色谱2024，Vol.42Issue(7)：632-645,14.DOI:10.3724/SP.J.1123.2023.12025
基于测序技术的核糖核酸修饰定位分析方法研究进展
Advances in mapping analysis of ribonucleic acid modifications through sequencing
摘要
Abstract
Over 170 chemical modifications have been discovered in various types of ribonucle-ic acids(RNAs),including messenger RNA(mRNA),ribosomal RNA(rRNA),transfer RNA(tRNA),and small nuclear RNA(snRNA).These RNA modifications play crucial roles in a wide range of biological processes such as gene expression regulation,RNA stability mainte-nance,and protein translation.RNA modifications represent a new dimension of gene expres-sion regulation known as the"epitranscriptome".The discovery of RNA modifications and the relevant writers,erasers,and readers provides an important basis for studies on the dynamic regulation and physiological functions of RNA modifications.Owing to the development of de-tection technologies for RNA modifications,studies on RNA epitranscriptomes have progressed to the single-base resolution,multilayer,and full-coverage stage.Transcriptome-wide methods help discover new RNA modification sites and are of great importance for elucidating the molec-ular regulatory mechanisms of epitranscriptomics,exploring the disease associations of RNA modifications,and understanding their clinical applications.The existing RNA modification sequencing technologies can be categorized according to the pretreatment approach and sequen-cing principle as direct high-throughput sequencing,antibody-enrichment sequencing,enzyme-assisted sequencing,chemical labeling-assisted sequencing,metabolic labeling sequencing,and nanopore sequencing technologies.These methods,as well as studies on the functions of RNA modifications,have greatly expanded our understanding of epitranscriptomics.In this review,we summarize the recent progress in RNA modification detection technologies,focusing on the basic principles,advantages,and limitations of different methods.Direct high-throughput se-quencing methods do not require complex RNA pretreatment and allow for the mapping of RNA modifications using conventional RNA sequencing methods.However,only a few RNA modifi-cations can be analyzed by high-throughput sequencing.Antibody enrichment followed by high-throughput sequencing has emerged as a crucial approach for mapping RNA modifications,sig-nificantly advancing the understanding of RNA modifications and their regulatory functions in different species.However,the resolution of antibody-enrichment sequencing is limited to ap-proximately 100-200 bp.Although chemical crosslinking techniques can achieve single-base res-olution,these methods are often complex,and the specificity of the antibodies used in these methods has raised concerns.In particular,the issue of off-target binding by the antibodies re-quires urgent attention.Enzyme-assisted sequencing has improved the accuracy of the localiza-tion analysis of RNA modifications and enables stoichiometric detection with single-base resolu-tion.However,the enzymes used in this technique show poor reactivity,specificity,and se-quence preference.Chemical labeling sequencing has become a widely used approach for profi-ling RNA modifications,particularly by altering reverse transcription(RT)signatures such as RT stops,misincorporations,and deletions.Chemical-assisted sequencing provides a sequence-independent RNA modification detection strategy that enables the localization of multiple RNA modifications.Additionally,when combined with the biotin-streptavidin affinity method,low-abundance RNA modifications can be enriched and detected.Nevertheless,the specificity of many chemical reactions remains problematic,and the development of specific reaction probes for particular modifications should continue in the future to achieve the precise localization of RNA modifications.As an indirect localization method,metabolic labeling sequencing specific-ally localizes the sites at which modifying enzymes act,which is of great significance in the study of RNA modification functions.However,this method is limited by the intracellular labe-ling of RNA and cannot be applied to biological samples such as clinical tissues and blood sam-ples.Nanopore sequencing is a direct RNA-sequencing method that does not require RT or the polymerase chain reaction(PCR).However,challenges in analyzing the data obtained from nanopore sequencing,such as the high rate of false positives,must be resolved.Discussing se-quencing analysis methods for various types of RNA modifications is instructive for the future development of novel RNA modification mapping technologies,and will aid studies on the func-tions of RNA modifications across the entire transcriptome.关键词
表观转录组学/核糖核酸修饰/定位分析/高通量测序/单碱基分辨率Key words
epitranscriptome/RNA modification/mapping analysis/high-throughput sequen-cing/single-base resolution分类
化学化工引用本文复制引用
熊军,冯甜,袁必锋..基于测序技术的核糖核酸修饰定位分析方法研究进展[J].色谱,2024,42(7):632-645,14.基金项目
国家重点研发项目(2022YFC3400700) （2022YFC3400700）
国家自然科学基金项目(22277093,22074110,22207090).National Key R&D Program of China(No.2022YFC3400700) （22277093,22074110,22207090）
National Natural Science Foundation of China(Nos.22277093,22074110,22207090). （Nos.22277093,22074110,22207090）
