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Main Title: A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases
Author(s): Kaspar, Felix
Giessmann, Robert T.
Krausch, Niels
Neubauer, Peter
Wagner, Anke
Gimpel, Matthias
Type: Article
Is Supplemented By: 10.5281/zenodo.3245012
Language Code: en
Abstract: Efficient 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.
Issue Date: 15-Jul-2019
Date Available: 14-Aug-2019
DDC Class: 540 Chemie und zugeordnete Wissenschaften
570 Biowissenschaften; Biologie
Subject(s): UV/Vis spectroscopy
spectral unmixing
discontinuous assay
enzymatic reaction monitoring
Sponsor/Funder: DFG, 390540038, EXC 2008: UniSysCat
Journal Title: Methods and Protocols
Publisher: MDPI
Publisher Place: Basel
Volume: 2
Issue: 3
Article Number: 60
Publisher DOI: 10.3390/mps2030060
EISSN: 2409-9279
Appears in Collections:FG Bioverfahrenstechnik » Publications

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