Department of Chemistry and International Institute for Nanotechnology, Northwestern University, Evanston, IL 60208.
Department of Chemistry and International Institute for Nanotechnology, Northwestern University, Evanston, IL 60208
Proc Natl Acad Sci U S A. 2014 Jul 8;111(27):9739-44. doi: 10.1073/pnas.1409431111. Epub 2014 Jun 19.
Small interfering RNA (siRNA) is a powerful and highly effective method to regulate gene expression in vitro and in vivo. However, the susceptibility to serum nuclease-catalyzed degradation is a major challenge and it remains unclear whether the strategies developed to improve the stability of siRNA free in serum solution are ideal for siRNA conjugated to nanoparticle surfaces. Herein, we use spherical nucleic acid nanoparticle conjugates, consisting of gold nanoparticles (AuNPs) with siRNA chemisorbed to the surface, as a platform to study how a model siRNA targeting androgen receptor degrades in serum (SNA-siRNAAR). In solutions of 10% (vol/vol) FBS, we find rapid endonuclease hydrolysis at specific sites near the AuNP-facing terminus of siRNAAR, which were different from those of siRNAAR free in solution. These data indicate that the chemical environment of siRNA on a nanoparticle surface can alter the recognition of siRNA by serum nucleases and change the inherent stability of the nucleic acid. Finally, we demonstrate that incorporation of 2'-O-methyl RNA nucleotides at sites of nuclease hydrolysis on SNA-siRNAAR results in a 10-fold increase in siRNA lifetime. These data suggest that strategies for enhancing the serum stability of siRNA immobilized to nanoparticles must be developed from a dedicated analysis of the siRNA-nanoparticle conjugate, rather than a reliance on strategies developed for siRNA free in solution. We believe these findings are important for fundamentally understanding interactions between biological media and oligonucleotides conjugated to nanoparticles for the development of gene regulatory and therapeutic agents in a variety of disease models.
小干扰 RNA（siRNA）是一种强大且高效的方法，可在体外和体内调节基因表达。然而，易受血清核酸酶催化降解的影响是一个主要的挑战，目前尚不清楚为提高游离于血清溶液中的 siRNA 稳定性而开发的策略是否适用于与纳米粒子表面偶联的 siRNA。在此，我们使用球形核酸纳米粒子缀合物作为平台，该缀合物由金纳米粒子（AuNPs）组成，表面化学吸附有 siRNA，研究靶向雄激素受体的模型 siRNA 在血清中（SNA-siRNAAR）降解的情况。在含有 10%（体积/体积）胎牛血清的溶液中，我们发现 siRNAAR 在 AuNP 面向末端的特定部位发生快速内切酶水解，这与游离于溶液中的 siRNAAR 不同。这些数据表明，纳米粒子表面上 siRNA 的化学环境可以改变血清核酸酶对 siRNA 的识别，并改变核酸的固有稳定性。最后，我们证明在 SNA-siRNAAR 上的核酸酶水解部位掺入 2'-O-甲基 RNA 核苷酸可使 siRNA 的寿命延长 10 倍。这些数据表明，必须从对 siRNA-纳米粒子缀合物的专门分析中开发出增强固定于纳米粒子上的 siRNA 的血清稳定性的策略，而不能依赖于为游离于溶液中的 siRNA 开发的策略。我们相信，这些发现对于从根本上理解生物介质与连接到纳米粒子的寡核苷酸之间的相互作用非常重要，因为这对于在各种疾病模型中开发基因调控和治疗剂具有重要意义。
