Structure and Function of Nucleic Acid Therapeutics

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治疗剂;（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）DNA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。我们将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亲和力和大肠杆菌RNase H对反义RNA底物结合和识别的起源。将确定选定的第2代和第3代AON以及第1代siRNA修饰的晶体结构。结构数据将与它们的热力学稳定性、核酸酶抗性、对E.大肠杆菌RNase H降解（AON）和总体疗效，与Isis Pharmaceuticals Inc.（AON）和Alnylam Pharmaceuticals（siRNA）。此外，我们将试图回答为什么RNase H结合双链RNA（dsRNA），但不切割与RNA配对的RNA链（不像与DNA配对的RNA链）的问题，通过解决DNA的晶体结构：RNA-RNase H和dsRNA-RNase H复合物。在第二个项目中，我们将分析具有较短主链的天然核酸的替代物的晶体结构;即，苏呋喃糖基和来苏吡喃糖基核酸（分别为TNA和LPNA），使用部分或全部修饰的寡核苷酸。第三个项目是我们以前努力的延伸，以最大限度地提高DNA晶体结构的分辨率，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，分析具有代表性的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）对第二代和第三代AON修饰进行晶体学分析，并将其结构与现有的体外和体内功能数据相关联：（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的结构研究（4）DNA的晶体结构：RNARNase H和dsRNA-RNase H复合物;（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酯的DNA的高分辨率X射线结构。

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.