Effects of condensing agent and nuclease on the extent of ejection from phage lambda

We have recently demonstrated, that DNA ejection from bacteriophage lambda can be partially or completely suppressed in vitro by external osmotic pressure. This suggests that DNA ejection from phage is driven by an internal mechanical force consisting of DNA bending and DNA-DNA electrostatic repulsion energies. In the present work we investigate the extent to which DNA ejection is incomplete at zero osmotic external pressure when phage is opened with its receptor in vitro. The DNA fragment remaining in the capsid and the tail that is no longer bent or compressed -and hence for which there is no internal driving force for ejection- is shown not to be ejected. We also demonstrate that DNA can be "pulled" out from the capsid by DNase I acting as a DNA binding protein or spermine acting as a DNA condensing agent. In particular, cryo electron microscopy and gel electrophoresis experiments show the following: (i) DNA ejection from bacteriophage lambda incubated in vitro with its receptor is incomplete at zero external osmotic force, with several persistence lengths of DNA remaining inside the phage capsid, if no nuclease (DNase I) or DNA condensing agent (spermine) is present in the host solution; (ii) in the presence of both DNase I and spermine in the host solution, 60% (approximately 29 kbp) of wild-type lambda DNA (48.5 kbp) remains unejected inside the phage capsid, in the form of an unconstrained toroidal condensate; (iii) with DNase I added, but no spermine, the ejection is complete; (iv) with spermine, but without DNase I added, all the DNA is again ejected, and organized as a toroidal condensate outside.     

Determination of nucleic acids using calcein-neodymium complex as a fluorescence probe.

A novel fluorometric method has been developed for rapid determination of DNA and RNA with calcein-neodymium complex as a fluorescence probe. The method is based on the fluorescence enhancement of calcein-Nd(III) complex in the presence of DNA or RNA, with maximum excitation and emission wavelength at 489 nm and 514 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.5 - 3.0 microg/ml for both DNA and yeast RNA, 0.4 - 2.0 microg/ml for fish sperm DNA (FS DNA) and 0 - 3.0 microg/ml for calf thymus DNA (CT DNA). The corresponding detection limits are 15.1 ng/ml for DNA, 21.2 ng/ml for yeast RNA, 10.5 ng/ml for FS DNA and 8.9 ng/ml for CT DNA. The interaction mechanism for the binding of calcein-Nd(III) complex to DNA is also studied. The results of absorption spectra, fluorescence polarization measurements and thermal denaturation experiments, suggested that the interaction between calcein-Nd(III) complex and DNA is an electrostatic interaction.     

Topology effect for DNA structure of cisplatin: topological transformation of cisplatin-closed circular DNA adducts by DNA topoisomerase I.

The reaction of cis-Pt(NH3)2Cl2(cis-DDP)-closed circular DNA adducts with DNA topoisomerse I(topo I) were studied by electron microscopy. We identified unique topoisomers such as a singly-linked catenane (2(1)2), trefoil (3(1), and dimetric catenane (2(1)2), etc., by analysis with electron micrographs. These unique recombination products resulted from cis-DDP-intra-twisting looped DNA adducts by DNA topo I, and the products could be explained a new mechanism based on an odd-even number rule. Our results suggest a new model on the working mechanisms for DNA topology of cis-DDP which enhances the recombination of DNA. Based on our results, we propose the topological idea that the yields of a mini closed circular DNA and pseudo trefoil DNA, etc., can be expected by reaction of cis-DDP-DNA-histone complexes with DNA topo I in the body.     

Spectroscopic and voltammetric study on the binding of aluminium(III) to DNA.

Cyclic voltammetry (CV) and ultraviolet (UV) spectroscopy were used, for the first time, to study the interaction between aluminium(III) and calf thymus DNA under neutral pH conditions. Thus obtained data confirmed the existence of a relatively strong interaction between Al(III) and DNA. The binding site for aluminium(III) on DNA chains is not the bases, but the phosphate groups on the DNA backbones, the same as that for [Co(phen)3](3+/2+) that binds non-specifically and electrostatically to the deoxyribose phosphate backbone of DNA. When coexisting, Al(III) binds more favorably to DNA than [Co(phen)3](3+/2+), which implies the relatively strong binding of Al(III) to the phosphate backbone of DNA under neutral pH conditions. The nature of the binding of Al(III) to DNA is also discussed.     

