A Green Chemosensor for Colorimetric Determination of Phosphate Ion in Soil, Bone, and Water Samples Using Curcumin Nanoparticles.

This article presents a sensitive and straightforward colorimetric chemosensor for the determination of phosphate ion utilizing curcumin nanoparticles (CUNPs) as the sensing system. The color of as-prepared CUNPs can be changed from yellow to orange upon adding iron(III) ions due to the formation of a complex with CUNPs. However, in the presence of phosphate ions, iron(III) ions prefer to bind to phosphate ions and, subsequently the color of CUNPs is selectively recovered because of releasing the iron(III) ions from the CUNPs-iron(III) complex. Therefore, in this work the selective color changing of the CUNPs-iron(III) system upon the addition of phosphate ions was used for the quantitative sensing of phosphate ions. Various factors, such as the pH, concentration of iron(III) and volume of CUNPs, were examined and the optimum conditions were established. A linear calibration graph over the range of 10 - 400 ng mL-1 for phosphate (r = 0.9995) was achieved using the optimal conditions. The limit of detection (LOD) of the proposed method for phosphate was 7.1 ng mL-1 and the relative standard deviation (RSD) for measuring 50 ng mL-1 of phosphate was 3.7% (n = 8). The developed method was applied for the measurement of phosphate in water, soil, and bone samples. Satisfactory results were obtained.

A novel RT-QPCR-based assay for the relative quantification of residue specific m6A RNA methylation.

N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3'UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic ß-cells exposed to inflammatory stimuli.

Magnetic bead/capture DNA/glucose-loaded nanoliposomes for amplifying the glucometer signal in the rapid screening of hepatitis C virus RNA.

A digital detection strategy based on a portable personal glucometer (PGM) was developed for the simple, rapid, and sensitive detection of hepatitis C virus (HCV) RNA, involving the release of glucose-loaded nanoliposomes due to coupling-site-specific cleavage by the endonuclease BamHI. The glucose-loaded nanoliposomes were synthesized using a reversed-phase evaporation method and provided an amplified signal at the PGM in the presence of HCV RNA. Initially, a 21-mer oligonucleotide complementary to HCV RNA was covalently conjugated to a magnetic bead through the amino group at the 5' end of the oligonucleotide, and then bound to a glucose-loaded liposome by typical carbodiimide coupling at its 3' end. In the presence of the target HCV RNA, the target hybridized with the oligonucleotide to form double-stranded DNA. The symmetrical duplex sequence 5'-GGATCC-3' between guanines was then catalytically cleaved by BamHI, which detached the glucose-loaded liposome from the magnetic bead. Following magnetic separation of the bead, the detached glucose-loaded liposome was lysed using Triton X-100 to release the glucose molecules within it, which were then detected as an amplified signal at the digital PGM. Under optimal conditions, the PGM signal increased with increasing HCV RNA, and displayed a strongly linear dependence on the level of HCV RNA for concentrations ranging from 10 pM to 1.0 µM. The detection limit (LOD) of the system was 1.9 pM. Good reproducibility and favorable specificity were achieved in the analysis of the target HCV RNA. Human serum samples containing HCV RNA were analyzed using this strategy, and the developed sensing platform was observed to yield satisfactory results based on a comparison with the corresponding results from a Cobas® Amplicor HCV Test Analyzer. Graphical abstract A digital detection strategy utilizing a personal glucometer was developed for the detection of hepatitis C virus RNA. The strategy involved the use of the endonuclease BamHI along with a 21-mer oligonucleotide conjugated to both a magnetic bead and a glucose-loaded nanoliposome. Hybridization of the nucleotide with the target RNA triggered the coupling-site-specific cleavage of the duplex by BamHI, leading to the release of the glucose-loaded nanoliposome. Following separation of the magnetic bead, the free nanoliposome was dissolved, liberating the glucose molecules within it, which in turn were detected as an amplified signal by the glucometer.

Probing the role of interfacial waters in protein-DNA recognition using a hybrid implicit/explicit solvation model.

