Evaluation of the Stability of DNA i‐Motifs in the Nuclei of Living Mammalian Cells

Džatko, Šimon; Krafčíková, Michaela; Hänsel‐Hertsch, Robert; Fessl, Tomáš; Fiala, Radovan; Loja, Tomáš; Krafčík, Daniel; Mergny, Jean‐Louis; Foldynová-Trantírková, Silvie; Trantı́rek, Lukáš

doi:10.1002/ange.201712284

Cited by 54 publications

(72 citation statements)

References 31 publications

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“…Although solution‐state in‐cell NMR investigation of transfected oligonucleotides has been carried out before, the ASO investigated here did not produce any signal detectable by this technique. This suggests that the amount of free intracellular ASO is low and that the ASO instead exists in the form of macromolecular complexes with mRNA and/or different proteins, as might be expected from the literature .…”

Section: Figurementioning

confidence: 76%

“…In‐cell NMR spectroscopy is a tag‐free technique and allows the study, at atomic resolution, of structural properties of, for example, proteins and small DNA/RNA hairpins in cells. Therefore, the ASO was delivered into HEK 293T cells by electroporation, as used previously for solution‐state in‐cell NMR studies, to ensure robust uptake, and a classical in‐cell solution‐state NMR spectrum was acquired. However, in the 31 P NMR spectrum no ASO signal could be detected (Figure A).…”

Section: Figurementioning

confidence: 99%

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Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

et al. 2019

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Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of a drug's function and efficiency. However, there are currently no reliable methods for studying 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. 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 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., size, stability and sensitivity) and of visualization techniques, in which (e.g., fluorescent) tagging of the ASO decreases its activity. The capability to detect an untagged, active drug, interacting in its natural environment, represents a first step towards studying molecular mechanisms in intact cells.

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Section: Figurementioning

confidence: 76%

Section: Figurementioning

confidence: 99%

Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

et al. 2019

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“…Such failures are often linked to poor membrane permeability and/or to the lack of binding specificity in the cellular environment. Nuclear magnetic resonance (NMR) spectroscopy applied to living cells has the potential to overcome these critical bottlenecks, as it can directly observe macromolecule–ligand interactions at atomic resolution within the cellular environment . Herein, we report an approach to perform protein‐observed ligand screening by NMR directly in the cytosol of living human cells.…”

Section: Figurementioning

confidence: 99%

Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency

et al. 2020

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Structure‐based drug development is often hampered by the lack of in vivo activity of promising compounds screened in vitro, due to low membrane permeability or poor intracellular binding selectivity. Herein, we show that ligand screening can be performed in living human cells by “intracellular protein‐observed” NMR spectroscopy, without requiring enzymatic activity measurements or other cellular assays. Quantitative binding information is obtained by fast, inexpensive 1H NMR experiments, providing intracellular dose‐ and time‐dependent ligand binding curves, from which kinetic and thermodynamic parameters linked to cell permeability and binding affinity and selectivity are obtained. The approach was applied to carbonic anhydrase and, in principle, can be extended to any NMR‐observable intracellular target. The results obtained are directly related to the potency of candidate drugs, that is, the required dose. The application of this approach at an early stage of the drug design pipeline could greatly increase the low success rate of modern drug development.

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“…i-motif formation is pH-dependent, and the pH of mid-transition from i-motif to unfolded structure depends on the C-tract length. The interest in i-motifs was boosted by recent reports showing that i-motifs can form in vitro in at physiological pH [5], in the genomic DNA of the nuclei of fixed cells [6] and in DNA constructs introduced in the nuclei of living cells [7].…”

Section: Introductionmentioning

confidence: 99%

Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process

Khristenko

Amato

Livet

et al. 2019

J. Am. Soc. Mass Spectrom.

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Ion mobility spectrometry (IMS) has become popular to characterize biomolecule folding.Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C-H + -C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~5.5 over a wide range of ammonium acetate concentrations (15 mM to 100 mM). The control experiments consisted of either the same sequence at pH ~7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and droplet surface, which in turn influences the probability of being ionized via a charged residue-type pathway or a chain extrusion-type pathway.

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Evaluation of the Stability of DNA i‐Motifs in the Nuclei of Living Mammalian Cells

Cited by 54 publications

References 31 publications

Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency

Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process

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