A novel spectrofluorimetric method for the determination of DNA

A new simple, selective and sensitive fluorescence quenching method was developed to determine nucleic acids (DNA) with the 9-anthracenecarboxylic acid (ACA)-cetyl trimethyl-ammonium bromide (CTAB) system. The fluorescence intensity of ACA was decreased by the addition (CTAB). However, the fluorescence intensity of the system increased dramatically when DNA was added to the solution. The fluorescence enhancement is probably based on the DNA interaction with CTAB. Under the optimum conditions, the changes of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.08-1.0 microg mL(-1) for CT (calf thymus) DNA or FS (fish sperm) DNA. Its detection limits are 0.02 microg mL(-1) for CT DNA and 0.019 microg mL(-1) for FS DNA. Based on this approach, a new quantitative method for DNA assay is presented in this paper.     

Construction and control of plasmid DNA network

The influences of different cations on plasmid DNA network structures on a mica substrate were investigated by atomic force microscopy (AFM). Interactions between the DNA strands and mica substrate, and between the DNA strands themselves were more strongly influenced by the complex cations (Fe(phen)3(2+), Ni(phen)3(2+), and Co(phen)3(3+)) than by the simple cations (Mg2+, Mn2+, Ni2+, Ca2+, Co3+). The mesh height of the plasmid DNA network was higher when the complex cations were added to DNA samples. The mesh size decreased with increasing DNA concentration and increased with decreasing DNA concentration in the same cation solution sample. Hence, plasmid DNA network height can be controlled by selecting different cations, and the mesh size can be controlled by adjusting plasmid DNA concentration.     

Flow-injection chemiluminescence study of Ce(IV)-Na2SO3-Tb(III)-fluoquinolone antibiotic system with DNA

A novel flow injection chemiluminescence (CL) system is developed to determine DNA. According to the fact that DNA linearly quenches the CL intensity of Ce(IV)-Na(2)SO(3)-Tb(III)-fluoquinolone antibiotic (FLUQ) system, DNA concentration is determined. The calibration graphs are linear in the range of 0.04-10 microg/ml (for both natural and denatured DNA), and the 3sigma limits of detection are 7.8 ng/ml (natural DNA) and 9.5 ng/ml (denatured DNA). According to fluorescence spectrum and CL spectrum, and through studying the reaction of Ce(IV)-Na(2)SO(3)-Tb(III)-FLUQ with nucleotides and bases, we conclude that DNA counteracts the energy transfer from FLUQ to Tb(III), and this function is related to both bases and phosphate groups in DNA.     

Pressure-induced transport of DNA confined in narrow capillary channels

Pressure-induced transport of double-stranded DNA (dsDNA) from 10 base pairs (bp) to 1.9 mega base pairs (Mbp) confined in a 750-nm-radius capillary was studied using a hydrodynamic chromatographic technique and four distinct length regions (rod-like, free-coiled, constant mobility, and transition regions) were observed. The transport behavior varied closely with region changes. The rod-like region consisted of DNA shorter than the persistence length (～150 bp) of dsDNA, and these molecules behaved like polymer rods. Free-coiled region consisted of DNA from ～150 bp to ～2 kilo base pairs (kbp), and the effective hydrodynamic radius R(HD) of these DNA scaled to L(0.5) (L is the DNA length in kbp), a characteristic property of freely coiled polymers. Constant mobility region consisted of DNA longer than ～100 kbp, and these DNA had a constant hydrodynamic mobility and could not be resolved. Transition region existed between free-coiled and constant mobility regions. The transport mechanism of DNA in this region was complicated, and a general empirical equation was established to relate the mobility with DNA length. Understanding of the fundamental principles of DNA transport in narrow capillary channels will be of great interest in the development of "lab-on-chip" technologies and nongel DNA separations.     

Electrochemical measurement of phenothiazine-interacted DNA

The amount of DNA was measured by using thioridazine, which would be attached to the DNA, as an electrochemical indicator. An indicator (thioridazine) solution, a test solution (DNA solution), and a poly-l-lysine solution were successively placed on a glassy carbon electrode, and the electrode was allowed to dry; DNA was immobilized on an electrode surface by the electrostatic binding between DNA and poly-l-lysine. The electrode was immersed into a buffer solution for 15 min, and then differential pulse voltammetry (DPV) was carried out: the oxidation current peak of thioridazine was observed, and its magnitude depended on the amount of DNA in the solution which was used for preparing the electrode. It could be estimated between 0.2 microg DNA (corresponds to 630 pmol nucleotides) to 20 microg DNA (63 nmol nucleotides) from the oxidation peak current of DPV.     

