Zinc finger nucleases (ZFN) have been used to direct precise modifications to the genetic information in living cells at high efficiency. An important consideration in the design of ZFNs is the number of zinc fingers that are required for efficient and specific cleavage. We examined dimeric ZFNs composed of 1+1, 2+2, 3+3, 4+4, 5+5 and 6+6 zinc fingers, targeting 6, 12, 18, 24, 30 and 36 bp, respectively. We found that 1+1 and 2+2 fingers supported neither in vitro cleavage nor single-strand annealing in a cell-based recombination assay. An optimum of ZFN activity was observed for 3+3 and 4+4 fingers. Surprisingly, 5+5 and 6+6 showed significantly reduced activity. While the extra fingers were not found to dramatically increase toxicity, directly inhibit recombination, or perturb the ZFN target site, we demonstrate the ability of subsets of three fingers in six-finger arrays to bind independently to regions of the target site, possibly explaining the decrease in activity. These results have important implications for the design of new ZFNs, as they show that in some cases an excess of fingers may actually decrease the performance of engineered multi-finger proteins. Maximum ZFN activity will require an optimization of both DNA binding affinity and specificity.

Zinc finger nucleases can be engineered to create highly efficient and precise changes to the genetic information within living cells. We report the investigation of an important parameter that defines the type of target site the nuclease can cleave. The active nuclease is a dimer, requiring that the DNA target site contain two zinc finger binding sites separated by a short spacer. Using a plasmid-based recombination assay in HEK 293T cells, we show that a 6 amino acid linker between the zinc finger DNA-binding domain and the FokI cleavage domain restricts nuclease activity to sites containing a 6 bp spacer. These observations concur with other recent studies, suggesting this information will be useful in the design of new potent and accurate zinc finger nucleases.Keywords gene therapy; gene targeting; homologous recombination; targeted mutagenesis; protein engineering; zinc finger nuclease The C2H2 class of zinc finger proteins has proven to be the most useful scaffold for engineering custom DNA-binding proteins. 1, 2 Several elegant and successful zinc finger engineering methods have been and continue to be developed. 3-8 The Modular Assembly methodology developed by Barbas and co-workers 3, 4 was one of the earliest and easiest to use, and has therefore played a seminal role in the development of number of targetable gene regulation and gene modification applications, including artificial transcription factors 9 and zinc finger nucleases (ZFNs). 10, 11 Among these, ZFNs have generated particular excitement because of their potential to catalyze the targeted disruption or correction of genetic information within living cells, at efficiencies that can not currently be achieved using other methods. 12ZFNs create double-strand breaks (DSBs) on DNA in cells, stimulating endogenous DNA repair mechanisms to repair the breaks. In mammalian cells, such breaks are primarily repaired by a nonhomologous end-joining pathway (NHEJ), which frequently leads to mutations at the cleavage site. Mutagenesis in as highs as 54% of targeted loci has been reported, 13 presenting ZFNs as a potent tool for targeted gene disruption. The presence of a repair template, typically an exogenous DNA with homology to the target locus, additional allows some of the breaks to be repaired by homologous recombination (HR). Targeted replacements and gene insertions in as high as 50% of target loci have been reported, 8, 14 representing a >100,000-fold *To whom correspondence should be addressed: David J. Segal, Ph.D., University of California, Davis, Genome Center/Pharmacology, 4513 GBSF, 451 E. Health Sciences Dr., Davis, CA 95616,, Email: E-mail: djsegal@ucdavis.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production proces...

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New bone is generated at an early stage of bone formation using BG for bone augmentation.

Transformations of enantioenriched donor‐acceptor (D−A) cyclopropylcarbinols to enantioenriched 1‐arylnaphthalenes that bear an ortho‐substituent on a benzene ring provided a successful chirality‐exchange method with a high level of stereoinduction. A central chirality‐transfer step, i. e., a Lewis‐acid‐mediated ring‐opening cyclization of enantioenriched D‐A cyclopropylcarbinols (97 to >99 % ee), afforded 1‐aryl‐1,2‐dihydronaphthalenes with an ortho‐substituent (Me, Br, OMe, OBn, or OiPr) on the benzene ring with high enantioselectivity (97 to >99 % ee). The central‐to‐axial chirality‐exchange step, i. e., the dehydrogenation of the obtained enantioenriched 1‐aryl‐1,2‐dihydronaphthalenes using DDQ, furnished the axially chiral 1‐arylnaphthalenes with high enantioselectivity (90 to >99 % ee). Importantly, we developed a highly enantioselective synthesis of a 1‐arylnaphthalene with an ortho‐alkoxy group on the benzene ring. Moreover, we improved the chirality exchange to furnish a 1‐arylnaphthalene with an ortho‐alkoxy‐substituted benzene ring in high yield with high ee.