Necessity of Flanking Repeats R1' and R8' of Human Pumilio1 Protein for RNA Binding.

Human Pumilio (hPUM) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1' and R8', however, remain unclear.

Genome sequence analysis of new plum pox virus isolates from Japan.

Objective: To find mutations that may have recently occurred in Plum pox virus (PPV), we collected six PPV-infected plum/peach trees from the western part of Japan and one from the eastern part. After sequencing the full-length PPV genomic RNAs, we compared the amino acid sequences with representative isolates of each PPV strain.

Results: All new isolates were found to belong to the PPV-D strain: the six isolates collected from western Japan were identified as the West-Japan strain while the one collected from eastern Japan as the East-Japan strain.

Development of a method to rapidly assess resistance/susceptibility of Micro-Tom tomatoes to Tomato yellow leaf curl virus via agroinoculation of cotyledons.

Objective: Tomato yellow leaf curl virus (TYLCV) is one of the pathogens severely damaging tomato crops. Therefore, methods to treat or prevent TYLCV infection need to be developed. For this purpose, a method to conveniently and quickly assess infection of tomatoes by TYLCV is desired.

Site-Specific Integration by Recruitment of a Complex of ΦC31 Integrase and Donor DNA to a Target Site by Using a Tandem, Artificial Zinc-Finger Protein.

To solve the problem of uncontrolled therapeutic gene integration, which is a critical drawback of retroviral vectors for gene therapy, the integration sites of exogenous genes should be precisely controlled not to perturb endogenous gene expression. To accomplish this, we explored the possibility of site-specific integration using two six-finger artificial zinc-finger proteins (AZPs) tandemly conjugated via a flexible peptide linker (designated "Tandem AZP"). A Tandem AZP in which two AZPs recognize specific 19 bp targets in a donor and acceptor DNA was expected to site-specifically recruit the donor DNA to the acceptor DNA.

Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma.

is a transcription factor essential for early mammalian development and for the maintenance of stem cells. Recently, was identified as a lineage specific oncogene, recurrently amplified and activated in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/.

Use of peptide aptamers, cationic peptides and artificial zinc finger proteins to generate resistance to plant viruses.

Various RNA/DNA viruses have caused severe infectious diseases in plants as well as animals, including humans, and been a threat to the production of agricultural crops. Therefore, prevention of plant virus infections is a major objective in crop protection. One attractive approach is to inhibit functions of viral proteins responsible for virus infections.

Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein-staphylococcal nuclease hybrid.

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired.

Inhibition of DNA replication of human papillomavirus by using zinc finger-single-chain FokI dimer hybrid.

Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously.

Sandwiched zinc-finger nucleases demonstrating higher homologous recombination rates than conventional zinc-finger nucleases in mammalian cells.

We previously reported that our sandwiched zinc-finger nucleases (ZFNs), in which a DNA cleavage domain is inserted between two artificial zinc-finger proteins, cleave their target DNA much more efficiently than conventional ZFNs in vitro. In the present study, we compared DNA cleaving efficiencies of a sandwiched ZFN with those of its corresponding conventional ZFN in mammalian cells. Using a plasmid-based single-strand annealing reporter assay in HEK293 cells, we confirmed that the sandwiched ZFN induced homologous recombination more efficiently than the conventional ZFN; reporter activation by the sandwiched ZFN was more than eight times that of the conventional one.

[Application of artificial DNA-binding proteins and artificial nucleases to prevention of virus infection: development of virus-resistant plants and protein-based anti-viral drugs].

Various DNA viruses are known to cause severe infectious diseases in both plants and mammals, including humans. For many of these infectious diseases, we have yet to find an effective prevention or treatment. Therefore, new methodologies for the prevention of virus infections in both agricultural crops and humans have been vigorously sought for a long time.

Gene- and protein-delivered zinc finger-staphylococcal nuclease hybrid for inhibition of DNA replication of human papillomavirus.

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome.

Inhibition of binding of tomato yellow leaf curl virus rep to its replication origin by artificial zinc-finger protein.

Previously we demonstrated that inhibition of replication-associated protein (Rep) binding to its replication origin by artificial zinc-finger proteins (AZPs) is a powerful method to prevent plant virus infection in vivo. In the present study, we applied the AZP technology to Tomato yellow leaf curl virus (TYLCV), which is a limiting factor in tomato cultivation worldwide. First, we determined 5'-ATCGGTGT ATCGGTGT-3' in the 195-bp intergenic region of the TYLCV-Israel strain, a strain reported first among TYLCV strains, as the Rep-binding site by gel shift assays.

Unidirectional cloning by cleaving heterogeneous sites with a single sandwiched zinc finger nuclease.

