的其他方法是合成有机化学，生化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：一个基于结构的全面计划，可分析和改善RNA亲和力，化学稳定性以及化学修饰的反义和siRNA寡核苷酸的功效，对药物发现核酸疗法的药物的影响，从而洞悉自我生产和与核酸酸性的核酸和/或rna的核酸酸性和/或rna的核酸源和/或rna的核酸核酸核和/或rNA的核酸核酸核和/或rna的核酸核酸构成核酸核和/或rna。化学修饰的核苷酸，从而研究并确认有关RNase H核酸内切酶和Y级DNA聚合酶的RNase H核酸内切酶和核苷酸插入底物识别的化学和结构碱基的现有假设。