Propidium Iodide and PicoGreen as Dyes for the DNA Fluorescence Correlation Spectroscopy Measurements

Many experimental designs, in which nucleic acid conformational changes are of interest, require reliable fluorescence labeling. The appropriate fluorescence probe should have suitable optical properties and, more importantly, should not interfere with the investigated processes. In order to avoid chemical modifications the fluorescence label needs to be associated with nucleic acid via weak non-covalent interactions. There are a number of fluorescent probes that change their fluorescent properties (i.e. their quantum yield and/or spectral characteristics) upon association with nucleic acid. Such probes are frequently used to detect, visualize and follow processes involving nucleic acid and its conformational changes. In order to obtain reliable data regarding macromolecule or aggregate topology a detailed knowledge of probe–nucleic acid interactions on the molecular level is needed. In this paper we show that the association of propidium iodide with DNA alters its conformation and that it selectively labels plasmid fragments and/or its subpopulations in a concentration-dependent meaner. Another dye, PicoGreen, exhibits better properties. It labels nucleic acid uniformly and without any concentration-dependent artifacts.     

YOYO as a Dye to Track Penetration of LK15 DNA Complexes in Spheroids: Use and Limits

To elucidate the reasons underlying the poor penetration of non-viral vectors in tissues, relating transport properties to physico-chemical parameters of vectors may be crucial. These properties can be influenced by the presence of multiples labels that are used. Therefore utilizing a vector with minimum of labels preferably not more than one is important to studying penetration in tissues. The cell impermeant bisintercalating dye YOYO-1 was found suitable to both monitor the formation of complexes between DNA and an amphipathic peptide LK15 and, to track their penetration in HCT116 spheroids by confocal microscopy. The results revealed a limited decrease of fluorescence ascribed to the high affinity of YOYO-1 to bind DNA. The residual fluorescence of complexes can be exploited to monitor penetration into spheroids, after correction for YOYO-1 attenuation, and to revealing hyaluronidase-induced reduced binding. Hence high affinity dyes such as YOYO-1 with inefficiently quenched fluorescence may be important to establish a relation between novel medicines characteristics and penetration in tissues.     

Effect of marine derived deoxyribonucleic acid on nonlinear optical properties of PicoGreen dye

We have investigated the effect of DNA on nonlinear absorption of PicoGreen dye using single beam open aperture Z-scan technique in nanosecond regime. We observed reverse saturable absorption at 532 nm for PicoGreen without DNA. In the presence of DNA, the sample begins to behave like saturable absorbers and this effect increased as the concentration of DNA was increased. The dye-intercalated DNA showed SA characteristics near the focus but exhibited RSA characteristics at the focus. Theoretical analysis has been performed using a two-photon absorption model based on nonlinear absorption coefficient and saturation intensity. Such tailoring of optical nonlinear absorption in PicoGreen makes it a potential candidate for photonic application.     

Direct immobilization and hybridization of DNA on group III nitride semiconductors

A key concern for group III-nitride high electron mobility transistor (HEMT) biosensors is the anchoring of specific capture molecules onto the gate surface. To this end, a direct immobilization strategy was developed to attach single-stranded DNA (ssDNA) to AlGaN surfaces using simple printing techniques without the need for cross-linking agents or complex surface pre-functionalization procedures. Immobilized DNA molecules were stably attached to the AlGaN surfaces and were able to withstand a range of pH and ionic strength conditions. The biological activity of surface-immobilized probe DNA was also retained, as demonstrated by sequence-specific hybridization experiments. Probe hybridization with target ssDNA could be detected by PicoGreen fluorescent dye labeling with a minimum detection limit of 2 nM. These experiments demonstrate a simple and effective immobilization approach for attaching nucleic acids to AlGaN surfaces which can further be used for the development of HEMT-based DNA biosensors.     

Enhanced Emission Induced by FRET from a Long-Lifetime, Low Quantum Yield Donor to a Long-Wavelength, High Quantum Yield Acceptor

We report observation of high quantum yield, long-lifetime fluorescence from a red dye BO-PRO-3 excited by resonance energy transfer (RET). The acceptor fluorescence was highly enhanced upon binding to the donor-labeled DNA. A ruthenium complex (Ru) was chosen as a donor in this system because of its long fluorescence lifetime. Both donor and acceptor were non-covalently bound to DNA. Emission from the donor-acceptor system (DA) at wavelengths exceeding 600 nm still preserves the long-lifetime component of the Ru donor, retaining average fluorescence lifetimes in the range of 30–50 ns. Despite the low quantum yield of the Ru donor in the absence of acceptor, its overall quantum yield of the DA pair was increased by energy transfer to the higher quantum yield acceptor BO-PRO-3. The wavelength-integrated intensity of donor and acceptor bound to DNA was many-fold greater than the intensity of the donor and acceptor separately bound to DNA. The origin of this effect is due to an efficient energy transfer from the donor, competing with non-radiative depopulation of the donor excited state. The distinctive features of DA complexes can be used in the development of a new class of engineered luminophores that display both long lifetime and long-wavelength emission. Similar DA complexes can be applied as proximity indicators, exhibiting strong fluorescence of adjacently located donors and acceptors over the relatively weak fluorescence of separated donors and acceptors.     

