Gateway to Understanding Argonaute Loading of Single-Stranded RNAs: Preparation of Deep Sequencing Libraries with In Vitro Loading Samples.

Identification of sequences preferred by individual RNA-binding proteins (RBPs) has been accelerated by recent advances in the quantitative analysis of protein-RNA interactions on a massive scale, and such experiments have even revealed hidden sequence specificity of RBPs that were assumed to be non-specific. Argonaute (AGO) proteins bind diverse guide small RNAs and were believed to have no sequence specificity besides the preference for particular bases at the 5' nucleotide. However, we recently showed that short single-stranded RNAs (ssRNAs) are loaded to AGOs in vivo and in cell extracts with detectable sequence preferences. To study the sequence specificity, we established a protocol for preparing the oligo-specific deep-sequencing library. The protocol includes in vitro loading assay that uses RNA oligos containing randomized nucleotides at the first five positions and also splinted-ligation that specifically amplifies the introduced oligo RNA species from a complex mixture of endogenous small RNAs and exogenously introduced RNA oligos. With the current sequencing depth, this procedure will allow quantitative profiling of interactions between the AGO and ~1000 ssRNA species with different sequences. The method would aid in studying the mechanism behind the selective loading of ssRNAs to AGOs and may potentially be applied to study interactions between RNA and other RNA-binding proteins.