2'-Aminoethoxy-2-amino-3-methylpyridine in triplex-forming oligonucleotides: high affinity, selectivity and resistance to enzymatic degradation

The phosphoramidite monomer of the C-nucleoside 2'-aminoethoxy-2-amino-3-methylpyridine (AE-MAP) has been synthesized for the first time and incorporated into triplex-forming oligonucleotides (TFOs). Ultraviolet melting and DNase I footprinting studies show that AE-MAP is a potent triplex-stabilizing monomer that is selective for GC base pairs. TFOs containing AE-MAP bind with high affinity to duplexes but only weakly to single stranded DNA. In addition, AE-MAP confers high nuclease resistance on oligonucleotides. TFOs containing AE-MAP have potential for gene knock-out and gene expression studies.     

2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH

Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.     

Synthesis,characterization, and DNA interaction of mononuclear copper(II) and zinc(II) complexes having a hard–soft NS donor ligand

Knoevenagel condensate-based Schiff base ligands (L) containing N and S donor sites have been designed and synthesized [L = 3-cinnamalideneacetylacetonethiosemicarbazone (CAT)/3-cinnamalideneacetylacetoneethylthiosemicarbazone (CAET)/3-cinnamalideneacetylacetonephenylthiosemicarbazone (CAPT)]. They afford complexes of the type [ML] [M = Cu(II) and Zn(II)]. Both the ligands and their complexes were characterized by analytical and spectral data. Intercalative binding of these complexes with DNA has been investigated by electronic absorption spectroscopy, viscosity measurements, cyclic voltammetry, and differential pulse voltammetry. Electrophoretic study of the complexes indicates that they efficiently cleave supercoiled pUC19 DNA in the presence of hydrogen peroxide.     

Synthesis and crystal structure of the first dinuclear zinc complex containing 1,4,7-triazacyclononane and biological properties of the protonated ligand

Abstract

A new binuclear complex, [Zn₂L₂Cl₄]·2H₂O {L?=?N-aldehyde-N-(4-(benzyloxy)benzyl)-1,4,7triazacyclononane}, was synthesized and characterized by X-ray, elemental analysis, infrared and electronic spectroscopy, and mass spectrometry. The central ion is bridged by the L and lies in a tetrahedral configuration with Zn···Zn distance of 6.283 Å. The complex crystallizes in the triclinic space group Pī. ESI-MS of the complex indicates that the protonated ligand HL⁺ is the active species. The interaction of HL⁺ with calf thymus–DNA (CT–DNA) and bovine serum albumin (BSA) was studied by means of various spectroscopic methods, which revealed that HL⁺ could interact with CT–DNA through groove-binding mode and could quench the intrinsic fluorescence of BSA in a static quenching process. DNA–cleavage experiments indicate that HL⁺ exhibits efficient DNA–cleavage activity in the presence of H₂O₂, hydroxyl radical (HO^?) may serve as the major cleavage active species, and the pseudo-Michaelis–Menten kinetic parameters (K_cat, K_M, V_max); 2.47?h^?1, 2.70?×?10^?4 M and 6.68?×?10^?4 Mh^?1.     

Synthesis,characterization, in-vitro biocidal and nuclease activity of some coordination compounds

Complexes of Cu(II), Fe(II) and Fe(III) have been synthesized with 2-nitro-3,3′-benzylidene bis[4-hydroxycoumarin]/4-chloro-3,3′-benzylidene bis[4-hydroxycoumarin]/4-hydroxy-3,3′-benzylidene bis[4-hydroxycoumarin]. They have been characterized using ¹H-NMR, ¹³C-NMR, IR spectra, electronic spectra, magnetic measurements, elemental analyses and screened for their in-vitro biocidal activity against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi, and Serratia marcescens bacterial strains and for their in-vitro antifungal activity against Aspergillus niger, Aspergillus flavus and Lasiodiplodia theobromae. The metal complexes exhibit good activity against bacterial strains compared to parent compounds, but no significant antifungal activity against fungal strains. In-vitro nuclease activity has been carried out using agarose gel electrophoresis. The synthesized compounds show effective nuclease activity.     

