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

Abstract

Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of ad rug's functionand efficiency. However, there are currently no reliable methods forstudying untagged biomolecules in macromolecular complexes in intact human cells. Here we have studied an antisense oligonucleotide (ASO) drug in HEK 293T and HeLa cells by NMR spectroscopy.U sing ac ombination 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 quantitative reverse transcription polymerase chain reaction (qRT-PCR). By applying DNP NMR to frozen cells, we overcame limitations both of solution-state in-cell NMR spectroscopy( e.g.,s ize, stabilitya nd sensitivity) and of visualization techniques, in which (e.g.,f luorescent)t agging of the ASO decreases its activity.T he capability to detecta nu ntagged, active drug, interacting in itsn atural environment, represents af irst step towards studying molecular mechanisms in intact cells.Understanding physiological processes in detail, as well as their inhibition by drugs, is an important topic of research. High-resolution structural techniques allow us to study complex biomoleculars ystems; however,t hesem ethods are mostly applicable to in vitro samples and cannot recapitulate cellular context,t hus indicating that these structures might differ from those in living cells. In contrast, functional data are routinelyo btainedi nt issues or cells through the use of visualization techniques such as confocal microscopy,w hich requires taggingf or visualization by chemical modification of the molecule of interest. Thus, the context in which data are acquired is highly relevant, but at agged system might exhibit altered behaviour: cellular trafficking and/or function, for example. Therefore, ac ellular structural biology approachi sn eeded, combining the advantage of the biologically relevant context of the cell with an atomic-resolution technique.In this work we studied danvatirsen, ah igh-affinity 16-nucleotides ynthetic antisense oligonucleotide (ASO) with ag apmer design and ap hosphorothioate (PS) backbone throughout the sequence [1] (Section 1.1 and Figure S01 in the Supporting Information). The phosphorothioate backbone is as tandard meanso fi ncreasingr esistance to degradation by nucleases with preservationo fRNaseH1 activity. [2] Danvatirsen downregulates the mRNA of the transcriptionf actor STAT3, and is currently in clinicalt rials, showing antitumour activity in lymphoma, non-small-cell lung cancer [1] and non-Hodgkin'sl ymphoma. [3] However,u nderstanding of intracellular trafficking and targeting processes is currentlyabottleneck in ASO drug development, [4,5] and until now no reliable methodh as allowed the study of untagged oligonucleotides or oligonucleotide structures in their active macromolecular complexes in human cells. Here we show the challenges of currently existing approaches to studyingA SOs in cells and present an ...

DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular environments

“…Finally, while experimental specificity is an important consideration for samples that are homogenously doped with polarization agents where the analyte is diluted in an environment containing concentrated chemically similar natural abundance molecules, covalent attachment of the polarization to the target would effectively eliminate the specificity limit. (Viennet, Viegas et al 2016, Lim, Ackermann et al 2020) Likewise, experimental specificity is not a concern for molecules with chemical shifts that are distinct from those of the components of the cellular biomass (Schlagnitweit, Sandoz et al 2019, Bertarello, Berruyer et al 2022).…”

Production of Structured RNA Fragments by In Vitro Transcription and HPLC Purification

“…RNAs are becoming increasingly important as regulators (Dethoff, Petzold, Chugh, Casiano‐Negroni, & Al‐Hashimi, 2012), drug targets (Dethoff et al., 2012; Wacker et al., 2020), or even as vaccines (Mulligan et al., 2020) and drugs (Schlagnitweit et al., 2019). Experiments performed to elucidate these RNA functions and the correlation to their structures rely heavily on samples prepared in vitro, mostly through transcription with the bacteriophage enzyme T7 RNA polymerase (T7 in vitro transcription [IVT]; Milligan & Uhlenbeck, 1989; Milligan, Groebe, Witherell, & Uhlenbeck, 1987).…”