Photoactivatable Ribonucleoside_Enhanced Crosslinking and Immunoprecipitation

PAR-CLIP maps RBP sites on the target RNAs (Hafner et al., 2008). This approach is similar to HITS-CLIP and CLIP-Seq, but it uses much more efficient crosslinking to stabilize the protein-RNA complexes. The requirement to introduce a photoactivatable ribonucleoside limits this approach to cell culture and in vitro systems.

In this method, 4-SU and 6-thioguanosine (6-SG) are incorporated into transcripts of cultured cells. UV irradiation crosslinks 4-SU/6-SG_labeled transcripts to interacting RBPs. The targeted complexes are immunoprecipitated and digested with RNase T1, followed by proteinase K, before RNA extraction. The RNA is reverse-transcribed to cDNA and sequenced. Deep sequencing of cDNA accurately maps RBPs interacting with labeled transcripts.


  • Highly accurate mapping of RNA-protein interactions
  • Labeling with 4-SU/6-SG improves crosslinking efficiency
  • Legitimately crosslinked sequences can be identified based on the presence of mutated bases, and mispriming can be filtered bioinformatically (Gillen et al., 2016)


  • Antibodies not specific to the target may precipitate nonspecific complexes
  • Limited to cell culture and in vitro systems
  • Photoreactive nucleosides can be cytotoxic (Kloetgen et al., 2015)
  • High concentrations of 4-SU can inhibit rRNA synthesis and induce a nucleolar stress response (Burger et al., 2013)


Illumina Library prep and Array Kit Selector


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