Showing papers on "Deoxyribozyme published in 2001"
TL;DR: The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes and these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.
Abstract: The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2′-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.
137 citations
TL;DR: This work illustrates how the incorporation of protein-like functionalities in nucleic acids can bridge the gap between proteins and oligonucleotides underscoring the potential for using nucleic acid scaffolds in the development of new materials and improved catalysts for use in chemistry and medicine.
Abstract: Two synthetically modified nucleoside triphosphate analogues (adenosine modified with an imidazole and uridine modified with a cationic amine) are enzymatically polymerized in tandem along a degenerate DNA library for the combinatorial selection of an RNAse A mimic. The selected activity is consistent with both electrostatic and general acid/base catalysis at physiological pH in the absence of divalent metal cations. The simultaneous use of two modified nucleotides to enrich the catalytic repertoire of DNA-based catalysts has never before been demonstrated and evidence of general acid/base catalysis at pH 7.4 for a DNAzyme has never been previously observed in the absence of a divalent metal cation or added cofactor. This work illustrates how the incorporation of protein-like functionalities in nucleic acids can bridge the gap between proteins and oligonucleotides underscoring the potential for using nucleic acid scaffolds in the development of new materials and improved catalysts for use in chemistry and medicine.
136 citations
TL;DR: These constructs demonstrate that antisense conformational triggers based on molecular beacons can be used to initiate catalytic events and have potential for the construction of deoxyribozyme‐based drug delivery tools specifically activated in cells containing somatic mutations.
Abstract: We have constructed catalytic molecular beacons from a hammerhead-type deoxyribozyme by a modular design. The deoxyribozyme was engineered to contain a molecular beacon stem-loop module that, when closed, inhibits the deoxyribozyme module and is complementary to a target oligonucleotide. Binding of target oligonucleotides opens the beacon stem-loop and allosterically activates the deoxyribozyme module, which amplifies the recognition event through cleavage of a doubly labeled fluorescent substrate. The customized modular design of catalytic molecular beacons allows for any two single-stranded oligonucleotide sequences to be distinguished in homogenous solution in a single step. Our constructs demonstrate that antisense conformational triggers based on molecular beacons can be used to initiate catalytic events. The selectivity of the system is sufficient for analytical applications and has potential for the construction of deoxyribozyme-based drug delivery tools specifically activated in cells containing somatic mutations.
133 citations
TL;DR: A copper-dependent self-cleaving DNA that was isolated by in vitro selection has been minimized to its smallest active domain using both in vitroselection and rational design methods.
114 citations
Patent•
25 Jul 2001
TL;DR: The present paper relates to nucleic acid molecules, including antisense and enzymatic nucleic acids molecules, such as hammerhead ribozymes, DNAzymes, allozymes and antisense, which modulate the expression of epidermal growth factor receptor genes as mentioned in this paper.
Abstract: The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, allozymes and antisense, which modulate the expression of epidermal growth factor receptor genes.
111 citations
TL;DR: Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.
Abstract: The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Taq polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by XbaI, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.
104 citations
TL;DR: The Bipartite DNAzyme represents a new and potentially useful reagent, both for the processing of RNA transcripts in vitro and for mRNA ablation procedures in vivo, and may share similar catalytic strategies with the hepatitis delta virus ribozyme.
92 citations
TL;DR: RNA and DNA molecules with catalytic properties have been isolated by in vitro selection from combinatorial nucleic acid libraries by enhancing the catalytic performance of nucleic acids by the incorporation of additional functional groups.
64 citations
TL;DR: The ability of RNA and DNA to form branched three-way junctions to regulate the RNA-cleaving activity of the in vitro selected "10-23" DNAzyme by three orders of magnitude is utilized.
49 citations
TL;DR: This work describes a method that identifies RNA regions that are accessible to oligonucleotides and shows that construction of hammerhead ribozymes targeted to the identified regions results in catalytic activities that are consistently and substantially greater than those of Ribozymes designed on the basis of computer modeling.
Abstract: Although ribozymes offer tremendous potential for posttranscriptionally controlling expression of targeted genes, their utility is often limited by the accessibility of the targeted regions within the RNA transcripts. Here we describe a method that identifies RNA regions that are accessible to oligonucleotides. Based on this selection protocol, we show that construction of hammerhead ribozymes targeted to the identified regions results in catalytic activities that are consistently and substantially greater than those of ribozymes designed on the basis of computer modeling. Identification of accessible sites should also be widely applicable to design of antisense oligonucleotides and DNAzymes.
41 citations
TL;DR: Two mono- and a di-RNA-cleaving DNA enzymes with the 10-23 catalytic motif were synthesized that were targeted to cleave at the conserved site/sites of the X gene of the hepatitis B virus, showing specific inhibition of X-gene-mediated transactivation of reporter-gree expression.
