Abstract
Antisense oligonucleotides (ASOs) are widely used for the identification of gene functions and regulation of genes involved in different diseases for therapeutic purposes. For in vitro evaluation of the knockdown activity of gapmer ASOs, we often use lipofection or electroporation to deliver gapmer ASOs into the cells. Here, we describe a method for evaluating the knockdown activity of gapmer ASOs by a cell-free uptake mechanism, termed as gymnosis, using MALAT1 gapmer ASOs modified with 2′-O-methoxyethyl RNA (2′-MOE) or 2′-O,4′-C-ethylene–bridged nucleic acid (ENA). This method is robust because it does not involve the use of any transfection reagent and has minimal effects on cell growth. Further, we describe a convenient technique for performing one-step reverse transcription and real-time qPCR using cell lysates without RNA extraction. Data for up to 96 samples can be obtained following these methods.
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References
Crooke ST, Witztum JL, Bennett CF et al (2018) RNA-targeted therapeutics. Cell Metab 27:714–739
Geary RS, Norris D, Yu R, Bennett CF (2015) Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv Drug Deliv Rev 87:46–51
Koizumi M (2007) True antisense oligonucleotides with modified nucleotides restricted in the N-conformation. Curr Top Med Chem 7:661–665
Raal FJ, Santos RD, Blom D et al (2010) Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial. Lancet 375:998–1006
Ackermann EJ, Guo S, Booten S et al (2012) Clinical development of an antisense therapy for the treatment of transthyretin-associated polyneuropathy. Amyloid 1:43–44
Gaudet D, Alexander VJ, Baker BF et al (2015) Antisense inhibition of apolipoprotein C-III in patients with hypertriglyceridemia. N Engl J Med 373:438–447
Morita K, Hasegawa C, Kaneko M et al (2002) 2’-O,4’-C-ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug. Bioorg Med Chem Lett 12:73–76
Morita K, Koizumi M (2018) Synthesis of ENA nucleotides and ENA oligonucleotides. Curr Protoc Nucleic Acid Chem 72:4.79.1–4.79.21
Takagi M, Morita K, Nakai D et al (2004) Enhancement of the inhibitory activity of oatp antisense oligonucleotides by incorporation of 2’-O,4’-C-ethylene-bridged nucleic acids (ENA) without a loss of subtype selectivity. Biochemistry 43:4501–4510
Koizumi M, Takagi-Sato M, Okuyama R et al (2006) Direct comparison of in vivo antisense activity of ENA oligonucleotides targeting PTP1B mRNA with that of 2’-O-(2-methoxy)ethyl-modified oligonucleotides. Oligonucleotides 16:253–262
Yagi M, Takeshima Y, Surono A et al (2004) Chimeric RNA and 2′-O,4′-C-ethylene-bridged nucleic acids have stronger activity than phosphorothioate oligodeoxynucleotides in induction of exon 19 skipping in dystrophin mRNA. Oligonucleotides 14:33–40
Lee T, Awano H, Yagi M et al (2017) 2’-O-methyl RNA/ethylene-bridged nucleic acid chimera antisense oligonucleotides to induce dystrophin exon 45 skipping. Genes (Basel) 8:E67
Stein CA, Hansen JB, Lai J et al (2010) Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res 38:e3
Hung G, Xiao X, Peralta R et al (2013) Characterization of target mRNA reduction through in situ RNA hybridization in multiple organ systems following systemic antisense treatment in animals. Nucleic Acid Ther 23:369–378
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Iwashita, S., Shoji, T., Koizumi, M. (2020). Evaluating the Knockdown Activity of MALAT1 ENA Gapmers In Vitro. In: Yokota, T., Maruyama, R. (eds) Gapmers. Methods in Molecular Biology, vol 2176. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0771-8_11
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DOI: https://doi.org/10.1007/978-1-0716-0771-8_11
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