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