ABSTRACT
A method is described for the determination of the activity of endonuclease. It based on the deaggregation of gold nanoparticles (AuNPs) aggregated by the action of poly(diallyldimethylammonium chloride) (PDDA). A single-stranded DNA (ssDNA) is released after enzymatic cleavage catalyzed by endonuclease. The released fragments bind electrostatically to PDDA and inhibit the PDDA-induced aggregation of AuNPs. This is accompanied by a color change from blue to red and a decrease in the absorption ratio (A630/A520). Under the optimal conditions, this ratio increases linearly in the 0.001 to 1 U·µL-1 EcoRI endonuclease activity range. The detection limit is of 2 × 10-4 U·µL-1 which is much better or at least comparable to previous reports. The method is deemed to have wide scope in that it may be used to study other endonuclease activity (such as BamHI) by simply changing the specific recognition site of the hairpin-like DNA probe. The assay may also be employed to screening for inhibitors of EcoRI endonuclease. Graphical abstract Schematic presentation of the colorimetric assay based on the deaggregation of AuNPs for the detection of endonuclease activity. A single-stranded sequence (ssDNA) is released by the EcoRI cleavage, which electrostatically binds to PDDA and inhibits the PDDA-induced aggregation of AuNPs accompanying with a color change from blue to red.
Subject(s)
Colorimetry/methods , DNA Probes/chemistry , Endonucleases/metabolism , Gold , Inverted Repeat Sequences , Metal Nanoparticles/chemistry , Colorimetry/standards , Deoxyribonuclease EcoRI/antagonists & inhibitors , Deoxyribonuclease EcoRI/metabolism , Endonucleases/antagonists & inhibitors , Limit of Detection , Polyethylenes/chemistry , Polyethylenes/metabolism , Quaternary Ammonium Compounds/chemistry , Quaternary Ammonium Compounds/metabolismABSTRACT
Endonuclease cleavage of DNA plays an important role in biological and medicinal chemistry. This study aimed to develop a reliable and sensitive method for nuclease activity assay by combining the high specificity of DNA cleavage reactions with ultrahigh fluorescence turn-on abilities of guanine-rich (G-rich) DNA sequences in proximity to silver nanoclusters (Ag NCs). The DNA-templated Ag NC (DNA-Ag NC) probe with endonuclease recognition sequence consists of NC and a G-rich probe. The NC probe was designed by adding Ag NC nucleation sequence at the 5'-end. The G-rich probe is the complementary DNA sequence modified by adding a G-rich overhang sequence at the 3'-end. Thus, the fluorescence of DNA-Ag NC probe was activated because of DNA hybridization. When these DNA-Ag NC probes were exposed to the targeted endonucleases, specific DNA cleavages occurred, and pieces of G-rich DNA fragments separated from Ag NCs, resulting in fluorescence turn-off. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. Detection was demonstrated by assaying EcoRI activity. Under optimized conditions, the fluorescence reduction efficiency was linear with the EcoRI concentration in the range of 5.0×10(-4) U µL(-1) to 3.0×10(-3) U µL(-1), with a detection limit of 3.5×10(-4) U µL(-1), which is much better than or at least comparable with that in previous reports. The potential application of the proposed method for screening endonuclease inhibitors was also demonstrated. The presented assay protocol proved to be convenient, effective, sensitive, and easy in preparing the fluorescent probe.
Subject(s)
Biosensing Techniques/methods , DNA Probes/chemistry , Deoxyribonuclease EcoRI/antagonists & inhibitors , Deoxyribonuclease EcoRI/metabolism , Escherichia coli/enzymology , Nanostructures/chemistry , Silver/chemistry , Base Sequence , Deoxyribonuclease EcoRI/analysis , Enzyme Assays/methods , Enzyme Inhibitors/pharmacology , Fluorescence , Limit of DetectionABSTRACT
DNA cleavage by endonucleases plays an important role in many biological events such as DNA replication, recombination, and repair and is used as a powerful tool in medicinal chemistry. However, conventional methods for assaying endonuclease activity and inhibition by gel electrophoresis and chromatography techniques are time-consuming, laborious, not sensitive, or costly. Herein, we combine the high specificity of DNA cleavage reactions with the benefits of quantum dots (QDs) and ultrahigh quenching abilities of inter- and intramolecular quenchers to develop highly sensitive and specific nanoprobes for multiplexed detection of endonucleases. The nanoprobe was prepared by conjugating two sets of DNA substrates carrying quenchers onto the surface of aminated QDs through direct assembly and DNA hybridization. With this new design, the background fluorescence was significantly suppressed by introducing inter- and intramolecular quenchers. When these nanoprobes are exposed to the targeted endonucleases, specific DNA cleavages occur and pieces of DNA fragments are released from the QD surface along with the quenchers, resulting in fluorescence recovery. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. The detection was accomplished with a single excitation light. Multiplexed detection was demonstrated by simultaneously assaying EcoRI and BamHI (as model analytes) using two different emissions of QDs. The limits of detection were 4.0 × 10(-4) U/mL for EcoRI and 8.0 × 10(-4) U/mL for BamHI, which were at least 100 times more sensitive than traditional gel electrophoresis and chromatography assays. Moreover, the potential application of the proposed method for screening endonuclease inhibitors has also been demonstrated. The assay protocol presented here proved to be simple, sensitive, effective, and easy to carry out.
