Institute of Molecular Medicine, Peking University, Beijing, China.
FASEB J. 2010 Dec;24(12):4844-55. doi: 10.1096/fj.09-142398. Epub 2010 Aug 23.
Double-stranded small interfering RNAs (siRNAs) are important modulators of biological processes and hold great promise for therapeutic applications. However, serum processing of synthetic siRNAs is still largely unknown. To address this issue, serum degradation assays of 125 siRNAs were first performed in this study. Four siRNA categories of distinct serum stability were identified, including a group of siRNAs that were stable in their native form for both in vitro and in vivo assays. Fine mapping of the cleavage events occurring in serum treatment demonstrated that most occurred at two vulnerable sites, leading to a speculation that rational modification of these sites might protect most siRNAs from serum degradation. For proof of concept, an exhaustive siRNA modification study was performed. In addition to the consistent stabilization pattern revealed at these sites, our study further showed that a single modification made at the cleavage site stabilized the siRNAs to a large extent, highlighting the importance of these sites in siRNA degradation. In summary, the present study provided a comprehensive picture of serum processing of siRNA as well as a starting point for a rational siRNA modification strategy, both of which are of great importance to in vivo and therapeutic applications of siRNA.
双链小干扰 RNA（siRNAs）是生物过程的重要调节剂，在治疗应用方面具有很大的潜力。然而，合成 siRNAs 的血清处理在很大程度上仍不为人知。为了解决这个问题，本研究首先对 125 种 siRNA 的血清降解进行了测定。鉴定出了具有不同血清稳定性的 4 种 siRNA 类别，包括一组在体外和体内测定中以其天然形式稳定的 siRNA。对血清处理中发生的切割事件的精细定位表明，大多数切割发生在两个脆弱的位点，这使得人们推测对这些位点进行合理修饰可能会保护大多数 siRNA 免受血清降解。为了验证这一概念，进行了详尽的 siRNA 修饰研究。除了在这些位点揭示的一致稳定模式外，我们的研究还进一步表明，在切割位点进行单一修饰在很大程度上稳定了 siRNA，突出了这些位点在 siRNA 降解中的重要性。总之，本研究提供了 siRNA 血清处理的全面图景以及合理的 siRNA 修饰策略的起点，这两者对于 siRNA 的体内和治疗应用都非常重要。
