ArticleNucleic acids research2009
Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting.
Article in Nucleic acids research, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 43 citations in OpenAlex.
- Flow Cytometry: The Next Revolution.Cells · 2023Review
- Protein and peptide engineering for chemical exchange saturation transfer imaging in the age of synthetic biology.NMR in biomedicine · 2023Review
- Computationally designed dual-color MRI reporters for noninvasive imaging of transgene expression.Nature biotechnology · 2022Article
- Quantification and tracking of genetically engineered dendritic cells for studying immunotherapy.Magnetic resonance in medicine · 2018Article
- Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant.PloS one · 2017Article
- Quantitative Detection of Nucleoside Analogues by Multi-enzyme Biosensors using Time-Resolved Kinetic Measurements.ChemMedChem · 2016Article
- Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently.Chemical Society reviews · 2015Review
- A new screening method for the directed evolution of thermostable bacteriolytic enzymes.Journal of visualized experiments : JoVE · 2012Article
- Enzymes to die for: exploiting nucleotide metabolizing enzymes for cancer gene therapy.Current gene therapy · 2012Review
- Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis.Future medicinal chemistry · 2011Article
- Computational design of orthogonal nucleoside kinases.Chemical communications (Cambridge, England) · 2010Article
- Synthesis of fluorescent nucleoside analogs as probes for 2'-deoxyribonucleoside kinases.Bioorganic & medicinal chemistry letters · 2010Article
- Exploring protein fitness landscapes by directed evolution.Nature reviews. Molecular cell biology · 2009Review
- Engineering Kinases to Phosphorylate Nucleoside Analogs for Antiviral and Cancer Therapy.Chimia · 2009Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
Nucleoside analogs (NAs) represent an important category of prodrugs for the treatment of viral infections and cancer, yet the biological potency of many analogs is compromised by their inefficient activation through cellular 2'-deoxyribonucleoside kinases (dNKs). We herein report the directed evolution and characterization of an orthogonal NA kinase for 3'-deoxythymidine (ddT), using a new FACS-based screening protocol in combination with a fluorescent analog of ddT. Four rounds of random mutagenesis and DNA shuffling of Drosophila melanogaster 2'-deoxynucleoside kinase, followed by FACS analysis, yielded an orthogonal ddT kinase with a 6-fold higher activity for the NA and a 20-fold k(cat)/K(M) preference for ddT over thymidine, an overall 10,000-fold change in substrate specificity. The contributions of individual amino acid substitutions in the ddT kinase were evaluated by reverse engineering, enabling a detailed structure-function analysis to rationalize the observed changes in performance. Based on our results, kinase engineering with fluorescent NAs and FACS should prove a highly versatile method for evolving selective kinase:NA pairs and for studying fundamental aspects of the structure-function relationship in dNKs.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.