NMR AND UV STUDIES OF ANTISENSE OLIGONUCLEOTIDES

反义寡核苷酸(AON)是迄今为止最具序列特异性的试剂，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，还可以作为治疗药物来抑制基因表达的启动事件，具有巨大的生物医学意义。继续研究的目的是阐明AON及其与RNA的杂交物作为合成骨架和/或碱基修饰的功能的结构和稳定性，并为单链AON和靶RNA分子在反义杂交中的结构效应提供基础。尽管目前文献资料不足，但我们正在进行的反义领域的计划已经提供了关于影响一组含有单一骨架修饰的AON双链的结构和相对稳定性的因素的有价值的信息。在这项建议中，研究将扩展到包括针对人巨细胞病毒(HCMV)基因或环RNA中选定的RNA位点的AON，这是在自然RNA和许多生物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二聚体合成子将被合成并在多个位置结合到AON中。研究将使用高分辨率核磁共振、结构计算、紫外光谱和酶消化分析。这些分析将集中在：(A)AON.RNA杂交物的结构和特性。分析将集中在：(A)含有HCMV靶标和多重修饰的AON的AON.RNA杂交物的结构和特征，(B)位于环区的AON.RNA杂交物的结构和特征以及给定AON序列的优选RNA环大小，(C)单链AON的结构和特征与其骨架和碱基修饰的函数以及这些AON的结构与这些AON的核溶解稳定性的结构相关性，(D)单链靶RNA的结构以及这些单链结构对AON.RNA杂交的影响，(E)含有系统化学结构变化的反义双链/络合物的相对热力学稳定性。在这些复杂分子的研究中，我们将使用本实验室最近报道的一种用于研究大分子的有效且经济的同位素标记方法--位点特定的碱基氚作为核磁共振谱简化的主要手段。这项拟议的研究补充了目前在化学合成和生物实验方面进行的密集工作。这些研究的结果将有助于对反义原理的基本理解，有助于不断增长的不同核酸结构的数据库，并有助于开发有效的反义基因调节剂的下一代有效的反义基因调节剂，以改进癌症、病毒感染性疾病和广泛的遗传疾病的治疗。

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.