In-cell NMR: from metabolites to macromolecules

Lippens, Guy; Cahoreau, Edern; Millard, Pierre; Charlier, Cyril; López, Juan M.; Hanoulle, Xavier; Portais, Jean‐Charles

doi:10.1039/c7an01635b

Cited by 22 publications

(111 citation statements)

References 146 publications

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“…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.…”

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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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: 99%

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

et al. 2019

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“…For Hahn-echo based15 N R 2 measurement or CPMG-based 15 N R 2 relaxation dispersion experiments at near physiological conditions that aim at the characterization of µs-ms dynamics, we however recommend the use of a very low D 2 O content in the sample buffer, as low as 1% molar fraction or, alternatively, the use of an external deuterium reference. This applies both to in vitro or in-cell NMR experiments[84][85][86][87][88][89] and is most important for intrinsically disordered proteins that are characterized by low 15 N R 2 rate constants and where even small R ex contributions can lead to large changes in the measured 15 N R 2 rate constant.…”

mentioning

confidence: 99%

15N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on 15N resulting from solvent exchange

et al. 2018

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15 N R 2 relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide-water exchange can severely skew Hahn-echo based 15 N R 2 relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the 15 N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent 15 N R 2 rate constants  which should not be mistaken with protein inherent chemical exchange contributions, R ex , to 15 N R 2. For measurements of 15 N R 2 rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D 2 O molar fraction in the sample buffer, as low as 1%, or the use of an external D 2 O reference along with a modified 15 N R 2 Hahn-echo based experiment. This combination allows for the measurement of R ex contributions to 15 N R 2 originating from conformational exchange in a time window from µs to ms.

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“…More recently, a bioreactor for measuring cellular metabolism by both fluorescence lifetime imaging microscopy and 13 C pyruvate MRI was reported (23) as well as an NMR tube bioreactor for 31 P cell viability measurements (24). Review articles on the use of bioreactors in NMR and MRI have been published recently (25,26). In this paper, we have shown that in vitro MRI of MCF-7/Her2 and MCF7/Neo4 cells at 9.4 T allows monitoring of the cellular response to treatment with Trastuzumab.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of MR relaxation times following trastuzumab treatment of breast cancer cells in a 3D bioreactor

Bartusik‐Aebisher¹,

Aebisher²,

Czmil³

2020

Acta Poloniae Pharmaceutica - Drug Research

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The three-dimensional tumor environment can be mimicked with the use of cells cultured within a hollow-fiber bioreactor (HFBR). In this work, magnetic resonance imaging (MRI) was used to monitor changes in relaxation time in breast cancer cells following treatment with Trastuzumab in an HFBR system. Breast cancer cells were inoculated into the HFBR system and cultured over a period of 4 weeks. Relaxation time maps were generated according to MRI techniques in three dimensional (3D) cultures of MCF7/Her2 breast cancer and MCF7/Neo4 control cells. MRI measurements showed a variation in values of spin-lattice (T 1) and spinspin (T 2) relaxation times related to cell density. Differences in both values (T 1 and T 2) were noted between untreated cells and cells treated with trastuzumab in the HFBR device. Additionally, 1 H MRI was able to provide information about drug penetration in breast cancer cell culture organized in 3D. In conclusion, MRI in vitro can provide direct, noninvasive visualization of cell density in 3D geometry and cell viability as a function of drug uptake. Both T 1 and T 2 values were higher for lower cell density and accordingly both values, T 1 and T 2 , were lower for higher cell density.

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In-cell NMR: from metabolites to macromolecules

Cited by 22 publications

References 146 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

15N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on 15N resulting from solvent exchange

Evaluation of MR relaxation times following trastuzumab treatment of breast cancer cells in a 3D bioreactor

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