Catalytic activity control of restriction endonuclease--triplex forming oligonucleotide conjugates.

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.

[1]  P. Nielsen,et al.  High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers , 2009, Nucleic acids research.

[2]  S. Mirkin,et al.  Triplex DNA structures. , 1995, Annual review of biochemistry.

[3]  Erin L. Doyle,et al.  Targeting DNA Double-Strand Breaks with TAL Effector Nucleases , 2010, Genetics.

[4]  David A Rusling,et al.  Selectivity and affinity of triplex-forming oligonucleotides containing 2'-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine for recognizing AT base pairs in duplex DNA. , 2004, Nucleic acids research.

[5]  P. Duchateau,et al.  Meganucleases and Other Tools for Targeted Genome Engineering: Perspectives and Challenges for Gene Therapy , 2011, Current gene therapy.

[6]  A. Jeltsch,et al.  Plasmid DNA cleavage by MunI restriction enzyme: single-turnover and steady-state kinetic analysis. , 1999, Biochemistry.

[7]  V. Šikšnys,et al.  Allosteric communication network in the tetrameric restriction endonuclease Bse634I. , 2006, Journal of molecular biology.

[8]  J. Concordet,et al.  Sequence-specific DNA cleavage mediated by bipyridine polyamide conjugates , 2008, Nucleic acids research.

[9]  S. Tonegawa,et al.  Creation of a large genomic deletion at the T-cell antigen receptor beta-subunit locus in mouse embryonic stem cells by gene targeting. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Toni Cathomen,et al.  Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases , 2007, Nature Biotechnology.

[11]  A. Pingoud,et al.  Photocaged variants of the MunI and PvuII restriction enzymes. , 2011, Biochemistry.

[12]  Aymeric Duclert,et al.  Generation of redesigned homing endonucleases comprising DNA-binding domains derived from two different scaffolds , 2009, Nucleic acids research.

[13]  Jia Liu,et al.  Targeted Gene Knock In and Sequence Modulation Mediated by a Psoralen-linked Triplex-forming Oligonucleotide* , 2008, Journal of Biological Chemistry.

[14]  K. Nakayama,et al.  Design and synthesis of photochemically controllable restriction endonuclease BamHI by manipulating the salt-bridge network in the dimer interface. , 2004, The Journal of organic chemistry.

[15]  R. Huber,et al.  Crystal structure of the Bse634I restriction endonuclease: comparison of two enzymes recognizing the same DNA sequence. , 2002, Nucleic acids research.

[16]  S. Neidle,et al.  DNA sequence specificity of triplex-binding ligands. , 2003, European journal of biochemistry.

[17]  Adam James Waite,et al.  An improved zinc-finger nuclease architecture for highly specific genome editing , 2007, Nature Biotechnology.

[18]  V. Šikšnys,et al.  Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: oligomeric structure-stability-function correlations. , 2005, Journal of molecular biology.

[19]  R. Huber,et al.  Crystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 Å resolution , 1999, The EMBO journal.

[20]  Yang Wang,et al.  Four base recognition by triplex-forming oligonucleotides at physiological pH , 2005, Nucleic acids research.

[21]  Dana Carroll,et al.  Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. , 2002, Genetics.

[22]  B. Spengler,et al.  Controlling the enzymatic activity of a restriction enzyme by light , 2009, Proceedings of the National Academy of Sciences.

[23]  Susan Carpenter,et al.  Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes , 2011, Nucleic acids research.

[24]  K. Nakayama,et al.  A hydrophilic azobenzene-bearing amino acid for photochemical control of a restriction enzyme BamHI. , 2005, Bioconjugate chemistry.

[25]  T. Brown,et al.  DNA triple helix formation at target sites containing several pyrimidine interruptions: stabilization by protonated cytosine or 5-(1-propargylamino)dU. , 1999, Biochemistry.

[26]  Elo Leung,et al.  Targeted Genome Editing Across Species Using ZFNs and TALENs , 2011, Science.

[27]  E. Balciunaite,et al.  DNA binding specificity of MunI restriction endonuclease is controlled by pH and calcium ions: involvement of active site carboxylate residues. , 1997, Biochemistry.

[28]  William J. Blake,et al.  Creation of a type IIS restriction endonuclease with a long recognition sequence , 2009, Nucleic acids research.

[29]  A. Jeltsch,et al.  Developing a programmed restriction endonuclease for highly specific DNA cleavage , 2005, Nucleic acids research.

[30]  T. Brown,et al.  Cg Base Pair Recognition Within Dna Triple Helices Using N -Methyl-3 H -Pyrrolo[2,3- d ]Pyrimidin-2(7 H )-One Nucleoside Analogues , 2007, Nucleosides, nucleotides & nucleic acids.

[31]  A. Janulaitis,et al.  CAATTG-specific restriction-modification munI genes from Mycoplasma: sequence similarities between R.MunI and R.EcoRI. , 1994, Gene.

[32]  David A Rusling,et al.  Stable recognition of TA interruptions by triplex forming oligonucleotides containing a novel nucleoside. , 2005, Biochemistry.

[33]  T. Brown,et al.  CG base pair recognition within DNA triple helices by modified N-methylpyrrolo-dC nucleosides. , 2010, Organic & biomolecular chemistry.

[34]  K. Nakayama,et al.  Photochemical regulation of the activity of an endonuclease BamHI using an azobenzene moiety incorporated site-selectively into the dimer interface. , 2004, Chemical communications.

[35]  P. Duchateau,et al.  Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. , 2006, Journal of molecular biology.

[36]  J. Alves,et al.  A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity , 2004, Biological chemistry.

[37]  R. Kucherlapati,et al.  Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination , 1985, Nature.

[38]  M. Bibikova,et al.  Efficient Gene Targeting in Drosophila With Zinc-Finger Nucleases , 2006, Genetics.

[39]  S Chandrasegaran,et al.  Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[40]  C. Bailly,et al.  Exploring the Cellular Activity of Camptothecin-Triple-Helix-Forming Oligonucleotide Conjugates , 2006, Molecular and Cellular Biology.

[41]  Frédéric Pâques,et al.  Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. , 2007, Current gene therapy.