{"title":"Enhancing the performance of an in vitro RNA biosensor through iterative design of experiments.","authors":"Rochelle Aw, Karen Polizzi","doi":"10.1002/btpr.70005","DOIUrl":null,"url":null,"abstract":"<p><p>The quality control of RNA has become increasingly crucial with the rise of mRNA-based vaccines and therapeutics. However, conventional methods such as LC-MS often require specialized equipment and expertise, limiting their applicability to high throughput experiments. Here, we optimize a previously characterized RNA integrity biosensor, that provides a simple colorimetric output, using Design of Experiments (DoE). Through iterative rounds of a Definitive Screening Design (DSD) and experimental validation, we systematically explored different assay conditions to enhance the biosensor's performance. Optimization led to a 4.1-fold increase in dynamic range and reduced RNA concentration requirements by one-third, significantly improving usability. Notable modifications included reducing the concentrations of reporter protein and poly-dT oligonucleotide and increasing DTT concentration, suggesting a reducing environment for optimal functionality. Importantly, the optimized biosensor retained its ability to discriminate between capped and uncapped RNA even at lower RNA concentrations. Overall, our improved biosensor offers enhanced performance and reduced sample requirements, paving the way for rapid, cost-effective RNA quality control in diverse settings, including resource-limited environments.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70005"},"PeriodicalIF":2.5000,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology Progress","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1002/btpr.70005","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
The quality control of RNA has become increasingly crucial with the rise of mRNA-based vaccines and therapeutics. However, conventional methods such as LC-MS often require specialized equipment and expertise, limiting their applicability to high throughput experiments. Here, we optimize a previously characterized RNA integrity biosensor, that provides a simple colorimetric output, using Design of Experiments (DoE). Through iterative rounds of a Definitive Screening Design (DSD) and experimental validation, we systematically explored different assay conditions to enhance the biosensor's performance. Optimization led to a 4.1-fold increase in dynamic range and reduced RNA concentration requirements by one-third, significantly improving usability. Notable modifications included reducing the concentrations of reporter protein and poly-dT oligonucleotide and increasing DTT concentration, suggesting a reducing environment for optimal functionality. Importantly, the optimized biosensor retained its ability to discriminate between capped and uncapped RNA even at lower RNA concentrations. Overall, our improved biosensor offers enhanced performance and reduced sample requirements, paving the way for rapid, cost-effective RNA quality control in diverse settings, including resource-limited environments.
期刊介绍:
Biotechnology Progress , an official, bimonthly publication of the American Institute of Chemical Engineers and its technological community, the Society for Biological Engineering, features peer-reviewed research articles, reviews, and descriptions of emerging techniques for the development and design of new processes, products, and devices for the biotechnology, biopharmaceutical and bioprocess industries.
Widespread interest includes application of biological and engineering principles in fields such as applied cellular physiology and metabolic engineering, biocatalysis and bioreactor design, bioseparations and downstream processing, cell culture and tissue engineering, biosensors and process control, bioinformatics and systems biology, biomaterials and artificial organs, stem cell biology and genetics, and plant biology and food science. Manuscripts concerning the design of related processes, products, or devices are also encouraged. Four types of manuscripts are printed in the Journal: Research Papers, Topical or Review Papers, Letters to the Editor, and R & D Notes.