Principle and Application of Ultraviolet Crosslinking Immunoprecipitation Technology
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 visually assessed at the early stage of the experiment.The irCLIP technique replaces radioisotopes with infrared dyes and greatly reduces the initial number of cells required for the experiment;however,an infrared imaging scanner is essential for the irCLIP application.To address more particular scientific issues,derivative CLIP-related techniques such as PAPERCLIP,cTag-PAPERCLIP,hiCLIP,and tiCLIP have also been developed in recent years.In practice,the aforementioned CLIP approaches have their advantages and disadvantages.When deciding on a technical strategy,we should take into account our experimental objectives and conditions,such as whether we need to precisely define the RNA site for binding to RBP;whether we have the necessary experimental conditions for working with radioisotopes or performing infrared imaging;the amount of initial sample size,and so on.In addition,the CLIP technique has a relatively large number of procedures and can be divided into several successive experimental modules.We can try to combine modules from different mainstream CLIP technologies to meet our experimental requirements,which also gives us more opportunities to improve and refine them and to build more targeted derivative CLIP technologies according to our research objectives.
UV cross-linking immunoprecipitationhigh-throughput sequencingRNA-binding protein
NETL
NSTL
万方数据
维普