Subject(s)
Deoxyribonuclease BamHI/metabolism , Deoxyribonuclease EcoRI/metabolism , Quantum Dots , Spectrometry, Fluorescence , DNA/metabolism , Deoxyribonuclease BamHI/antagonists & inhibitors , Deoxyribonuclease EcoRI/antagonists & inhibitors , Nucleic Acid HybridizationABSTRACT
We discovered inhibitors of the restriction enzymes EcoRI, BamHI and HindIII by screening our library of compounds with a phenethylphenylphthalimide skeleton, based on α-glucosidase inhibitors and liver X receptor antagonists derived from thalidomide. Structural development afforded the potent restriction enzyme inhibitors 25 and 26.
Subject(s)
Deoxyribonuclease BamHI/antagonists & inhibitors , Deoxyribonuclease EcoRI/antagonists & inhibitors , Deoxyribonuclease HindIII/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Isoindoles/chemistry , Phthalimides/chemistry , Thalidomide/chemistry , Deoxyribonuclease BamHI/metabolism , Deoxyribonuclease EcoRI/metabolism , Deoxyribonuclease HindIII/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Glycoside Hydrolase Inhibitors , Isoindoles/chemical synthesis , Isoindoles/pharmacology , Liver X Receptors , Orphan Nuclear Receptors/antagonists & inhibitors , Orphan Nuclear Receptors/metabolism , Phthalimides/chemical synthesis , Phthalimides/pharmacology , alpha-Glucosidases/metabolismABSTRACT
We have developed a highly sensitive and selective fluorescence polarization assay method based on the specificity of the DNA cleavage reaction with the enhancement of gold nanoparticles (AuNPs) for assaying endonuclease activity and inhibition. This assay can detect EcoRI endonuclease down to 5.0×10(-4) U mL(-1) with a detection range from 5.0×10(-4) to 10 U mL(-1).
Subject(s)
Deoxyribonuclease EcoRI/antagonists & inhibitors , Deoxyribonuclease EcoRI/metabolism , Enzyme Inhibitors/pharmacology , Fluorescence Polarization , Gold/chemistry , Metal Nanoparticles/chemistry , Deoxyribonuclease EcoRI/chemistry , Enzyme Activation/drug effectsABSTRACT
The interaction of EcoRI with different oligodeoxyribonucleotides (ODNs) was analyzed using the method of the slow step-by-step simplification in their complexity. Orthophosphate (KI = 31 mM), 2-deoxyribose 5-phosphate (KI = 4.6 mM) and different dNMPs (KI = 2.1-2.5 mM) were shown to be the minimal ligands of the enzyme. The lengthening of a nonspecific d(pN)n (n = 1-6) by one nucleotide unit resulted in the increase of their affinity by a factor of approximately 2.0. Weak nonspecific electrostatic contacts of EcoRI with internucleotide phosphate groups of ODNs can account for about 5 orders of magnitude in the ligand affinity, whereas the contribution of specific interactions between EcoRI and d(pN)n is no more than 2 orders of magnitude of a total ODN's affinity.
Subject(s)
Deoxyribonuclease EcoRI/metabolism , Oligodeoxyribonucleotides/metabolism , Base Sequence , Binding Sites , Deoxyribonuclease EcoRI/antagonists & inhibitors , Deoxyribonuclease EcoRI/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Kinetics , Ligands , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/pharmacology , Static Electricity , Substrate SpecificityABSTRACT
BACKGROUND: Many enzymes are active only in a dimeric form, including a variety of type II restriction endonucleases. Disruption of subunit interactions is therefore a potential method for multimeric enzyme inhibition. EcoRI is a homodimeric restriction endonuclease, the dimeric interface of which consists of a four-helix bundle. We set out to design helical peptides to interact with this interface and block dimer formation, thus rendering EcoRI inactive. RESULTS: Here we describe two synthetic, helical peptides based on the interfacial region of EcoRI. Both peptides inhibit the enzyme, but the peptide derived from the alpha 4 helix of EcoRI had both a higher helical content and better efficacy than a variant peptide, alpha 4(Leu), that has three Ile-->Leu mutations (IC50 values of 27 microM and 90 microM, and helical contents of 29% and 10%, respectively). Size-exclusion chromatography confirmed that the alpha 4 peptide disrupted dimerization of EcoRI, and circular dichroism indicated that EcoRI remained folded upon binding to alpha 4. Inhibition with alpha 4 and alpha 4(Leu) was shown to be specific for EcoRI, as the dimeric restriction enzyme PvuII was not affected by the peptides. CONCLUSIONS: Interfacial peptide inhibitors of the dimeric EcoRI were obtained that both inhibit dimerization and endonuclease activity. The peptide sequence with a preference for a helical conformation was a more effective inhibitor, presumably because the more preorganized state enhanced interactions with the helical interface of EcoRI. The specific nature of this endonuclease-peptide interaction was also confirmed. The potential of this strategy for inhibiting other enzyme classes is currently being addressed.
