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1.
Photodiagnosis Photodyn Ther ; 45: 103898, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38008301

ABSTRACT

We synthesized a new silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose (SGlc). Conjugation with SGlc improved A549 cellular uptake without significant changes in the photophysical and photochemical properties and subcellular localization. This improved cellular uptake led to enhanced photodynamic activity. Furthermore, conjugation with SGlc suppressed dark toxicity. These advantages were not observed for a conjugate with a glucose molecule. These results indicated that the conjugation with SGlc is a promising strategy for enhancing photodynamic efficacy.


Subject(s)
Photochemotherapy , Porphyrins , Humans , Photosensitizing Agents/pharmacology , Photochemotherapy/methods , A549 Cells , Glucose , Porphyrins/pharmacology
2.
Anal Biochem ; 662: 115000, 2023 02 01.
Article in English | MEDLINE | ID: mdl-36470466

ABSTRACT

In this study, a double-stranded DNA (dsDNA) fluorescent labeling method was developed using the fusion proteins of fluorescent protein (FP), and 7 kDa DNA-binding family members including Sso7d from Sulfolobus solfataricus, Aho7c from Acidianus hospitalis, ATSV7 from Acidianus tailed spindle virus and Sto7 from Sulfolobus tokodaii. Using this fluorescent DNA labeling method, we succeeded in single-molecule imaging of bacteriophage λDNA molecules stretched on glass surfaces. The fluorescence of the λDNA with FP fusion proteins decayed 2.4- to 6.4-fold slower than that of the typical intercalating method with SYTOX Green (SxG). In addition, the dynamic behaviors of FP-fused Aho7c-λDNA were relaxed and stretched with and without buffer flow, respectively, in microflow channels and were similar to that with typical intercalating dye, such as YOYO-1 and SxG. this fluorescent DNA labeling method. This fluorescent DNA labeling method can solve the problem of rapid fluorescence decay due to the intercalating dyes and therefore can be expected as an alternative to compound-based fluorescent dye. Thus, this study establishes FP fusion proteins as useful fluorescent DNA probes at the single-molecule level.


Subject(s)
DNA , Fluorescent Dyes , DNA/chemistry , Fluorescent Dyes/chemistry , Staining and Labeling , Organic Chemicals
3.
Bioorg Med Chem Lett ; 52: 128391, 2021 11 15.
Article in English | MEDLINE | ID: mdl-34601028

ABSTRACT

Sulfoquynovosylacyl propanediol (SQAP; 1) has been developed as a radiosensitizer (anti-cancer agent) for solid tumors, but it was easily cleaved in vivo and had a problem of short residence time. We synthesized a novel compound of a SQAP derivative (3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside: ODSG; 2) to solve these problems not easily cleaved by lipase. ODSG (2) cytotoxicity was investigated in vitro, resulting in low toxicity like SQAP (1).


Subject(s)
Lipase/metabolism , Radiation-Sensitizing Agents/pharmacology , A549 Cells , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Molecular Structure , Radiation-Sensitizing Agents/chemistry , Radiation-Sensitizing Agents/metabolism , Structure-Activity Relationship
4.
Molecules ; 26(4)2021 Feb 17.
Article in English | MEDLINE | ID: mdl-33671359

ABSTRACT

DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.


Subject(s)
DNA/chemistry , Single Molecule Imaging/methods , Electricity , Optical Imaging , Optical Tweezers , Rheology
5.
ACS Omega ; 6(1): 533-543, 2021 Jan 12.
Article in English | MEDLINE | ID: mdl-33458505

ABSTRACT

Using bovine pancreatic ribonuclease A (RNase A) and cholesterol, we synthesized cholesteryl-conjugated ribonuclease A (CHRNase A) to evaluate the influence of a conjugated hydrophobic moiety on protein function. Nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight spectrometry suggested that one cholesteryl group was conjugated to RNase A. Differential scanning calorimetry indicated that CHRNase A was denatured in the solid state but was folded in phosphate buffer (0.05 mol/L, pH 6.5). CHRNase A resembled RNase A in its secondary structure, but circular dichroism (CD) spectra revealed that the helical content of CHRNase A was decreased and the tertiary structure of CHRNase A differed from that of RNase A. Furthermore, fluorescence measurements, CD spectra, an 8-anilino-1-naphthalenesulfonic acid ammonium salt-based assay, and surface tension measurements suggested that cholesterol was conjugated to a tyrosine residue on the protein surface. The relative activity of CHRNase A to RNase A was 79 ± 7%, and the enzyme activity of CHRNase A by adding ß-cyclodextrin (ß-CyD) increased to 129 ± 7%. Therefore, we considered that the cholesteryl group interacted with substrate (cytidine 2'3'-cyclic monophosphate monosodium salt) to inhibit the enzyme reaction. Finally, the environment around tyrosine residues in CHRNase A in dimethyl sulfoxide was similar to that of native RNase A in phosphate buffer (0.05 mol/L, pH 6.5). These results suggest that cholesterol conjugation to RNase A altered RNase A functionality, including improvement of RNase A resistance to dimethyl sulfoxide and modulation of the ability of ß-CyD to control RNase A enzymatic activity.

