DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli.
DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli.
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DOI：
10.1186/1472-6750-10-85
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发表时间：
2010-12-06
影响因子：
3.5
通讯作者：
Fox GE
中科院分区：
文献类型：
--
作者：
Liu Y;Stepanov VG;Strych U;Willson RC;Jackson GW;Fox GE
通过在细菌宿主中过表达来制造大量重组RNA受到它们在细胞内环境中的不稳定性的阻碍。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，以使所得嵌合构建体在细胞质中积累到足够高的水平。作为补充，从细胞中分离嵌合体和从稳定支架中回收重组RNA的成本效益的程序，这种策略可能成为现有的化学或酶促RNA合成方法的可行替代方案。将编码71个核苷酸的重组RNA的序列插入到溶蛋白弧菌5S rRNA基因的质粒携带的缺失突变体中，代替原始5S rRNA的螺旋III -环C片段。转化大肠杆菌后，在大肠杆菌中组成型表达了嵌合RNA（3×pen aRNA）。coli rrnB P1和P2启动子。RNA嵌合体的积累水平超过了宿主的5S rRNA。建立了一种基于细胞组分液-固分离的细菌总RNA提取方法。该方案避免了有毒化学物质，因此比传统方法更适合大规模RNA纯化。一对生物素化的8-17 DNA酶用于从嵌合体中定量切除71-nt重组RNA。重组RNA通过序列特异性捕获法分离，DNA酶通过生物素亲和层析回收。证明了使用“5S rRNA支架”策略在体内制造大量小RNA的基于发酵的方法的可行性。该方法提供了一种经济的方法，用于大规模生产小RNA，包括shRNA，siRNA和适体，用于临床和生物医学研究。
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.
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影响因子：
2.9
作者：
BLUM, H;BEIER, H;GROSS, HJ
通讯作者：
GROSS, HJ
影响因子：
--
作者：
BREAKER, RR;JOYCE, GF
通讯作者：
JOYCE, GF
影响因子：
46.9
作者：
Breaker, RR
通讯作者：
Breaker, RR
影响因子：
3.5
作者：
INOUE, H;HAYASE, Y;OHTSUKA, E
通讯作者：
OHTSUKA, E
DOI：
10.1128/aem.24.3.430-436.1972
发表时间：
1972-01-01
期刊：
APPLIED MICROBIOLOGY
影响因子：
--
作者：
LAGO, BD;BIRNBAUM, J;DEMAIN, AL
通讯作者：
DEMAIN, AL