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1.
Mol Biotechnol ; 60(5): 339-349, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29524201

RESUMO

There have been many attempts to unveil the therapeutic potential of antisense molecules during the last decade. Due to its specific role in canonical Wnt signalling, ß-catenin is a potential target for an antisense-based antitumour therapy. In order to establish such a strategy with peptide nucleic acids, we developed a reporter assay for quantification of antisense effects. The luciferase-based assay detects splice blocking with high sensitivity. Using this assay, we show that the splice donor of exon 13 of ß-catenin is particularly suitable for an antisense strategy, as it results in a truncated protein which lacks transactivating functions. Since the truncated proteins retain the interactions with Tcf/Lef proteins, they act in a dominant negative fashion competing with wild-type proteins and thus blocking the transcriptional activity of ß-catenin. Furthermore, we show that the truncation does not interfere with binding of cadherin and α-catenin, both essential for its function in cell adhesion. Therefore, the antisense strategy blocks Wnt signalling with high efficiency but retains other important functions of ß-catenin.


Assuntos
Técnicas de Silenciamento de Genes/métodos , Ácidos Nucleicos Peptídicos/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Éxons , Células HEK293 , Células HeLa , Humanos , Sítios de Splice de RNA/efeitos dos fármacos , Fatores de Transcrição TCF/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismo
2.
BMC Biotechnol ; 18(1): 1, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29316906

RESUMO

BACKGROUND: The application of antisense molecules, such as morpholino oligonucleotides, is an efficient method of gene inactivation in vivo. We recently introduced phosphonic ester modified peptide nucleic acids (PNA) for in vivo loss-of-function experiments in medaka embryos. Here we tested novel modifications of the PNA backbone to knockdown the medaka tcf3 gene. RESULTS: A single tcf3 gene exists in the medaka genome and its inactivation strongly affected eye development of the embryos, leading to size reduction and anophthalmia in severe cases. The function of Tcf3 strongly depends on co-repressor interactions. We found interactions with Groucho/Tle proteins to be most important for eye development. Using a dominant negative approach for combined inactivation of all groucho/tle genes also resulted in eye phenotypes, as did interference with three individual tle genes. CONCLUSIONS: Our results show that side chain modified PNAs come close to the knockdown efficiency of morpholino oligonucleotides in vivo. A single medaka tcf3 gene combines the function of the two zebrafish paralogs hdl and tcf3b. In combination with Groucho/Tle corepressor proteins Tcf3 acts in anterior development and is critical for eye formation.


Assuntos
Olho/embriologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Técnicas de Silenciamento de Genes/métodos , Oryzias/embriologia , Animais , Animais Geneticamente Modificados , Anoftalmia/genética , Embrião não Mamífero/fisiologia , Anormalidades do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Morfolinos/genética , Oryzias/genética , Ácidos Nucleicos Peptídicos/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
BMC Biotechnol ; 12: 50, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22901024

RESUMO

BACKGROUND: Synthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications. RESULTS: We generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes. CONCLUSIONS: PNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.


Assuntos
Proteínas do Olho/genética , Proteínas de Peixes/genética , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Oryzias/genética , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/genética , Animais , Sequência de Bases , DNA Antissenso/síntese química , DNA Antissenso/química , DNA Antissenso/genética , Técnicas de Inativação de Genes , Dados de Sequência Molecular , Estrutura Molecular , Conformação de Ácido Nucleico , Ácidos Nucleicos Peptídicos/síntese química , Proteína Homeobox SIX3
4.
Mol Med ; 18: 111-22, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22105607

RESUMO

Although rapidly becoming a valuable tool for gene silencing, regulation or editing in vitro, the direct transfer of small interfering ribonucleic acids (siRNAs) into cells is still an unsolved problem for in vivo applications. For the first time, we show that specific modifications of antisense oligomers allow autonomous passage into cell lines and primary cells without further adjuvant or coupling to a cell-penetrating peptide. For this reason, we termed the specifically modified oligonucleotides "cell membrane-crossing oligomers" (CMCOs). CMCOs targeted to various conserved regions of human immunodeficiency virus (HIV)-1 were tested and compared with nontargeting CMCOs. Analyses of uninfected and infected cells incubated with labeled CMCOs revealed that the compounds were enriched in infected cells and some of the tested CMCOs exhibited a potent antiviral effect. Finally, the CMCOs did not exert any cytotoxicity and did not inhibit proliferation of the cells. In vitro, our CMCOs are promising candidates as biologically active anti-HIV reagents for future in vivo applications.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV/efeitos dos fármacos , HIV/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Replicação Viral/efeitos dos fármacos , Fármacos Anti-HIV/química , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Oligonucleotídeos Antissenso/química
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