NMR AND UV STUDIES OF ANTISENSE OLIGONUCLEOTIDES

反义寡核苷酸的 NMR 和 UV 研究

• 批准号: 6180370
• 负责人: XIAOLIAN GAO
• 金额: $ 21.89万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180370
• 关键词: antisense nucleic acid; binding sites; bioimaging /biomedical imaging; chemical binding; chemical stability; chemical structure function; complementary RNA; computer simulation; conformation; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; nucleic acid hybridization; nucleic acid sequence; nucleic acid structure; nucleotide analog; oligonucleotides; structural biology; thermodynamics; ultraviolet spectrometry

Antisense oligonucleotides (AONs) are by far the most sequence-specific reagents, which are able to recognize more than ten designated residues in RNA sequences. Therefore, the use of AONs only as probes for gene functions but also as therapeutic agents for inhibition of the initiating events of gene expression as immense biomedical significance. The objectives of the proposed continued research are to elucidate the structure and the stabilities of AONs and their hybrids with RNA as a function of synthetic backbone and/or base modifications and to provide a basis for the structural effects of the single stranded AONs and the target RNA molecules on antisense hybridization. Although only insufficient literature information is present, our ongoing program in the antisense area has provided valuable information concerning the factors that affect the structures and the relative stabilities of a set of AON duplexes containing a single backbone modification. In this proposal, the studies will be extended to includes AON targeting a selected RNA site in human cytomegalovirus (HCMV) gene or loop RNAs, which are models for the loop motifs frequently observed in natural RNAs and in numerous biological functions. DNA dimer synthons containing an important group of achiral backbone, sugar and/or base modifications will be synthesized and incorporated into AONs at multiple positions. Studies will be undertaken using high resolution NMR, structure computation, UV spectroscopy and enzymatic digestion assays. The analyses will focus on: (a) structures and characteristics of AON.RNA hybrids digestion assays. The analyses will focus on: (a) structures and characteristics of AON.RNA hybrids containing the HCMV target and multiply modified AONs, (b) structures and characteristics of the AON.RNA hybrids when located in a loop region and the preferred RNA loop size for a given AON sequence, (c) structures and characteristics of single stranded AONs as a function of their backbone and base modifications and the structural correlations with the nucleolytic stability of these AONs, (d) structures of the single stranded target RNAs and the effects of these single stranded structures on AON.RNA hybridization, (e) the relative thermodynamic stabilities of the antisense duplexes/complexes containing systematic chemical structural variations. In the studies of these complex molecules, we will use site specific nucleobase deuteration, which is an efficient and cost effective isotope labeling method recently reported by this laboratory for the studies of large nucleic acid molecules, as a primary means for NMR spectral simplification. The proposed research complements the current intensive efforts in chemical synthesis and biological experimentation of AONs. The results of these studies should contribute to the fundamental understanding of the antisense principles, to the growing database of diverse nucleic acid structures, and to the development of the next generation of effective antisense gene regulation of effective antisense gene regulation agents for improved treatment of cancer, viral infectious diseases and a wide range of genetic disorders.

反义寡核苷酸是迄今为止最具序列特异性的试剂，它能够识别RNA序列中的十多个指定残基。因此，AON不仅可以作为基因功能的探针，而且可以作为抑制基因表达起始事件的治疗剂，具有巨大的生物医学意义。拟议继续研究的目标是阐明的结构和稳定性的AON和它们的杂交与RNA作为一个功能的合成骨架和/或碱基修饰，并提供一个基础的单链AON和靶RNA分子的反义杂交的结构效应。虽然只有不足的文献信息是目前，我们正在进行的计划在反义领域提供了有价值的信息有关的因素，影响的结构和相对稳定性的一组AON双链体含有一个单一的骨干修改。在这项提议中，研究将扩展到包括AON靶向人巨细胞病毒（HCMV）基因或环RNA中选定的RNA位点，这些RNA是在天然RNA和许多生物学功能中经常观察到的环基序的模型。含有一组重要的非手性骨架、糖和/或碱基修饰的DNA二聚体寡核苷酸将被合成并在多个位置掺入AON中。将使用高分辨率NMR、结构计算、UV光谱和酶消化测定进行研究。分析的重点是：（a）AON的结构和特点。RNA杂交酶切分析。分析将侧重于：（a）含有HCMV靶和多重修饰的AON的AON·RNA杂交体的结构和特征，（B）当AON·RNA杂交体位于环区时的结构和特征以及给定AON序列的优选RNA环大小，（c）第（1）款单链AON的结构和特性作为其骨架和碱基修饰的函数，以及与这些AON的溶核稳定性的结构相关性。AON，（d）单链靶RNA的结构和这些单链结构对AON.RNA杂交的影响，（e）含有系统化学结构变异的反义双链体/复合物的相对热力学稳定性。在这些复杂分子的研究中，我们将使用位点特异性核碱基氘化，这是一个有效的和成本效益的同位素标记方法，最近由本实验室报告的大核酸分子的研究，作为NMR光谱简化的主要手段。拟议的研究补充了目前AON的化学合成和生物实验的密集努力。这些研究的结果应该有助于对反义原理的基本理解，对不断增长的不同核酸结构的数据库，以及对下一代有效的反义基因调控的开发，有效的反义基因调控剂用于改善癌症、病毒感染性疾病和广泛的遗传疾病的治疗。