Preparation of a Conditional RNA Switch

  • Paul Zakrevsky
  • Lorena Parlea
  • Mathias Viard
  • Eckart Bindewald
  • Kirill A. Afonin
  • Bruce A. ShapiroEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1632)


RNA has gained great interest for use in biomedical and therapeutic applications. This is due in part to RNA’s ability to perform multiple functions, including the regulation of endogenously expressed genes. However, the ability of RNA based drugs to distinguish target diseased cells from healthy tissue remains challenging. Here we present methods for the production of a recently developed conditional RNA switch that releases a Dicer substrate RNA in response to interaction with a specific RNA biomarker.

Key words

RNA Toehold Nanotechnology Nanomedicine RNase H RNA switch Conditional RNA 



This work has been funded in whole or in part with Federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under Contract No. HHSN261200800001E. This research was supported [in part] by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US Government.


  1. 1.
    Grabow WW, Jaeger L (2014) RNA self-assembly and RNA nanotechnology. Acc Chem Res 47(6):1871–1880CrossRefPubMedGoogle Scholar
  2. 2.
    Guo P (2010) The emerging field of RNA nanotechnology. Nat Nanotechnol 5(12):833–842CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Batey RT, Rambo RP, Doudna JA (1999) Tertiary motifs in RNA structure and folding. Angew Chem Int Ed Engl 38(16):2326–2343CrossRefPubMedGoogle Scholar
  4. 4.
    Butcher SE, Pyle AM (2011) The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks. Acc Chem Res 44(12):1302–1311CrossRefPubMedGoogle Scholar
  5. 5.
    Parlea L et al (2016) Ring Catalog: a resource for designing self-assembling RNA nanostructures. Methods 103:128–137CrossRefPubMedGoogle Scholar
  6. 6.
    Bindewald E, Hayes R, Yingling YG, Kasprzak W, Shapiro BA (2008) RNAJunction: a database of RNA junctions and kissing loops for three-dimensional structural analysis and nanodesign. Nucleic Acids Res 36(Database issue):D392–D397CrossRefPubMedGoogle Scholar
  7. 7.
    Parlea LG, Sweeney BA, Hosseini-Asanjan M, Zirbel CL, Leontis NB (2016) The RNA 3D motif atlas: computational methods for extraction, organization and evaluation of RNA motifs. Methods 103:99–119CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Grabow WW et al (2011) Self-assembling RNA nanorings based on RNAI/II inverse kissing complexes. Nano Lett 11(2):878–887CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Severcan I, Geary C, Verzemnieks E, Chworos A, Jaeger L (2009) Square-shaped RNA particles from different RNA folds. Nano Lett 9(3):1270–1277CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zhou J et al (2013) Functional in vivo delivery of multiplexed anti-HIV-1 siRNAs via a chemically synthesized aptamer with a sticky bridge. Mol Ther 21(1):192–200CrossRefPubMedGoogle Scholar
  11. 11.
    Afonin KA et al (2015) Triggering of RNA interference with RNA-RNA, RNA-DNA, and DNA-RNA nanoparticles. ACS Nano 9(1):251–259CrossRefPubMedGoogle Scholar
  12. 12.
    Delebecque CJ, Lindner AB, Silver PA, Aldaye FA (2011) Organization of intracellular reactions with rationally designed RNA assemblies. Science 333(6041):470–474CrossRefPubMedGoogle Scholar
  13. 13.
    Afonin KA et al (2010) In vitro assembly of cubic RNA-based scaffolds designed in silico. Nat Nanotechnol 5(9):676–682CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Yu J, Liu Z, Jiang W, Wang G, Mao C (2015) De novo design of an RNA tile that self-assembles into a homo-octameric nanoprism. Nat Commun 6:5724CrossRefPubMedGoogle Scholar
  15. 15.
    Zalatan JG et al (2015) Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160(1–2):339–350CrossRefPubMedGoogle Scholar
  16. 16.
    Afonin KA et al (2016) The use of minimal RNA toeholds to trigger the activation of multiple functionalities. Nano Lett 16(3):1746–1753CrossRefPubMedGoogle Scholar
  17. 17.
    Hochrein LM, Schwarzkopf M, Shahgholi M, Yin P, Pierce NA (2013) Conditional Dicer substrate formation via shape and sequence transduction with small conditional RNAs. J Am Chem Soc 135(46):17322–17330Google Scholar
  18. 18.
    Bindewald E et al (2016) Multistrand structure prediction of nucleic acid assemblies and design of RNA switches. Nano Lett 16(3):1726–1735CrossRefPubMedGoogle Scholar
  19. 19.
    Zakrevsky P, Bindewald E, Shapiro BA (2016) RNA toehold interactions initiate conditional gene silencing. DNA RNA Nanotechnol 3(1):11–13CrossRefGoogle Scholar
  20. 20.
    Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15(21):8783–8798CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Zhu X, Li T, Dang Y, Feng Y, Huang P (2005) A novel in vitro transcription method for producing siRNAs without specific sequence requirements. Mol Biotechnol 31(3):187–192CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Paul Zakrevsky
    • 1
  • Lorena Parlea
    • 1
  • Mathias Viard
    • 5
    • 6
  • Eckart Bindewald
    • 2
  • Kirill A. Afonin
    • 3
    • 4
  • Bruce A. Shapiro
    • 1
    Email author
  1. 1.RNA Structure and Design Section, RNA Biology LaboratoryNational Cancer Institute, National Institutes of HealthFrederickUSA
  2. 2.Frederick National Laboratory for Cancer ResearchLeidos Biomedical ResearchFrederickUSA
  3. 3.Nanoscale Science Program, Department of ChemistryUniversity of North Carolina at CharlotteCharlotteUSA
  4. 4.The Center for Biomedical Engineering and ScienceUniversity of North Carolina at CharlotteCharlotteUSA
  5. 5.RNA Structure and Design Section, RNA Biology LaboratoryCenter for Cancer Research, National Cancer Institute, National Institutes of HealthFrederickUSA
  6. 6.Leidos Biomedical Research Inc.Center for Cancer Research, National Cancer Institute, National Institutes of HealthFrederickUSA

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