When proteins bind to their DNA target sites, ordered water molecules are often present at the protein-DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily nonspecific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein-DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA-bound water molecules, while treating the majority of the solvent implicitly. Comparing the performance of this model with that of its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein side-chain placement and DNA sequence recovery. Base-by-base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large-scale statistical evidence for the role of ordered water for protein-DNA recognition, together with detailed examination of several well-characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems.

Toward detection of DNA-bound proteins using solid-state nanopores: insights from computer simulations.

Through all-atom molecular dynamics simulations, we explore the use of nanopores in thin synthetic membranes for detection and identification of DNA binding proteins. Reproducing the setup of a typical experiment, we simulate electric field driven transport of DNA-bound proteins through nanopores smaller in diameter than the proteins. As model systems, we use restriction enzymes EcoRI and BamHI specifically and nonspecifically bound to a fragment of dsDNA, and streptavidin and NeutrAvidin proteins bound to dsDNA and ssDNA via a biotin linker. Our simulations elucidate the molecular mechanics of nanopore-induced rupture of a protein-DNA complex, the effective force applied to the DNA-protein bond by the electrophoretic force in a nanopore, and the role of DNA-surface interactions in the rupture process. We evaluate the ability of the nanopore ionic current and the local electrostatic potential measured by an embedded electrode to report capture of DNA, capture of a DNA-bound protein, and rupture of the DNA-protein bond. We find that changes in the strain on dsDNA can reveal the rupture of a protein-DNA complex by altering both the nanopore ionic current and the potential of the embedded electrode. Based on the results of our simulations, we suggest a new method for detection of DNA binding proteins that utilizes peeling of a nicked double strand under the electrophoretic force in a nanopore.

Ultrasensitive and selective electrochemical identification of hepatitis C virus genotype 1b based on specific endonuclease combined with gold nanoparticles signal amplification.

This work proposes a new strategy for the electrochemical detection of hepatitis C virus (HCV) RNA level and identification of HCV-1b genotype based on the site-specific cleavage of BamHI endonuclease combined with gold nanoparticles (AuNPs) signal amplification. The assay procedures include the reverse transcription, polymerase chain reaction (PCR) amplification, and electrochemical detection. The samples of 244 mer sequence of HCV RNA from the highly conserved region of HCV-1a, HCV-1b, HCV-1, and HCV-6a, respectively, were first reverse transcribed into complementary cDNA and amplified by PCR. The PCR-amplified samples were then analyzed using a synthetic 21 mer DNA probe, which has been assembled on the electrode surface via a bifunctional molecule of p-aminobenzoic acid (ABA). The results demonstrated that the developed approach can be used for specifically identification of the HCV-1b genotype and selective and sensitive detection of HCV-1b cDNA (244 mer) with a detection limit as low as (3.1 ± 0.8) × 10(-22) M (less than 200 molecules; the concentration refers to the one before PCR amplification). Moreover, the developed method has an ability to discriminate the HCV-1b cDNA sequence from even single-base mismatched DNA sequence, to assay the HCV-1b cDNA level precisely from the mixture of HCV-1, HCV-1b, HCV-1a, and HCV-6a, and to detect HCV in real clinical samples. The protocol has high potential application in molecular diagnostics of HCV in clinical environments.

Asymmetric DNA recognition by the OkrAI endonuclease, an isoschizomer of BamHI.

Restriction enzymes share little or no sequence homology with the exception of isoschizomers, or enzymes that recognize and cleave the same DNA sequence. We present here the structure of a BamHI isoschizomer, OkrAI, bound to the same DNA sequence (TATGGATCCATA) as that cocrystallized with BamHI. We show that OkrAI is a more minimal version of BamHI, lacking not only the N- and C-terminal helices but also an internal 3(10) helix and containing ß-strands that are shorter than those in BamHI. Despite these structural differences, OkrAI recognizes the DNA in a remarkably similar manner to BamHI, including asymmetric contacts via C-terminal 'arms' that appear to 'compete' for the minor groove. However, the arms are shorter than in BamHI. We observe similar DNA-binding affinities between OkrAI and BamHI but OkrAI has higher star activity (at 37°C) compared to BamHI. Together, the OkrAI and BamHI structures offer a rare opportunity to compare two restriction enzymes that work on exactly the same DNA substrate.

