Cotranscriptional Production of Chemically Modified RNA Nanoparticles

  • Maria L. KireevaEmail author
  • Kirill A. Afonin
  • Bruce A. Shapiro
  • Mikhail Kashlev
Part of the Methods in Molecular Biology book series (MIMB, volume 1632)


RNA nanoparticles consisting of multiple RNA strands of different sequences forming various three-dimensional structures emerge as promising carriers of siRNAs, RNA aptamers, and ribozymes. In vitro transcription of a mixture of dsDNA templates encoding all the subunits of the RNA nanoparticle may result in cotranscriptional self-assembly of the nanoparticle. Based on our experience with production of RNA nanorings, RNA nanocubes, and RNA three-way junctions, we propose a strategy for optimization of the cotranscriptional production of chemically modified ribonuclease-resistant RNA nanoparticles.

Key words

In vitro transcription T7 RNA polymerase T7 R&DNA polymerase 2′-F-dUTP 2′-F-dCTP RNA nanorings RNA nanocubes Cotranscriptional assembly 



We thank Lorena Parlea and Paul Zakrevsky for fruitful discussions, and Josh Turek-Herman for proofreading the manuscript. The contents of this publication do not necessarily reveal the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial product, or organizations imply endorsement by the US Government. This work was supported by the Intramural Research Program of the National Institutes of Health, Center for Cancer Research, the National Cancer Institute.


