用 Simple Western 方法检测 mRNA 体外转录过程中使用的残余 T7 RNA 聚合酶。

IF 3 3区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Yvonne Shieh, Andrew R. Swartz, Richard R. Rustandi
{"title":"用 Simple Western 方法检测 mRNA 体外转录过程中使用的残余 T7 RNA 聚合酶。","authors":"Yvonne Shieh,&nbsp;Andrew R. Swartz,&nbsp;Richard R. Rustandi","doi":"10.1002/elps.202400044","DOIUrl":null,"url":null,"abstract":"<p>Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99 kDa protein with high binding affinity for the promoter sequence and a low error rate. Post-IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high-throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3 ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires &lt;10 µL of sample volume, and consists of minimal sample handling and hands-on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"45 19-20","pages":"1834-1839"},"PeriodicalIF":3.0000,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elps.202400044","citationCount":"0","resultStr":"{\"title\":\"Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western\",\"authors\":\"Yvonne Shieh,&nbsp;Andrew R. Swartz,&nbsp;Richard R. Rustandi\",\"doi\":\"10.1002/elps.202400044\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99 kDa protein with high binding affinity for the promoter sequence and a low error rate. Post-IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high-throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3 ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires &lt;10 µL of sample volume, and consists of minimal sample handling and hands-on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.</p>\",\"PeriodicalId\":11596,\"journal\":{\"name\":\"ELECTROPHORESIS\",\"volume\":\"45 19-20\",\"pages\":\"1834-1839\"},\"PeriodicalIF\":3.0000,\"publicationDate\":\"2024-06-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elps.202400044\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ELECTROPHORESIS\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/elps.202400044\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ELECTROPHORESIS","FirstCategoryId":"99","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/elps.202400044","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

摘要

治疗性信使 RNA(mRNA)已被证明是一种可扩展的多功能疫苗平台,可用于快速开发和制造新的候选疫苗。mRNA 是利用噬菌体 T7 RNA 聚合酶(T7 RNAP)通过体外转录(IVT)酶法合成的,T7 RNAP 是一种 99 kDa 蛋白,与启动子序列的结合亲和力高,出错率低。IVT 后,mRNA 会被纯化以去除杂质,但如果 T7 RNAP 未被充分清除,可能会导致不良的临床副作用。因此,对 IVT 和处理中间产物中的 T7 RNAP 浓度进行量化以了解下游纯化过程中的清除情况非常重要。利用毛细管免疫测定技术 Simple Western (SW) 开发了一种高通量 T7 RNAP 检测方法,可定量检测低至 5.3 纳克/毫升的浓度,且具有良好的精确度和准确性。与现有的 T7 RNAP 免疫测定或总蛋白测定(如双喹啉酸测定或布拉德福德)相比,SW T7 RNAP 测定法对 T7 RNAP 具有特异性,只需对 T7 RNAP 进行定量即可。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western

Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western

Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99 kDa protein with high binding affinity for the promoter sequence and a low error rate. Post-IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high-throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3 ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires <10 µL of sample volume, and consists of minimal sample handling and hands-on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
ELECTROPHORESIS
ELECTROPHORESIS 生物-分析化学
CiteScore
6.30
自引率
13.80%
发文量
244
审稿时长
1.9 months
期刊介绍: ELECTROPHORESIS is an international journal that publishes original manuscripts on all aspects of electrophoresis, and liquid phase separations (e.g., HPLC, micro- and nano-LC, UHPLC, micro- and nano-fluidics, liquid-phase micro-extractions, etc.). Topics include new or improved analytical and preparative methods, sample preparation, development of theory, and innovative applications of electrophoretic and liquid phase separations methods in the study of nucleic acids, proteins, carbohydrates natural products, pharmaceuticals, food analysis, environmental species and other compounds of importance to the life sciences. Papers in the areas of microfluidics and proteomics, which are not limited to electrophoresis-based methods, will also be accepted for publication. Contributions focused on hyphenated and omics techniques are also of interest. Proteomics is within the scope, if related to its fundamentals and new technical approaches. Proteomics applications are only considered in particular cases. Papers describing the application of standard electrophoretic methods will not be considered. Papers on nanoanalysis intended for publication in ELECTROPHORESIS should focus on one or more of the following topics: • Nanoscale electrokinetics and phenomena related to electric double layer and/or confinement in nano-sized geometry • Single cell and subcellular analysis • Nanosensors and ultrasensitive detection aspects (e.g., involving quantum dots, "nanoelectrodes" or nanospray MS) • Nanoscale/nanopore DNA sequencing (next generation sequencing) • Micro- and nanoscale sample preparation • Nanoparticles and cells analyses by dielectrophoresis • Separation-based analysis using nanoparticles, nanotubes and nanowires.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信