6.
Molecules ; 25(16)2020 Aug 14.
Article in English | MEDLINE | ID: mdl-32823837

ABSTRACT

Hydrophobic interaction is important for protein conformation. Conjugation of a hydrophobic group can introduce intermolecular hydrophobic contacts that can be contained within the molecule. It is possible that a strongly folded state can be formed in solution compared with the native state. In this study, we synthesized cholesteryl conjugated lysozyme (CHLysozyme) using lysozyme and cholesterol as the model protein and hydrophobic group, respectively. Cholesteryl conjugation to lysozyme was confirmed by nuclear-magnetic resonance. Differential-scanning calorimetry suggested that CHLysozyme was folded in solution. CHLysozyme secondary structure was similar to lysozyme, although circular dichroism spectra indicated differences to the tertiary structure. Fluorescence measurements revealed a significant increase in the hydrophobic surface of CHLysozyme compared with that of lysozyme; CHLysozyme self-associated by hydrophobic interaction of the conjugated cholesterol but the hydrophobic surface of CHLysozyme decreased with time. The results suggested that hydrophobic interaction changed from intramolecular interaction to an intermolecular interaction. Furthermore, the relative activity of CHLysozyme to lysozyme increased with time. Therefore, CHLysozyme likely forms a folded state with an extended durability of activity. Moreover, lysozyme was denatured in 100% DMSO but the local environment of tryptophan in CHLysozyme was similar to that of a native lysozyme. Thus, this study suggests that protein solution stability and resistance to organic solvents may be improved by conjugation of a hydrophobic group.


Subject(s)
Cholesterol/chemistry , Models, Molecular , Muramidase/chemistry , Animals , Chickens , Cholesterol/metabolism , Hydrophobic and Hydrophilic Interactions , Muramidase/metabolism , Protein Conformation
7.
Xenobiotica ; 49(3): 346-362, 2019 Mar.
Article in English | MEDLINE | ID: mdl-29543539

ABSTRACT

Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits angiogenesis in vivo and results in increased oxigenation and reduced tumor volume. We investigated the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a human pulmonary carcinoma, Lu65. For the metabolism analysis, a 2 mg (2.98 MBq)/kg of [glucose-U-14C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body. The degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-14C]-SQAP (CP-3635) was administered through intravenous injection of a 1 mg (3.48 MBq)/kg dose followed by whole body autoradiography of the mice. The autoradiography analysis demonstrated that SQAP rapidly distributed throughout the whole body and then quickly decreased within 4 hours except the tumor and excretion organs such as liver, kidney. Retention of SQAP was longer in tumor parts than in other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the tumor had also completely disappeared within 72 hours.


Subject(s)
Glycolipids/pharmacokinetics , Radiation-Sensitizing Agents/pharmacokinetics , Administration, Intravenous , Animals , Autoradiography , Chromatography, High Pressure Liquid , Chromatography, Liquid , Glycolipids/administration & dosage , Glycolipids/therapeutic use , Humans , Lung Neoplasms/drug therapy , Mice, Nude , Radiation-Sensitizing Agents/administration & dosage , Radiation-Sensitizing Agents/therapeutic use , Tandem Mass Spectrometry
8.
Sensors (Basel) ; 18(3)2018 Mar 05.
Article in English | MEDLINE | ID: mdl-29510585

ABSTRACT

Continued advancement of protein array, bioelectrode, and biosensor technologies is necessary to develop methods for higher amount and highly oriented immobilization activity of proteins. In pursuit of these goals, we developed a new immobilization method by combining electrostatic transport and subsequent molecular diffusion of protein molecules. Our developed immobilization method is based on a model that transports proteins toward the substrate surface due to steep concentration gradient generated by low-frequency AC electric field. The immobilization of the maximum amounts can be obtained by the application of the AC voltage of 80 Vpp, 20 Hz both for His-tagged Green Fluorescent Protein (GFP) and Discosoma sp. Red Fluorescent Protein (DsRed), used as model proteins. The amounts of the immobilized His-tagged GFP and DsRed were approximately seven-fold higher than that in the absence of the application of low-frequency AC electric field. Furthermore, the positively and negatively charged His-tagged GFP at acidic and alkaline pH were immobilized by applying of low-frequency AC electric field, whereas the non-charged His-tagged GFP at the pH corresponding to its isoelectric point (pI) was not immobilized. Therefore, unless the pH is equal to pI, the immobilization of electrically charged proteins was strongly enhanced through electrostatic transport and subsequent molecular diffusion.

