Quantitative fluorescence imaging determines the absolute number of locked nucleic acid oligonucleotides needed for suppression of target gene expression

Abstract

Locked nucleic acid based antisense oligonucleotides (LNA-ASOs) can reach their intracellular RNA targets without delivery modules. Functional cellular uptake involves vesicular accumulation followed by translocation to the cytosol and nucleus. However, it is yet unknown how many LNA-ASO molecules need to be delivered to achieve target knock down. Here we show by quantitative fluorescence imaging combined with LNA-ASO microinjection into the cytosol or unassisted uptake that ∼105 molecules produce >50% knock down of their targets, indicating that a substantial amount of LNA-ASO escapes from endosomes. Microinjected LNA-ASOs redistributed within minutes from the cytosol to the nucleus and remained bound to nuclear components. Together with the fact that RNA levels for a given target are several orders of magnitude lower than the amounts of LNA-ASO, our data indicate that only a minor fraction is available for RNase H1 mediated reduction of target RNA. When non-specific binding sites were blocked by co-administration of non-related LNA-ASOs, the amount of target LNA-ASO required was reduced by an order of magnitude. Therefore, dynamic processes within the nucleus appear to influence the distribution and activity of LNA-ASOs and may represent important parameters for improving their efficacy and potency.

Deciphering the role of miR-71 in Echinococcus multilocularis early development in vitro

“…We also attempted knockdown of miR-71 using LNA anti-miR but neither phenotypic alterations nor changes in mRNA levels were observed although miR-71 levels were decreased as measured by RT-qPCR. One possible explanation for these results is that most LNA molecules were retained in endosomes [48] and were not able to interact with cytosolic miR-71. The LNA anti-miR retained in endosomes could have been released after cell lysis to form strong heteroduplex with the miRNA [49], which is characteristic of this type of oligonucleotides and often causes false results in RT-qPCR.…”

Nuclear and Cytoplasmatic Quantification of Unconjugated, Label-Free Locked Nucleic Acid Oligonucleotides

“…At present, most studies on intracellular presence and trafficking rely on covalent attachment of a fluorophore onto the oligonucleotide [38][39][40][41][42][43]. These studies involving fluorescently labeled oligonucleotides are beneficial to understanding the distribution, and some constructs mimic very well the situation of unconjugated AON [44]. However, chemically modified AONs will not, or cannot, act exactly in vitro and in vivo as the unconjugated AON, and some reports have shown that certain modifications or fluorescent tags alter the properties of an oligonucleotide significantly [45][46][47][48].…”

The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells

“…As we were finishing this manuscript, an article was published in which the nuclear concentration needed for knockdown of the abundant long noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) using locked nucleic acid (LNA) gapmers was determined (48). Approximately 100,000 LNA molecules were required for 50% knockdown of this transcript with a copy number of ;2500 per cell (based on literature values).…”

“…Thus, areas that showed highly fluorescent spots from other focal planes than the nucleus were distinguished from those nuclei that had a homogeneous fluorescence distribution. For each of the 3 independent uptake experiments, between 57 and 103 nuclei were measured (untreated control [38][39][40][41][42][43][44][45][46][47][48]. A 1-way ANOVA was performed to test whether there was a treatment effect.…”