Reinvestigation of the molecular influence of hypoxanthine on the DNA cleavage efficiency of restriction endonucleases BglII, EcoRI and BamHI.

Hypoxanthine (Hyp), a deaminated base of adenine (Ade), can be employed as a good probe molecule to reveal the significance of the minor groove of guanine (Gua) in biomolecular interactions because Hyp possesses a similar structure to Gua lacking its 2-amino group. In this study, we examined cleavage efficiencies of restriction endonuclease enzymes on DNA substrates with Hyp in their recognition sequences. As a substrate for BglII, EcoRI and BamHI, 24-mer DNA oligomer with Hyp (in place of Gua) was prepared together with its complementary sequences with cytosine (Cyt) or thymine (Thy) as the counter base. At 37 degrees C incubation for 1 h, BglII and EcoRI showed higher DNA cleavage reactivity on Hyp-containing DNA substrates than on normal ones, whereas BamHI showed lower values on Hyp-containing substrates. Such high cleavage performance of BglII and EcoRI on Hyp-containing DNA substrates is in contrast to the results obtained 20 years ago, in which short DNA substrates (8- or 10-mer) and low reaction temperatures (15-20 degrees C) were employed. These new results suggest that the lack of the exocyclic 2-amino group of Gua could contribute to enhanced recognition access of BglII and EcoRI to DNA substrates.

A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections.

Herein, a method is described to increase the information density of sequencing experiments used to deconvolute nucleic acid selections. The method is facile and should be applicable to any selection experiment. A critical feature of this method is the use of biotinylated primers to amplify and encode a BamHI restriction site on both ends of a PCR product. After amplification, the PCR reaction is captured onto streptavidin resin, washed, and digested directly on the resin. Resin-based digestion affords clean product that is devoid of partially digested products and unincorporated PCR primers. The product's complementary ends are annealed and ligated together with T4 DNA ligase. Analysis of ligation products shows formation of concatemers of different length and little detectable monomer. Sequencing results produced data that routinely contained three to four copies of the library. This method allows for more efficient formulation of structure-activity relationships since multiple active sequences are identified from a single clone.

Probing the two-metal ion mechanism in the restriction endonuclease BamHI.

The choreography of restriction endonuclease catalysis is a long-standing paradigm in molecular biology. Bivalent metal ions are required almost for all PD..D/ExK type enzymes, but the number of cofactors essential for the DNA backbone scission remained ambiguous. On the basis of crystal structures and biochemical data for various restriction enzymes, three models have been developed that assign critical roles for one, two, or three metal ions during the phosphodiester hydrolysis. To resolve this apparent controversy, we investigated the mechanism of BamHI catalysis using quantum mechanical/molecular mechanical simulation techniques and determined the activation barriers of three possible pathways that involve a Glu-113 or a neighboring water molecule as a general base or an external nucleophile that penetrated from bulk solution. The extrinsic mechanism was found to be the most favorable with an activation free energy of 23.4 kcal/mol, in reasonable agreement with the experimental data. On the basis of the effect of the individual metal ions on the activation barrier, metal ion A was concluded to be pivotal for the reaction, while the enzyme lacking metal ion B still has moderate efficiency. Thus, we propose that the catalytic scheme of BamHI does not involve a general base for nucleophile generation and requires one obligatory metal ion for catalysis that stabilizes the attacking nucleophile and coordinates it throughout the nucleophilic attack. Such a model may also explain the variation in the number of metal ions in the crystal structures and thus could serve as a framework for a unified catalytic scheme of type II restriction endonucleases.

Metal-binding sites at the active site of restriction endonuclease BamHI can conform to a one-ion mechanism.

