紫外交联免疫沉淀技术原理及其应用  

Principle and Application of Ultraviolet Crosslinking Immunoprecipitation Technology

作　　者：杜亚琼 王琬瑶 高帆 徐旸[2] 时文涛 WANG Wan-Yao;GAO Fan;XU Yang;SHI Wen-Tao(Department of Genetics,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China;School of Medicine,Nankai University,Tianjin 300071,China)

机构地区：[1]天津医科大学基础医学院遗传学系,天津300070 [2]南开大学医学院,天津300071

出　　处：《生物化学与生物物理进展》2024年第1期136-144,共9页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(32170649,81802091)资助项目。

摘　　要：紫外交联免疫沉淀(UV cross-linking immunoprecipitation,CLIP)技术最初建立于2003年。通过紫外交联、免疫沉淀、逆转录及后续的高通量测序等步骤,可在全转录组范围鉴定特定RNA结合蛋白(RNA-binding proteins,RBP)的靶标RNA序列和结合位点。在近20年的应用过程中,该技术被不断改进和完善,可操作性、实验结果的准确性都有所提升,技术的应用范围也有所拓展。本文对CLIP技术的基本原理、实验方法、实际应用进行介绍,着重比较几种主流CLIP技术的异同,并对如何选择具体的技术路线提出建议。The UV cross-linking immunoprecipitation(CLIP)technique was first established in 2003.Sequences of target RNAs and binding sites of specific RNA-binding proteins(RBPs)were identified within the entire transcriptome by UV cross-linking,immunoprecipitation,reverse transcription,and subsequent high-throughput sequencing.Over the last 20 years,CLIP has been continuously modified and improved.Advanced operability and accuracy have extended its application category.Currently,the widely used CLIP technologies include high-throughput sequencing with crosslinking-immunoprecipitation(HITS-CLIP),photoactivatable-ribonucleoside-enhanced CLIP(PAR-CLIP),individual nucleotide resolution CLIP(iCLIP),enhanced CLIP(eCLIP),infrared-CLIP(irCLIP),etc.HITS-CLIP combines high-throughput sequencing with UV cross-linking immunoprecipitation.The 254 nm UV cross-linking and RNAase digestion steps allow the technology to capture transient intracellular RBP-RNA interactions.However,there are limitations in the efficiency of UV cross-linking,with low resolution and high intrinsic background noise.For PAR-CLIP,photoactivatable ribonucleoside was incorporated into RNA molecules,and RBP cross-linked with RNA by 365 nm UV light to improve cross-linking efficiency and resolution.Cross-linking mediated single-base mutations provide more accurate binding site information and reduce interference from background sequences.Long-term alternative nucleotide incorporation,on the other hand,can be cytotoxic and may skew experimental results.iCLIP can identify RBP-RNA cross-linking sites at the single nucleotide level through cDNA circularization and subsequent re-linearization steps,but it has more experimental procedures,and partial cDNAs lost in the circularization step are inevitable.eCLIP discards the radioisotope labeling procedure and reduces RNA loss by ligating adaptors in two separate steps,greatly improving the library-building efficiency,and reducing bias associated with PCR amplification;however,the efficiency of immunoprecipitation cannot be v

关 键 词：紫外交联免疫沉淀 高通量测序 RNA结合蛋白 

分 类 号：Q513[生物学—生物化学] Q522