  1. 1.
    Castanotto D, Rossi JJ (2009) The promises and pitfalls of RNA-interference-based therapeutics. Nature 457(7228):426–433. doi: 10.1038/nature07758 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Zuckerman JE, Davis ME (2015) Clinical experiences with systemically administered siRNA-based therapeutics in cancer. Nat Rev Drug Discov 14(12):843–856. doi: 10.1038/nrd4685 CrossRefPubMedGoogle Scholar
  3. 3.
    Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31(3):230–232. doi: 10.1038/nbt.2507 CrossRefPubMedGoogle Scholar
  4. 4.
    Kelley ML, Strezoska Z, He K, Vermeulen A, Smith A (2016) Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing. J Biotechnol 233:74–83. doi: 10.1016/j.jbiotec.2016.06.011 CrossRefPubMedGoogle Scholar
  5. 5.
    Li L, Allen C, Shivakumar R, Peshwa MV (2013) Large volume flow electroporation of mRNA: clinical scale process. Methods Mol Biol 969:127–138. doi: 10.1007/978-1-62703-260-5_9 CrossRefPubMedGoogle Scholar
  6. 6.
    Preskey D, Allison TF, Jones M, Mamchaoui K, Unger C (2016) Synthetically modified mRNA for efficient and fast human iPS cell generation and direct transdifferentiation to myoblasts. Biochem Biophys Res Commun 473(3):743–751. doi: 10.1016/j.bbrc.2015.09.102 CrossRefPubMedGoogle Scholar
  7. 7.
    Grabow WW, Jaeger L (2014) RNA self-assembly and RNA nanotechnology. Acc Chem Res 47(6):1871–1880. doi: 10.1021/ar500076k CrossRefPubMedGoogle Scholar
  8. 8.
    Haque F, Guo P (2015) Overview of methods in RNA nanotechnology: synthesis, purification, and characterization of RNA nanoparticles. Methods Mol Biol 1297:1–19. doi: 10.1007/978-1-4939-2562-9_1 CrossRefPubMedGoogle Scholar
  9. 9.
    Shukla GC, Haque F, Tor Y, Wilhelmsson LM, Toulme JJ, Isambert H, Guo P, Rossi JJ, Tenenbaum SA, Shapiro BA (2011) A boost for the emerging field of RNA nanotechnology. ACS Nano 5(5):3405–3418. doi: 10.1021/nn200989r CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Afonin KA, Kasprzak WK, Bindewald E, Kireeva M, Viard M, Kashlev M, Shapiro BA (2014) In silico design and enzymatic synthesis of functional RNA nanoparticles. Acc Chem Res 47(6):1731–1741. doi: 10.1021/ar400329z CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Afonin KA, Kireeva M, Grabow WW, Kashlev M, Jaeger L, Shapiro BA (2012) Co-transcriptional assembly of chemically modified RNA nanoparticles functionalized with siRNAs. Nano Lett 12(10):5192–5195. doi: 10.1021/nl302302e CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Afonin KA, Grabow WW, Walker FM, Bindewald E, Dobrovolskaia MA, Shapiro BA, Jaeger L (2011) Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine. Nat Protoc 6(12):2022–2034. doi: 10.1038/nprot.2011.418 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Afonin KA, Bindewald E, Yaghoubian AJ, Voss N, Jacovetty E, Shapiro BA, Jaeger L (2010) In vitro assembly of cubic RNA-based scaffolds designed in silico. Nat Nanotechnol 5(9):676–682. doi: 10.1038/nnano.2010.160 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Afonin KA, Viard M, Tedbury P, Bindewald E, Parlea L, Howington M, Valdman M, Johns-Boehme A, Brainerd C, Freed EO, Shapiro BA (2016) The use of minimal RNA toeholds to trigger the activation of multiple functionalities. Nano Lett 16(3):1746–1753. doi: 10.1021/acs.nanolett.5b04676 CrossRefPubMedGoogle Scholar
  15. 15.
    Afonin KA, Lin YP, Calkins ER, Jaeger L (2012) Attenuation of loop-receptor interactions with pseudoknot formation. Nucleic Acids Res 40(5):2168–2180. doi: 10.1093/nar/gkr926 CrossRefPubMedGoogle Scholar
  16. 16.
    Geary C, Rothemund PW, Andersen ES (2014) RNA nanostructures. A single-stranded architecture for cotranscriptional folding of RNA nanostructures. Science 345(6198):799–804. doi: 10.1126/science.1253920 CrossRefPubMedGoogle Scholar
  17. 17.
    Milligan JF, Uhlenbeck OC (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol 180:51–62CrossRefPubMedGoogle Scholar
  18. 18.
    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
  19. 19.
    Sousa R, Padilla R (1995) A mutant T7 RNA polymerase as a DNA polymerase. EMBO J 14(18):4609–4621PubMedPubMedCentralGoogle Scholar
  20. 20.
    Wyatt JR, Walker GT (1989) Deoxynucleotide-containing oligoribonucleotide duplexes: stability and susceptibility to RNase V1 and RNase H. Nucleic Acids Res 17(19):7833–7842CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Conrad F, Hanne A, Gaur RK, Krupp G (1995) Enzymatic synthesis of 2′-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates. Nucleic Acids Res 23(11):1845–1853CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Gopalakrishna S, Gusti V, Nair S, Sahar S, Gaur RK (2004) Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase. RNA 10(11):1820–1830. doi: 10.1261/rna.5222504 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Afonin KA, Viard M, Kagiampakis I, Case CL, Dobrovolskaia MA, Hofmann J, Vrzak A, Kireeva M, Kasprzak WK, KewalRamani VN, Shapiro BA (2015) Triggering of RNA interference with RNA-RNA, RNA-DNA, and DNA-RNA nanoparticles. ACS Nano 9(1):251–259. doi: 10.1021/nn504508s CrossRefPubMedGoogle Scholar
  24. 24.
    Holmes SF, Foster JE, Erie DA (2003) Kinetics of multisubunit RNA polymerases: experimental methods and data analysis. Methods Enzymol 371:71–81. doi: 10.1016/S0076-6879(03)71005-2 CrossRefPubMedGoogle Scholar
  25. 25.
    Parlea L, Bindewald E, Sharan R, Bartlett N, Moriarty D, Oliver J, Afonin KA, Shapiro BA (2016) Ring catalog: a resource for designing self-assembling RNA nanostructures. Methods 103:128–137. doi: 10.1016/j.ymeth.2016.04.016 CrossRefPubMedGoogle Scholar
  26. 26.
    Afonin KA, Viard M, Martins AN, Lockett SJ, Maciag AE, Freed EO, Heldman E, Jaeger L, Blumenthal R, Shapiro BA (2013) Activation of different split functionalities on re-association of RNA-DNA hybrids. Nat Nanotechnol 8(4):296–304. doi: 10.1038/nnano.2013.44 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lubkowska L, Kireeva ML (2015) Direct competition assay for transcription fidelity. Methods Mol Biol 1276:153–164. doi: 10.1007/978-1-4939-2392-2_8 CrossRefPubMedGoogle Scholar
  28. 28.
    Goedken ER, Marqusee S (2001) Co-crystal of Escherichia coli RNase HI with Mn2+ ions reveals two divalent metals bound in the active site. J Biol Chem 276(10):7266–7271. doi: 10.1074/jbc.M009626200 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Maria L. Kireeva
    • 1
    Email author
  • Kirill A. Afonin
    • 1
    • 2
    • 3
  • Bruce A. Shapiro
    • 1
    • 4
  • Mikhail Kashlev
    • 1
  1. 1.RNA Biology LaboratoryNational Cancer Institute, National Institutes of HealthFrederickUSA
  2. 2.Nanoscale Science Program, Department of ChemistryUniversity of North Carolina at CharlotteCharlotteUSA
  3. 3.The Center for Biomedical Engineering and ScienceUniversity of North Carolina at CharlotteCharlotteUSA
  4. 4.RNA Structure and Design SectionRNA Biology Laboratory, National Cancer Institute, National Institutes of HealthFrederickUSA

Personalised recommendations