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

  • Eling Goh
  • Katsutomo OkamuraEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1680)


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.

Key words

Argonaute Single-stranded RNA High-throughput sequencing In-vitro 



We thank Gregory Hannon for sharing the tagged AGO2 plasmid. Research in K.O.’s group was supported by the National Research Foundation, Prime Minister’s Office, Singapore under its NRF Fellowship Programme (NRF2011NRF-NRFF001-042).


  1. 1.
    Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14(7):447–459. doi: 10.1038/nrg3462 CrossRefPubMedGoogle Scholar
  2. 2.
    Kobayashi H, Tomari Y (2016) RISC assembly: coordination between small RNAs and Argonaute proteins. Biochim Biophys Acta 1859(1):71–81. doi: 10.1016/j.bbagrm.2015.08.007 CrossRefPubMedGoogle Scholar
  3. 3.
    Suzuki HI, Katsura A, Yasuda T, Ueno T, Mano H, Sugimoto K, Miyazono K (2015) Small-RNA asymmetry is directly driven by mammalian Argonautes. Nat Struct Mol Biol 22(7):512–521. doi: 10.1038/nsmb.3050 CrossRefPubMedGoogle Scholar
  4. 4.
    Chak LL, Okamura K (2014) Argonaute-dependent small RNAs derived from single-stranded, non-structured precursors. Front Genet 5:172. doi: 10.3389/fgene.2014.00172 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Okamura K, Ladewig E, Zhou L, Lai EC (2013) Functional small RNAs are generated from select miRNA hairpin loops in flies and mammals. Genes Dev 27(7):778–792. doi: 10.1101/gad.211698.112 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Winter J, Link S, Witzigmann D, Hildenbrand C, Previti C, Diederichs S (2013) Loop-miRs: active microRNAs generated from single-stranded loop regions. Nucleic Acids Res 41(10):5503–5512. doi: 10.1093/nar/gkt251 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lima WF, Prakash TP, Murray HM, Kinberger GA, Li W, Chappell AE, Li CS, Murray SF, Gaus H, Seth PP, Swayze EE, Crooke ST (2012) Single-stranded siRNAs activate RNAi in animals. Cell 150(5):883–894. doi: 10.1016/j.cell.2012.08.014 CrossRefPubMedGoogle Scholar
  8. 8.
    Chorn G, Klein-McDowell M, Zhao L, Saunders MA, Flanagan WM, Willingham AT, Lim LP (2012) Single-stranded microRNA mimics. RNA 18(10):1796–1804. doi: 10.1261/rna.031278.111 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Liu J, Yu D, Aiba Y, Pendergraff H, Swayze EE, Lima WF, Hu J, Prakash TP, Corey DR (2013) ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy. Nucleic Acids Res 41(20):9570–9583. doi: 10.1093/nar/gkt693 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hu J, Liu J, Narayanannair KJ, Lackey JG, Kuchimanchi S, Rajeev KG, Manoharan M, Swayze EE, Lima WF, Prakash TP, Xiang Q, Martinez C, Corey DR (2014) Allele-selective inhibition of mutant atrophin-1 expression by duplex and single-stranded RNAs. Biochemistry 53(28):4510–4518. doi: 10.1021/bi500610r CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Matsui M, Corey DR (2016) Non-coding RNAs as drug targets. Nat Rev Drug Discov. doi: 10.1038/nrd.2016.117
  12. 12.
    Okamura K, Liu N, Lai EC (2009) Distinct mechanisms for microRNA strand selection by Drosophila Argonautes. Mol Cell 36(3):431–444. doi: S1097-2765(09)00687-X [pii] 10.1016/j.molcel.2009.09.027CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Czech B, Malone CD, Zhou R, Stark A, Schlingeheyde C, Dus M, Perrimon N, Kellis M, Wohlschlegel J, Sachidanandam R, Hannon G, Brennecke J (2008) An endogenous siRNA pathway in Drosophila. Nature 453:798–802CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Zhang Z, Lee JE, Riemondy K, Anderson EM, Yi R (2013) High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing. Genome Biol 14(10):R109. doi: 10.1186/gb-2013-14-10-r109 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Maroney PA, Chamnongpol S, Souret F, Nilsen TW (2008) Direct detection of small RNAs using splinted ligation. Nat Protoc 3(2):279–287. doi: 10.1038/nprot.2007.530 CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Temasek Life Sciences LaboratorySingaporeSingapore
  2. 2.School of Biological SciencesNanyang Technological UniversitySingaporeSingapore

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