A fluorescent molecular switch for room temperature operation based on oligonucleotide hybridization without labeling of probes or targets

A molecular switch was prepared by self-assembly. Neutravidin served as a template that allowed for a biotinylated probe oligonucleotide to be placed adjacent to a biotinylated long-chain linker that was terminated with thiazole orange (TO). Hybridization of probe oligonucleotide with target to form double-stranded DNA resulted in intercalation of the adjacent TO probe. This was a reversible process that could be tracked by fluorescence intensity changes. Formamide was used as a denaturant for double-stranded DNA, and could be used to depress thermal denaturation temperatures. In this work formamide had a dual function, providing for control of hybridization selectivity at room temperature, while concurrently ameliorating non-specific adsorption to improve signal-to-noise when using thiazole orange as a fluorescence signalling agent to determine oligonucleotide hybridization. Room temperature single nucleotide polymorphism (SNP) discrimination for oligonucleotide targets was achieved both in solution and for molecular switches that were immobilized onto optical fibers. In solution, a concentration of 18.5% formamide provided greater than 40-fold signal difference between single-stranded DNA and double-stranded DNA, in contrast to only a 2-fold difference in the absence of formamide. Selectivity for SNP determination in solution was demonstrated using targets of varying lengths including a 141-base PCR amplicon. The improved signal-to-noise achieved by use of formamide is likely due to preferential displacement of dye molecules that are otherwise electrostatically bound to the polyanionic nucleic acid backbone.     

Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation.     

Intercalation interactions between dsDNA and acridine studied by single molecule force spectroscopy

In this letter, we report on the direct measurement of the intercalation interactions between acridine and double-stranded DNA (dsDNA) using single molecule force spectroscopy. The interaction between acridine and dsDNA is broken by force of 36 pN at a loading rate of 5.0 nN/s. The most probable rupture force between acridine and dsDNA is dependent on the loading rate, indicating that the binding of acridine and dsDNA is a dynamic process. The combination of SMFS experimental data with the theoretical model clearly suggests the presence of two energy barriers along with an unbinding trajectory of acridine-dsDNA.     

酸性条件下的DNA构象变化加速DNA变性解链

调控DNA的变性解链过程是DNA扩增与检测的关键步骤。对于传统的热循环DNA扩增策略,由于变温过程中热量分布不均一以及变温速度慢等不利因素,会直接影响DNA变性解链过程,从而降低DNA检测放大的效果、延长检测的时长。因此,探索快速、高效的调控DNA变性解链的方法具有重要的研究意义。本文发展了以胞嘧啶在酸性条件下的质子化反应为基础,通过改变溶液的pH值,来诱导DNA构象在Watson-Crick（WC）碱基对与Hoogsteen（HG）碱基对之间的分子构象转换,从而实现精准、快速、高效的DNA变性解链调控目标。结果表明,相较于传统的温控方法,pH调控方法能显著提高DNA变性的速率约6倍以上。本文发现pH调控方法通过降低双链DNA的反应焓约160 kJ/mol,从而提高双链DNA变性速率和效率。该方法具有用于DNA信号放大与检测等相关应用的潜力。     

Effect of spermine conjugation on the interaction of acridine with alternating purine-pyrimidine oligodeoxyribonucleotides studied by CD, fluorescence and absorption spectroscopies

We studied by electronic spectroscopies the interaction between double-stranded oligonucleotides containing either adenine-thymine or guanine-cytosine alternating sequences and N(1)-(acridin-9-yl)-1,16-diamino-4,8,13-triazahexadecane, which is a conjugated molecule formed by the covalent binding of spermine and 9-aminoacridine moieties via a trimethylene chain. Solutions containing the oligonucleotides and the conjugate, at different molar ratios, were studied by using electronic absorption, fluorescence emission and circular dichroism. Calculated association constants and fluorescence emission spectra showed that spermine conjugation induces sequence selectivity. The orientation of the intercalated acridine rings with respect to the oligonucleotide base planes was deduced from the electronic circular dichroism spectra. Evidence of the formation of spermine-induced aggregated structures, with potential applications to DNA packaging, gene therapy and anti-tumor therapy, was also achieved. Our data demonstrates that this spermine-acridine conjugate adds several specific characteristics provided by the polyamine moiety, as sequence selectivity, to the interesting properties of acridine derivatives.     

