Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases.
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.
Volná
Petra
P
Department of Pediatrics, University of Washington, Box 359300-CW, Seattle WA 98195, USA.
Jarjour
Jordan
J
Baxter
Sarah
S
Roffler
Steve R
SR
Monnat
Raymond J
RJ
Stoddard
Barry L
BL
Scharenberg
Andrew M
AM
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002469:Cell Separation; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D008280:Magnetics; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins / Q000032:analysis; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases