High resolution biosensor to test the capping level and integrity of mRNAs.
High resolution biosensor to test the capping level and integrity of mRNAs.
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DOI：
10.1093/nar/gkaa955
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发表时间：
2020-12-16
影响因子：
14.9
通讯作者：
Polizzi K
中科院分区：
文献类型：
--
作者：
Moya-Ramírez I;Bouton C;Kontoravdi C;Polizzi K
5' 帽结构在真核 mRNA 上普遍存在，对于转录后加工、翻译起始和稳定性至关重要。在这里，我们描述了一种生物传感器，旨在检测 mRNA 上帽结构的存在，该传感器对 mRNA 降解也敏感，因此可以一步检测到未帽或降解的 mRNA。该生物传感器基于嵌合蛋白，将识别和转导作用结合在单个分子中。该传感器的主要特点是其简单性，可以用最少的仪器对上限水平进行半定量分析。该生物传感器被证明可以检测几种体外转录 mRNA 的加帽水平。对于大小从 250 nt 到约 2700 nt 的 RNA，其灵敏度和动态范围保持恒定，并且生物传感器能够以至少 20% 的增量检测加帽水平的变化，检测限为 2.4 pmol。值得注意的是，它还可以应用于更复杂的分析物，例如 mRNA 疫苗和体内转录的 mRNA。该生物传感器是能够检测分析上具有挑战性的结构（例如 mRNA 帽）的技术的创新示例。它可以应用于多种场景，从疫苗等基于 mRNA 的产品的质量分析到体外加帽反应的优化。
5′ Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing, translation initiation and stability. Here we describe a biosensor designed to detect the presence of cap structures on mRNAs that is also sensitive to mRNA degradation, so uncapped or degraded mRNAs can be detected in a single step. The biosensor is based on a chimeric protein that combines the recognition and transduction roles in a single molecule. The main feature of this sensor is its simplicity, enabling semi-quantitative analyses of capping levels with minimal instrumentation. The biosensor was demonstrated to detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic range remained constant with RNAs ranging in size from 250 nt to approximately 2700 nt and the biosensor was able to detect variations in the capping level in increments of at least 20%, with a limit of detection of 2.4 pmol. Remarkably, it also can be applied to more complex analytes, such mRNA vaccines and mRNAs transcribed in vivo. This biosensor is an innovative example of a technology able to detect analytically challenging structures such as mRNA caps. It could find application in a variety of scenarios, from quality analysis of mRNA-based products such as vaccines to optimization of in vitro capping reactions.
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DOI：
10.1261/rna.056614.116
发表时间：
2016-09
期刊：
RNA (New York, N.Y.)
影响因子：
--
作者：
Fuchs AL;Neu A;Sprangers R
通讯作者：
Sprangers R
影响因子：
10.8
作者：
Blakney, Anna K.;McKay, Paul F.;Shattock, Robin J.
通讯作者：
Shattock, Robin J.
影响因子：
4.3
作者：
Beverly, Michael;Dell, Amy;Houghton, Leslie
通讯作者：
Houghton, Leslie
影响因子：
4.9
作者：
Garcia-Fernandez, Emilio;Carmen Gonzalez-Garcia, M.;Orte, Angel
通讯作者：
Orte, Angel
影响因子：
14.9
作者：
Cawley A;Warwicker J
通讯作者：
Warwicker J