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. 2019 Jan 25;47(2):953-969.
doi: 10.1093/nar/gky1158.

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

Affiliations

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

Annette Buntz et al. Nucleic Acids Res. .

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.

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Figures

Figure 1.
Figure 1.
Delivery of LNA-ASOs by microinjection and single cell knock down analysis. The number of LNA-ASO molecules required for target knock down was determined by delivering a defined amount of LNA-ASOs into the cytosol via microinjection and subsequent single cell knock down analysis in injected cells (A). Calibration experiments were performed to determine the number of injected molecules. The fluorescence signal of a tracer molecule was used to measure intracellular concentrations via fluorescence correlation spectroscopy (FCS) and photon counting imaging (B). Target knock down was detected in injected cells on RNA and protein level by fluorescence in-situ hybridization and immunofluorescence, respectively. RNA and protein levels were assessed by quantitative image analysis. Using an automated analysis pipeline, cell nuclei were first identified by image segmentation. Thereafter, mean fluorescence intensities of tracer and target signals in the segmented cell nuclei were calculated. Injected cells were distinguished from non-injected cells using the tracer signal (C).
Figure 2.
Figure 2.
MALAT1 RNA knock down in microinjected cells. MCF-7 cells were microinjected with a solution of 10 μM dextran-AF488 as tracer + 1000 nM/100 nM/10 nM unlabelled MALAT1 LNA-ASOs. Cells were incubated for 4 h, and fixed with 4% PFA. MALAT1 RNA was detected via fluorescence in situ hybridization. Fluorescence microscopy revealed knock down of MALAT1 RNA in injected cells at high LNA-ASO concentrations. White arrows indicate injected cells. Scale bars: 50 μm (A). The cellular fluorescence signal originating from MALAT1 RNA staining was quantified. Injected and non-injected cells were distinguished by defining a threshold of the tracer signal (B). Intracellular concentrations of LNA-ASOs after microinjection were estimated from calibration experiments. Mean values ± standard error of the mean (SEM) of cellular MALAT1 RNA levels are depicted. 20 000–200 000 LNA-ASO molecules need to be injected into the cells to observe significant knock down of MALAT1 RNA. Statistical significance was assessed with a two-way ANOVA and Tukey posttest. The degree of significance is ****P < 0.0001.
Figure 3.
Figure 3.
Competition experiment using unrelated LNA-ASOs. MCF-7 cells were microinjected with a solution of 10 μM dextran-AF488 as tracer + target LNA-ASOs (MALAT1) + unrelated LNA-ASOs at the indicated concentrations. After microinjection, cells were incubated for 4 h, and fixed with 4% PFA. MALAT1 RNA was detected via fluorescence in situ hybridization. White arrows indicate injected cells. Scale bars: 50 μm (A). Knock down of target RNA was assessed by quantitative image analysis. Scatter plot representation of relative MALAT1 RNA levels versus relative tracer signals (B). Mean values ± standard error of the mean (SEM) of cellular MALAT1 RNA levels are depicted (C).
Figure 4.
Figure 4.
HIF1A protein knock down in microinjected cell. MCF-7 cells were microinjected with a solution of 10 μM dextran-AF488 as tracer + 1/0.1 μM unlabelled HIF1A LNA-ASOs. Cells were incubated with 100 μM deferoxamine for 48 h, and fixed with 4% PFA. HIF1A protein was detected via HIF1A-antibody immunocytochemistry. Fluorescence microscopy revealed knock down of HIF1A protein in injected cells at high LNA-ASO concentrations. White arrows indicate injected cells. Scale bars: 50 μm (A). Knock down analysis in injected cells was performed using quantitative image analysis. The cellular fluorescence signal originating from HIF1A protein staining was quantified. Injected cells were distinguished from non-injected cells using the tracer signal (B). Mean values ± standard error of the mean (SEM) of cellular HIF1A protein levels in injected and non-injected cells are depicted. Statistical significance was assessed with a two-way ANOVA and Tukey posttest. The degree of significance is ****P < 0.0001 (C).
Figure 5.
Figure 5.
Rapid nuclear accumulation of LNA-ASOs after microinjection. MCF-7 cells were co-injected with 5 μM TMR-dextran (70 kDa) as tracer + 5 μM MALAT1 LNA-AF488 (LNA-ASO-AF488). Directly after injection confocal time lapse imaging was started. LNA rapidly accumulates in the nucleus whereas the tracer remains in the cytosol. Scale bar: 20 μm (A). To assess the influence of active transport on nuclear accumulation, cellular ATP pools were depleted before injection. Cells were kept under starvation conditions over night and incubated with 6 mM deoxyglucose and 10 mM sodium azide for 45 min prior to injection. Scale bar: 20 μm (B).
Figure 6.
Figure 6.
Restricted diffusion of LNA-ASOs inside the nucleus. Fluorescence recovery after photobleaching (FRAP) experiments were performed in microinjected cells using FAM-HIF1A-LNA-ASOs. MCF-7 cells were injected with a solution of 20 μM FAM-LNA-ASOs and incubated for 30 min. Diffusion of the labelled oligonucleotides was assessed by photobleaching a defined region of interest and monitoring the recovery of fluorescence signal within the photobleached area. Scale bars: 10 μm (A). The time trace of fluorescence intensity within the photobleached area was recorded, normalized and fit to an exponential model (see Material and Methods). Mean diffusion coefficient ± SD was calculated from the half-life time of fluorescence recovery. Three independent experiments were performed and 30 cells were analyzed in total (B). Nucleoli were identified via incubation with 1 mM EU for 1 h, which is incorporated into freshly synthesized RNA. EU-treated cells were injected with a solution of 5 μM LNA-ATTO647N (LNA-ASO-ATTO647). Cells were fixed 20 min after injection and incorporated EU was detected via Click-reaction with AF594-N3. Scale bar: 5 μm (C). Diffusion of FAM-HIF1A-LNA-ASOs was assessed in aqueous solution performing FRAP experiments under similar conditions as in the nuclear environment. To account for faster recovery kinetics, the radius of the bleached area was increased (D).
Figure 7.
Figure 7.
Detection of nuclear LNA-ASOs after gymnotic delivery. MCF-7 cells were incubated with 5 μM HIF1A LNA-AF594 (LNA-AF594 oligonucleotides) for 72 h. Live cell imaging was carried out on a confocal microscope set to photon counting mode. Using a standard curve, photons counts were translated into concentrations and presented as false-color heat map. A logarithmic color scale was applied to visualize differences in the low concentration range. Scale bar: 40 μm (A). Vesicular LNA-ASO concentrations were calculated using automated image segmentation (B). Nuclei were identified manually using transmission images. LNA-ASO concentrations were measured from average gray values in nuclear areas free of signal contamination originating from vesicles (C). Bars represent mean values ± SD. Quantitative analysis of three independent experiments.

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