Design and Preparation of Aptamer–siRNA Chimeras (AsiCs) for Targeted Cancer Therapy

  • Sven Kruspe
  • Paloma H. GiangrandeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1632)


Conjugation of cell-internalizing DNA or RNA aptamers to tumor-suppressing siRNAs represents a novel promising approach for cancer therapy. Here we describe how to employ RNA aptamers that bind to cell surface receptors as carriers for cell-targeted siRNA (or miRNA) delivery. This protocol was optimized to improve the efficiency of the aptamer–siRNA/miRNA conjugates and facilitate its implementation in most molecular biology labs. The single working steps include (1) outlining the optimal sequences of the RNA strands bearing the aptamer and siRNA sequence, for which further (2) a dsDNA template is synthesized and then (3) transcribed into an RNA that will be (4) folded and annealed to a chemically synthesized siRNA complementary strand. Moreover, we reference recent examples and advances in the aptamer delivery field.

Key words

Aptamers siRNA miRNA RNAi Targeted delivery Primer extension In vitro transcription Targeted cancer therapy 



This work was supported by grants to P.H.G. from the National Institutes of Health (R01CA138503 and R21DE019953), Mary Kay Foundation (9033-12 and 001-09), Elsa U Pardee Foundation (E2766), and the Roy J Carver Charitable Trust (RJCCT 01-224); and to S.K. from the German Research Fundation (Deutsche Forschungsgemeinschaft, DFG).


  1. 1.
    Keefe AD, Pai S, Ellington A (2010) Aptamers as therapeutics. Nat Rev Drug Discov 9(7):537–550. doi: 10.1038/nrd3141 CrossRefPubMedGoogle Scholar
  2. 2.
    Zhou J, Rossi JJ (2014) Cell-type-specific, aptamer-functionalized agents for targeted disease therapy. Mol Ther Nucleic Acids 3:e169. doi: 10.1038/mtna.2014.21 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kruspe S, Mittelberger F, Szameit K, Hahn U (2014) Aptamers as drug delivery vehicles. ChemMedChem 9(9):1998–2011. doi: 10.1002/cmdc.201402163 CrossRefPubMedGoogle Scholar
  4. 4.
    McNamara JO II, Andrechek ER, Wang Y, Viles KD, Rempel RE, Gilboa E, Sullenger BA, Giangrande PH (2006) Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat Biotechnol 24(8):1005–1015. doi: 10.1038/nbt1223 CrossRefPubMedGoogle Scholar
  5. 5.
    Dassie JP, Liu XY, Thomas GS, Whitaker RM, Thiel KW, Stockdale KR, Meyerholz DK, McCaffrey AP, McNamara JO II, Giangrande PH (2009) Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors. Nat Biotechnol 27(9):839–849. doi: 10.1038/nbt.1560 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Dassie JP, Giangrande PH (2013) Current progress on aptamer-targeted oligonucleotide therapeutics. Ther Deliv 4(12):1527–1546. doi: 10.4155/tde.13.118 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Berezhnoy A, Brenneman R, Bajgelman M, Seales D, Gilboa E (2012) Thermal stability of siRNA modulates aptamer-conjugated siRNA inhibition. Mol Ther Nucleic Acids 1:e51. doi: 10.1038/mtna.2012.41 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bonner G, Patra D, Lafer EM, Sousa R (1992) Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure. EMBO J 11(10):3767–3775PubMedPubMedCentralGoogle Scholar
  9. 9.
    Sousa R, Rose JP, Chung YJ, Lafer EM, Wang BC (1989) Single crystals of bacteriophage T7 RNA polymerase. Proteins 5(4):266–270. doi: 10.1002/prot.340050403 CrossRefPubMedGoogle Scholar
  10. 10.
    Gilboa-Geffen A, Hamar P, Le MT, Wheeler LA, Trifonova R, Petrocca F, Wittrup A, Lieberman J (2015) Gene knockdown by EpCAM aptamer-siRNA chimeras suppresses epithelial breast cancers and their tumor-initiating cells. Mol Cancer Ther 14(10):2279–2291. doi: 10.1158/1535-7163.MCT-15-0201-T CrossRefPubMedGoogle Scholar
  11. 11.
    Padilla R, Sousa R (1999) Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2′-groups using a mutant T7 RNA polymerase (RNAP). Nucleic Acids Res 27(6):1561–1563CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhou J, Swiderski P, Li H, Zhang J, Neff CP, Akkina R, Rossi JJ (2009) Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells. Nucleic Acids Res 37(9):3094–3109. doi: 10.1093/nar/gkp185 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Milligan JF, Uhlenbeck OC (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol 180:51–62CrossRefPubMedGoogle Scholar
  14. 14.
    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
  15. 15.
    Tang GQ, Bandwar RP, Patel SS (2005) Extended upstream A-T sequence increases T7 promoter strength. J Biol Chem 280(49):40707–40713. doi: 10.1074/jbc.M508013200 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Internal MedicineUniversity of IowaIowa CityUSA
  2. 2.Department of Radiation OncologyUniversity of IowaIowa CityUSA
  3. 3.Molecular and Cellular Biology ProgramUniversity of IowaIowa CityUSA
  4. 4.Medical Scientist Training ProgramUniversity of IowaIowa CityUSA
  5. 5.Abboud Cardiovascular Research CenterUniversity of IowaIowa CityUSA

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