A breathing wormlike chain model on DNA denaturation and bubble: effects of stacking interactions

DNA stably exists as a double-stranded structure due to hydrogen-bonding and stacking interactions between bases. The stacking interactions are strengthened when DNA is paired, which results in great enhancement of bending rigidity. We study the effects of this stacking-induced stiffness difference on DNA denaturation and bubble formations. To this end, we model double-stranded DNA as a duplex of two semiflexible chains whose persistence length varies depending on the base-pair distance. Using this model, we perform the Langevin dynamics simulation to examine the characteristics of the denaturation transition and the statistics of the bubbles. We find that the inclusion of the stacking interactions causes the denaturation transition to be much sharper than otherwise. At physiological temperature, the stacking interactions prohibit the initiation of bubble formation but promote bubbles, once grown, to retain the large size.     

Electrochemical DNA Biosensors Applicable to the Study of Interactions Between DNA and DNA Intercalators

Electrochemical DNA biosensors, based either on carbon paste electrode (CPE) or hanging mercury drop electrode (HMDE) were prepared. These biosensors were used in the study of interaction between double stranded DNA (dsDNA) and single stranded DNA (ssDNA) and acridine orange, a well known DNA intercalator. The different electrochemical behaviors were compared in the article.     

Active uptake of drugs into photosensitive liposomes and rapid release on UV photolysis

Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-L-alpha-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4'-n-butylphenyl)azo-4'-(gamma-phenylbutyroyl))-glycero-3- phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30 degrees C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photo-induced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.     

Energy transfer of ionic dyes in mixed surfactant vesicle

Stable vesicles composed of cationic and anionic single-tailed-surfactant were prepared, and their image obtained by electron microscopy with negative staining technique. Significant fluorescence enhancement for acridine orange in vesicle with regards to water has been observed. In heterogeneous vesicle solution composed of mixed cationic and anionic surfactants for the energy transfer between acridine orange (D) and pyronine (A), the Förster dipole-dipole model was valid, and it is interesting to note that the energy transfer rate constant (k_ET) was smaller than that in homogeneous aqueous solution. On the inside and outside of the stable vesicle, immiscible water solution of acridine orange and pyronine could be obtained, and the distance calculated from the energy transfer between D and A separated by the bilayer membrane implied that the location of ionic dye molecules was in the Gouy-Chapman layers of the vesicles. Furthermore, due to the electrostatic absorption of the dye molecules to charged headgroups of surfactants, acridine orange and pyronine accumulated and aggregated to the vesicle bilayer membrane.     

Real-time surface-enhanced Raman spectroscopy monitoring of surface pH during electrochemical melting of double-stranded DNA

The application of a negative potential ramp at a double-stranded DNA (dsDNA) functionalized electrode surface results in the gradual denaturation of the DNA in a process known as electrochemical melting. The underlying physical chemistry behind electrochemically driven DNA denaturation is not well understood, and one possible mechanism is a change in local pH at the electrode surface. We demonstrate that by coimmobilization of p-mercaptobenozic acid at a dsDNA-functionalized electrode surface, it is possible to monitor both DNA denaturation and the local pH simultaneously using surface-enhanced Raman spectroscopy. We find that the local pH at the electrode surface does not change as the applied potential is scanned negative and the dsDNA denatures. We therefore conclude that in these experiments electrochemical melting is not caused by electrochemically driven local pH changes.     

Strand invasion of mixed-sequence B-DNA by acridine-linked, gamma-peptide nucleic acid (gamma-PNA)

Peptide nucleic acid (PNA) is a synthetic mimic of DNA and RNA that can recognize double-stranded B-DNA through direct Watson-Crick base-pairing. Although promising, PNA recognition is presently limited to mostly purine- and pyrimidine-rich targets, because mixed-sequence PNA, in general, does not have sufficient binding free energy to invade B-DNA. In this Article, we show that conformationally preorganized gamma-peptide nucleic acid (gamma-PNA) containing an acridine moiety covalently linked at the C-terminus can invade mixed-sequence B-DNA in a sequence-specific manner. Recognition occurs through direct Watson-Crick base-pairing. This finding is significant because it demonstrates that the same principles that guide the recognition of single-stranded DNA and RNA can also be applied to double-stranded B-DNA.     

An integrated method for mutation detection using on-chip sample preparation, single-stranded conformation polymorphism, and heteroduplex analysis

This work integrates rapid techniques for mutation detection by producing single-stranded DNA and (renatured) double-stranded DNA on-chip, labeling these with fluorescent DNA stains and then performing two complementary methods of mutation detection-single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis (HA). This involves the denaturation of double-stranded polymerase chain reaction (PCR) product into single-stranded DNA, the mutation analysis of the single-stranded DNA by SSCP and the rehybridized double-stranded DNA by HA. These steps were performed entirely on-chip within several minutes of operation. The combination of these two mutation detection methods on-chip provides a highly sensitive method of mutation detection for either genotyping or screening. Many mutation analysis methods rely upon fluorescently labeled samples from a PCR with fluorescently labeled primers. By labeling on-chip we not only attain improved signal strength, but the method is considerably more versatile. Although we used PCR products in this work, this method could be used to analyze DNA from any source. We believe that this combination of several procedures on a single chip represents a significant step in the development of higher levels of integration upon microfluidic devices.     