Abstract: Two mono- and a di-RNA-cleaving DNA enzymes with the 10-23 catalytic motif were synthesized that were targeted to cleave at the conserved site/sites of the X gene of the hepatitis B virus In each case, protein-independent but Mg(2+)-dependent cleavage of in vitro-synthesized full-length X RNA was obtained Specific cleavage products were obtained with two different mono- and a di-DNA enzyme, with the latter giving rise to multiple RNA fragments that retained the cleavage specificity of the mono-DNA enzymes A relatively less efficient cleavage was also obtained under simulated physiological conditions by the two mono-DNA enzymes but the efficiency of the di-DNA enzyme was significantly reduced A single nucleotide change (G to C) in the 10-23 catalytic motif of the DNA enzyme 307 abolished its ability to cleave target RNA completely Both, mono- and di-DNA enzymes, when introduced into a mammalian cell, showed specific inhibition of X-gene-mediated transactivation of reporter-gene expression This decrease was due to the ability of these DNA enzymes to cleave X RNA intracellularly, which was also reflected by significant reduction in the levels of X protein in a liver-specific cell line, HepG2 Ribonuclease protection assay confirmed the specific reduction of X RNA in DNA-enzyme-treated cells Potential in vivo applications of mono- and di-DNA enzymes in interfering specifically with the X-gene-mediated pathology are discussed
Patent•
19 Oct 2001
TL;DR: In this paper, the authors present an invention related to nucleic acid molecules (e.g., ribozymes, DNAzymes, antisense oligonucleotides, triplex forming oligonuclideans, 2,5-A oligonules, decoys) that modulate the expression and/or replication of West Nile Virus (WNV).
Abstract: The present invention relates to nucleic acid molecules (e.g., ribozymes, DNAzymes, antisense oligonucleotides, triplex forming oligonucleotides, 2,5-A oligonucleotides, decoys) that modulate the expression and/or replication of West Nile Virus (WNV). The present invention also relates to chemical compositions, and methods of treatment of WNV infection and associated diseases, and conditions.
TL;DR: In this paper, the deoxyribozymes that can catalyze the formation of such phosphorothioester internucleotide linkages were selected to enhance the rate of ligation in part through the provision of a template that aligns the ligation junction.
TL;DR: These findings suggest that DNA constructs could be engineered to efficiently catalyze other self-modifying reactions, including ATP-dependent DNA ligation, which is of significant interest in self- modification reactions, given the fundamental role that DNA serves in modern living systems.
TL;DR: The phosphorothioate modification of the DNAzyme significantly increased its stability in cells without a substantial loss of kinetic efficiency in vitro and indicated the potential use of DNAzyme technology for gene function study and cancer therapy.
Patent•
02 Feb 2001TL;DR: The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acids molecules, such as hammerhead ribozymes, DNAzymes, and antisense, which modulate the expression of the Chk-1 gene.
Abstract: The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, and antisense, which modulate the expression of the Chk-1 gene
Patent•
08 Feb 2001
TL;DR: The present invention relates to enzymatic nucleic acid molecules, including ribozymes and nucleozymes (DNA catalysts, DNA enzymes), methods of synthesis, and uses thereof as mentioned in this paper.
Abstract: The present invention relates to enzymatic nucleic acid molecules, including ribozymes and nucleozymes (DNA catalysts, DNA enzymes), methods of synthesis, and uses thereof.
Patent•
16 May 2001
TL;DR: Nucleic acid molecules, including antisense and enzymatic nucleic acid molecule, such as hammerhead ribozymes, DNAzymes, and GeneBlocs, can modulate the expression of ERG (Ets-related gene) as discussed by the authors.
Abstract: Nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, and GeneBlocs, which modulate the expression of ERG (Ets-related gene).
Patent•
09 Feb 2001
TL;DR: In this article, the present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acids molecules, such as hammerhead ribozymes, DNAzymes, and antisense which modulate the expression of the CD20 and/or NOGO genes.
Abstract: The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, and antisense, which modulate the expression of the CD20 and/or NOGO genes. Diagnostic systems and methods for detecting the presence of nucleic acids are further disclosed.
Patent•
09 Aug 2001
TL;DR: Nucleic acid molecules, including antisense and enzymatic nucleic acids, such as hammerhead ribozymes, DNAzymes, and GeneBlocs, which modulate the expression of calcium activated chloride channels (CLCA1, CLCA2, CRLCA3, and CRCLA4) as discussed by the authors.
Abstract: Nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, and GeneBlocs, which modulate the expression of calcium activated chloride channels (CLCA1, CLCA2, CLCA3, and CLCA4)
Patent•
05 Apr 2001
TL;DR: In this article, the present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acids molecules, such as hammerhead ribozymes, DNAzymes, and antisense, which modulate the expression of NOGO receptor genes.
Abstract: The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, and antisense, which modulate the expression of NOGO and NOGO receptor genes.
Patent•
09 Feb 2001
TL;DR: The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acids molecules, such as hammerhead ribozymes, DNAzymes, and antisense, which modulate the expression of NOGO gene.
Abstract: The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, and antisense, which modulate the expression of NOGO gene.
TL;DR: In this article, the development of functional nucleic acids and peptides by combinatorial chemistry and downsizing methods is described, and the metal ion effect on their activity is discussed.
Abstract: Development of functional nucleic acids and peptides by combinatorial chemistry and downsizing methods is described. First, catalytic short RNAs (ribozymes) and DNAs (deoxyribozymes) are made and the metal ion effect on their activity is discussed. Surprisingly, these small nucleic acids can cleave RNA substrates at only one site precisely. Second, a novel and only 24 residue peptide derived from RecA protein can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA). A new developed support with this peptide recognizes ssDNA but not dsDNA. Finally, a novel screening method of combinatorial chemistry and its application for studying interactions between life molecules including sugars are reviewed.
Patent•
23 Feb 2001
TL;DR: In this paper, the present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acids molecules, such as hammerhead ribozymes, DNAzymes, and antisense which modulate the expression of the GRID (Grb2-related with Insert Domain) gene.
Abstract: The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, and antisense, which modulate the expression of the GRID (Grb2-related with Insert Domain) gene.