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Cited by 52RNA Interference and Gene DeliveryDNA and Nucleic Acid ChemistryAdvanced biosensing and bioanalysis techniques

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

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Buntz, Annette, Killian, Tobias, Schmid, Daniela, Seul, Heike, Brinkmann, Ulrich, Ravn, Jacob, Lindholm, Marie, Knoetgen, Hendrik, Haucke, Volker, Mundigl, Olaf·2019·Nucleic Acids Research
DOI 10.1093/nar/gky1158PMC PMC6344898
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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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  • ∼2000 cytoplasmic siRNAs are estimated to achieve maximal silencing, whereas ∼50 000 are required for an ASO.

    “∼2000 cytoplasmic siRNAs are estimated to achieve maximal silencing, whereas ∼50 000 are required for an ASO [54, 55].”

    From Elucidating brain transport pathways and cell type-dependent gene silencing of a durable lipid–siRNA conjugate administered into cerebrospinal fluid (2025) · cites this paper as [55]

    The Discussion (page unavailable) reports efficient downregulation with fewer than 2,000 siRNA molecules per cell, but does not describe that as maximal silencing or specify a cytoplasmic count. The Results (page unavailable) report that approximately 100,000 LNA-ASO molecules produced more than 50% knockdown, not that 50,000 ASOs were required for maximal silencing. (LLM, conf=0.90)

    Experimental — accuracy not yet measured

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Metadata

Authors
Buntz, A., Killian, T., Schmid, D., Seul, H., Brinkmann, U., Ravn, J., Lindholm, M., Knoetgen, H., Haucke, V., & Mundigl, O.
Affiliations
  • Roche Pharma AG (Germany) (DE)
  • Roche (Denmark) (DK)
  • F. Hoffmann-La Roche AG (CH)
  • Leibniz-Forschungsinstitut für Molekulare Pharmakologie (DE)
Corresponding author
Mundigl, Olaf
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2018-11-20
Volume
47
Issue
2
Pages
953-969
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gky1158
PMC
PMC6344898
OpenAlex
W2900920733
Cited by
52
References
3
Citations by year
2026: 8 · 2025: 5 · 2024: 4 · 2023: 6 · 2022: 7 · 2021: 9 · 2020: 5 · 2019: 8
FWCI
1.96
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Roche Postdoc Fellowship Program (RPF_ID_398)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
Keywords
gene knockdown, RNA interference, endosomal escape, nuclear translocation, RNase H1, non-specific binding, microinjection, quantitative fluorescence imaging
MeSH terms
Cell Nucleus, Humans, Microinjections, Microscopy, Fluorescence, Oligonucleotides, Oligonucleotides, Antisense, Fluorescence Recovery After Photobleaching, Gene Knockdown Techniques, MCF-7 Cells

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