染料和聚电解质间的相互作用——在染料-磺化聚苯乙烯络合物中的能量转移和染料荧光强度的增强

本工作对磺化聚苯乙烯-吖啶橙体系的光物理行为进行了研究。发现吖啶橙的荧光强度强烈地依赖于所用染料和聚电解质的当量比(P/D)。在P/D=100时观察到有最大的荧光强度。经过校正,此值和Fox等研究苯乙烯和乙烯基共聚物的能量转移中所得结果基本一致。这表明在高分子电解质-染料络合物中能发生有效的能量转移。 本工作还研究了亚甲蓝对吖啶橙荧光的猝灭效应。观察到当体系中加入聚电解质时猝灭效应得以增强。     

Preparation of DNA-loaded polysulfone microspheres by liquid-liquid phase separation and its functional utilization

DNA-loaded polysulfone (PSf) microspheres were fabricated by means of a liquid-liquid phase separation technique. The porous microspheres were then used to remove DNA-binding intercalating materials--ethidium bromide, acridine orange, and endocrine disruptors. The DNA-loaded PSf microspheres are stable in water. The stability of the DNA-loaded microspheres and/or the release rate of DNA from the microspheres can be controlled by manipulating the microsphere structure. Increasing the polymer concentration, which causes lower porosity and smaller pores on the outer surface of the microspheres, led to increased stability of the microspheres and decreased release rate of DNA. Additionally, the drying temperature also affected the stability of the microspheres. The DNA-loaded PSf microspheres could effectively accumulate harmful DNA-intercalating pollutants and endocrine disruptors, such as ethidium bromide, acridine orange, biphenyl, dibenzofuran, and dibenzo-p-dioxin. The amount of pollutants removed by the microspheres is dependent on the amount of incorporated DNA and on the microsphere structure. The DNA-loaded microspheres have the potential to be used in environmental applications.     

Effect of denaturation on the photochemistry of pyrimidine bases in isolated DNA

The influence of denaturation on DNA photochemistry was studied by quantifying the yield of formation of all possible bipyrimidine photolesions within isolated genomic DNA samples exposed to UVC radiation. Effects of DNA melting was studied either by carrying out irradiation over a wide range of temperature (0-90 degrees C) or by decreasing the ionic strength of the solution at 30 degrees C. A first observation was a much larger decrease in the photoreactivity upon increasing the temperature in single-stranded than in double-stranded DNA. Secondly, formation of trans,syn cyclobutane dimers and, to a lesser extent, modification in the ratio between the yields of cyclobutane dimers and (6-4) photoproducts, were found to be other main features associated with denaturation. These results emphasize the modulating role of structure in the yield and nature of UV-induced DNA damage.     

DNA sensor: A novel electrochemical gene detection method using carbon electrode immobilized DNA probes

Abstract

The authors have developed a novel, rapid, convenient, and specific gene detection method, named the ‘DNA sensor,’ using a graphite electrode loaded with DNA probes. Synthesized oligonucleotide (5-TGCAGTTCCGGTGGCTGATC-3′) complementary to oncogene v-myc was employed for a model probe. The oligonucleotide was chemically adsorbed on a basal plane pyrolytic graphite (BPPG) electrode. The sensor was able to be applied to a hybridization reaction (40°C) in a linearized pVM623 solution carrying the Pst I fragment of v-myc (1.5 kbp).

After the hybridization reaction, the sensor was immersed into an acridine orange solution (1 μM) and washed with a phosphate buffer (pH 7.0). Acridine orange intercalated between base pairs of the formed double stranded DNAs on the electrode. The anodic peak potential of acridine orange that interacted with the DNAs on the electrode was measured. The positive shift of the peak potential increased in proportional to the pVM623 concentration in the hybridization reaction. 10^?1 g/ml of pVM623 was able to be detected in the buffer solution using the sensor. This gene detection was completed within an hour.     

