Abstract
Oligonucleotides can be designed or evolved to bind to specific DNA, RNA, protein, or small molecule targets and thereby alter the biological function of the target. The therapeutic potential of oligonucleotides targeted to intracellular molecules will depend largely on their ability to be taken up by the cells of interest, as well as their subsequent subcellular distribution. Here we describe methods to characterize the extent and mechanism of cellular uptake of AS1411, an aptamer oligonucleotide that has progressed to human clinical trials and which is also widely used by researchers as a cancer-targeting ligand.
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References
Crooke ST, Wang S, Vickers TA, Shen W, Liang XH (2017) Cellular uptake and trafficking of antisense oligonucleotides. Nat Biotechnol 35(3):230–237. https://doi.org/10.1038/nbt.3779
Juliano RL (2016) The delivery of therapeutic oligonucleotides. Nucleic Acids Res 44(14):6518–6548. https://doi.org/10.1093/nar/gkw236
Bates PJ, Laber DA, Miller DM, Thomas SD, Trent JO (2009) Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Exp Mol Pathol 86(3):151–164. https://doi.org/10.1016/j.yexmp.2009.01.004
Reyes-Reyes EM, Teng Y, Bates PJ (2010) A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism. Cancer Res 70(21):8617–8629. https://doi.org/10.1158/0008-5472.CAN-10-0920
Yang NJ, Hinner MJ (2015) Getting across the cell membrane: an overview for small molecules, peptides, and proteins. Methods Mol Biol 1266:29–53. https://doi.org/10.1007/978-1-4939-2272-7_3
Doherty GJ, McMahon HT (2009) Mechanisms of endocytosis. Annu Rev Biochem 78:857–902. https://doi.org/10.1146/annurev.biochem.78.081307.110540
Mayor S, Parton RG, Donaldson JG (2014) Clathrin-independent pathways of endocytosis. Cold Spring Harb Perspect Biol 6(6). https://doi.org/10.1101/cshperspect.a016758
Ivanov AI (2014) Pharmacological inhibitors of exocytosis and endocytosis: novel bullets for old targets. Methods Mol Biol 1174:3–18. https://doi.org/10.1007/978-1-4939-0944-5_1
Ivanov AI (2008) Pharmacological inhibition of endocytic pathways: is it specific enough to be useful? Methods Mol Biol 440:15–33. https://doi.org/10.1007/978-1-59745-178-9_2
Vassilieva EV, Nusrat A (2008) Vesicular trafficking: molecular tools and targets. Methods Mol Biol 440:3–14. https://doi.org/10.1007/978-1-59745-178-9_1
Bates PJ, Kahlon JB, Thomas SD, Trent JO, Miller DM (1999) Antiproliferative activity of G-rich oligonucleotides correlates with protein binding. J Biol Chem 274(37):26369–26377
Bates PJ, Reyes-Reyes EM, Malik MT, Murphy EM, O'Toole MG, Trent JO (2017) G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: uses and mechanisms. Biochim Biophys Acta Gen Subj 1861(5 Pt B):1414–1428. https://doi.org/10.1016/j.bbagen.2016.12.015
Rosenberg JE, Bambury RM, Van Allen EM, Drabkin HA, Lara PN Jr, Harzstark AL, Wagle N, Figlin RA, Smith GW, Garraway LA, Choueiri T, Erlandsson F, Laber DA (2014) A phase II trial of AS1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal cell carcinoma. Investig New Drugs 32(1):178–187. https://doi.org/10.1007/s10637-013-0045-6
Choi EW, Nayak LV, Bates PJ (2010) Cancer-selective antiproliferative activity is a general property of some G-rich oligodeoxynucleotides. Nucleic Acids Res 38(5):1623–1635. https://doi.org/10.1093/nar/gkp1088
Dapić V, Bates PJ, Trent JO, Rodger A, Thomas SD, Miller DM (2002) Antiproliferative activity of G-quartet-forming oligonucleotides with backbone and sugar modifications. Biochemistry 41(11):3676–3685. https://doi.org/10.1021/bi0119520
Commisso C, Davidson SM, Soydaner-Azeloglu RG, Parker SJ, Kamphorst JJ, Hackett S, Grabocka E, Nofal M, Drebin JA, Thompson CB, Rabinowitz JD, Metallo CM, Vander Heiden MG, Bar-Sagi D (2013) Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature 497(7451):633–637. https://doi.org/10.1038/nature12138
Reyes-Reyes E, Salipur FR, Shams M, Forsthoefel MK, Bates PJ (2015) Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation. Mol Oncol 9(7):1392–1405. https://doi.org/10.1016/j.molonc.2015.03.012
Horbach SP, Halffman W (2017) The ghosts of HeLa: how cell line misidentification contaminates the scientific literature. PLoS One 12(10):e0186281. https://doi.org/10.1371/journal.pone.0186281
de Diesbach P, N’kuli F, Delmée M, Courtoy PJ (2003) Infection by mycoplasma hyorhinis strongly enhances uptake of antisense oligonucleotides: a reassessment of receptor-mediated endocytosis in the HepG2 cell line. Nucleic Acids Res 31(3):886–892. https://doi.org/10.1093/nar/gkg181
Rosenblatt MN, Burns JR, Duncan VE, Hughes JA (2000) Infection of the macrophage cell line NR8383 with Mycobacterium tuberculosis (H37Ra) leads to an increase in oligodeoxynucleotide accumulation. Antisense Nucleic Acid Drug Dev 10(1):1–9. https://doi.org/10.1089/oli.1.2000.10.1
Li L, Wan T, Wan M, Liu B, Cheng R, Zhang R (2015) The effect of the size of fluorescent dextran on its endocytic pathway. Cell Biol Int 39(5):531–539. https://doi.org/10.1002/cbin.10424
Bates PJ, Choi EW, Nayak LV (2009) G-rich oligonucleotides for cancer treatment. Methods Mol Biol 542:379–392. https://doi.org/10.1007/978-1-59745-561-9_21
Remaut K, Lucas B, Braeckmans K, Sanders NN, De Smedt SC, Demeester J (2005) FRET-FCS as a tool to evaluate the stability of oligonucleotide drugs after intracellular delivery. J Control Release 103(1):259–271. https://doi.org/10.1016/j.jconrel.2004.11.019
Acknowledgments
The work described was funded by NIH grant R01CA122383 (Bates). The authors are named coinventors on issued patents or patent applications pertaining to AS1411
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Reyes-Reyes, E.M., Bates, P.J. (2019). Characterizing Oligonucleotide Uptake in Cultured Cells: A Case Study Using AS1411 Aptamer. In: Gissberg, O., Zain, R., Lundin, K. (eds) Oligonucleotide-Based Therapies. Methods in Molecular Biology, vol 2036. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9670-4_10
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DOI: https://doi.org/10.1007/978-1-4939-9670-4_10
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