DNAzyme approach for simultaneous mRNA cap and poly(A) tail length analysis: A one-step method to multiple quality attributes

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis Pub Date : 2025-01-23 DOI:10.1016/j.jpba.2025.116695

Ying Wang , Li Li , John Kong , Ravikiran Yerabolu , Kari Hullen , Kaixi Zhao , Emily Wen , Matthew J. Gunsch , David Foley , Yu He

{"title":"DNAzyme approach for simultaneous mRNA cap and poly(A) tail length analysis: A one-step method to multiple quality attributes","authors":"Ying Wang , Li Li , John Kong , Ravikiran Yerabolu , Kari Hullen , Kaixi Zhao , Emily Wen , Matthew J. Gunsch , David Foley , Yu He","doi":"10.1016/j.jpba.2025.116695","DOIUrl":null,"url":null,"abstract":"<div><div>The dynamic landscape of mRNA technology highlights the need for innovative quality control (QC) strategies. In this study, we described an efficient one-step digestion approach for concurrent generation of 5’- and 3’-end fragments, enabling simultaneous mRNA capping and poly(A) tail analysis. Tailored 10–23-type DNAzymes, designed from 5’- and 3’-Untranslated Regions (UTRs), selectively cleaved mRNA to release both the 5’-Capped or uncapped short fragments and 3’-Poly(A) tail cleavage products. Polyacrylamide gel electrophoresis (PAGE) and ion pair reversed-phase liquid chromatography (IP-RP LC) analyses confirmed the production of 5’- and 3’-cleavage fragments in a single-step reaction, and LC-mass spectrometry (LC MS) validated these findings. The DNAzyme-mediated cleavage offers notable advantages over other assays for mRNA cap and tail characterization. Direct and simultaneous analysis of both capping efficiency and poly(A) tail length post-cleavage by DNAzymes, without additional purification steps and costly MS analysis, markedly streamlines the sample preparation and analysis process, making it highly suitable for QC testing.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"257 ","pages":"Article 116695"},"PeriodicalIF":3.1000,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of pharmaceutical and biomedical analysis","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0731708525000366","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}

引用次数: 0

Abstract

The dynamic landscape of mRNA technology highlights the need for innovative quality control (QC) strategies. In this study, we described an efficient one-step digestion approach for concurrent generation of 5’- and 3’-end fragments, enabling simultaneous mRNA capping and poly(A) tail analysis. Tailored 10–23-type DNAzymes, designed from 5’- and 3’-Untranslated Regions (UTRs), selectively cleaved mRNA to release both the 5’-Capped or uncapped short fragments and 3’-Poly(A) tail cleavage products. Polyacrylamide gel electrophoresis (PAGE) and ion pair reversed-phase liquid chromatography (IP-RP LC) analyses confirmed the production of 5’- and 3’-cleavage fragments in a single-step reaction, and LC-mass spectrometry (LC MS) validated these findings. The DNAzyme-mediated cleavage offers notable advantages over other assays for mRNA cap and tail characterization. Direct and simultaneous analysis of both capping efficiency and poly(A) tail length post-cleavage by DNAzymes, without additional purification steps and costly MS analysis, markedly streamlines the sample preparation and analysis process, making it highly suitable for QC testing.

查看原文本刊更多论文

求助全文

约1分钟内获得全文求助全文

来源期刊

Journal of pharmaceutical and biomedical analysis 医学-分析化学

CiteScore

6.70

自引率

5.90%

发文量

588

审稿时长

37 days

期刊介绍： This journal is an international medium directed towards the needs of academic, clinical, government and industrial analysis by publishing original research reports and critical reviews on pharmaceutical and biomedical analysis. It covers the interdisciplinary aspects of analysis in the pharmaceutical, biomedical and clinical sciences, including developments in analytical methodology, instrumentation, computation and interpretation. Submissions on novel applications focusing on drug purity and stability studies, pharmacokinetics, therapeutic monitoring, metabolic profiling; drug-related aspects of analytical biochemistry and forensic toxicology; quality assurance in the pharmaceutical industry are also welcome. Studies from areas of well established and poorly selective methods, such as UV-VIS spectrophotometry (including derivative and multi-wavelength measurements), basic electroanalytical (potentiometric, polarographic and voltammetric) methods, fluorimetry, flow-injection analysis, etc. are accepted for publication in exceptional cases only, if a unique and substantial advantage over presently known systems is demonstrated. The same applies to the assay of simple drug formulations by any kind of methods and the determination of drugs in biological samples based merely on spiked samples. Drug purity/stability studies should contain information on the structure elucidation of the impurities/degradants.