Abstract
Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca2+ enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ∼100 nm in size are found in Ca2+-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory.
Highlights
The development of robust technologies to decipher the roles of yet-to-be-annotated cellular transcripts and proteins is a major challenge
To further elucidate the mechanism of the Ca2+ enrichment of medium (CEM) effect, we investigated whether the inclusion of various concentrations of CaCl2 led to the formation of particles when added to the culture medium (DMEM with 10% fetal bovine serum (FBS)) in the absence of oligonucleotide
This report describes a key phenomenon in which Ca2+ enrichment of medium potentiates the activity of a broad range of oligonucleotides in a wide range of cell types (Figures 1 and 5, Supplementary Figure S2 and S4)
Summary
The development of robust technologies to decipher the roles of yet-to-be-annotated cellular transcripts and proteins is a major challenge. Oligonucleotide-based technologies (such as antisense oligonucleotides (ASOs), aptamers, siRNAs, anti-miRNAs and more recently single guide RNAs (sgRNAs) for CRISPR-Cas systems) have increasingly gained attention in terms of their extraordinary ability to precisely recognize biological targets in a sequence-dependent and/or -independent manner. These methodologies have permitted successful modification of gene expression and protein activity in vitro to elucidate biological and pathological mechanisms. Recent innovations in the oligonucleotide chemistry have improved the potency and pharmacokinetics of oligonucleotide-based therapeutics [1,2,3] These modifications provide robust systemic oligonucleotide activity even in the absence of delivery vehicles, directly expanding their applicability as therapeutic agents. Systemically active naked oligonucleotides modified with 2 ,4 -bridged nucleic acid (2 ,4 -BNA) ( known as locked nucleic acid, LNA) chemistry [8,9] have recently been found to be taken up by various cell lines, including human cells, without the use of transfection reagents [10]
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