Understanding the structure and function of nucleic acids in their native environment is crucial to structural biology and one focus of in-cell NMR spectroscopy. Many challenges hamper in-cell NMR in human cell lines, e.g. sample decay through cell death and RNA degradation. The resulting low signal intensities and broad line widths limit the use of more complex NMR experiments, reducing the possible structural and dynamic information that can be extracted. Here, we optimize the detection of imino proton signals, indicators of base-pairing and therefore secondary structure, of a double-stranded DNA oligonucleotide in HeLa cells, using selective excitation. We demonstrate the reproducible quantification of in-cell selective longitudinal relaxation times (selT1), which are reduced compared to the in vitro environment, as a result of interactions with the complex cellular environment. By measuring the intracellular selT1, we optimize the existing proton pulse sequences, and shorten measurement time whilst enhancing the signal gained per unit of time. This exemplifies an advantage of selective excitation over conventional methods like jump-return water suppressionÂ*for in-cell NMR. Furthermore, important experimental controls are discussed, including intracellular quantification, supernatant control measurements, as well as the processing of lowly concentrated in-cell NMR samples. We expect that robust and fast in-cell NMR experiments of nucleic acids will facilitate the study of structure and dynamics and reveal their functional correlation.
A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins
A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins
Abstract
Introducing the flow through the bioreactor has revolutionized in-cell NMR spectroscopy by prolonging the measurement time available to acquire spectral information about biomacromolecules in metabolically active cells. Bioreactor technology relies on immobilizer matrices, which secure cells in the active volume of the NMR coil and enable uniform perfusion of the growth medium, supplying fresh nutrients to the cells while removing toxic byproducts...
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09-10-2023 02:39 AM
[NMR paper] A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins
A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins
Introducing the flow through the bioreactor has revolutionized in-cell NMR spectroscopy by prolonging the measurement time available to acquire spectral information about biomacromolecules in metabolically active cells. Bioreactor technology relies on immobilizer matrices, which secure cells in the active volume of the NMR coil and enable uniform perfusion of the growth medium, supplying fresh nutrients to the cells while removing toxic byproducts of their metabolism. The main drawbacks of...
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[NMR paper] Observation of nucleic acids inside living human cells by in-cell NMR spectroscopy.
Observation of nucleic acids inside living human cells by in-cell NMR spectroscopy.
Observation of nucleic acids inside living human cells by in-cell NMR spectroscopy.
Biophys Physicobiol. 2020;17:36-41
Authors: Yamaoki Y, Nagata T, Sakamoto T, Katahira M
Abstract
The intracellular environment is highly crowded with biomacromolecules such as proteins and nucleic acids. Under such conditions, the structural and biophysical features of nucleic acids have been thought to be different from those in vitro. To obtain high-resolution...
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10-29-2020 10:21 AM
[NMR paper] Recent progress of in-cell NMR of nucleic acids in living human cells.
Recent progress of in-cell NMR of nucleic acids in living human cells.
Related Articles Recent progress of in-cell NMR of nucleic acids in living human cells.
Biophys Rev. 2020 Mar 06;:
Authors: Yamaoki Y, Nagata T, Sakamoto T, Katahira M
Abstract
The inside of living cells is highly crowded with biological macromolecules. It has long been considered that the properties of nucleic acids and proteins, such as their structures, dynamics, interactions, and enzymatic activities, in intracellular environments are different from those...
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03-09-2020 01:12 PM
[NMR paper] Accurate ab initio prediction of NMR chemical shifts of nucleic acids and nucleic acids/protein complexes.
Accurate ab initio prediction of NMR chemical shifts of nucleic acids and nucleic acids/protein complexes.
Related Articles Accurate ab initio prediction of NMR chemical shifts of nucleic acids and nucleic acids/protein complexes.
Nucleic Acids Res. 2014 Nov 17;
Authors: Victora A, Möller HM, Exner TE
Abstract
NMR chemical shift predictions based on empirical methods are nowadays indispensable tools during resonance assignment and 3D structure calculation of proteins. However, owing to the very limited statistical data basis,...
Optimising in-cell NMR acquisition for nucleic acids
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