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

24Citations
Citations of this article
19Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

LAGLIDADG homing endonucleases (LHEs) cleave 18-24bp 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. © 2007 The Author(s).

Cite

CITATION STYLE

APA

Volná, P., Jarjour, J., Baxter, S., Roffler, S. R., Monnat, R. J., Stoddard, B. L., & Scharenberg, A. M. (2007). Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases. Nucleic Acids Research, 35(8), 2748–2758. https://doi.org/10.1093/nar/gkm182

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free