Structure and Function of Nucleic Acid Therapeutics

核酸疗法的结构和功能

基本信息

批准号：
8292045
负责人：
MARTIN EGLI
金额：
$ 29.76万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-02-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8292045
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-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 Pancreatic ribonuclease 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 Shapes Small Interfering RNA Specificity Structure Substrate Specificity System Techniques Testing Therapeutic Thermodynamics Thymine Time Transfer RNA 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 nucleotidyltransferase prevent programs research and development ribonuclease H1 three dimensional structure tool uptake

项目摘要

ABSTRACT 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 as thermodynamics, kinetics and single-crystal NMC.

抽象的化学修饰核酸 (CNA) 具有潜在的反基因、反义或 RNA 干扰作用基于 (RNAi) 的药物，作为天然 DNA 和 RNA 的模型系统，作为诊断和应用中的化学探针高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。该应用程序是我们针对 CNA 研究的延续，优化其结构和活性以供未来反义应用的长期目标寡核苷酸 (AON) 和小干扰 RNA (siRNA) 疗法，以设计核酸的病因学结构，并通过选定的 DNA 和 RNA 处理确定底物识别的起源酶。我们提出了四个具有广泛生物学意义的目标，以理解对核酸结构和稳定性进行化学修饰，并通过探测核酸-蛋白质相互作用通过 CNA 的结构和功能，提供底物识别和处理的原理 RNase H 以及 A 类和 Y 类 DNA 聚合酶。目标（1）侧重于构象的研究与发现和评估有关的修饰的稳定性和功效的特征下一代 AON 和 siRNA 疗法的开发。这项工作将在与核酸药物研发领域的两家世界领先企业 Alnylam Pharmaceuticals Inc. 和 Isis 合作 Pharmaceuticals, Inc. 在目标 (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) 更深入地研究核酸结构的各个方面已经逃避了使用标准技术的表征，例如水分子的方向和核糖22-羟基。 Aim (3) 中的工作针对 RNase H，这是一种核酸内切酶，在通过破坏某些 AON 靶向的 mRNA 进行反义应用。通过3D结构具有结合但未切割的双链体的复合物的数据，我们将探测核酸的特征识别的核心。酶耐受的 RNA 相对链的构象范围为通过 AON/RNA 杂合体复合物的 3D 结构进行测量。在目标 (4) 中，我们将解决最近的问题假设某些 DNA 聚合酶似乎更依赖于形状而不是氢键来获得准确的结果和高效的复制。以我们最近含有 2,4-二氟甲苯（F，一种非极性物质）的 CNA 结构为基础 T 模拟）和 F 修饰模板与跨损伤（Y 类）DNA Pol 的复合物，我们将确定含有复制型（A 类）DNA Pol 的 F 或 dFTP 的三元 Pol-DNA-dNTP 复合物的结构，以及将这些数据与稳态前和稳态的活动数据相关联。主要使用的工具是X射线晶体学。我们将依靠的其他方法来实现我们的目标是合成有机化学，生化和分子生物学工具以及热力学、动力学和单晶 NMC。

项目成果

期刊论文数量（61）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Correlating structure and stability of DNA duplexes with incorporated 2'-O-modified RNA analogues.

将 DNA 双链体的结构和稳定性与掺入的 2-O-修饰的 RNA 类似物相关联。

DOI：
10.1021/bi980392a
发表时间：
1998
期刊：
Biochemistry.
影响因子：
0
作者：
Tereshko,V;Portmann,S;Tay,EC;Martin,P;Natt,F;Altmann,KH;Egli,M
通讯作者：
Egli,M

The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding.

大肠杆菌的 yrdC 基因产物的结构揭示了一种新的折叠，并表明其在 RNA 结合中的作用。

DOI：
10.1110/ps.9.12.2557
发表时间：
2000
期刊：
Protein science : a publication of the Protein Society
影响因子：
0
作者：
Teplova,M;Tereshko,V;Sanishvili,R;Joachimiak,A;Bushueva,T;Anderson,WF;Egli,M
通讯作者：
Egli,M

Unexpected origins of the enhanced pairing affinity of 2'-fluoro-modified RNA.

DOI：
10.1093/nar/gkq1270
发表时间：
2011-04
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Pallan PS;Greene EM;Jicman PA;Pandey RK;Manoharan M;Rozners E;Egli M
通讯作者：
Egli M

Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides.

DOI：
10.1021/ja207086x
发表时间：
2011-10-19
期刊：
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
影响因子：
15
作者：
Egli, Martin;Pallan, Pradeep S.;Allerson, Charles R.;Prakash, Thazha P.;Berdeja, Andres;Yu, Jinghua;Lee, Sam;Watt, Andrew;Gaus, Hans;Bhat, Balkrishen;Swayze, Eric E.;Seth, Punit P.
通讯作者：
Seth, Punit P.

Unique gene-silencing and structural properties of 2'-fluoro-modified siRNAs.

DOI：
10.1002/anie.201006519
发表时间：
2011-03-01
期刊：
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
影响因子：
16.6
作者：
Manoharan, Muthiah;Akinc, Akin;Pandey, Rajendra K.;Qin, June;Hadwiger, Philipp;John, Matthias;Mills, Kathy;Charisse, Klaus;Maier, Martin A.;Nechev, Lubomir;Greene, Emily M.;Pallan, Pradeep S.;Rozners, Eriks;Rajeev, Kallanthottathil G.;Egli, Martin
通讯作者：
Egli, Martin