Come On In, Antisense Oligonucleotides

by Harald Zähringer Labtimes 06/2015

Antisense oligonucleotide related research and therapies depend on efficient oligonucleotide transfer into cells. Photo: The Children’s Hospital of Philadelphia

Gene silencing techniques taking advantage of antisense oligonucleotides and RNAi, rely on the efficient uptake of constructed oligonucleotides into the cells. The oligo transfer may be greatly enhanced by simply adding CaCl2 to the cell culture medium.

Oligonucleotide-based gene silencing and editing techniques, such as Antisense Oligonucleotides (ASOs), Aptamers, siRNAs, miRNAs and CRISPR-Cas are widely employed in life science research to modify gene expression and protein function. The nature of the individual oligonucleotides applied for gene silencing may slightly differ, however, they all share the same fundamental mode of action: they bind to their complementary target sequence and trigger the cleavage or transcriptional repression of the target mRNA. The major difference between ASOs and siRNAs is how they approach their target. ASOs are delivered into the cells as single strands and have to find the complementary sequence on their own. siRNAs are smuggled into the cells as duplexes and are guided to their targets by Argonaute proteins, which are part of a cell’s RNAi silencing complex.

It is pretty obvious that both methods largely depend on the efficient uptake of the applied oligonucleotides into the cells, which – despite sophisticated transfection strategies – is not always the case, especially for naked oligonucleotides. Cultured primary cells, for example, lose their uptake capability for ASOs one or two days after isolation. Similarly, the uptake efficiency of naked oligonucleotides modified with 2’,4’-bridged nucleic acid (2’,4’-BNA or locked nucleic acid, LNA), which are taken up by cells without transfection reagents, strongly depends on the host cell line.

Add some CaCl2

The entry of ASOs into cells may, however, be enhanced by a simple trick: Satoshi Obika’s group at the Graduate School of Pharmaceutical Sciences in Osaka, Japan, reports in a recent paper that “the in-vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications” significantly increases by adding CaCl2 to the cell culture medium (Hori et al., Nucleic Acids Research, doi: 10.1093/nar/gkv626). The group refers to the new transfection strategy as “Ca2+ enrichment of medium” (CEM). The assumption of the Japanese group is supported by several experiments. They first examined the knockdown activity of a ZsGreen1-ASO in human hepatoma cells (HLE) stably expressing a ZsGreen1 reporter in the presence of CaCl2 or MgCl2. To the Japanese researchers’ surprise, the knockdown activity increased in proportion to the CaCl2 concentration with a maximal activity at 9 mM. The addition of MgCl2 showed no effect.

Enhanced knockdown activity

The CaCl2 trick also worked for ­endogenous targets. Obika’s team constructed a 2’,4’-BNA-ASO against endogenous apolipoprotein B (ApoB) mRNA and incubated a human hepatoma cell line with the ApoB-targeting ASO. Similarly to the previous experiment, knockdown activity of the ApoB-ASO increases in the presence of CaCl2 in a concentration-dependent manner. The same holds true for a further experiment investigating the silencing of Survivin mRNA in three different cell lines, applying a Survivin-targeting ASO, stemming from a clinical trial. The 9 mM CaCl2 enhances the down-regulation of Survivin in all three cell lines and shows no toxic effect to the cells, even at high concentrations of 20 mM.

Localisation experiments with Cy3-labelled ApoB-ASO confirmed that the increase in ASO activity is mainly due to enhanced uptake of the ASO into the cell. Interestingly, the CEM method also facilitates the transfer of siRNAs but has no effect on the uptake of plasmids as well as cationic lipid-ASO complexes, which are generated, for example, during Lipofectamine transfection.

So, the question is: what causes the increase in ASO uptake efficiency? Hori et al. found monodispersed nanoparticle-complexes with diameters of approximately 100 nanometres – which are obviously built of smaller 15 nanometre-sized particles – upon addition of CaCl2. They speculate that the nanoparticles may be critical for efficient ASO transfer. But that’s basically an academic question. Gene silencing practitioners will leave this question to basic researchers and are just happy with an increased efficiency of their oligonucleotide-based gene silencing experiments, by simply adding CaCl2 to the cell culture medium.

Last Changed: 22.11.2015

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