9.
J Biomol Struct Dyn ; 36(1): 32-44, 2018 01.
Article in English | MEDLINE | ID: mdl-27928933

ABSTRACT

Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.


Subject(s)
Antigens, Polyomavirus Transforming/metabolism , DNA Replication , DNA, Superhelical/metabolism , DNA, Viral/metabolism , Antigens, Polyomavirus Transforming/chemistry , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Humans , Magnetics , Microscopy, Fluorescence , Models, Molecular , Optical Tweezers , Protein Binding , Replication Origin/genetics
10.
Chem Pharm Bull (Tokyo) ; 65(4): 373-380, 2017.
Article in English | MEDLINE | ID: mdl-28381678

ABSTRACT

We observed that uncoated furosemide tablets turned yellow in a light-shielded automatic packaging machine and discoloration of the furosemide tablets was heterogeneity and occurred on the surface of the tablets only. The machine was equipped with an internal blower to maintain a constant temperature. Therefore, we investigated the effect of air flow on the discoloration of the furosemide tablets using a blower in a dark environment. The color difference (ΔE) of the furosemide tablets increased linearly as the blowing time increased. We performed structural analysis of the yellow compound in the furosemide tablets by LC-MS and identified the compound as a hydrolysate of furosemide. This suggested that furosemide hydrolysis was accelerated by the air flow. The furosemide tablets were prepared with the most stable furosemide polymorph, form I. X-Ray powder diffractometry and IR spectroscopy showed that during tablet preparation, no crystal transition occurred to an unstable furosemide polymorph. Furthermore, IR spectroscopy showed that the crystal form of furosemide in the yellow portion of the tablets was form I. To elucidate the factors producing the discoloration, we investigated the effect of humidity and atmosphere (air, oxygen, and nitrogen) on the discoloration of the furosemide tablets. The results suggested that the discoloration of the furosemide tablets was accelerated by oxidation, although humidity did not affect the hydrolysis. Therefore, we concluded that the discoloration of the furosemide tablets in the automatic packing machine was caused by acceleration of oxidative degradation by air flow.


Subject(s)
Color , Furosemide/chemistry , Light , Air , Nitrogen/chemistry , Oxygen/chemistry , Tablets
11.
Chem Pharm Bull (Tokyo) ; 63(8): 617-27, 2015.
Article in English | MEDLINE | ID: mdl-26235169

ABSTRACT

The aim of this study was to identify the chemical structure of the photodegradation products of furosemide in a water-acetonitrile mixture (1 : 1). Furosemide solution was irradiated with a D65 fluorescent lamp and the products were isolated by preparative HPLC. The fractions were evaporated to dryness in vacuo. The purity of the photodegradation products was measured by HPLC. The purity of products 1, 3, and 4 was greater than 90%, whereas that of product 2 was 13%, therefore, photodegradation product 2 was unstable. We identified photodegradation products 1 and 3 as 4-chloro-5-sulfamoylanthranilic acid and 4-hydroxy-N-furfuryl-5-sulfamoylanthranilic acid, respectively, by LC/MS and NMR. Additionally, we assumed that photodegradation product 4 was methyl 2-((furan-2-ylmethyl)amino)-4-hydroxy-3-(methyleneamino)-5-sulfamoylbenzoate by LC/MS and NMR. This showed that furosemide underwent hydrolysis and substitution, and reacted with the acetonitrile under the light of a D65 fluorescent lamp. We were furthermore able to determine the elution times of the photodegradation products of furosemide by applying the Japanese Pharmacopoeia chromatographic method for related substances to the isolated products.


Subject(s)
Furosemide/chemistry , Photolysis , Acetonitriles/chemistry , Chromatography, High Pressure Liquid , Hydrolysis , Magnetic Resonance Spectroscopy , Mass Spectrometry , Water/chemistry , ortho-Aminobenzoates/analysis
12.
Anal Chem ; 87(6): 3490-7, 2015 Mar 17.
Article in English | MEDLINE | ID: mdl-25697222

ABSTRACT

Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA.


