Tu Mei-Juan, Wright Halley K, Batra Neelu, Yu Ai-Ming
Department of Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, CA, USA.
Methods Mol Biol. 2021;2323:249-265. doi: 10.1007/978-1-0716-1499-0_18.
Research on RNA function and therapeutic potential is dominated by the use of chemoengineered RNA mimics. Recent efforts have led to the establishment of novel technologies for the production of recombinant or bioengineered RNA molecules, which should better recapitulate the structures, functions and safety profiles of natural RNAs because both are produced and folded in living cells. Herein, we describe a robust approach for reproducible fermentation production of bioengineered RNA agents (BERAs) carrying warhead miRNAs, siRNAs, aptamers, or other forms of small RNAs, based upon an optimal hybrid tRNA/pre-miRNA carrier. Target BERA/sRNAs are readily purified by fast protein liquid chromatography (FPLC) to a high degree of homogeneity (>97%). This approach offers a consistent high-level expression (>30% of total bacterial RNAs) and large-scale production of ready-to-use BERAs (multiple to tens milligrams from 1 L bacterial culture).
RNA功能及治疗潜力的研究主要依赖于化学工程改造的RNA模拟物。最近的研究成果催生了用于生产重组或生物工程RNA分子的新技术，这些技术有望更好地模拟天然RNA的结构、功能和安全性，因为它们都是在活细胞中产生并折叠的。在此，我们描述了一种基于优化的杂交tRNA/前体miRNA载体，可重复性发酵生产携带弹头miRNA、siRNA、适体或其他形式小RNA的生物工程RNA制剂（BERA）的稳健方法。目标BERA/小RNA可通过快速蛋白质液相色谱（FPLC）轻松纯化至高度均一性（>97%）。该方法可实现稳定的高水平表达（>细菌总RNA的30%），并大规模生产即用型BERA（从1升细菌培养物中可获得数毫克至数十毫克）。
