Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease.
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PMID:Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1. 850 50

A new generation of PNAs, so-called pseudocomplementary PNAs (pcPNAs), which are able to target the designated sites on duplex DNA with mixed sequence of purines and pyrimidines via double-duplex invasion mode, has recently been introduced. It has been demonstrated that appropriate pairs of decameric pcPNAs block an access of RNA polymerase to the corresponding promoter. Here, we show that this type of PNAs protects selected DNA sites containing all four nucleobases from the action of restriction enzymes and DNA methyltransferases. We have found that pcPNAs as short as octamers form stable and sequence-specific complexes with duplex DNA in a very salt-dependent manner. In accord with a strand-invasion mode of complex formation, the pcPNA binding proceeds much faster with supercoiled than with linear plasmids. The double-duplex invasion complexes selectively shield specific DNA sites from BclI restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate DNA duplexes in a virtually sequence-unrestricted manner.
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PMID:Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs. 1097 78

Data on five single-nucleotide polymorphisms (SNPs) per gene are estimated to allow association of disease risks or pharmacogenetic parameters with individual genes. Efficient technologies for rapidly detecting SNPs will therefore facilitate the mining of genomic information. Known methods for SNP analysis include restriction-fragment-length polymorphism polymerase chain reaction (PCR), allele-specific oligomer hybridization, oligomer-specific ligation assays, minisequencing, direct sequencing, fluorescence-detected 5'-exonuclease assays, and hybridization with PNA probes. Detection by mass spectrometry (MS) offers speed and high resolution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) can detect primer extension products, mass-tagged oligonucleotides, DNA created by restriction endonuclease cleavage, and genomic DNA. We have previously reported MALDI-TOF-monitored nuclease selections of modified oligonucleotides with increased affinity for targets. Here we use nuclease selections for genotyping by treating DNA to be analyzed with oligonucleotide probes representing known genotypes and digesting probes that are not complementary to the DNA. With phosphodiesterase I, the target-bound, complementary probe is largely refractory to nuclease attack and its peak persists in mass spectra (Fig. 1A). In optimized assays, both alleles of a heterozygote were genotyped with six nonamer DNA probes (> or = 125 fmol each) and asymmetrically amplified DNA from exon 10 of the cystic fibrosis transmembrane regulatory gene (CFTR).
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PMID:Rapid genotyping by MALDI-monitored nuclease selection from probe libraries. 1106 45

This study evaluates the potential of pseudocomplementary peptide nucleic acids (pcPNAs) for sequence-specific modification of enzyme activity towards double-stranded DNA (dsDNA). To this end, we analyze the ability of pcPNA-dsDNA complexes to site-selectively interfere with the action of four type IIs restriction enzymes. We have found that pcPNA-dsDNA complexes exhibit a different degree of DNA protection against cleaving/nicking activity of various isoschizomeric endonucleases under investigation (PleI, MlyI and N.BstNBI) depending on their type and mutual arrangement of PNA-binding and enzyme recognition/cleavage sites. We have also found that the pcPNA targeting to closely located PleI or BbsI recognition sites on dsDNA generates in some cases the nicking activity of these DNA cutters. At the same time, MlyI endonuclease, a PleI isoschizomer, does not exhibit any DNA nicking/cleavage activity, being completely blocked by the nearby pcPNA binding. Our results have general implications for effective pcPNA interference with the performance of DNA-processing proteins, thus being important for prospective applications of pcPNAs.
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PMID:Pseudocomplementary PNAs as selective modifiers of protein activity on duplex DNA: the case of type IIs restriction enzymes. 1285 8

Three chimeric dimer synthons (oeg_t(NH)T, oeg_up(NH)T and oeg_uh(NH)T) containing thymine (t), 5-(1-propynyl)-uracil (up) and 5-(1-hexyn-1-yl)-uracil (uh) PNA units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized in solution and incorporated into T20 oligonucleotide analogues, using standard P-amidite chemistry. Insertion of dimer blocks led to destabilization of duplexes with dA20 target. The smallest Tm drops were found for chimeras containing oeg_up(NH)T dimers. Incorporation of the chimeric synthons into the 3'-end of T20 brought about growing resistance to 3'-exonucleolytic (SV PDE) cleavage in the order of oeg_t(NH)T < oeg_up(NH)T < oeg_uh(NH)T. Due to different endonuclease activities of 3'- and 5'-exonucleases applied, placing of five consecutive dimers at the 5'-terminus resulted in a relatively smaller, but also side-chain dependent, stabilization towards the hydrolysis by 5'-exonuclease (BS PDE). Neither exonucleases (SV and BS PDE) nor an endonuclease (Nuclease P1) could hydrolyse the unnatural phosphodiester bond linking the 3'-OH of thymidine to the terminal OH of N-(2-hydroxyethyl)glycine PNA backbone.
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PMID:PNA-DNA chimeras containing 5-alkynyl-pyrimidine PNA units. Synthesis, binding properties, and enzymatic stability. 1460 35

The current WHO classification of mastocytosis defines one major and four minor diagnostic criteria for systemic mastocytosis (SM). One of the minor criteria is the detection of the "gain-of-function" mutation D816V of the c-kit proto-oncogene in extracutaneous organs. The receptor molecule KIT is a potential therapeutic target for tyrosine kinase inhibitors. KIT mutations have been described in more than 80% of SM, but only in the minority of cutaneous mastocytoses (CM). Usually exon 17 amplicons generated by polymerase chain reaction are analyzed for the detection of c-kit mutations. Most frequently the method of restriction fragment length polymorphism (RFLP) analysis using the endonuclease Hinf I is used. Another well-established technique utilizes melting point analysis of amplification products with specific hybridization probes. Recently, also allele-specific PCR assays have been described. The technique used for the detection of c-kit mutations in mastocytosis is dependent on the kind of material to be analyzed and the laboratory equipment available. In this chapter the techniques of PNA-mediated PCR-clamping in combination with melting point analysis for the genotyping of amplification products are described for mutational analysis in total DNA and microdissected cells from formalin-fixed paraffin-embedded bone marrow trephine biopsies.
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PMID:c-kit mutational analysis in paraffin material. 2366 90