AGO-CLIP

AGO-CLIP

Argonaute-crosslinking and immunoprecipitation (AGO-CLIP) is a protocol that maps miRNA binding to AGO sites in C. elegans. 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.

Pros:

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

Cons:

• Low efficiency in hybrid capture
• Unable to provide quantitative analysis of the strength of miRNA-target interactions¹

• AGO-CLIP: Grosswendt S., Filipchyk A., Manzano M., Klironomos F., Schilling M., et al. (2014) Unambiguous identification of miRNA:target site interactions by different types of ligation reactions. Mol Cell 54: 1042-1054
• 1. 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. 2014;advance online publication:

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

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5. 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
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