A Magnetic Bead-Integrated Chip for the Large Scale Manufacture of Normalized esiRNAs

The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications.

化学合成的小干扰RNA（small interfering RNA, siRNA）双链体已成为RNA干扰（RNA interference, RNAi）功能缺失研究中一款强大且应用广泛的工具，但其存在脱靶效应较高的问题。近年来，核糖核酸内切酶制备的小干扰RNA（endoribonulease-prepared siRNA, esiRNA）因脱靶效应更低且成本效益更优，成为极具吸引力的替代方案。然而，当前esiRNA的制备工艺较为复杂，大规模生产中的纯化与归一化步骤尤为突出。本研究开发了一款磁珠集成芯片，可将扩增或转录产物固定于磁珠表面，并以稳定便捷的方式完成转录、酶切、归一化与纯化流程。该芯片可实现即开即用型esiRNA的大规模制备。本研究从转录、翻译与功能层面验证了所制备esiRNA的沉默特异性与效率。本研究成功在单孔内完成多款归一化esiRNA的制备，其中包括靶向沉默PARP1与BRCA1的esiRNA；随后利用这些esiRNA有效探究了其对细胞活力的协同影响。本研究构建了靶向68个酪氨酸激酶基因的小型esiRNA文库用于功能缺失研究，并筛选出4个调控海拉细胞迁移能力的基因。我们认为，相较于现有工艺，本方法为esiRNA制备提供了更稳定且更具成本效益的选择，因此这类异质性RNA链可在多数高强度、大规模应用中发挥效用。