Argonaute-Crosslinking and Immunprecipitation

Argonaute-crosslinking and immunoprecipitation (AGO-CLIP) is a protocol that maps miRNA binding to AGO sites in C. elegans (Grosswendt et al., 2014). In this method, the photoreactive nucleoside 4-SU is first incorporated into C. elegans RNA in vivo. The 4-SU_labeled RNA is crosslinked to bound protein, and the extracted lysate is treated with RNase T1, which shortens some miRNAs. After immunoprecipitation and washing, crosslinked RNA is treated with PNK, T4 RNA ligase, and finally proteinase K. The RNA is extracted, reverse-transcribed to cDNA, and sequenced. Deep sequencing provides single base-pair resolution of miRNA and AGO binding sites.


  • Maps miRNA binding to AGO sites using CLIP
  • Provides single base-pair resolution of the binding sites


  • Low efficiency in hybrid capture
  • Unable to provide quantitative analysis of the strength of miRNA-target interactions (Imig et al., 2014)


Illumina Library prep and Array Kit Selector


Schirle N. T., Sheu-Gruttadauria J. and MacRae I. J. Structural basis for microRNA targeting. Science. 2014;346:608-613


Hamilton M. P., Rajapakshe K. I., Bader D. A., et al. The Landscape of microRNA Targeting in Prostate Cancer Defined by AGO-PAR-CLIP. Neoplasia. 2016;18:356-370

Imig J., Brunschweiger A., Brummer A., et al. miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction. Nat Chem Biol. 2015;11:107-114

Luna J. M., Scheel T. K., Danino T., et al. Hepatitis C Virus RNA Functionally Sequesters miR-122. Cell. 2015;160:1099-1110

Wang P., Ning S., Zhang Y., et al. Identification of lncRNA-associated competing triplets reveals global patterns and prognostic markers for cancer. Nucleic Acids Res. 2015;

Grosswendt S., Filipchyk A., Manzano M., et al. Unambiguous identification of miRNA:target site interactions by different types of ligation reactions. Mol Cell. 2014;54:1042-1054

Newie I., Sokilde R., Persson H., et al. The HER2-encoded miR-4728-3p regulates ESR1 through a non-canonical internal seed interaction. PLoS One. 2014;9:e97200