Subject(s)
Deoxyribonuclease EcoRI/antagonists & inhibitors , Peptides/pharmacology , Protein Structure, Secondary , Chromatography, Gel , Circular Dichroism , Deoxyribonuclease EcoRI/chemistry , Deoxyribonuclease EcoRI/metabolism , Dimerization , Enzyme Activation/drug effects , Models, Molecular , Mutagenesis, Site-Directed , Peptides/chemical synthesis , Protein Conformation/drug effects , Substrate SpecificityABSTRACT
Two new methyltransferase inhibitors were isolated from the culture filtrate of Streptomyces sp. strain No. 1513 and named 1513-DMIa and 1513-DMIb. 1513-DMIa and 1513-DMIb were distinguished in certain properties from DMI-1, DMI-2, DMI-3 and DMI-4 previously reported. The molecular weight of 1513-DMIa and 1513-DMIb were estimated to be 576 and 8400 from the results of FAB-MS and gel filtration, respectively. The inhibitory activities of 1513-DMIa and 1513-DMIb were shown to be pH- and temperature-dependent and both inhibited M. EcoRI in an uncompetitive manner with respect to DNA or S-adenosylmethionine (SAM).
Subject(s)
Enzyme Inhibitors/chemistry , Site-Specific DNA-Methyltransferase (Adenine-Specific)/antagonists & inhibitors , Streptomyces/metabolism , Chromatography, Gel , DNA-Cytosine Methylases/antagonists & inhibitors , Deoxyribonuclease EcoRI/antagonists & inhibitors , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Kinetics , Molecular Structure , Molecular Weight , Nuclear Magnetic Resonance, Biomolecular , Spectrometry, Mass, Fast Atom Bombardment , Streptomyces/chemistryABSTRACT
EcoRI DNA methyltransferase (MTase) is rapidly inactivated by N-ethylmaleimide with concomitant incorporation of 2 mol of N-ethyl[2-3H]maleimide/mol of functional monomer. Preincubation of the enzyme with either S-adenosylmethionine or DNA reduces the rate of activity loss, whereas preincubation with DNA and the S-adenosylmethionine analog sinefungin completely protects the enzyme from inactivation. An endo proteinase Glu-C digest of N-ethyl[2-3H]maleimide-modified enzyme was prepared and separated by high pressure liquid chromatography. Modified and unmodified cysteine-containing peptides were located and identified by radioactivity, mass spectrometry, and tandem mass spectrometry. In the absence of any ligands, cysteines 25, 116, and 223 are modified by N-ethylmaleimide; in the presence of DNA and sinefungin, Cys-223 is essentially unmodified. Thus, N-ethylmaleimide modification of Cys-223 in EcoRI DNA MTase is responsible for the loss of enzyme activity. Cys-223 is preceded by Asn, and this (or Cys-Asn) occurs with high frequency in adenine and cytosine (N-4) DNA MTases. Direct involvement of cysteine in methyl transfer reactions to adenine N-6 and cytosine N-4 is supported by the similarity of the reactions catalyzed by adenine N-6 and cytosine N-4 DNA MTases, the frequent presence of Asn-flanking Cys, and the importance of Cys-223 to EcoRI MTase function.
Subject(s)
Cysteine , Deoxyribonuclease EcoRI/metabolism , Amino Acid Sequence , Chromatography, High Pressure Liquid , DNA Restriction Enzymes/genetics , Deoxyribonuclease EcoRI/antagonists & inhibitors , Deoxyribonuclease EcoRI/genetics , Dipeptides/analysis , Escherichia coli/enzymology , Ethylmaleimide/pharmacology , Kinetics , Mass Spectrometry/methods , Molecular Sequence Data , Peptide Fragments/isolation & purification , Staphylococcus aureus/metabolismABSTRACT
The ability of oligopyrimidines to inhibit, through triple helix formation, the specific protein-DNA interactions of the EcoRI restriction and modification enzymes (EcoRI and MEcoRI) with their recognition sequence (GAATTC) was studied. The oligonucleotides (CTT)4 and (CTT)8 formed triplexes in plasmids at (GAA)n repeats containing EcoRI sites. Cleavage and methylation of EcoRI sites within these sequences were specifically inhibited by the oligonucleotides, whereas an EcoRI site adjacent to a (GAA)n sequence was inhibited much less. Also, other EcoRI sites within the plasmid, or in exogenously added lambda DNA, were not inhibited. These results demonstrate the potential of using triplex-forming oligonucleotides to block protein-DNA interactions at specific sites, and thus this technique may be useful in chromosome mapping and in the modulation of gene expression.