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

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dc.contributor.authorDzurov, Matejcs
dc.contributor.authorPospíšilová, Šárkacs
dc.contributor.authorKrafčíková, Michaelacs
dc.contributor.authorTrantírek, Lukášcs
dc.contributor.authorVojtová, Lucycs
dc.contributor.authorRyneš, Jáncs
dc.coverage.issue5cs
dc.coverage.volume77cs
dc.date.issued2023-09-09cs
dc.description.abstractIntroducing 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.en
dc.formattextcs
dc.format.extent1-13cs
dc.format.mimetypeapplication/pdfcs
dc.identifier.citationJournal of Biomolecular NMR. 2023, vol. 77, issue 5, p. 1-13.en
dc.identifier.doi10.1007/s10858-023-00422-7cs
dc.identifier.issn1573-5001cs
dc.identifier.orcid0000-0003-0620-8817cs
dc.identifier.orcid0000-0001-5281-7045cs
dc.identifier.other185238cs
dc.identifier.researcheridD-7762-2012cs
dc.identifier.scopus12039667200cs
dc.identifier.urihttp://hdl.handle.net/11012/244175
dc.language.isoencs
dc.publisherSpringer Naturecs
dc.relation.ispartofJournal of Biomolecular NMRcs
dc.relation.urihttps://link.springer.com/article/10.1007/s10858-023-00422-7cs
dc.rightsCreative Commons Attribution 4.0 Internationalcs
dc.rights.accessopenAccesscs
dc.rights.sherpahttp://www.sherpa.ac.uk/romeo/issn/1573-5001/cs
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/cs
dc.subjectPLA-PEG-PLAen
dc.subjectThermosensitive hydrogelen
dc.subjectCell immobilizationen
dc.subjectBioreactoren
dc.subjectIn-cell NMRen
dc.titleA thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteinsen
dc.type.driverarticleen
dc.type.statusPeer-revieweden
dc.type.versionpublishedVersionen
sync.item.dbidVAV-185238en
sync.item.dbtypeVAVen
sync.item.insts2025.02.03 15:51:02en
sync.item.modts2025.01.17 16:42:15en
thesis.grantorVysoké učení technické v Brně. Středoevropský technologický institut VUT. Pokročilé biomateriálycs
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