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 commonly used matrices include the inability to recover intact cells post-measurement for additional analyses and/or requirements for specific operating temperatures. Here, we report on the development and characterization of a set of thermosensitive and nontoxic triblock copolymers based on poly(D,L-lactide)-b-poly(ethylene glycol)-b-poly(D,L-lactide) (PLA-PEG-PLA). Here, we show for the first time that these copolymers are suitable as immobilizer matrices for the acquisition of in-cell NMR spectra of nucleic acids and proteins over a commonly used sample temperature range of 15â??40Â*°C and, importantly, allow recovery of cells after completion of in-cell NMR spectra acquisition. We compared the performances of currently used matrices in terms of cell viability (dye exclusion assays), cellular metabolism (1D 31P NMR), and quality of in-cell NMR spectra of two model biomacromolecules (hybrid double-stranded/i-motif DNA and ubiquitin). Our results demonstrate the suitability and advantages of PLA-PEG-PLA copolymers for application in bioreactor-assisted in-cell NMR.
[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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[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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Findingthe Right Candidate for the Right Position: A Fast NMR-Assisted CombinatorialMethod for Optimizing Nucleic Acids Binders
Findingthe Right Candidate for the Right Position: A Fast NMR-Assisted CombinatorialMethod for Optimizing Nucleic Acids Binders
Ester Jime?nez-Moreno, Laura Montalvillo-Jime?nez, Andre?s G. Santana, Ana M. Go?mez, Gonzalo Jime?nez-Ose?s, Francisco Corzana, Agatha Bastida, Jesu?s Jime?nez-Barbero, Francisco Javier Can?ada, Irene Go?mez-Pinto, Carlos Gonza?lez and Juan Luis Asensio
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/jacs.6b00328/20160511/images/medium/ja-2016-00328a_0006.gif
Journal of the American Chemical Society
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[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,...
A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins
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