A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins

0581740 - BFÚ 2024 RIV NL eng J - Článek v odborném periodiku
Dzurov, M. - Pospíšilová, S. - Krafčíková, Michaela - Trantírek, L. - Vojtová, L. - Ryneš, J.
A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins.
Journal of Biomolecular NMR. Roč. 77, č. 5-6 (2023), s. 203-215. ISSN 0925-2738. E-ISSN 1573-5001
Grant CEP: GA MŠMT EF15_003/0000477
Institucionální podpora: RVO:68081707
Klíčová slova: peg-plga copolymer * drug-delivery * hydrogels * spectroscopy * stability * agarose * dna * pla-peg-pla * Thermosensitive hydrogel * Cell immobilization * Bioreactor * In-cell NMR
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 2.7, rok: 2022
Způsob publikování: Open access
https://link.springer.com/article/10.1007/s10858-023-00422-7

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 15-40 & DEG,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.
Trvalý link: https://hdl.handle.net/11104/0350097