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Nucleic Acids Research Advance Access originally published online on May 27, 2009
Nucleic Acids Research 2009 37(13):4472-4481; doi:10.1093/nar/gkp400
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Nucleic Acids Research, 2009, Vol. 37, No. 13 4472-4481
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting

Lingfeng Liu, Yongfeng Li, Dennis Liotta and Stefan Lutz*

Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA

*To whom correspondence should be addressed. Tel: +1 404 712 2170; Fax: +1 404 727 6586; Email: sal2{at}emory.edu

Received April 8, 2009. Revised May 2, 2009. Accepted May 3, 2009.

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 kcat/KM 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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