Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Volná, Petra, Jarjour, Jordan, Baxter, Sarah, Roffler, Steve R., Monnat, Raymond J., Stoddard, Barry L., Scharenberg, Andrew M.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885675/
https://www.ncbi.nlm.nih.gov/pubmed/17426121
http://dx.doi.org/10.1093/nar/gkm182
_version_ 1782133643161894912
author Volná, Petra
Jarjour, Jordan
Baxter, Sarah
Roffler, Steve R.
Monnat, Raymond J.
Stoddard, Barry L.
Scharenberg, Andrew M.
author_facet Volná, Petra
Jarjour, Jordan
Baxter, Sarah
Roffler, Steve R.
Monnat, Raymond J.
Stoddard, Barry L.
Scharenberg, Andrew M.
author_sort Volná, Petra
collection PubMed
description 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.
format Text
id pubmed-1885675
institution National Center for Biotechnology Information
language English
publishDate 2007
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-18856752007-06-07 Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases Volná, Petra Jarjour, Jordan Baxter, Sarah Roffler, Steve R. Monnat, Raymond J. Stoddard, Barry L. Scharenberg, Andrew M. Nucleic Acids Res Nucleic Acid Enzymes 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. Oxford University Press 2007-04 2007-04-10 /pmc/articles/PMC1885675/ /pubmed/17426121 http://dx.doi.org/10.1093/nar/gkm182 Text en © 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.
spellingShingle Nucleic Acid Enzymes
Volná, Petra
Jarjour, Jordan
Baxter, Sarah
Roffler, Steve R.
Monnat, Raymond J.
Stoddard, Barry L.
Scharenberg, Andrew M.
Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases
title Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases
title_full Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases
title_fullStr Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases
title_full_unstemmed Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases
title_short Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases
title_sort flow cytometric analysis of dna binding and cleavage by cell surface-displayed homing endonucleases
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885675/
https://www.ncbi.nlm.nih.gov/pubmed/17426121
http://dx.doi.org/10.1093/nar/gkm182
work_keys_str_mv AT volnapetra flowcytometricanalysisofdnabindingandcleavagebycellsurfacedisplayedhomingendonucleases
AT jarjourjordan flowcytometricanalysisofdnabindingandcleavagebycellsurfacedisplayedhomingendonucleases
AT baxtersarah flowcytometricanalysisofdnabindingandcleavagebycellsurfacedisplayedhomingendonucleases
AT rofflerstever flowcytometricanalysisofdnabindingandcleavagebycellsurfacedisplayedhomingendonucleases
AT monnatraymondj flowcytometricanalysisofdnabindingandcleavagebycellsurfacedisplayedhomingendonucleases
AT stoddardbarryl flowcytometricanalysisofdnabindingandcleavagebycellsurfacedisplayedhomingendonucleases
AT scharenbergandrewm flowcytometricanalysisofdnabindingandcleavagebycellsurfacedisplayedhomingendonucleases