Synthesis of Dumbbell‐Shaped Cyclic RNAs for RNA Interference

Naoko Abe1, Hiroshi Abe1, Yoshihiro Ito1

1 Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, Saitama, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 16.4
DOI:  10.1002/0471142700.nc1604s48
Online Posting Date:  March, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


RNA interference (RNAi) is a potent and highly specific gene‐silencing phenomenon that was first reported for the nematode Caenorhabditis elegans. It has been discovered that genes could be silenced by introducing double‐stranded RNAs (dsRNAs) complementary to the messenger RNA sequences. Since then, RNAi has been shown as an evolutionarily well‐conserved process that plays an important role in host defense and in regulation of gene expression. Much effort has been dedicated to the application of the short dsRNA species (short interfering RNAs; siRNAs) as therapeutic agents, as they were shown to be effective in mammalian cells. Recently, we altered the structure of a siRNA molecule and produced dumbbell‐shaped nanocircular RNAs. RNA dumbbells were shown to be stabilized in serum compared with its siRNA counterpart, despite their natural RNA strand. It has also been found that RNA dumbbells containing a 23‐bp stem and two 9‐nt loops exhibit a prolonged RNAi effect in cultured mammalian cells. In this unit, we describe the synthesis of RNA dumbbells from the design, its enzymatic synthesis, and to the purification. Curr. Protoc. Nucleic Acid Chem. 48:16.4.1‐16.4.11. © 2012 by John Wiley & Sons, Inc.

Keywords: cyclic RNA; RNA synthesis; short interfering RNA; RNA interference; dumbbell‐shaped structure; ligase

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Synthesis of RNA Oligomers
  • Basic Protocol 2: Enzymatic Synthesis of RNA Dumbbells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Synthesis of RNA Oligomers

  • RNA oligomers (synthesized and purified)
  • T4 RNA ligase (e.g., from Takara Bio) containing:
    • 10 × T4 RNA Ligation Reaction buffer: 500 mM Tris⋅Cl, pH 7.5, 100 mM MgCl 2, 1 mM dithiothreitol (DTT), 10 mM ATP
    • 0.1% Bovine serum albumin (BSA)
  • 60% PEG6000 solution (to prepare, add 450 µL of water to 600 mg of PEG6000 in a 2‐mL polypropylene tube with a hinged lid; heat the tube at 90°C to dissolve and mix the solution well before use)
  • Milli‐Q water (Millipore), autoclaved (we use autoclaved Milli‐Q water for these experiments)
  • Chloroform
  • 3 M sodium acetate (pH 5.2; autoclaved)
  • Isopropyl alcohol
  • 80% (v/v) ethanol
  • 2× formamide loading solution (see recipe)
  • Ammonium peroxodisulfate (APS)
  • N,N,N′,N′‐tetramethylethylenediamine (TEMED)
  • Gel solution (see recipe)
  • 1× TBE buffer ( appendix 2A)
  • Stains‐All dye (Sigma‐Aldrich)
  • N,N‐dimethylformamide (DMF)
  • 2 × formamide loading solution without dyes (80% formamide, 10 mM EDTA, pH 8.0)
  • 10 mM EDTA (pH 8.0) solution, autoclaved ( appendix 2A)
  • 1.5‐mL microcentrifuge tubes, sterilized 2‐mL microcentrifuge tubes with a hinged lid, sterilized
  • 37° and 90°C heating blocks
  • Vortex mixer
  • Centrifuge
  • –30°C freezer
  • Glass plates for PAGE (20 × 22 cm)
  • Spacers and a comb for PAGE (1‐mm thickness)
  • Electrophoresis apparatus
  • Power supply system
  • Tray (to stain gels with Stains‐All)
  • Plastic film
  • Gel photography equipment
  • Fluorescent thin‐layer chromatographic (TLC) plate
  • Hand‐held ultraviolet (UV) lamp
  • Marker pen
  • Razor blade
  • 200‐µL pipet tips
  • Rotating microtube mixer
  • Filtration device (0.45‐µm pore size)
  • Microcon Ultracel YM‐3 (Millipore)
  • UV‐VIS spectrometer
  • Additional reagents and equipment for denaturing PAGE ( appendix 3B)
PDF or HTML at Wiley Online Library