Subject(s)
Bacteriophage lambda , DNA, Superhelical/chemistry , Models, Molecular , Nucleic Acid Denaturation , Time Factors
13.
Anal Biochem ; 457: 24-30, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24751469

ABSTRACT

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.


Subject(s)
Exodeoxyribonucleases/metabolism , Microfluidic Analytical Techniques/methods , Fluorescent Dyes , Nucleic Acid Denaturation , Organic Chemicals , Time Factors
14.
Sensors (Basel) ; 14(3): 5174-82, 2014 Mar 12.
Article in English | MEDLINE | ID: mdl-24625741

ABSTRACT

Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (-flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (-flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (-flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (-flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3'-5'exo-) was promoted by DNA stretching with buffer flow.


Subject(s)
DNA, Single-Stranded/biosynthesis , Luminescent Proteins/metabolism , Microfluidics/methods , Replication Protein A/metabolism , Bacterial Proteins/metabolism , DNA Polymerase I/metabolism , Fluorescence , Time Factors
15.
J Fluoresc ; 23(4): 635-40, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23471630

ABSTRACT

We developed two labeling methods for the direct observation of single-stranded DNA (ssDNA), using a ssDNA binding protein and a ssDNA recognition peptide. The first approach involved protein fusion between the 70-kDa ssDNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). The second method used the ssDNA binding peptide of Escherichia coli RecA labeled with Atto488 (ssBP-488; Atto488-IRMKIGVMFGNPETTTGGNALKFY). The labeled ssλDNA molecules were visualized over time in micro-flow channels. We report substantially different dynamics between these two labeling methods. When ssλDNA molecules were labeled with RPA-YFP, terminally bound fusion proteins were sheared from the free ends of the ssλDNA molecules unless 25-mer oligonucleotides were annealed to the free ends. RPA-YFP-ssλDNA complexes were dissociated by the addition of 0.2 M NaCl, although complex reassembly was possible with injection of additional RPA-YFP. In contrast to the flexible dynamics of RPA-YFP-ssλDNA complexes, the ssBP-488-ssλDNA complexes behaved as rigid rods and were not dissociated even in 2 M NaCl.


Subject(s)
Bacteriophage lambda , DNA, Single-Stranded/metabolism , DNA, Viral/metabolism , Fluorescent Dyes/metabolism , Microfluidic Analytical Techniques , Staining and Labeling/methods , Amino Acid Sequence , Base Sequence , DNA, Single-Stranded/genetics , DNA, Viral/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Fluorescent Dyes/chemistry , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Rec A Recombinases/chemistry
16.
J Fluoresc ; 21(3): 1189-94, 2011 May.
Article in English | MEDLINE | ID: mdl-21225324

ABSTRACT

Direct observation studies of single molecules have revealed molecular behaviors usually hidden in the ensemble and time-averaging of bulk experiments. Direct single DNA molecule analysis of DNA metabolism reactions such as DNA replication, repair, and recombination is necessary to fully understand these essential processes. Intercalation of fluorescent dyes such as YOYO-1 and SYTOX Orange has been the standard method for observing single molecules of double-stranded DNA (dsDNA), but effective fluorescent dyes for observing single molecules of single-stranded DNA (ssDNA) have not been found. To facilitate direct single-molecule observations of DNA metabolism reactions, it is necessary to establish methods for discriminating ssDNA and dsDNA. To observe ssDNA directly, we prepared a fusion protein consisting of the 70 kDa DNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). This fusion protein had ssDNA-binding activity. In our experiments, dsDNA was stained by SYTOX Orange and ssDNA by RPA-YFP, and we succeeded in staining ssDNA and dsDNA by using RPA-YFP and SYTOX Orange simultaneously.


Subject(s)
DNA, Single-Stranded/analysis , DNA, Single-Stranded/metabolism , Replication Protein A , Binding Sites , DNA-Binding Proteins , Luminescent Proteins , Methods , Recombinant Fusion Proteins
17.
Anal Biochem ; 400(1): 145-7, 2010 May 01.
Article in English | MEDLINE | ID: mdl-20085744

ABSTRACT

A simple molecular combing method for analysis of biochemical reactions, called the moving droplet method, has been developed. In this method, small droplets containing DNA molecules run down a sloped glass substrate, and this creates a moving interface among the air, droplet, and substrate that stretches the DNA molecules. This method requires a much smaller volume of sample solution than other established combing methods, allowing wider application in various fields. Using this method, lambdaDNA molecules were stretched and absorbed to a glass substrate, and single-molecule analysis of DNA synthesis by DNA polymerases was performed.


