Large Scale Purification of RNA Nanoparticles by Preparative Ultracentrifugation

  • Daniel L. Jasinski
  • Chad T. Schwartz
  • Farzin Haque
  • Peixuan GuoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1297)


Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE). Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation.

Key words

RNA Nanoparticles Ultracentrifugation Nanotechnology Nanobiotechnology RNA nanotechnology RNA therapeutics Large-scale purification 



The research was supported by NIH grants R01-EB003730 and U01-CA151648 to P.G. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. Funding to Peixuan Guo’s Endowed Chair in Nanobiotechnology position is from the William Fairish Endowment Fund. PG is a cofounder of Kylin Therapeutics, Inc., RNA Nano, LLC., and Biomotor and Nucleic Acid Nanotechnology Development Corp., Ltd.


  1. 1.
    Guo P, Zhang C, Chen C et al (1998) Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation. Mol Cell 2:149–155CrossRefGoogle Scholar
  2. 2.
    Mitra S, Shcherbakova IV, Altman RB et al (2008) High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis. Nucleic Acids Res 36:e63CrossRefGoogle Scholar
  3. 3.
    Guo P, Haque F, Hallahan B et al (2012) Uniqueness, advantages, challenges, solutions, and perspectives in therapeutics applying RNA nanotechnology. Nucleic Acid Ther 22: 226–245Google Scholar
  4. 4.
    Khisamutdinov EF, Jasinski DL, Guo P (2014) RNA as a boiling-resistant anionic polymer material to build robust structures with defined shape and stoichiometry. ACS Nano 8:4771–4781CrossRefGoogle Scholar
  5. 5.
    Khisamutdinov E, Li H, Jasinski D et al (2014) Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square, and pentagon nanovehicles. Nucleic Acids Res 42:9996–10004CrossRefGoogle Scholar
  6. 6.
    Jasinski D, Khisamutdinov EF, Lyubchenko YL et al (2014) Physicochemically tunable poly-functionalized RNA square architecture with fluorogenic and ribozymatic properties. ACS Nano 8:7620–7629CrossRefGoogle Scholar
  7. 7.
    Shu Y, Haque F, Shu D et al (2013) Fabrication of 14 different rna nanoparticles for specific tumor targeting without accumulation in normal organs. RNA 19:766–777CrossRefGoogle Scholar
  8. 8.
    Shu Y, Shu D, Haque F et al (2013) Fabrication of pRNA nanoparticles to deliver therapeutic RNAs and bioactive compounds into tumor cells. Nat Protoc 8:1635–1659CrossRefGoogle Scholar
  9. 9.
    Shu Y, Cinier M, Shu D et al (2011) Assembly of multifunctional phi29 pRNA nanoparticles for specific delivery of siRNA and other therapeutics to targeted cells. Methods 54:204–214CrossRefGoogle Scholar
  10. 10.
    Liu J, Guo S, Cinier M et al (2010) Fabrication of stable and RNase-resistant RNA nanoparticles active in gearing the nanomotors for viral DNA packaging. ACS Nano 5:237–246CrossRefGoogle Scholar
  11. 11.
    Haque F, Shu D, Shu Y et al (2012) Ultrastable synergistic tetravalent RNA nanoparticles for targeting to cancers. Nano Today 7:245–257CrossRefGoogle Scholar
  12. 12.
    Shukla GC, Haque F, Tor Y et al (2011) A boost for the emerging field of RNA nanotechnology. ACS Nano 5:3405–3418CrossRefGoogle Scholar
  13. 13.
    Shu Y, Pi F, Sharma A et al (2014) Stable RNA nanoparticles as potential new generation drugs for cancer therapy. Adv Drug Deliv Rev 66C:74–89CrossRefGoogle Scholar
  14. 14.
    Leontis N, Sweeney B, Haque F et al (2013) Conference scene: advances in RNA nanotechnology promise to transform medicine. Nanomedicine 8:1051–1054CrossRefGoogle Scholar
  15. 15.
    Trautmann L, Janbazian L, Chomont N et al (2006) Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med 12:1198–1202CrossRefGoogle Scholar
  16. 16.
    Guo P (2010) The emerging field of RNA nanotechnology. Nat Nanotechnol 5:833–842CrossRefGoogle Scholar
  17. 17.
    Zassenhaus HP, Butow RA, Hannon YP (1982) Rapid electroelution of nucleic-acids from agarose and acrylamide gels. Anal Biochem 125:125–130CrossRefGoogle Scholar
  18. 18.
    Anderson AC, Scaringe SA, Earp BE et al (1996) HPLC purification of RNA for crystallography and NMR. RNA 2:110–117Google Scholar
  19. 19.
    Glisin V, Crkvenjar R, Byus C (1974) Ribonucleic-acid isolated by cesium-chloride centrifugation. Biochemistry 13:2633–2637CrossRefGoogle Scholar
  20. 20.
    Ali A, Roossinck MJ (2007) Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation. J Virol Methods 141:84–86CrossRefGoogle Scholar
  21. 21.
    Higashi K, Narayana KS, Adams HR et al (1966) Utilization of citric acid procedure and zonal ultracentrifugation for mass isolation of nuclear RNA from Walker 256 carcinosarcoma. Cancer Res 26:1582–1590Google Scholar
  22. 22.
    Lin CX, Perrault SD, Kwak M et al (2013) Purification of DNA-origami nanostructures by rate-zonal centrifugation. Nucleic Acids Res 41:e40CrossRefGoogle Scholar
  23. 23.
    Eikenber EF, Bickle TA, Traut RR et al (1970) Separation of large quantities of ribosomal subunits by zonal ultracentrifugation. Eur J Biochem 12:113–116CrossRefGoogle Scholar
  24. 24.
    Patsch JR, Sailer S, Kostner G et al (1974) Separation of main lipoprotein density classes from human plasma by rate-zonal ultracentrifugation. J Lipid Res 15:356–366Google Scholar
  25. 25.
    Guo P, Erickson S, Anderson D (1987) A small viral RNA is required for in vitro packaging of bacteriophage phi29 DNA. Science 236:690–694CrossRefGoogle Scholar
  26. 26.
    Guo P, Shu Y, Binzel D et al (2012) Synthesis, conjugation, and labeling of multifunctional pRNA nanoparticles for specific delivery of siRNA, drugs and other therapeutics to target cells. Methods Mol Biol 928:197–219Google Scholar
  27. 27.
    Shu D, Moll WD, Deng Z et al (2004) Bottom-up assembly of RNA arrays and superstructures as potential parts in nanotechnology. Nano Lett 4:1717–1723CrossRefGoogle Scholar
  28. 28.
    Shu D, Huang L, Hoeprich S et al (2003) Construction of phi29 DNA-packaging RNA (pRNA) monomers, dimers and trimers with variable sizes and shapes as potential parts for nano-devices. J Nanosci Nanotechnol 3:295–302CrossRefGoogle Scholar
  29. 29.
    Ando H, Watanabe S, Ohwaki T, Miyake Y (1985) Crystallization of excipients in tablets. J Pharm Sci 74:128–131Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Daniel L. Jasinski
    • 1
  • Chad T. Schwartz
    • 2
  • Farzin Haque
    • 1
  • Peixuan Guo
    • 1
    Email author
  1. 1.Nanobiotechnology Center, Markey Cancer Center, Department of Pharmaceutical SciencesUniversity of KentuckyLexingtonUSA
  2. 2.Beckman Coulter, Inc.IndianapolisUSA

Personalised recommendations