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Open AccessArticle

A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases

1
Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstraße 76, ACK24, D-13355 Berlin, Germany
2
BioNukleo GmbH, Ackerstraße 76, D-13355 Berlin, Germany
*
Author to whom correspondence should be addressed.
The authors contributed equally to the work.
Methods Protoc. 2019, 2(3), 60; https://doi.org/10.3390/mps2030060
Received: 13 June 2019 / Revised: 10 July 2019 / Accepted: 10 July 2019 / Published: 15 July 2019
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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. View Full-Text
Keywords: UV/Vis spectroscopy; spectral unmixing; discontinuous assay; enzymatic reaction monitoring; nucleosides; nucleobases UV/Vis spectroscopy; spectral unmixing; discontinuous assay; enzymatic reaction monitoring; nucleosides; nucleobases
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Kaspar, F.; Giessmann, R.T.; Krausch, N.; Neubauer, P.; Wagner, A.; Gimpel, M. A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases. Methods Protoc. 2019, 2, 60.

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