Please use this identifier to cite or link to this item: http://dx.doi.org/10.14279/depositonce-8825
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dc.contributor.authorKaspar, Felix-
dc.contributor.authorGiessmann, Robert T.-
dc.contributor.authorKrausch, Niels-
dc.contributor.authorNeubauer, Peter-
dc.contributor.authorWagner, Anke-
dc.contributor.authorGimpel, Matthias-
dc.date.accessioned2019-08-14T10:36:56Z-
dc.date.available2019-08-14T10:36:56Z-
dc.date.issued2019-07-15-
dc.identifier.urihttps://depositonce.tu-berlin.de/handle/11303/9795-
dc.identifier.urihttp://dx.doi.org/10.14279/depositonce-8825-
dc.description.abstractEfficient reaction monitoring is crucial for data acquisition in kinetic and mechanistic studies. However, for conversions of nucleosides to their corresponding nucleobases, as observed in enzymatically catalyzed nucleoside phosphorylation reactions, the current analytical arsenal does not meet modern requirements regarding cost, speed of analysis and high throughput. Herein, we present a UV/Vis spectroscopy-based assay employing an algorithm for spectral unmixing in a 96-well plate format. The algorithm relies on fitting of reference spectra of nucleosides and their bases to experimental spectra and allows determination of nucleoside/nucleobase ratios in solution with high precision. The experimental procedure includes appropriate dilution of a sample into aqueous alkaline solution, transfer to a multi-well plate, measurement of a UV/Vis spectrum and subsequent in silico spectral unmixing. This enables data collection in a high-throughput fashion and reduces costs compared to state-of-the-art HPLC analyses by approximately 5-fold while being 20-fold faster and offering comparable precision. Additionally, the method is robust regarding dilution and sample transfer errors as it only considers spectral form and not absolute intensity. It can be applied to all natural nucleosides and nucleobases and even unnatural ones as demonstrated by several examples.en
dc.description.sponsorshipDFG, 390540038, EXC 2008: UniSysCaten
dc.language.isoenen
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/en
dc.subject.ddc540 Chemie und zugeordnete Wissenschaftende
dc.subject.ddc570 Biowissenschaften; Biologiede
dc.subject.otherUV/Vis spectroscopyen
dc.subject.otherspectral unmixingen
dc.subject.otherdiscontinuous assayen
dc.subject.otherenzymatic reaction monitoringen
dc.subject.othernucleosidesen
dc.subject.othernucleobasesen
dc.titleA UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobasesen
dc.typeArticleen
dc.date.updated2019-08-14T02:29:09Z-
dc.relation.issupplementedby10.5281/zenodo.3245012-
dc.relation.issupplementedby10.5281/zenodo.3243376-
tub.accessrights.dnbfreeen
tub.publisher.universityorinstitutionTechnische Universität Berlinen
dc.identifier.eissn2409-9279-
dc.type.versionpublishedVersionen
dcterms.bibliographicCitation.doi10.3390/mps2030060en
dcterms.bibliographicCitation.journaltitleMethods and Protocolsen
dcterms.bibliographicCitation.originalpublisherplaceBaselen
dcterms.bibliographicCitation.volume2en
dcterms.bibliographicCitation.originalpublishernameMDPIen
dcterms.bibliographicCitation.issue3en
dcterms.bibliographicCitation.articlenumber60en
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