Application of magdala red as a fluorescence probe in the determination of nucleic acids

A fluorescence quenching method was developed for the rapid determination of DNA and RNA using magdala red as fluorescence probe. In weakly acidic medium, the fluorescence of magdala red (lambdaex/lambdaem = 540/555 nm) can be largely quenched by DNA or RNA. The calibration graphs are linear over the range 0.01-1.2 microg/mL for both calf thymus DNA (CT DNA) and salmon DNA (SM DNA), and 0.015-1.0 microg/mL for yeast RNA, respectively. The corresponding detection limits are 6.0 ng/mL for CT DNA, 7.0 ng/mL for SM DNA and 15.0 ng/mL for yeast RNA, respectively. CT DNA could be determined in the presence of 20% (w/w) yeast RNA, and the relative standard deviation of six replicate measurements is 3.18% for 400 ng/mL of CT DNA. Interference from coexisting substances in the determination of DNA was also examined. Real samples were determined with satisfactory results.     

Metallobleomycin-mediated cleavage of DNA not involving a threading-intercalation mechanism.

The DNA cleavage properties of metallobleomycins conjugated to three solid supports were investigated using plasmid DNA, relaxed covalently closed circular DNA, and linear duplex DNA as substrates. Cleavage of pBR322 and pSP64 plasmid DNAs by Fe(II).BLM A(5)-CPG-C(2) was observed with efficiencies not dissimilar to that obtained using free Fe(II).BLM A(5). Similar results were observed following Fe(II).BLM A(5)-CPG-C(2)-mediated cleavage of a relaxed plasmid, a substrate that lacks ends or negative supercoiling capable of facilitating strand separation. BLMs covalently tethered to solid supports, including Fe(II).BLM A(5)-Sepharose 4B, Fe(II).BLM A(5)-CPG-C(6), and Fe(II).BLM A(5)-CPG-C(2), cleaved a 5'-(32)P end labeled linear DNA duplex with a sequence selectivity identical to that of free Fe(II).BLM A(5); cleavage predominated at 5'-G(82)T(83)-3' and 5'-G(84)T(85)-3'. To verify that these results could also be obtained using other metallobleomycins, supercoiled plasmid DNA and a linear DNA duplex were employed as substrates for Co(III).BLM A(5)-CPG-C(2). Free green Co(III).BLM A(5) was only about 2-fold more efficient than green Co(III).BLM A(5)-CPG-C(2) in effecting DNA cleavage. A similar result was obtained using Cu(II).BLM A(5)-CPG-C(2) + dithiothreitol. In addition, the conjugated Co.BLM A(5) and Cu.BLM A(5) cleaved the linear duplex DNA with a sequence selectivity identical to that of the respective free metalloBLMs. Interestingly, when supercoiled plasmid DNA was used as a substrate, conjugated Fe.BLM A(5) and Co.BLM A(5) were both found to produce Form III DNA in addition to Form II DNA. The formation of Form III DNA by conjugated Fe.BLM A(5) was assessed quantitatively. When corrected for differences in the intrinsic efficiencies of DNA cleavage by conjugated vs free BLMs, conjugated Fe.BLM A(5) was found to produce Form III DNA to about the same extent as the respective free Fe.BLM A(5), arguing that this conjugated BLM can also effect double-strand cleavage of DNA. Although previous evidence supporting DNA intercalation by some metallobleomycins is convincing, the present evidence indicates that threading intercalation is not a requirement for DNA cleavage by Fe(II).BLM A(5), Co(III).BLM A(5), or Cu(I).BLM A(5).     