We previously developed a novel type of zinc finger nucleases (ZFNs), sandwiched ZFNs that can discriminate DNA substrates from cleavage products and thus cleave DNA much more efficiently than conventional ZFNs as well as perform with multiple turnovers like restriction endonucleases. In the present study, we used the sandwiched ZFN to unidirectionally clone exogenous genes into target vectors by cleaving heterogeneous sites that contained heterogeneous spacer DNAs between two zinc-finger protein binding sites with a single sandwiched ZFN. We demonstrated that the sandwiched ZFN cleaved a 40-fold excess of both insert and vector plasmids within 1h and confirmed by sequencing that the resulting recombinants harbored the inserted DNA fragment in the desired orientation.

Generation of cell-permeable artificial zinc finger protein variants.

Designed or artificial zinc finger proteins (ZFPs) are one of the most promising DNA-binding proteins that target genomic sequences of interest in vitro and in vivo. Conjugation of other functional domains such as transcriptional regulatory domains and endonucleases to ZFPs provided powerful molecular tools to modulate endogenous gene expression and genetic information. These ZFP variants have been introduced into cells as DNA-encoding ZFP variants by using plasmids or viral vectors.

Self-propagating artificial transcription factors to enhance upregulation of target genes.

Zinc-finger-based artificial transcription factors (ATFs) have been used to regulate expression of target genes both in vitro and in vivo. However, if we develop ATF expression further, target gene regulation by ATFs may be more effective. Here, we report a new transcriptional regulation system in which an ATF that is designed to upregulate a target gene also activates itself.

Hypoxia-specific downregulation of endogenous human VEGF-A gene by hypoxia-driven expression of artificial transcription factor.

Repression of vascular endothelial growth factor A (VEGF-A) is an attractive approach to cancer therapy. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF-A and constitutive expressions of ATFs were previously shown to downregulate the endogenous VEGF-A gene expression, repression of VEGF-A specifically in hypoxic tumors is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-driven expression of the ATF for hypoxia-specific repression of human VEGF-A gene.

Hypoxia-specific upregulation of the endogenous human VEGF-A gene by hypoxia-driven expression of artificial transcription factor.

Activation of vascular endothelial growth factor A (VEGF-A) is an attractive approach to treatment of ischemic diseases. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF-A and constitutive expression of ATFs upregulated the endogenous VEGF-A gene expression, activation of VEGF-A specifically in ischemic tissues is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-specific activation of human VEGF-A gene by hypoxia-driven expression of the ATF.

Sandwiched zinc-finger nucleases harboring a single-chain FokI dimer as a DNA-cleavage domain.

Zinc-finger nucleases (ZFNs) are a powerful tool for manipulation of genomic DNA. Recently, we reported a new ZFN composed of one artificial zinc-finger protein (AZP) and a single-chain FokI dimer (scFokI) that refines ZFN technology. While AZP-scFokI cleaved DNA specifically around the AZP-target site, several nucleotide positions were cleaved due to the mobility of the scFokI domain.

Regulation of vascular endothelial growth factor gene under hypoxia by using artificial transcription factors.

The vascular endothelial growth factor A (VEGF-A) gene is an attractive therapeutic target because both activation and repression of the gene are useful for treatment or cure of many diseases related to abnormal angiogenesis. To up- or downregulate the endogenous gene expression at will, we previously designed a 6-finger AZP to recognize a 19-bp target DNA in the VEGF-A gene, and fused the AZP with a nuclear localization signal and a transcriptional regulatory domain to generate artificial transcription factors (ATFs) for VEGF-A. Using the ATFs, we previously demonstrated efficient modulation of the endogenous VEGF-A expression under normoxia.

Construction of plants resistant to TYLCV by using artificial zinc-finger proteins.

Previously, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to tomato yellow leaf curl virus (TYLCV) causing serious damage to an important agricultural crop, tomato.

Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease.

To refine zinc-finger nuclease (ZFN) technology, we constructed a sandwiched ZFN, in which a DNA cleavage enzyme was sandwiched with two artificial zinc-finger proteins (AZPs). Because the sandwiched ZFN is designed to cleave the DNA between the two AZP-binding sites, the sandwiched ZFN is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To prove the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two 3-finger AZPs.

Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer.

Zinc-finger-FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP-scFokI). DNA cleavage assays revealed that the AZP-scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP-FokI dimer.

Zinc-finger-based artificial transcription factors and their applications.

Artificial transcription factors (ATFs) are potentially a powerful molecular tool to modulate endogenous target gene expression in living cells and organisms. To date, many DNA-binding molecules have been developed as the DNA-binding domains for ATFs. Among them, ATFs comprising Cys(2)His(2)-type zinc-finger proteins (ZFPs) as the DNA-binding domain have been extensively explored.

Enhanced cleavage of double-stranded DNA by artificial zinc-finger nuclease sandwiched between two zinc-finger proteins.

To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs.

Generation of plants resistant to tomato yellow leaf curl virus by using artificial zinc-finger proteins.

Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed and constructed an AZP binding to the direct repeat to block the TYLCV Rep binding.