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Nucleic Acids Research Advance Access originally published online on April 10, 2007
Nucleic Acids Research 2007 35(8):2748-2758; doi:10.1093/nar/gkm182
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Nucleic Acids Research, 2007, Vol. 35, No. 8 2748-2758
© 2007 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

Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases

Petra Volná1,2, Jordan Jarjour2,3, Sarah Baxter4, Steve R. Roffler5, Raymond J. Monnat, Jr4, Barry L. Stoddard6 and Andrew M. Scharenberg1,2,3,*

1Department of Pediatrics, University of Washington, Box 359300-CW, Seattle WA 98195, USA, 2Children's Hospital, & Regional Medical Center, 307 Westlake Ave N Suite 300, Seattle WA 98109, USA, 3Department of Immunology, University of Washington, 1959 Pacific Street NE, Seattle WA 98195, USA, 4Department of Pathology, University of Washington, Seattle WA 98195, USA, 5Institute of Biochemical Sciences, Academia Sinica, 128 Yen-chiu-yuan Rd., sec. 2, Tapei, Taiwan, 6Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, A3-025, Seattle WA 98109, USA

*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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