CHiTA: A scarless High-Throughput pipeline for characterization of ribozymes.

IF 4.2 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Methods Pub Date : 2024-12-09 DOI:10.1016/j.ymeth.2024.12.007
Lauren N McKinley, Philip C Bevilacqua
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引用次数: 0

Abstract

Small self-cleaving ribozymes are catalytic RNAs that cleave their phosphodiester backbone rapidly and site-specifically without the assistance of proteins. Their catalytic properties make them ideal targets for applications in RNA pharmaceuticals and bioengineering. Consequently, computational pipelines that predict or design thousands of self-cleaving ribozyme candidates have been developed. Traditional experimental techniques for verifying the activity of these putative ribozymes, however, are low-throughput and time intensive. High-throughput (HT) pipelines that employ next-generation sequencing (NGS) analyze the activity of these thousands of ribozymes simultaneously. Until recently, the application of these HT pipelines has been limited to studying all single and double mutants of a select representative ribozyme. Unfortunately, this prevents the exploration of candidates having different lengths, circular permutations, and auxiliary stem-loops. Moreover, pipelines that analyze ribozymes en masse often include transcription of non-native flanking sequences that preclude accurate assessment of the intrinsic rate of ribozyme self-cleavage. To overcome these limitations, we developed a HT pipeline, "Cleavage High-Throughput Assay (CHiTA)", which employs NGS and massively parallel oligonucleotide synthesis (MPOS) to characterize ribozyme activity for thousands of candidates in a scarless fashion. Herein, we describe detailed strategies and protocols to implement CHiTA to measure the activity of putative ribozymes from a wide range of ribozyme classes.

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来源期刊
Methods
Methods 生物-生化研究方法
CiteScore
9.80
自引率
2.10%
发文量
222
审稿时长
11.3 weeks
期刊介绍: Methods focuses on rapidly developing techniques in the experimental biological and medical sciences. Each topical issue, organized by a guest editor who is an expert in the area covered, consists solely of invited quality articles by specialist authors, many of them reviews. Issues are devoted to specific technical approaches with emphasis on clear detailed descriptions of protocols that allow them to be reproduced easily. The background information provided enables researchers to understand the principles underlying the methods; other helpful sections include comparisons of alternative methods giving the advantages and disadvantages of particular methods, guidance on avoiding potential pitfalls, and suggestions for troubleshooting.
期刊最新文献
Nanotechnological approaches to improve corticosteroids ocular therapy. Validation of plasmonic-based biosensors for rapid and in depth characterization of monoclonal antibodies directed against rabbit haemorrhagic and foot-and-mouth disease viruses in biological samples. CHiTA: A scarless High-Throughput pipeline for characterization of ribozymes. Cleaving the way for heterologous peptide production: An overview of cleavage strategies. Terahertz-based biosensors for biomedical applications: A review.
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