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Observing an antisense drug complex in intact human cells by in-cell NMR

Judith Schlagnitweit, Sarah Friebe Sandoz, Aleksander Jaworski, Ileana Guzzetti, Fabien Aussenac, Rodrigo J. Carbajo, Elisabetta Chiarparin, Andrew J. Pell, Katja Petzold
doi: https://doi.org/10.1101/589812
Judith Schlagnitweit
1Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
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  • For correspondence: judith.schlagnitweit@ki.se katja.petzold@ki.se
Sarah Friebe Sandoz
1Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
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Aleksander Jaworski
2Department of Materials and Environmental Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, Sweden
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Ileana Guzzetti
1Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
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Fabien Aussenac
3Bruker BioSpin, Wissembourg, France
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Rodrigo J. Carbajo
4Analytical & Structural Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, UK
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Elisabetta Chiarparin
4Analytical & Structural Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, UK
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Andrew J. Pell
2Department of Materials and Environmental Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, Sweden
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Katja Petzold
1Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
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  • For correspondence: judith.schlagnitweit@ki.se katja.petzold@ki.se
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Abstract

Gaining insight into the uptake of drugs into cells, trafficking and their target engagement enhances understanding of the drug’s function and efficiency. Here we study an antisense oligonucleotide drug (ASO) delivered into HEK293T and HeLa cells, by Nuclear Magnetic Resonance (NMR). Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by qRT-PCR, measuring downregulation of its target mSTAT3. Applying DNP NMR to frozen cells, we overcome limitations of traditional solution-state in-cell NMR (e.g. size, stability and sensitivity) as well as of visualization techniques, where (e.g. fluorescent) tagging of the ASO decreases its activity. The possibility to study an untagged, active drug, interacting in its natural environment, will increase insights into molecular mechanisms of delivery, intracellular trafficking and target engagement in intact cells.

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Posted March 27, 2019.
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Observing an antisense drug complex in intact human cells by in-cell NMR
Judith Schlagnitweit, Sarah Friebe Sandoz, Aleksander Jaworski, Ileana Guzzetti, Fabien Aussenac, Rodrigo J. Carbajo, Elisabetta Chiarparin, Andrew J. Pell, Katja Petzold
bioRxiv 589812; doi: https://doi.org/10.1101/589812
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Observing an antisense drug complex in intact human cells by in-cell NMR
Judith Schlagnitweit, Sarah Friebe Sandoz, Aleksander Jaworski, Ileana Guzzetti, Fabien Aussenac, Rodrigo J. Carbajo, Elisabetta Chiarparin, Andrew J. Pell, Katja Petzold
bioRxiv 589812; doi: https://doi.org/10.1101/589812

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