The number of metal ions required for phosphoryl transfer in restriction endonucleases is still an unresolved question in molecular biology. The two Ca(2+) and Mn(2+) ions observed in the pre- and post-reactive complexes of BamHI conform to the classical two-metal ion choreography. We probed the Mg(2+) cofactor positions at the active site of BamHI by molecular dynamics simulations with one and two metal ions present and identified several catalytically relevant sites. These can mark the pathway of a single ion during catalysis, suggesting its critical role, while a regulatory function is proposed for a possible second ion.

Analysis of plasmid DNA damage induced by melanin with capillary electrophoresis.

Dilute linear poly(N-isopropylacrylamide) (PNIPAM) in Tris-Mes-EDTA (TME) buffer was used as sieving matrix for capillary electrophoresis (CE) of plasmid DNA and plasmid topological isomers induced by melanin in uncoated capillary. At the optimized condition of 0.1% (w/v) PNIPAM in TME buffer, base line separation of the plasmid DNA ladder (2-12 kbp) was achieved within 15 min. Three positive clones with inserts of 468, 1147 and 1566 bp can be distinguished from the plasmid pUC 18 vector within 13 min. The migration order of the plasmid topological isomers in the dynamic coating matrix was confirmed by the enzymatically prepared and UV-induced plasmids. The covalently closed circular form appeared firstly, followed by the linear plasmid form and then the open circular form. The effect of bacterial melanin obtained from Pseudomonas maltophilia AT18 on plasmid pUC 18 was investigated by CE in uncoated capillary in vitro. Plasmid pUC 18 incubated with either melanin or copper ions alone sustained little DNA damage. The combination of melanin with Cu(II) can cause the plasmid pUC 18 conformational changes from covalently closed circular form to open form. Understanding the damage effect of melanin with copper ions on DNA would be important for the melanin-related application, such as photoprotective antioxidant in protecting the skin from cancer, pathophysiology research in clinic. The investigation of melanin induced plasmid conformational changes by CE in uncoated capillary also revealed that the application of the dynamic coating matrix could be extended to the study of plasmid conformational changes in other plasmid-based biological technologies.

Detection of the local structural changes in the dimer interface of BamHI initiated by DNA binding and dissociation using a solvatochromic fluorophore.

To detect the local structural change in an interface between proteins induced by the substrate binding and dissociation, a solvatochromic fluorescent N(beta)-L-alanyl-5-(N,N-dimethylamino)-naphthalene-1-sulfonamide (DanAla) was introduced into 132 position of the dimer interface in BamHI. Before addition of the substrate, the fluorescence from the normal planer excited state of DanAla moiety was observed as a main emission, and thereby the DanAla in the dimer interface is located in the hydrophobic microenvironment. The incubation with the substrate for 20 min induced the gradual increase in fluorescence intensity around 430 nm. The fact reflects that the polarity is reduced by the slight structural change initiated by the formation of the complex with the substrate. Furthermore, the incubation for more than 20 min caused the slight decrease in fluorescence around 430 nm and an appearance of fluorescence (560 nm) due to twisted intramolecular charge transfer (TICT) excited state. Therefore, the DanAla is exposed to comparative polar environment after the dissociation of the substrate. The fluorescence lifetime as a minor component, which is attributed to the TICT excited state, was reduced by addition of the substrate. The results provide that the hydrophobicity in the dimer interface is increased by the substrate binding. Interestingly, we found that the structure of an initial form is different from that of a refolded form after the dissociation of the substrate using a spectral subtraction technique. We have achieved detection of the changing structure induced by the substrate binding and dissociation using a steady-state and time-resolved fluorescence.

Differences in hydration coupled to specific and nonspecific competitive binding and to specific DNA Binding of the restriction endonuclease BamHI.

Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120-150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.

Interfacial water as a "hydration fingerprint" in the noncognate complex of BamHI.

