title: Catalytic activity control of restriction endonuclease--triplex forming oligonucleotide conjugates.
abstract: Targeting of individual genes in complex genomes requires endonucleases of extremely high specificity. To direct cleavage at the unique site(s) in the genome, both naturally occurring and artificial enzymes have been developed. These include homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and restriction or chemical nucleases coupled to a triple-helix forming oligonucleotide (TFO). The desired cleavage has been demonstrated both in vivo and in vitro for several model systems. However, to limit cleavage strictly to unique sites and avoid undesired reactions, endonucleases with controlled activity are highly desirable. In this study we present a proof-of-concept demonstration of two strategies to generate restriction endonuclease-TFO conjugates with controllable activity. First, we combined the restriction endonuclease caging and TFO coupling procedures to produce a caged MunI-TFO conjugate, which can be activated by UV-light upon formation of a triple helix. Second, we coupled TFO to a subunit interface mutant of restriction endonuclease Bse634I which shows no activity due to impaired dimerization but is assembled into an active dimer when two Bse634I monomers are brought into close proximity by triple helix formation at the targeted site. Our results push the restriction endonuclease-TFO conjugate technology one step closer to potential in vivo applications.
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Arunas
authors.initials: A
authors.lastname: Silanskas
authors.forename: Mindaugas
authors.initials: M
authors.lastname: Zaremba
authors.forename: Giedrius
authors.initials: G
authors.lastname: Sasnauskas
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D055162:Biocatalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D004720:Endonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D014466:Ultraviolet Rays
chemical_list: D009841:Oligonucleotides; D004247:DNA; D004720:Endonucleases