Advanced Fluorescence Imaging to Distinguish Between Intracellular Fractions of Antisense Oligonucleotides
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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.
Key wordsAntisense oligonucleotide Cellular uptake Confocal laser scanning microscopy Drug delivery Fluorescence correlation spectroscopy Fluorescence lifetime imaging microscopy
We thank Leica Microsystems for the use of the Leica TCS SP8 FALCON system, and are especially grateful to Johanna Berndt and Frank Hecht for support with the application and data analysis. This work was supported by the Prinses Beatrix Spierfonds (grant number W.OR14-19).
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