The molecular code of specific DNA recognition by proteins as a paradigm in molecular biology remains an unsolved puzzle primarily because of the subtle interplay between direct protein-DNA interaction and the indirect contribution from water and ions. Transformation of the nonspecific, low affinity complex to a specific, high affinity complex is accompanied by the release of interfacial water molecules. To provide insight into the conversion from the loose to the tight form, we characterized the structure and energetics of water at the protein-DNA interface of the BamHI complex with a noncognate sequence and in the specific complex. The fully hydrated models were produced with Grand Canonical Monte Carlo simulations. Proximity analysis shows that water distributions exhibit sequence dependent variations in both complexes and, in particular, in the noncognate complex they discriminate between the correct and the star site. Variations in water distributions control the number of water molecules released from a given sequence upon transformation from the loose to the tight complex as well as the local entropy contribution to the binding free energy. We propose that interfacial waters can serve as a "hydration fingerprint" of a given DNA sequence.

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway.

Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition. In order to elucidate the connection between the mechanics and the chemistry of DNA recognition and cleavage, we used a single-molecule approach to measure rate changes in the reaction pathway of EcoRV and BamHI as a function of DNA tension. We show that the induced-fit rate of EcoRV is strongly reduced by such tension. In contrast, BamHI is found to be insensitive, providing evidence that both substrate binding and hydrolysis are not influenced by this force. Based on these results, we propose a mechanochemical model of induced-fit reactions on DNA, allowing determination of induced-fit rates and DNA bend angles. Finally, for both enzymes a strongly decreased association rate is obtained on stretched DNA, presumably due to the absence of intradomain dissociation/re-association between non-specific sites (jumping). The obtained results should apply to many other DNA-associated proteins.

Enzymatic clipping of DNA wires coated with magnetic nanoparticles.

The study describes how DNA coated with magnetic nanoparticles remains biologically active and accessible to the BamH1 restriction enzyme. Long DNA molecules are coated with magnetic nanoparticles using electrostatic interactions. The coated, stretched, and surface-bound DNA is incubated in the restriction enzyme that specifically recognizes any strand containing the GGATCC base sequence and clips the DNA. We show that, despite the presence of the nanoparticles on the DNA, the enzyme is still able to recognize the cleavage site and effectively digest the assembly.

Photochemical regulation of the activity of an endonuclease BamHI using an azobenzene moiety incorporated site-selectively into the dimer interface.

Endonuclease BamHI mutants having an azophenylalanine residue in the dimer interface (azoAla-BamHI) were synthesized; while the activity was almost suppressed using trans-azoAla-BamHI, the cis-isomer generated with photoirradiation recovered its intrinsic activity.

Genetic organization of the biosynthetic gene cluster for the antitumor angucycline oviedomycin in Streptomyces antibioticus ATCC 11891.

The oviedomycin biosynthetic gene cluster from Streptomyces antibioticus ATCC 11891 has been sequenced and characterized. It contains all the necessary genes for oviedomycin biosynthesis, together with several genes for the generation of malonyl-CoA extender units. Production of this unusual angucyclinone in its natural host occurs only in solid cultures in parallel with aerial mycelium and spore formation. A mutant that did not produce oviedomycin was generated by disruption of the beta-ketoacyl synthase gene ovmK. No other physiological process in the mutant appears to be affected; this rules out a direct relationship between oviedomycin production and cell differentiation in S. antibioticus.

Design and synthesis of photochemically controllable restriction endonuclease BamHI by manipulating the salt-bridge network in the dimer interface.

The strategy for the design of photochemically controllable enzymes by manipulating the dimer interface is described. Employing a restriction endonuclease BamHI, the selective incorporation of amino acids having a photoremovable 6-nitroveratryl group into the specific position (Lys132) in the dimer interface of the BamHI mutant (H133A) was performed. The activity of the photofunctionalized BamHI mutant was significantly suppressed, and the following photoirradiation induced the recovery of the activity. In addition, uncaging of the 6-nitroveratryl group introduced to Lys132 did not seriously reduce the catalytic activity and affinity for the substrate. These results indicate that the activity of the enzyme can be effectively regulated by caging and uncaging of the specific amino acid in the dimer interface using the photoremovable group.