A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis

Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real-time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.     

Effect of coordination sphere of the copper center and Cu―Cu distance on catechol oxidase and nuclease activities of the copper complexes

To explore the effect of Cu―Cu distance in the structure of copper complexes on their catechol oxidase and nuclease activity, six copper complexes with a similar coordination sphere but different Cu―Cu distances were synthesized and characterized with elemental analysis, single‐crystal X‐ray diffraction, molar conductivity measurements, IR and UV–visible spectroscopy. Complex 1 is a binuclear copper complex and complex 4 is a polynuclear complex with a Z‐chain structure, as evidenced by their crystal structures. Complementary characterizations showed that complexes 2 and 3 have a similar binuclear structure to the complex 1 ; and complexes 5 and 6 are analogous to complex 4 . The catechol oxidase activity of complexes 1 , 2 , 3 is quite akin to that of 4 , 5 , 6 , suggesting that the catechol oxidase activity of the complexes was determined by the coordination environment of the copper center, when Cu―Cu distance is large. In contrast, DNA cleavage activity of the complexes 1 , 2 and 3 are much higher than that of 4 , 5 and 6 , indicating that the planar ligand structure in the complexes 4 , 5 and 6 is more critical than the copper coordination sphere and the Cu―Cu distance for their nuclease activity. Copyright © 2014 John Wiley & Sons, Ltd.     

核酸酶P1活性中心金属离子与氯化钴(Ⅱ)相互作用

核酸酶P1是一种重要的DNA与RNA水解酶。本文利用ICP、VIS、NMR、酶活性测定等分析方法，首次拓展研究了核酸酶P1与CoCl₂的直接相互作用。结果发现：核酸酶P1活性中心Zn(Ⅱ)离子可被外加CoCl₂中的Co(Ⅱ)部分取代，而Co(Ⅱ)进入酶的活性中心，生成相应的酶衍生物“Co(Ⅱ)-P1”，并影响了酶的催化活性。与此同时还获得了Co(Ⅱ)进入核酸酶P1活性中心Zn2位上的酶衍生物 ¹H NMR谱。     

Synthesis of the unnatural amino acid N-N-(ferrocene-1-acetyl)-l-lysine: A novel organometallic nuclease

The synthesis of a new unnatural amino acid, N^α-N^ε-(ferrocene-1-acetyl)-l-lysine, was achieved by coupling a ferroceneacetic acid molecule onto the side chain amine of a lysine. The structure of the compound provides options for incorporation of the molecule into peptides or large proteins. In addition, N^α-N^ε-(ferrocene-1-acetyl)-l-lysine exhibits nuclease activity. It is expected that incorporation of this ferrocenyl amino acid into any nucleic acid-binding protein will endow the protein with nuclease capability.     

Preparation,characterization and biological evaluation of two chiral binuclear copper(II) complexes

Two chiral Cu(II) complexes of [Cu₂(R‐L)₂](PF₆)₂·2C₂H₅OH ( 1 ) and [Cu₂(S‐L)₂](PF₆)₂·2C₂H₅OH ( 2 ) (HL = 2‐(Bis(quinolin‐2‐ylmethyl)amino)‐1‐propanol) were designed and synthesized to serve as chemical nucleases and anticancer drugs. X‐ray crystallography revealed that two complexes contain chiral binuclear cations and PF₆^? anions. The interaction of two complexes with CT‐DNA was researched via various spectroscopic techniques and viscosity measurement, indicating that the complexes were bound to CT‐DNA by a classical intercalation binding mode. In addition, the two complexes exhibited remarkable DNA cleavage activity with an optimal dosage of 10 μM in the absence of any exogenous oxidant agent. Both of the complexes showed excellent in vitro cytotoxicity on A549 cell lines with IC₅₀ values in the low micromolar range. Moreover, complex 2 could damage DNA of A549 cells into fragmentation and then induced cell apoptosis in a dose‐dependent manner, which was demonstrated by comet assay and Hoechst 33342 staining experiment. Further research showed that complex 2 could also induce G₂ and S phase cell cycle arrest.