Quantification of the gene silencing performances of rationally-designed synthetic small RNAs.
Quantification of the gene silencing performances of rationally-designed synthetic small RNAs.
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DOI：
10.1007/s11693-015-9177-7
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发表时间：
2015-09-01
期刊：
影响因子：
--
通讯作者：
Magni, Paolo
中科院分区：
文献类型：
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作者：
Massaiu, Ilaria;Pasotti, Lorenzo;Magni, Paolo
小分子RNA（small RNA，sRNA）是一种高效、特异性调控细菌靶基因表达的遗传工具。受自然发生的sRNA的启发，最近的工作提出了在合成生物学中使用人工sRNA来预测所需基因的抑制。它们在代谢工程和细菌生理学研究等几个应用领域中的潜力得到了证实。最近提出了合理设计新型sRNA的指导原则。根据这些指导原则，在这项工作中，合成的sRNA的设计，构建和定量表征大肠杆菌。靶向报告基因RFP的sRNA通过测量当RFP在不同转录水平下、在不同启动子的控制下、在不同菌株中以及在单基因或操纵子结构中表达时的特异性基因沉默来测试。通过使用维持在不同拷贝数的质粒来调节sRNA水平。结果表明RFP沉默以sRNA和mRNA表达依赖性方式如预期那样起作用。使用数学模型来支持sRNA表征并估计可用于比较所设计的sRNA的性能的效率相关参数。当RFP被置于双基因合成操纵子中时，基因沉默也是成功的，而操纵子中的非靶基因（GFP）没有受到显著影响。最后，针对靶向内源性乳酸脱氢酶基因的另一种设计的sRNA评估沉默。在这项工作中进行的定量研究阐明了有趣的性能相关和上下文相关的合成sRNAs的功能，这将有力地支持可预测的基因沉默在不同的基础或应用研究。
Small RNAs (sRNAs) are genetic tools for the efficient and specific tuning of target genes expression in bacteria. Inspired by naturally occurring sRNAs, recent works proposed the use of artificial sRNAs in synthetic biology for predictable repression of the desired genes. Their potential was demonstrated in several application fields, such as metabolic engineering and bacterial physiology studies. Guidelines for the rational design of novel sRNAs have been recently proposed. According to these guidelines, in this work synthetic sRNAs were designed, constructed and quantitatively characterized in Escherichia coli. An sRNA targeting the reporter gene RFP was tested by measuring the specific gene silencing when RFP was expressed at different transcription levels, under the control of different promoters, in different strains, and in single-gene or operon architecture. The sRNA level was tuned by using plasmids maintained at different copy numbers. Results demonstrated that RFP silencing worked as expected in an sRNA and mRNA expression-dependent fashion. A mathematical model was used to support sRNA characterization and to estimate an efficiency-related parameter that can be used to compare the performance of the designed sRNA. Gene silencing was also successful when RFP was placed in a two-gene synthetic operon, while the non-target gene (GFP) in the operon was not considerably affected. Finally, silencing was evaluated for another designed sRNA targeting the endogenous lactate dehydrogenase gene. The quantitative study performed in this work elucidated interesting performance-related and context-dependent features of synthetic sRNAs that will strongly support predictable gene silencing in disparate basic or applied research studies.