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Advanced Fluorescence Imaging to Distinguish Between Intracellular Fractions of Antisense Oligonucleotides
M Leontien van der Bent1,2, Derick G Wansink3, Roland Brock4
1Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Center, Nijmegen, The Netherlands.
Abstract:
Antisense oligonucleotides (AON) have been intensively studied as tools in molecular cell biology and as novel therapeutics in various diseases over the past two decades. Especially cellular uptake and endosomal release of AONs are topics of interest, as these are crucial steps in reaching the subcellular AON target sites and achieving biological activity. We used cell-penetrating peptides (CPPs) to enhance uptake and endosomal release of AONs, and monitored these two processes and the subsequent fate of the AONs by advanced fluorescence microscopy in living cells. In this chapter, we discuss the use of automated time-lapse confocal laser scanning microscopy (CLSM) to follow AON uptake and trafficking in time, fluorescence lifetime imaging microscopy (FLIM) to distinguish between free and AON-bound fluorophore, and fluorescence correlation spectroscopy (FCS) to measure subcellular AON concentrations and molecular associations. Additionally, we expand on the analysis of these microscopy data.
Insights
Cell-penetrating peptides (CPPs) improve the cellular uptake and endosomal escape of antisense oligonucleotides (AONs). Advanced microscopy techniques track AON trafficking and concentration within cells for therapeutic applications.
Area of Science:
- Molecular Cell Biology
- Biotechnology
- Drug Delivery Systems
Background:
- Antisense oligonucleotides (AONs) are valuable tools in molecular biology and potential therapeutics.
- Efficient cellular uptake and endosomal release are critical for AON biological activity.
- Understanding AON trafficking is essential for optimizing their therapeutic potential.
Purpose of the Study:
- To investigate the use of cell-penetrating peptides (CPPs) for enhancing AON cellular uptake and endosomal release.
- To monitor the intracellular fate and trafficking of AONs using advanced fluorescence microscopy.
- To analyze microscopy data for quantitative insights into AON delivery and localization.
Main Methods:
- Automated time-lapse confocal laser scanning microscopy (CLSM) for tracking AON uptake and trafficking dynamics.
- Fluorescence lifetime imaging microscopy (FLIM) to differentiate between free and AON-bound fluorophores.
- Fluorescence correlation spectroscopy (FCS) for measuring subcellular AON concentrations and molecular interactions.
Main Results:
- CPPs significantly enhanced the cellular uptake of AONs.
- CPPs facilitated the endosomal escape of AONs, improving their intracellular availability.
- Microscopy techniques provided real-time insights into AON trafficking and localization within living cells.
Conclusions:
- CPPs are effective agents for improving AON delivery into cells and endosomal release.
- Advanced fluorescence microscopy techniques offer powerful tools for studying AON cellular dynamics.
- This approach holds promise for the development of more effective AON-based therapeutics.
