title: LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells.
abstract: Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands;
authors.forename: Thomas W
authors.initials: TW
authors.lastname: van Ravesteyn
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands;
authors.forename: Marleen
authors.initials: M
authors.lastname: Dekker
authors.affiliation: Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Alexander
authors.initials: A
authors.lastname: Fish
authors.affiliation: Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Titia K
authors.initials: TK
authors.lastname: Sixma
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands;
authors.forename: Astrid
authors.initials: A
authors.lastname: Wolters
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands;
authors.forename: Rob J
authors.initials: RJ
authors.lastname: Dekker
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; h.t.riele@nki.nl.
authors.forename: Hein P J
authors.initials: HP
authors.lastname: Te Riele
mesh_terms: D000818:Animals; D020137:Base Pair Mismatch; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D053843:DNA Mismatch Repair; D004260:DNA Repair; D004277:DNA, Single-Stranded; D004926:Escherichia coli / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics
chemical_list: D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D004247:DNA
keywords: DNA mismatch repair; embryonic stem cells; locked nucleic acid; single-stranded oligonucleotides; subtle gene modification