Fluorescence Dynamics of DNA Condensed by the Molecular Crowding Agent Poly(Ethylene Glycol)

Condensation of extended DNA into compact structures is encountered in a variety of situations, both natural and artificial. While condensation of DNA has been routinely carried out by the use of multivalent cations, cationic lipids, detergents, and polyvalent cationic polymers, the use of molecular crowding agents in condensing DNA is rather striking. In this work, we have studied the dynamics of plasmid DNA condensed in the presence of a molecular crowding agent, polyethylene glycol (PEG). Steady-state and time-resolved fluorescence of the recently established condensation-indicating DNA binder, YOYO-1 [G. Krishnamoorthy, G. Duportail, and Y. Mely (2002), Biochemistry 41, 15277–15287] was used in inferring the dynamic aspects of DNA condensates. It is shown that DNA condensed by PEG is more flexible and less compact when compared to DNA condensed by binding agents such as polyethyleneimine. The relevance of such differences in dynamics toward functional aspects of condensed DNA is discussed.     

Novel,Monomeric Cyanine Dyes as Reporters for DNA Helicase Activity

The dimeric cyanine dyes, YOYO-1 and TOTO-1, are widely used as DNA probes because of their excellent fluorescent properties. They have a higher fluorescence quantum yield than ethidium homodimer, DAPI and Hoechst dyes and bind to double-stranded DNA with high affinity. However, these dyes are limited by heterogeneous staining at high dye loading, photocleavage of DNA under extended illumination, nicking of DNA, and inhibition of the activity of DNA binding enzymes. To overcome these limitations, seven novel cyanine dyes (Cyan-2, DC-21, DM, DM-1, DMB-2OH, SH-0367, SH1015-OH) were synthesized and tested for fluorescence emission, resistance to displacement by Mg²⁺, and the ability to function as reporters for DNA unwinding. Results show that Cyan-2, DM-1, SH-0367 and SH1015-OH formed highly fluorescent complexes with dsDNA. Of these, only Cyan-2 and DM-1 exhibited a large fluorescence enhancement in buffers, and were resistant to displacement by Mg²⁺. The potential of these two dyes to function as reporter molecules was evaluated using continuous fluorescence, DNA helicase assays. The rate of DNA unwinding was not significantly affected by either of these two dyes. Therefore, Cyan-2 and DM-1 form the basis for the synthesis of novel cyanine dyes with the potential to overcome the limitations of YOYO-1 and TOTO-1.     

Spectroscopic Study on the Interaction of Pyronine Y with Nucleic Acids and Its Analytical Application

ABSTRACT

The interaction of pyronine Y (PY) with nucleic acids was studied by resonance Rayleigh scattering (RRS) for nucleic acid detection. The enhanced RRS intensity of nucleic acids reacted with PY was proportional to the concentration of nucleic acids in the ranges of 27.0–625 ng ml^?1 for fish sperm DNA, 39.0–500 ng ml^?1 for calf thymus DNA, and 59.0–375 ng ml^?1 for yeast RNA. The limits of determination were 0.2 ng ml^?1 for fish sperm DNA, 0.6 ng ml^?1 for calf thymus DNA, and 0.7 ng ml^?1 for yeast RNA. The method had been successfully applied to the quick determination of nucleic acids in synthetic and natural samples.     

光学分子成像技术观察纳流芯片对DNA分子的研究

λ-DNA与荧光染料YOYO-1结合,利用荧光显微技术对DNA分子在毛细现象作用下进入宽40 nm、深60 nm的纳米沟道内,并在其内部被拉伸以及沿沟道移动的情形进行了观察.讨论了DNA分子在沟道内的运动情况.结合光学分子成像技术与该尺寸范围的纳流芯片,将有助于研究生物分子的动力学和静力学性质.     

