Specific Binding of Anionic Porphyrin and Phthalocyanine to the G-Quadruplex with a Variety of <em>i</em><em>n Vitro</em> and<em> </em><em>i</em><em>n Vivo</em> Applications

The G-quadruplex, a four-stranded DNA structure with stacked guanine tetrads (G-quartets), has recently been attracting attention because of its critical roles in vitro and in vivo. In particular, the G-quadruplex functions as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplex can show peroxidase-like activity with an anionic porphyrin, iron (III) protoporphyrin IX (hemin). Importantly, hemin binds to G-quadruplexes with high selectivity over single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is attributable to an electrostatic repulsion of phosphate groups in ssDNA and dsDNA. The G-quadruplex and hemin-G-quadruplex complex allow development of sensing techniques to detect DNA, metal ions and proteins. In addition to hemin, anionic phthalocyanines also bind to the G-quadruplex formed by human telomere DNA, specifically over ssDNA and dsDNA. Since the binding of anionic phthalocyanines to the G-quadruplex causes an inhibition of telomerase activity, which plays a role in the immortal growth of cancer cells, anionic phthalocyanines are promising as novel anticancer drug candidates. This review focuses on the specific binding of hemin and anionic phthalocyanines to G-quadruplexes and the applications in vitro and in vivo of this binding property.     

Identifying G-quadruplex-binding ligands using DNA-functionalized gold nanoparticles

The G-rich overhang of human telomere tends to form a G-quadruplex structure, and G-quadruplex formation can effectively inhibit telomerase activity in most cancer cells. Therefore, it is important to identify the formation and properties of the G-quadruplex, with the particular aim of selecting G-quadruplex-binding ligands that could potentially lead to the development of anticancer therapeutic agents. With this goal in mind, we report a fluorescence resonance energy transfer (FRET) assay system for the identification of G-quadruplex ligands using DNA-functionalized gold nanoparticles (DNA-GNPs) as the fluorescence quencher and a carboxyfluorescein (FAM)-tagged human telomeric sequence (F-GDNA) as the recognition probe. A thiolated complementary strand of human telomeric DNA (cDNA), which first adheres to the surface of the GNPs and then hybridizes with F-GDNA, results in the fluorescence quenching of F-GDNA by the GNPs. However, fluorescence is restored when single-stranded F-GDNA folds into a G-quadruplex structure upon the binding of quadruplex ligands, leading to the release of F-GDNA from the surface of the GNPs. Combined data from fluorescence measurements and CD spectroscopy indicated that ligands selected by this FRET method could induce GDNA to form a G-quadruplex. Therefore, this FRET G-quadruplex assay is a simple and effective approach to identify quadruplex-binding ligands, and, as such, it promises to provide a solid foundation for the development of novel anticancer therapeutic agents.     

N-fused porphyrin with pyridinium side-arms: a new class of aromatic ligand with DNA-binding ability

N-fused porphyrin (NFP) is a porphyrin analogue with an 18π tetrapyrrolic macrocycle, in which a unique tripentacyclic ring is embedded. While the optical properties of NFP of absorbing and emitting near-infrared (NIR) light around 1000 nm are promising for its application to NIR technology, its unique structure is also attractive as a platform to construct a novel class of DNA-binding ligands. Herein, we have synthesized a water-soluble derivative of NFP (pPyNFP) possessing four cationic pyridinium substituents and examined its acid/base behaviors and interactions with various forms of DNAs in aqueous solution. pPyNFP interacts with ssDNA and dsDNA electrostatically. pPyNFP also interacts with a G-quadruplex DNA derived from the human telomeric sequence and causes a characteristic spectral change of the G-quadruplex DNA, which suggests that pPyNFP modulates the Na(+)-induced (2 + 2) antiparallel G-quadruplex to the all-parallel structure.     

Detection of ferulic acid based on the plasmon resonance light scattering of silver nanoparticles

In this contribution, a plasmon resonance light scattering (PRLS) detection method of ferulic acid (FA) is proposed based on the formation of silver nanoparticles (NPs). It was found that, FA acted as a reducing agent in alkaline medium and could be oxidized by AgNO₃, resulting in the formation of silver NPs. The formed silver NPs, which were identified by measuring the plasmon resonance absorption spectra, PRLS spectra and transmission electron microscopy (TEM) image, display characteristic plasmon resonance optical absorption and PRLS band in the visible region. It was found that the PRLS intensity, which could be easily measured using a common spectrofluorometer, was in proportion to the concentration of FA over the range from 0.2 to 2.0 μmol l⁻¹ with the corresponding limits of determination (3σ) of 15.2 nmol l⁻¹. With that, ferulate sodium injection samples have been detected with R.S.D. lower than 3.0% and recoveries over the range of 101.2–104.5%. On the other hand, the present reaction maybe provides the basis of an environmentally friendly approach for the synthesization of silver NPs.     