Polyplexes of poly(methylaminophosphazene): energetics of DNA melting

The interaction of DNA with a synthetic biocompatible and biodegradable cationic polymer, poly(methylaminophosphazene) hydrochloride (PMAP·HCl), was investigated by high-sensitivity differential scanning calorimetry under conditions of strong and weak electrostatic interactions of the macroions. Thermodynamic parameters of the DNA double-helix melting were determined as a function of pH and the PMAP·HCl/DNA weight ratio. PMAP·HCL was shown to reveal two functions with respect to DNA: the polyelectrolyte function and the donor-acceptor one. The first function stabilizes the helical conformation of DNA, and the second one destabilizes it. The stabilizing effect of PMAP·HCl is of entropic origin, related to a displacement of mobile counterions from the DNA's nearest surroundings by the poly(methylaminophosphazene) charged groups. The donor-acceptor function of poly(methylaminophosphazene) dominates when its electrostatic interaction with DNA is either saturated (in the complex coacervate phase at high poly(methylaminophosphazene) concentrations) or completely suppressed (in a salt medium when the polycation carries a small charge). Under these conditions, poly(methylaminophosphazene) destabilizes DNA. It preferentially binds to the DNA coil form likely via the formation of multiple labile hydrogen bonds with the donor-acceptor groups of DNA.     

Raman spectroscopic study of microcosmic photodamage of the space structure of DNA sensitized by Yangzhou haematoporphyrin derivative and Photofrin II

After calf thymus DNA has been photodamaged by Yangzhou haematoporphyrin derivative (YHPD) or Photofrin II, the Raman characteristic frequencies and the intensities of the bands assigned to various groups of the components of DNA change considerably. As a result of damage, homogeneous B-form calf thymus DNA becomes a mixture containing: (1) modified B-form DNA, which is shorter than the original because of double-helical DNA scission; (2) single-stranded DNA due to the breakage of some H-bonds and the lack of some bases, etc.     

四环素-铜(Ⅱ)配合物与DNA相互作用的吸收光谱研究

用紫外光谱方法研究了四环素(TC) Cu(II)配合物与DNA的相互作用.吸收光谱研究表明,DNA能与四环素(TC)及Cu(II)形成的配合物发生反应,配合物与DNA的作用方式随着配合物类型及DNA浓度的不同而不尽相同:当四环素与铜形成1∶1型配合物时,较低浓度的DNA能与配合物以嵌插方式相互作用,而较高浓度的DNA与该配合物除了发生嵌插作用外,还存在另外的作用方式;当四环素与铜形成1∶2型配合物时,DNA与该配合物则主要以嵌插方式相互作用,并且这两种配合物与DNA的嵌插作用均是通过四环素配体插入的.     

样品稀释对毛细管电泳分离检测DNA片段的影响

通过理论推导和实验验证表明;适当稀释DNA样品溶液,采用流体力学进样或电动进样都不会较大地减低峰高,而DNA片段毛细管电泳的分离效率和分离度还能有所提高。采用稀释样品的方法可提高DNA样品的使用效率。采用羟乙基纤维素无胶筛分介质分离了DNA片段。用激光诱导荧光（氩离子激光器,488nm）电荷耦合器件检测。用低浓度的筛分介质（0．4％）分离了分子质量较大的ADNA－HindⅢ全部8个片段（12bp～23130bP）。用高浓度的筛分介质（1．6％）分离分子质量较小的pBR322－HaeⅢ22个片段（18bp～587bp）。     

Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker

This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.     

Magnetically trigged direct electrochemical detection of DNA hybridization using Au67 quantum dot as electrical tracer

A novel gold nanoparticle-based protocol for detection of DNA hybridization based on a magnetically trigged direct electrochemical detection of gold quantum dot tracers is described. It relies on binding target DNA (here called DNA1) with Au(67) quantum dot in a ratio 1:1, followed by a genomagnetic hybridization assay between Au(67)-DNA1 and complementary probe DNA (here called DNA2) marked paramagnetic beads. Differential pulse voltammetry is used for a direct voltammetric detection of resulting Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate on magnetic graphite-epoxy composite electrode. The characterization, optimization, and advantages of the direct electrochemical detection assay for target DNA are demonstrated. The two main highlights of presented assay are (1) the direct voltammetric detection of metal quantum dots obviates their chemical dissolution and (2) the Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate does not create the interconnected three-dimensional network of Au-DNA duplex-paramagnetic beads as previously developed nanoparticle DNA assays, pushing down the achievable detection limits.     

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.     

多吡啶钌配合物作为DNA结构探针的研究

本文对多吡啶钌配合物作为DNA荧光或结构探针的研究背景、研究技术及其特点、钌配合物与DNA的键合模式及其结合力大小的影响因素、钌配合物与DNA键合的异构选择性及不同键合速率、非放射性核酸标记及DNA分子光开关等方面进行了简要述评     

Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA > DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.