Orientation of dye molecules in DNA-based films with chain alignment and judgment of their DNA-binding modes

Novel composite films of chain-oriented DNA, which contain the DNA-binding dyes aligned in specific orientation, were successfully prepared by drying the solution under a horizontal magnetic field. Most of the dye-DNA composite films showed linear dichroism, as revealed by polarized ultraviolet-visible (UV-vis) spectroscopy. The intercalators, ethidium bromide and acridine orange, were fixed in chain-oriented DNA films in a similar binding manner as in solutions. Also, Hoechst 33258 and 4',6-diamidino-2-phenylindole were found to be aligned along the minor groove, even in the solid films. Thus, our new method of preparing dye-DNA composite films with chain orientation is useful for aligning small molecules, and it will provide views of the novel anisotropic materials expected in various application fields. We used this method to prepare composite DNA films with newly designed original compounds. Seven of nine dyes were judged to bind obviously to DNA as intercalators by polarized UV-vis spectroscopy. The DNA-binding manners were further analyzed by fluorescence anisotropy measurements. On the basis of the curves for the rotational angle dependence of the anisotropy, we were able to estimate the angles between the transition-dipole moments of dyes and the aligned chain axis of DNA. Interestingly, two original compounds were found to be in the tilted forms with regard to the plane of base pairs. We emphasize here that the method using aligned dye-DNA films is very convenient for identifying the binding modes of the compounds for double-stranded DNA.     

Influence of prior complex formation on the photoaddition of chlorpromazine to calf thymus deoxyribonucleic acid

Chlorpromazine (CPZ), 2-chloro-N-(3-dimethylaminopropyl) phenothiazine, causes cutaneous photosensitivity in man. The photoaddition of CPZ to deoxyribonucleic acid (DNA) may be an important mechanism for the phototoxicity. We have investigated the complexes formed between CPZ and calf thymus DNA prior to irradiation, related their formation to the photoaddition of CPZ to DNA and initiated studies to identify the photoadducts. In the presence of high concentrations of double-stranded DNA, the CPZ absorption maximum shifted from 305 to 340 nm with an isosbestic point at 323 nm. The CPZ fluorescence at 460 nm was quenched a maximum of 90%. The excitation and emission spectra for the unquenchable fluorescence are the same as those for free CPZ. These results together with those from flow dichroism measurements indicated that CPZ formed two complexes with double-stranded DNA. One complex involves intercalations, is non-fluorescent and absorbs at 345 nm. The second complex absorbs at 310 nm, fluoresces at 460 nm and has the phenothiazine ring parallel to the DNA axis. Non-covalent binding of CPZ to heat-denatured DNA did not shift the CPZ absorption spectrum but quenched 65% of the CPZ fluorescence. One complex between CPZ and denatured DNA will account for these results. CPZ photolysis was inhibited compared with that of free CPZ by binding to double-stranded DNA (more than 98%) or denatured DNA (65%). CPZ photoadded ten times more efficiently to denatured DNA than to double-stranded DNA. These results indicate that CPZ photolysis and photoaddition are quenched in the intercalation complex. The photoaddition to double-stranded DNA does not result from intercalated CPZ because the action spectrum maximized at 310 nm rather than at 340 nm.     

DNA分子结构的多态性研究 (Ⅱ) 吖啶橙凝聚体变化诱导的质粒DNA大分子构象的研究

利用吸收光谱和荧光光谱方法,研究了吖啶橙（ＡＯ）与质粒ＤＮＡ水溶液、以及含胶束介质的吖啶橙与质粒ＤＮＡ溶液体系的相互结合作用及减色效应。结果表明：吖啶橙对质粒ＤＮＡ的吸收光谱有减色效应;含十二烷基硫酸钠（ＳＤＳ）的ＡＯ水溶液体系中,随着ＳＤＳ浓度的增加,其光谱结果表现为由凝聚态向单体的转化。而在含十二烷基硫酸钠（ＳＤＳ）的ＡＯ与质粒ＤＮＡ溶液体系中,吖啶橙凝聚态随ＳＤＳ浓度的增加,对ＡＯ与质粒ＤＮＡ相互结合产生协同的减色效应,使质粒ＤＮＡ空间结构发生缩拢。进一步采用电泳法研究了ＡＯ凝聚态可能对质粒ＤＮＡ构象的影响,结果表明：在ＡＯ与质粒ＤＮＡ溶液体系中,ＡＯ浓度的增加对质粒ＤＮＡ构象未产生影响;而在含有ＳＤＳ的ＡＯ与质粒ＤＮＡ的溶液体系中,由于ＳＤＳ对ＡＯ凝聚态的解聚作用,以及ＳＤＳ对质粒ＤＮＡ减色效应的协同作用,使得质粒ＤＮＡ的构象发生变化,诱导质粒ＤＮＡ形成超螺旋构象     

胶体金在DNA分子识别中的应用——胶体金对罗丹明B与单双链DNA识别作用的影响

通过研究罗丹明B与单双链DNA作用过程中，胶体金加入前后的紫外可见吸收光谱的变化，发现在胶体金加入的前后，罗丹明B均以沟槽方式与单双链DNA相作用；胶体金的加入可以较大程度地增大化合物与DNA之间的相互作用．