X-ray photoelectron spectroscopic investigations of modifications in plasmid DNA after interaction with Hg nanoparticles

The electronic structure of plasmid DNA both prior to and after its conjugation with Hg nanoparticles (NP) has been investigated here. The DNA molecules undergo severe perturbation on their interaction with Hg-NP. The circular plasmid DNA becomes predominantly linearised on interaction with 10 mM Hg salt solution. These perturbations are also reflected in the electronic structure of C1s and O1s suggesting severe modifications in the exocyclic groups, of the nucleic acid bases, participating in the hydrogen bondings. The results reflect the rupturing of the purine-pyrimidine bonds after interaction with the NP. The study also suggests selective and specific interactions of DNA bases with the Hg nanoparticles leading to the formation of metal-purine-pyrimidine complexes through rupturing of G-C base pairs. Hg-NP exclusively interact with the nitrogen bases of the DNA molecule and do not cause any significant modification to the phosphate backbone of DNA.     

Determination of Nucleic Acids Sensitized by Emulsifier OP‐micelle Using Ethyl Rhodamine B as a Resonance Light‐Scattering Probe

Abstract

The resonance light scattering (RLS) spectra of ethyl rhodamine B with nucleic acid (calf thymus DNA and herring sperm DNA) have been studied. The effective factors and the optimum conditions have been studied, and the enhanced intensity of RLS is in proportion to the concentration of nucleic acids in the range 0～5.00 µg mL^?1 for calf thymus DNA (ctDNA) and 0～3.50 µg mL^?1 for herring sperm DNA (hsDNA). The limits of detection are 3.42 and 3.14 ng mL^?1, respectively. Based on this, a RLS method for the determination of nucleic acids sensitized by emulsifier OP‐micelle was accordingly established. The binding mode concerning the interactions of ethyl rhodamine B with nucleic acids was also studied, and this method has good selectivity and high sensitivity and it has been applied to the determination of DNA in synthetic samples and real samples with satisfactory results.     

Eu（Sm）－DBM－CPB体系的激光感生时间分辨荧光光谱的研究

用自装的激光感生时间分辨荧光光谱装置，研究了Ｅｕ、Ｓｉｎ－二苯甲酰甲烷（ＤＢＭ）体系在阳离子表面活性剂溴代十六烷基吡啶（ＣＰＢ）存在下的光谱特性和荧光衰减动力学特性。给出同时测定Ｅｕ和Ｓｉｎ的激光感生时间分辨荧光光谱分析方法，用于高纯氧化物中痕量Ｅｕ和Ｓｉｎ的测定，获得了满意的结果。     

SPECTRAL STUDIES ON THE BINDING OF A BISACRIDINIUM DERIVATIVE LUCIGENIN WITH DOUBLE HELIX DNA

ABSTRACT

In this paper, the noncovalent binding of the cationic reagent lucigenin (LC) to DNA was investigated using spectroscopic methods. The results from absorption, circular dichroism and fluorescence studies demonstrated that LC could intercalate into the helix of DNA. Polarization and melting studies further supported the intercalation binding of LC with DNA. The binding constant was obtained by varying the DNA concentration, while keeping the concentration of LC constant. It was of the order of 10⁴ mol^?1 L in DNA base pairs. The experiment also showed that electrostatic interaction played a significant role in the intercalation of LC with DNA. It is supposed to be because of being attracted first by anionic DNA that LC can be intercalated into the interior of the DNA double helix. This research offers a new intercalation functional group to DNA-targeted drug design.     

阴离子染料百里酚蓝与纳克级核酸的共振光散射光谱特性及其分析应用

在 p H6 .0 0— 7.0 0和离子强度低于 0 .0 5 0 mol/ L的条件下 ,研究了百里酚蓝 (TB) -十六烷基三甲基溴化铵 (CTMAB) -核酸体系的共振光散射光谱 (RLS) ,影响因素及最佳反应条件。在最佳条件下 ,体系的ΔIRL S与 y DNA、ct DNA和 fs DNA在一定的浓度范围内呈线性关系。检出限可达 1.30 ng/ m L。该方法简便、快速 ,具有较高的灵敏度和准确度 ,应用于合成样品中核酸的测定 ,结果令人满意     

核酸-桑色素-铝(Ⅲ)三元荧光体系的研究

基于核酸对桑色素 铝 (Ⅲ )配合物的荧光增强作用 ,以桑色素 铝 (Ⅲ )为荧光探针 ,考察该探针与核酸的结合反应 ,建立了新的准确测定核酸的方法 ,并研究了该三元荧光体系的作用机理。在 pH 8 5时 ,fsDNA ,ctDNA ,smDNA和 yRNA的浓度与桑色素 铝 (Ⅲ )的荧光强度成线性关系 ,响应范围分别为 0 2 5～1 5 0 ,0 2 5～ 2 0 0 ,0 10～ 1 6 0和 0 2 5～ 2 0 0 μg·mL-1,检测限 (3σ/K)分别为 3,2 ,2和 3ng·mL-1。测定了合成样品 ,回收率 93 3%～ 10 7 9% ,相对标准偏差小于 3 6 %     