Literature Cited

   Abe, N., Abe, H., and Ito, Y. 2007. Dumbbell‐shaped nanocircular RNAs for RNA interference. J. Am. Chem. Soc. 129:15108‐15109.
   Abe, N., Abe, H., Nagai, C., Harada, M., Hatakeyama, H., Harashima, H., Ohshiro, T., Nishihara, M., Furukawa, K., Maeda, M., Tsuneda, S., and Ito, Y. 2011. Synthesis, structure, and biological activity of dumbbell‐shaped nanocircular RNAs for RNA interference. Bioconjug. Chem. 22:2082‐2092.
   Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363‐366.
   Bohjanen, P.R., Colvin, R.A., Puttaraju, M., Been, M.D., and GarciaBlanco, M.A. 1996. A small circular TAR RNA decoy specifically inhibits Tat‐activated HIV‐1 transcription. Nucleic Acids Res. 24:3733‐3738.
   Brummelkamp, T.R., Bernards, R., and Agami, R. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550‐553.
   Choung, S., Kim, Y.J., Kim, S., Park, H.O., and Choi, Y.C. 2006. Chemical modification of siRNAs to improve serum stability without loss of efficacy. Biochem. Biophys. Res. Commun. 342:919‐927.
   Durand, M., Chevrie, K., Chassignol, M., Thuong, N.T., and Maurizot, J.C. 1990. Circular‐dichroism studies of an oligodeoxynucleotide containing a hairpin loop made of a hexaethylene glycol chain: Conformation and stability. Nucleic Acids Res. 18:6353‐6359.
   Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001. Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494‐498.
   Fire, A., Xu, S.Q., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double‐stranded RNA in Caenorhabditis elegans. Nature 391:806‐811.
   Hajeri, P.B. and Singh, S.K. 2009. siRNAs: Their potential as therapeutic agents: Part I. Designing of siRNAs. Drug Discov. Today 14:851‐858.
   Hall, A.H.S., Wan, J., Shaughnessy, E.E., Shaw, B.R., and Alexander, K.A. 2004. RNA interference using boranophosphate siRNAs: Structure‐activity relationships. Nucleic Acids Res. 32:5991‐6000.
   Harborth, J., Elbashir, S.M., Vandenburgh, K., Manninga, H., Scaringe, S.A., Weber, K., and Tuschl, T. 2003. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucl. Acid Drug Dev. 13:83‐105.
   Harrison, B. and Zimmerman, S.B. 1984. Polymer‐stimulated ligation: Enhanced ligation of oligo‐ and polynucleotides by T4 RNA ligase in polymer solutions. Nucleic Acids Res. 12:8235‐8251.
   Ho, C.K. and Shuman, S. 2002. Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains. Proc. Natl. Acad. Sci. U.S.A. 99:12709‐12714.
   Hoshika, S., Minakawa, N., Kamiya, H., Harashima, H., and Matsuda, A. 2005. RNA interference induced by siRNAs modified with 4′‐thioribonucleosides in cultured mammalian cells. FEBS Lett. 579:3115‐3118.
   Ma, M.Y.X., McCallum, K., Climie, S.C., Kuperman, R., Lin, W.C., Sumnersmith, M., and Barnett, R.W. 1993. Design and synthesis of RNA miniduplexes via a synthetic linker approach. 2. Generation of covalently closed, double‐stranded cyclic HIV‐1 TAR RNA analogs with high Tat‐binding affinity. Nucleic Acids Res. 21:2585‐2589.
   Miyagishi, M., Sumimoto, H., Miyoshi, H., Kawakami, Y., and Taira, K. 2004. Optimization of an siRNA‐expression system with an improved hairpin and its significant suppressive effects in mammalian cells. J. Gene Med. 6:715‐723.
   Naito, Y., Yamada, T., Ui‐Tei, K., Morishita, S., and Saigo, K. 2004. siDirect: Highly effective, target‐specific siRNA design software for mammalian RNA interference. Nucleic Acids Res. 32:W124‐W129.
   Rana, T.M. 2007. Illuminating the silence: Understanding the structure and function of small RNAs. Nat. Rev. Mol. Cell Biol. 8:23‐36.
   Sambrook, J. and Russell, D. 2001. Molecular Cloning: A Laboratory Manual; third ed. Cold Spring Harbor Laboratory Press, Woodbury, New York.
   Singh, S.K. and Hajeri, P.B. 2009. siRNAs: Their potential as therapeutic agents: Part II. Methods of delivery. Drug Discov. Today 14:859‐865.
   Uhlenbeck, O.C. and Gumport, R.I. 1982. The Enzymes: Nucleic Acids, Volume 15, Pt. B; 3rd ed. Academic Press, New York.
   Watts, J.K., Choubdar, N., Sadalapure, K., Robert, F., Wahba, A.S., Pelletier, J., Pinto, B.M., and Damha, M.J. 2007. 2′‐Fluoro‐4′‐thioarabino‐modified oligonucleotides: Conformational switches linked to siRNA activity. Nucleic Acids Res. 35:1441‐1451.
   Watts, J.K., Deleavey, G.F., and Damha, M.J. 2008. Chemically modified siRNA: Tools and applications. Drug Discov. Today 13:842‐855.
   Yuan, B.B., Latek, R., Hossbach, M., Tuschl, T., and Lewitter, F. 2004. siRNA Selection Server: An automated siRNA oligonucleotide prediction server. Nucleic Acids Res. 32:W130‐W134.
Internet Resources
  siRNA at Whitehead (Whitehead Institute) (Yuan et al., ).
  siDirect (The University of Tokyo) (Naito et al., ) Web site.
  siRNA Target Finder (Ambion from Applied Biosystems)
  BLOCK‐iT RNAi Designer (Invitrogen) Web page.
  RNAi design (Integrated DNA Technologies).
PDF or HTML at Wiley Online Library