Department of Biology and Biochemistry, University of Houston, Houston, TX, USA.
BMC Biotechnol. 2010 Dec 6;10:85. doi: 10.1186/1472-6750-10-85.
Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis.
Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse.
The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.
通过在细菌宿主中过度表达来大量制造重组 RNA 会受到其在细胞内环境中不稳定的阻碍。为了克服这个问题，可以将感兴趣的 RNA 融合到稳定的细菌 RNA 中，从而使嵌合构建体在细胞质中积累到足够高的水平。通过补充从细胞中分离嵌合体和从稳定支架中回收重组 RNA 的具有成本效益的程序，这种策略可能成为现有化学或酶促 RNA 合成方法的可行替代方法。
编码 71 个核苷酸的重组 RNA 的序列被插入到 V. proteolyticus 5S rRNA 基因的质粒缺失突变体中，取代了原始 5S rRNA 的螺旋 III-环 C 片段。转化到大肠杆菌后，嵌合 RNA（3×pen aRNA）从大肠杆菌 rrnB P1 和 P2 启动子组成型表达。RNA 嵌合体积累到超过宿主 5S rRNA 的水平。开发了一种依赖于细胞成分液-固分配的新方法来从细菌细胞中分离总 RNA。该方案避免了有毒化学品，因此比传统方法更适合大规模 RNA 纯化。使用一对生物素化的 8-17 DNA 酶来实现从嵌合体中定量切除 71 个核苷酸的重组 RNA。通过序列特异性捕获带有固定互补脱氧寡核苷酸的珠子来分离重组 RNA，而 DNA 酶通过生物素亲和层析回收用于重复使用。
使用“5S rRNA 支架”策略在体内制造大量小 RNA 的发酵方法的可行性得到了证明。该方法为包括 shRNA、siRNA 和适体在内的小 RNA 的大规模生产提供了一种经济的方法，可用于临床和生物医学研究。
