Semi-Automated High-Throughput Substrate Screening Assay for Nucleoside Kinases.

Katja F Hellendahl,Peter Neubauer,Maryke Fehlau,Anke Kurreck,Sebastian Hans

doi:10.3390/ijms222111558

Katja F Hellendahl, Peter Neubauer + Show 3 more

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https://doi.org/10.3390/ijms222111558

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Abstract

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and nucleotide analogue production in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening method for NKs is of great importance. Here, we report on the validation of a well-known luciferase-based assay for the detection of NK activity in a 96-well plate format. The assay was semi-automated using a liquid handling robot. Good linearity was demonstrated (r² > 0.98) in the range of 0–500 µM ATP, and it was shown that alternative phosphate donors like dATP or CTP were also accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplarily used for the comparison of the substrate spectra of four NKs using 20 (8 natural, 12 modified) substrates. The screening results correlated well with literature data, and additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

Highlights

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Nucleotides are essential biomolecules involved in all cellular processes as building blocks of nucleic acids, energy storage, cellular communication mediators, and cofactors for many enzymatic reactions
The relevance of nucleoside 50 -triphosphates (NTPs) is evident in mRNA vaccines, which have become increasingly important in the current COVID-19 pandemic [7]

Summary

Introduction

Modified nucleotides are used to label nucleic acids in PCR [1], for fluorescent in situ hybridization (FISH) [2], or microarray [3] applications. They improve nuclease stability of aptamers [4] and small interfering RNAs [5,6]. The relevance of nucleoside 50 -triphosphates (NTPs) is evident in mRNA vaccines, which have become increasingly important in the current COVID-19 pandemic [7]. By incorporating modified NTPs such as N1-methylpseudouridine triphosphates or pseudouridine triphosphates into therapeutic mRNAs, significantly less activation of the innate immune response compared to uridine is induced [8]. There is evidence that modified mRNA is translated more efficiently than unmodified mRNA

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