Subject(s)
DNA, Viral/biosynthesis , Animals , Carbocyanines/chemistry , DNA-Directed DNA Polymerase/metabolism , Fluorescent Dyes/chemistry , Glass , Rats
18.
J Fluoresc ; 19(1): 33-40, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18561002

ABSTRACT

Single-molecule studies have revealed molecular behaviors usually hidden in the ensemble and time averaging of bulk experiments. Single-molecule measurement that can control physical form of individual DNA molecules is a powerful method to obtain new knowledge about correlation between DNA-tension and enzyme activity. Here we study the effect of physical form of DNA on exonucleaseIII (ExoIII) reaction. ExoIII has a double-stranded DNA specific 3'-->5' exonuclease activity and the digestion is distributive. We observed the ExoIII digestion of individual stretched DNA molecules from the free ends. The sequentially captured photographs demonstrated that the digested DNA molecule linearly shortened with the reaction time. We also carried out the single-molecule observation under random coiled form by pausing the buffer flow. The digestion rates obtained from both single-molecule experiments showed that the digestion rate under the stretched condition was two times higher than the random coiled condition. The correlation between physical form of DNA and digestion rate of ExoIII was clearly demonstrated by single-molecule observations.


Subject(s)
DNA/chemistry , DNA/metabolism , Exodeoxyribonucleases/metabolism , Enzyme Activation , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Exodeoxyribonucleases/analysis , Fluorescence , Time Factors
19.
J Biomol Struct Dyn ; 25(5): 473-80, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18282002

ABSTRACT

Single-molecule DNA digestion by exonuclease III, which has 3' to 5' exonuclease activity, was analyzed using a micro-channel with two-layer laminar flow. First, a DNA-bead complex was optically trapped in one layer in the absence of exonuclease III permitted the DNA to be stretched by the laminar flow. The exonuclease III reaction was initiated by moving the trapped DNA-bead complex to another layer of flow, which contained exonuclease III. As the reaction proceeded, the fluorescently-stained DNA was observed to shorten. The process was photographed; examination of the photographs showed that the DNA molecule shortened in a linear fashion with respect to the reaction time. The digestion rate obtained from the single-molecule experiment was compared to that measured from a bulk experiment and was found to be ca. 28 times higher than the bulk digestion rate.


Subject(s)
DNA, Viral/metabolism , Escherichia coli Proteins/metabolism , Exodeoxyribonucleases/metabolism , Bacteriophage lambda/genetics , DNA, Viral/chemistry , Electrophoresis, Agar Gel , Escherichia coli Proteins/chemistry , Exodeoxyribonucleases/chemistry , Fluorescent Dyes/metabolism , Hydrolysis , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Optical Tweezers
20.
Arch Biochem Biophys ; 464(1): 28-35, 2007 Aug 01.
Article in English | MEDLINE | ID: mdl-17470356

ABSTRACT

Binding effect of the antitumor complex rhodium(II) acetate [Rh(2)(O(2)CCH(3))(4)] (Rh1) to the plasmid pUC19 DNA has been studied under different molar ratio of Rh1 compound to base pair of pUC19 DNA (R(f)) and reaction time. The Rh1 binding inhibited the activity of restriction enzyme. The binding effect was monitored using gel electrophoresis. The results indicate that at least one Rh1 binds with the recognition sequence and the binding has no preference between A-T and G-C pairs. At high value of R(f)=100, ICP-MS (Inductively Coupled Plasma Mass Spectrometry) measurement confirmed that 46% of Rh1 binds to DNA. PCR amplification of the DNA was also inhibited by the Rh1 binding. The transformation experiment using Escherichia coli suggested that the cell growth was inhibited after binding the Rh1 to the plasmid. These results indicated that DNA synthesis could be inhibited both in vitro and in vivo by the Rh(2)(O(2)CCH(3))(4) binding.


Subject(s)
Antineoplastic Agents/pharmacology , DNA Replication , DNA Restriction Enzymes/metabolism , Escherichia coli/metabolism , Rhodium/chemistry , Acetates/chemistry , Cell Line, Tumor , DNA Adducts/chemistry , DNA Primers/chemistry , DNA Restriction Enzymes/chemistry , Electrophoresis, Agar Gel , Humans , Nucleic Acids/chemistry , Plasmids/metabolism , Polymerase Chain Reaction , Protein Binding
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