RNA Scaffolds pp 169-179 | Cite as

Folding RNA–Protein Complex into Designed Nanostructures

  • Tomonori Shibata
  • Yuki Suzuki
  • Hiroshi Sugiyama
  • Masayuki Endo
  • Hirohide SaitoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1316)


RNA–protein (RNP) complexes are promising biomaterials for the fields of nanotechnology and synthetic biology. Protein-responsive RNA sequences (RNP motifs) can be integrated into various RNAs, such as messenger RNA, short-hairpin RNA, and synthetic RNA nano-objects for a variety of purposes. Direct observation of RNP interaction in solution at high resolution is important in the design and construction of RNP-mediated nanostructures. Here we describe a method to construct and visualize RNP nanostructures that precisely arrange a target protein on the RNA scaffold with nanometer scale. High-speed AFM (HS-AFM) images of RNP nanostructures show that the folding of RNP complexes of defined sizes can be directly visualized at single RNP resolution in solution.

Key words

Ribonucleoprotein Kink-turn RNA L7Ae RNA–protein complex RNA nanostructures RNA nanotechnology RNP High-speed atomic force microscopy 



We thank Hirohisa Ohno and Eriko Osada (Kyoto University) to initiate the projects. We also thank Peter Karagiannis (Kyoto University) for critically reading the manuscript. This work was supported by the New Energy and Industrial Technology Development Organization (09A02021a) and a Grant-in-Aid for Scientific Research on Innovative Areas “Molecular Robotics” (No. 24104002) from The Ministry of Education, Culture, Sport, Science, and Technology, Japan.


  1. 1.
    Bleichert F, Baserga SJ (2010) Ribonucleoprotein multimers and their functions. Crit Rev Biochem Mol Biol 45:331–350CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Staley JP, Woolford JL Jr (2009) Assembly of ribosomes and spliceosomes: complex ribonucleoprotein machines. Curr Opin Cell Biol 21:109–118CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Ramaswami M, Taylor JP, Parker R (2013) Altered ribostasis: RNA-protein granules in degenerative disorders. Cell 154:727–736CrossRefPubMedGoogle Scholar
  4. 4.
    Selmer M, Dunham CM, Murphy FV, Weixlbaumer A, Petry S, Kelley AC, Weir JR, Ramakrishnan V (2006) Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313:1935–1942CrossRefPubMedGoogle Scholar
  5. 5.
    Leung EK, Suslov N, Tuttle N, Sengupta R, Piccirilli JA (2011) The mechanism of peptidyl transfer catalysis by the ribosome. Annu Rev Biochem 80:527–555CrossRefPubMedGoogle Scholar
  6. 6.
    Ohno H, Kobayashi T, Kabata R, Endo K, Iwasa T, Yoshimura SH, Takeyasu K, Inoue T, Saito H (2011) Synthetic RNA-protein complex shaped like an equilateral triangle. Nat Nanotechnol 6:115–119Google Scholar
  7. 7.
    Klein DJ, Schmeing TM, Moore PB, Steitz TA (2001) The kink-turn: a new RNA secondary structure motif. EMBO J 20:4214–4221CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Rozhdestvensky TS, Tang TH, Tchirkova IV, Brosius J, Bachellerie JP, Huttenhofer A (2003) Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in archaea. Nucleic Acids Res 31:869–877CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Moore T, Zhang Y, Fenley MO, Li H (2004) Molecular basis of box C/D RNA-protein interactions; cocrystal structure of archaeal L7Ae and a box C/D RNA. Structure 12:807–818CrossRefPubMedGoogle Scholar
  10. 10.
    Turner B, Melcher SE, Wilson TJ, Norman DG, Lilley DM (2005) Induced fit of RNA on binding the L7Ae protein to the kink-turn motif. RNA 11:1192–1200CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Osada E, Suzuki Y, Hidaka K, Ohno H, Sugiyama H, Endo M, Saito H (2014) Engineering RNA–protein complexes with different shapes for imaging and therapeutic applications. ACS Nano 8:8130–8140CrossRefPubMedGoogle Scholar
  12. 12.
    Shinozaki Y, Sumitomo K, Tsuda M, Koizumi S, Inoue K, Torimitsu K (2009) Direct observation of ATP-induced conformational changes in single P2X(4) receptors. PLoS Biol 7:e1000103CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Kodera N, Yamamoto D, Ishikawa R, Ando T (2010) Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468:72–76CrossRefPubMedGoogle Scholar
  14. 14.
    Suzuki Y, Gilmore JL, Yoshimura SH, Henderson RM, Lyubchenko YL, Takeyasu K (2011) Visual analysis of concerted cleavage by type IIF restriction enzyme SfiI in subsecond time region. Biophys J 101:2992–2998CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Rajendran A, Endo M, Sugiyama H (2014) State-of-the-art high-speed atomic force microscopy for investigation of single-molecular dynamics of proteins. Chem Rev 114:1493–1520CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Tomonori Shibata
    • 2
  • Yuki Suzuki
    • 1
  • Hiroshi Sugiyama
    • 1
  • Masayuki Endo
    • 3
  • Hirohide Saito
    • 2
    Email author
  1. 1.Department of Chemistry, Graduate School of ScienceKyoto UniversityKyotoJapan
  2. 2.Center for iPS Cell Research and Application (CiRA)Kyoto UniversityKyotoJapan
  3. 3.Institute for Integrated Cell-Material Sciences (WPI-iCeMS)Kyoto UniversityKyotoJapan

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