Nucleic Acids Research Advance Access published online on April 10, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm182
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Nucleic Acid Enzymes |
Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases
1Department of Pediatrics, University of Washington, Box 359300-CW, Seattle WA 98195, USA; Children's Hospital, & Regional Medical Center, 307 Westlake Ave N Suite 300, Seattle WA 98109, USA, 2Department of Immunology, University of Washington, 1959 Pacific Street NE, Seattle WA 98195, USA; Children's Hospital & Regional Medical Center, 307 Westlake Ave N Suite 300, Seattle WA 98109, USA, 3Department of Pathology, University of Washington, Seattle WA 98195, USA, 4Institute of Biochemical Sciences, Academia Sinica, 128 Yen-chiu-yuan Rd., sec. 2, Tapei, Taiwan, 5Department of Pathology, University of Washington, Box 357705, Seattle WA 98195, USA, 6Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, A3-025, Seattle WA 98109, USA, and 7Department of Pediatrics, University of Washington, Box 359300-CW, Seattle WA 98195, USA; Department of Immunology, University of Washington, 1959 Pacific Street NE, Seattle WA 98195, USA; Children's Hospital & Regional Medical Center, 307 Westlake Ave N Suite 300, Seattle WA 98109, USA
*To whom correspondence should be addressed. To whom correpondence should be addressed. Tel: +1 206 987 7314; Fax: +1 206 987 7310; Email: andrewms{at}u.washington.edu
Received February 6, 2007. Revised March 12, 2007. Accepted March 13, 2007.
LAGLIDADG homing endonucleases (LHEs) cleave 18–24 bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE–dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities.
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