抗生物素蛋白与DNA相互作用的单分子研究

抗生物素蛋白(avidin)在生物单分子实验中被广泛用于DNA与修饰表面的连接,同时avidin也可作为一种DNA载体用于基因治疗中.本文利用原子力显微镜(AFM)、动态光散射(DLS)、单分子磁镊(MT)技术系统地研究了avidin与DNA之间的相互作用,以及avidin引起DNA凝聚的机理.首先通过AFM对avidin-DNA复合体形貌进行观察,发现不但有avidin导致DNA凝聚的环状形貌,同时也存在avidin自身聚集引起的DNA凝聚现象,通过定量分析,发现其凝聚尺寸越来越小,而当avidin浓度大于2 ng·μL~(-1)时,其凝聚尺寸又突然变大.DLS实验结果也显示了同样的规律,伴随着avidin浓度的升高,DNA的粒径大小从大约170 nm减小到125 nm左右,其电泳迁移率由-2.76(10~(-4)cm~2·V~(-1)·s~(-1))变化到-0.1(10~(-4)cm~2·V~(-1)·~(-1)).此外,通过MT技术的力谱曲线变化,发现avidin导致的DNA凝聚与其他多价离子相比,长度的变化曲线几乎呈线性变化,偶尔存在少而小的阶跃,这种变化趋势与组蛋白的变化曲线更相似.因此可以判断,avidin导致DNA凝聚是由avidin与DNA的静电吸引和avidin自身聚集两种相互作用引起的.     

Structural polymorphism of DNA-dendrimer complexes

DNA condensation in vivo relies on electrostatic complexation with small cations or large histones. We report a synchrotron x-ray study of the phase behavior of DNA complexed with synthetic cationic dendrimers of intermediate size and charge. We encounter unexpected structural transitions between columnar mesophases with in-plane square and hexagonal symmetries, as well as liquidlike disorder. The isoelectric point is a locus of structural instability. A simple model is proposed based on competing long-range electrostatic interactions and short-range entropic adhesion by counterion release.     

Direct Observation Method of Individual Single-Stranded DNA Molecules Using Fluorescent Replication Protein A

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.     

Characterization of Monoolein-Based Lipoplexes Using Fluorescence Spectroscopy

Lipoplexes are commonly used as delivery systems in vitro and in vivo, the role of a neutral lipid as helper being of extreme importance in these systems. Cationic liposomes composed of dioctadecyldimethylammonium bromide (DODAB) with monoolein (MO) as a helper, at different molar ratios (1:2; 1:1 and 1:0.5) were prepared, and subsequently titrated to DNA. The structural and physicochemical properties of the lipid/DNA complexes were assessed by ethidium bromide (EtBr) exclusion, 90° static light scattering (90° SLS) assays and fluorescence resonance energy transfer (FRET). In EtBr exclusion assays, the steady-state fluorescence spectra of EtBr were decomposed into the sum of two lognormal emissions, emanating from two different environments – H₂O and DNA, and the effect of charge ratio (+/-) was observed. 90° SLS assays gave an important contribution, detecting size variations in systems with different MO fractions on the lipoplexes. In FRET assays, 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-HPC) was used as donor and EtBr as acceptor. The DNA component previously calculated by EtBr exclusion, was used to determine the energy transfer efficiency, as an indirect measurement of the lipoplexes structural and physicochemical properties. Our results demonstrate that the inclusion of monoolein in the cationic liposomes formulation significantly modifies the rate of DNA complexation, being DODAB:MO (1:1) the system with higher DNA condensation efficiency.     

DNA and PNA as templates for building nanoassemblies via electrostatic complexation with gold nanoparticles

Organisation of nanoparticles on structurally well-defined templates is a first step towards creating nanomachines. In this respect, nucleic acids are ideal structural templates and a variety of secondary structures realizable from DNA/RNA––e.g., duplexes, hairpins, triplexes, cruciforms, tetraplexes can be exploited to engineer nanoparticle organization at will. We have used oligonucleotides and their analogues such as phosphorothioates and peptide nucleic acids to electrostatically encapsulate cationic-capped gold nanoparticles. This article describes synthesis and characterization of DNA/PNA-gold nanoparticle composites using TEM and UV-T_m techniques. These types of assemblies may have potential for creating nanowires and lithographic circuits.