Application of fluorescent nucleoside analogs in direct evolution of Drosophila melanogaster deoxyribonucleoside kinase Open Access

Zhang, Xiao (2009)

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Nucleoside analogs (NAs) are widely used as prodrugs, such as in highly active antiretroviral therapy (HAART) and chemotherapy. NAs require cellular enzymes to convert them into their active triphosphate form in order to be incorporated into DNA by low fidelity polymerase such as HIV reverse transcriptase or polymerase β in cancer cells during replication cycle, functioning as reverse transcriptase and polymerase inhibitor. Phosphorylation of NAs to monophosphates by deoxyribonucleoside kinases (dNKs) is usually the 'bottle neck' in prodrug activation. Engineering 2'-deoxyribonucleoside kinases with higher activity and orthogonal specificity for nucleoside analogs is under intensive study. However, the directed evolution of dNKs is hampered by a lack of efficient library screening techniques. A method combining fluorescent nucleoside analogs with fluorescent-active cell sorting (FACS) has been developed in our lab and evaluated by evolving Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) using fluorescent 2',3'-dideoxythymidine (fddT) as substrate. As part of my thesis, I have prepared a fluorescent 3'-fluoro-2'-deoxyuridine (fFT) and explored its use to evolve kinases that are specific to 3'-fluoro-2'-deoxyuridine (FT). My results show that the partial hydrogen bond interaction between the 3' fluoro moiety and active site residues is beneficial for enrichment of desirable Dm-dNK mutants by FACS. Seperately, I have investigated alternative fluorescent probes that do not clash with residues in the active site, employing 1, 3-dipolar cycloaddition of alkenophiles with diaryl-tetrazoles.

Table of Contents

1 Introduction...1

1.1 DNA replication...1
1.2 Nucleoside analogs and nucleoside kinases...2
1.3 Protein engineering of deoxyribonucleoside kinases...4
1.4 Library screening and fluorescent-active cell sorting...6

2 Results and Discussion...11

2.1 Synthesis of fluorescent 3-fluoro-2'-deoxyuridine (fFT)...11
2.2 Protein expression and purification...12
2.3 Kinetic assay for 3-fluoro-2'-deoxyuridine (FT)...13
2.4 Screening 1st-round of mutagenesis library by FACS...14
2.5 Synthesis of 5-vinyl-2'-deoxyuridine and tetrazole...15
2.6 Kinetic assay for 5-vinyl-2'-deoxyuridine...16
2.7 Photoinducible 1, 3-dipolar cycloaddition...17

3 Materials and Methods...19

Experimental procedure...20


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