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Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis
Published on: July 3, 2015
Analysis of oligonucleotide binding, internalization, and intracellular trafficking utilizing a novel radiolabeled
1Clinical Pharmacology Branch, NCI, NIH, Bethesda, Maryland.
Abstract:
Although antisense oligonucleotides have been widely used to inhibit gene expression, their mechanism of entry into cells and their site of action are still in some doubt. In this report, we describe a novel technique for kinetically analyzing oligonucleotide association with living cells as well as intracellular compartmentalization. The technique utilizes a photoactivatable, radiolabeled crosslinker, the Denny-Jaffe reagent. Oligonucleotides containing pendant amine groups were conjugated to this reagent, added to HL60 cells in culture, and photocrosslinked to associated proteins, which were analyzed electrophoretically. We find that several proteins are labeled, predominantly a 75 kD one that appears to be membrane-associated. Our results suggest that the majority of intracellular oligonucleotide is associated in vesicles with the same protein to which it bound on the cell surface, but only a small percentage of non-protein-bound cytosolic oligonucleotide can be detected. Additionally, oligonucleotides are readily accumulated by nuclei, and by treating whole nuclei, a unique set of nuclear binding proteins is detected.
Insights
Researchers developed a new method to track how antisense oligonucleotides enter cells. This technique reveals that oligonucleotides associate with proteins, forming vesicles and accumulating in cell nuclei.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Antisense oligonucleotides (ASOs) are crucial for gene expression inhibition.
- The cellular uptake mechanisms and intracellular action sites of ASOs remain unclear.
- Understanding ASO trafficking is vital for optimizing their therapeutic applications.
Purpose of the Study:
- To develop a novel technique for analyzing oligonucleotide-cell interactions.
- To investigate the kinetic association and intracellular localization of ASOs.
- To identify proteins involved in ASO binding and cellular uptake.
Main Methods:
- Utilized a photoactivatable, radiolabeled crosslinker (Denny-Jaffe reagent).
- Conjugated oligonucleotides with pendant amine groups to the crosslinker.
- Applied to HL60 cells, photocrosslinked associated proteins, and performed electrophoretic analysis.
Main Results:
- Identified several protein partners, notably a 75 kD membrane-associated protein.
- Demonstrated that intracellular ASOs are largely vesicle-associated, linked to cell surface proteins.
- Detected minimal free cytosolic oligonucleotides and significant nuclear accumulation.
Conclusions:
- The novel technique provides kinetic insights into ASO cellular association and compartmentalization.
- ASO cellular entry involves protein binding, vesicle formation, and nuclear uptake.
- Identified specific protein interactions relevant to ASO mechanism of action.
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