Egli Martin, Minasov George, Tereshko Valentina, Pallan Pradeep S, Teplova Marianna, Inamati Gopal B, Lesnik Elena A, Owens Steve R, Ross Bruce S, Prakash Thazha P, Manoharan Muthiah
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, USA.
Biochemistry. 2005 Jun 28;44(25):9045-57. doi: 10.1021/bi050574m.
The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the development of RNAi therapeutics.
本文介绍了10种新的RNA 2'-O修饰的合成方法、它们在寡核苷酸中的掺入情况，以及对其与反义活性相关的RNA亲和力和核酸酶抗性等性质的评估。所有修饰与天然磷酸骨架结合后，相对于第一代DNA硫代磷酸酯（PS-DNA），在与RNA杂交稳定性方面都有显著提高。特别是带有正电荷的2'-O修饰所提供的核酸酶抗性超过了PS-DNA。然而，小的带负电的2'-O取代基虽然增强了RNA亲和力，但不足以防止核酸酶降解。同样地，含有用相对较大的2'-O-[2-(苄氧基)乙基]取代基修饰的3'-末端残基的寡核苷酸会被外切核酸酶迅速降解，这证明了空间位阻通常会提高对核酸酶消化的保护作用这一假设是错误的。为了分析导致RNA亲和力和核酸酶抗性增强的因素，我们确定了每条链含有单个2'-O修饰胸苷的自互补A-DNA十聚体双链体的晶体结构。取代基的构象预组织、取代基与糖磷酸骨架之间有利的静电相互作用以及2'-O修饰附近稳定的水结构似乎都有助于提高RNA亲和力。在对核酸酶具有最有效保护作用的修饰结构中，观察到带正电的取代基与磷酸基团紧密结合。一些分析的2'-O修饰所表现出的有前景的性质可能值得对其在体内反义应用潜力进行更详细的评估。RNA的化学修饰也有望显著提高小干扰RNA（siRNA）的功效。因此，本文引入的2'-O修饰可能有利于RNAi治疗的发展。
