Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides.

IF 3.6 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
RNA Biology Pub Date : 2024-01-01 Epub Date: 2024-06-05 DOI:10.1080/15476286.2024.2363029
Francis Combes, Thanh-Huong Bui, Frida J Pettersson, Sjoerd Hak
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引用次数: 0

Abstract

Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3'-5' exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques.

使用多核苷酸磷酸化酶和多胸苷寡核苷酸快速、可扩展地检测合成 mRNA 副产品。
合成 mRNA 的生产和储存会产生各种副产品,从而降低 mRNA 疫苗和疗法的整体完整性和功能性。因此,mRNA 的完整性被指定为一项关键的质量属性,在临床使用前必须使用最先进的分析方法对其进行评估。目前的研究首先证明了热降解对转录本翻译能力的影响,然后介绍了一种新的酶解方法来评估常规 mRNA 和长自增 mRNA 的完整性。首先将寡核苷酸 T 与完整 mRNA 的聚(A)尾杂交,然后用 3'-5' 外切核酸酶消化未杂交的 RNA 片段,就能选择性地从 RNA 片段中释放出单个核苷酸。然后,这些核苷酸中的腺苷酸部分可转化为 ATP,并通过发光检测作为 mRNA 副产物的灵敏指标。我们开发了一种基于多核苷酸磷酸化酶(PNPase)的检测方法,可快速灵敏地评估 mRNA 的完整性(无论其长度如何),从而为基于色谱、电泳或测序的技术提供了一种新颖且完全可扩展的替代方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
RNA Biology
RNA Biology 生物-生化与分子生物学
CiteScore
8.60
自引率
0.00%
发文量
82
审稿时长
1 months
期刊介绍: RNA has played a central role in all cellular processes since the beginning of life: decoding the genome, regulating gene expression, mediating molecular interactions, catalyzing chemical reactions. RNA Biology, as a leading journal in the field, provides a platform for presenting and discussing cutting-edge RNA research. RNA Biology brings together a multidisciplinary community of scientists working in the areas of: Transcription and splicing Post-transcriptional regulation of gene expression Non-coding RNAs RNA localization Translation and catalysis by RNA Structural biology Bioinformatics RNA in disease and therapy
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