Evaluation of binding of perylene diimide and benzannulated perylene diimide ligands to dna by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the self-association, G-quadruplex DNA binding, and selectivity of a series of perylene diimides (PDIs) (PIPER, Tel01, Tel11, Tel12, and Tel18) or benzannulated perylene diimide ligands (Tel34 and Tel32). Fluorescence and resonance light scattering spectra of Tel01, Tel12, Tel32, and Tel34 reveal that these analogs undergo self-association in solution. UV-Vis and fluorescence titrations with G-quadruplex, duplex, or single-stranded DNA demonstrate that all the analogs, with the exception of Tel32, bind to G-quadruplex DNA, with those PDIs that are self-associated in solution showing the highest degree of selectivity for binding G-quadruplex DNA. Parallel ESI-MS analysis of the stoichiometries demonstrates the ability of the ligands, with the exception of Tel32, to bind to G-quadruplex DNA. While most ligands show major 1:1 and 2:1 binding stoichiometries as expected in the case of end-stacking, interestingly, three of the most quadruplex-selective ligands show a different behavior. Tel01 forms 3:1 complexes, while Tel12 and Tel32 only form 1:1 complexes. Collisional activation dissociation patterns are compatible with ligand binding to G-quadruplex DNA via stacking on the ends of the terminal G-tetrads. Experiments with duplex and single strand DNA were performed to assess the binding selectivities of the ligands. PIPER, Tel11, and Tel18 demonstrated extensive complexation with duplex DNA, while Tel11 and Tel18 bound to single strand DNA, confirming the lack of selectivity of these two ligands. Our results indicate that Tel01, Tel12, and Tel34 are the most selective for G-quadruplex DNA.     

The gold-nanoparticle-based surface plasmon resonance light scattering and visual DNA aptasensor for lysozyme

We developed a new simple and sensitive assay for lysozyme based on gold nanoparticle plasmon resonance light scattering (PRLS) measurement and naked-eye detection using for the first time the lysozyme DNA aptamer as the recognition element. Lysozyme DNA aptamer could stabilize gold nanoparticles (AuNPs) at high ionic strength. Introducing lysozyme to the system easily triggered the aggregation of AuNPs, producing a red-to-blue color change of the solution, red-shifted plasmon absorption, and enhanced plasmon resonance light scattering. The linear range was found to be 0.2∼4 nM for 0.7 nM AuNPs, 0.3∼6 nM for 1.4 nM AuNPs and 0.6∼8 nM for 2.1 nM AuNPs. About 0.1 nM lysozyme can produce an observable enhancement of PRLS signal. For visual detection, 1 nM lysozyme can produce a very distinctive color change. Satisfactory recoveries were obtained for simulated saliva and diluted urine samples, indicating that the method has potential for analyses of clinical samples. The simplicity and high sensitivity that are consistent with the resources and needs of many laboratories makes this method a good choice for routine analysis.     

多柔比星稀土金属离子配合物与DNA相互作用的研究

用紫外分光光度法和荧光光谱法研究了多柔比星( Adriamycin,ADM)稀土金属离子配合物(ADM-M)与DNA的相互作用.结果发现,在pH=7.0时,ADM与Eu3+、yb3+能形成稳定配合物,该配合物可使DNA的最大吸收产生明显的减色效应及红移,并能够竞争置换溴化乙锭(EB)与DNA的结合点.KI猝灭试验发现D...     

基于荧光探针噻唑橙与核酸适体构象变化的钾离子检测

建立了一种基于核酸适体(Aptamer)构象效应和荧光探针噻唑橙(TO)为荧光分子开关进行钾离子检测的光学方法.室温下钾离子可与Aptamer结合形成G-四面体结构,使双链解链变为四面体结构和单链,从而导致TO荧光强度降低.考察了TO浓度、反应温度及反应时间的影响.在最佳实验条件下,钾离子浓度在1.0×10-6 ～2....     

Quadruplex-to-duplex transition of G-rich oligonucleotides probed by cationic water-soluble conjugated polyelectrolytes

G-quartet DNA converts to duplex form in the presence of its complementary strand. This conformational change can be detected in real time by a homogeneous assay method based on the signal amplification of conjugated polyelectrolytes and the specific interaction of intercalating dyes with double-stranded DNA (dsDNA). The probe solution contains a cationic, conjugated polymer (CCP), G-quadruplex labeled with a fluorescein at the 5'-terminus (G-quadruplex-Fl), and ethidium bromide (EB). The addition of a complementary target results in the transition from G-quadruplex to duplex (dsDNA-Fl) and EB intercalation within the duplex structure. Excitation of the CCP leads to energy transfer from CCP to dsDNA-Fl (FRET-1) and then energy transfer from dsDNA-Fl to EB (FRET-2). Increasing the number of mismatched bases discourages dsDNA formation, which is detected in the assay.     

Hybridization detection of DNA by measuring organic small molecule amplified resonance light scattering signals

The interaction of organic small molecules (OSMs) with a biological molecule is very important. In this contribution, quinone-imine dyes including Acridine Yellow (AY), Neutral Red (NR), Acridine Orange (AO), Brilliant Cresyl Blue (BCB), Thionin (TN), Azur A (AA), Azur B (AB), and Methylene Blue (MB) respectively with double strand DNA (dsDNA) and single strand DNA (ssDNA) were investigated based on the measurements of enhanced resonance light scattering (RLS) and TEM. Mechanism investigations have shown that groove binding occurs between dsDNA and these OSMs, which depends on G-C sequences of dsDNA and the volumes of OSMs. With the amplified RLS signals resulting from the interactions of OSMs with DNA, a new technique has been proposed to detect the hybridization and mismatch of DNA labeling neither the target nor the probe DNA. The results have suggested that the extent of the amplified RLS signals of dsDNA by AY is the maximum among these eight OSMs, and therefore, it has been selected as a typical model system for further discussions.     

P(NIPAM-co-NVP)共聚物分子链水溶液中的可逆聚集研究

用溶液聚合方法合成了线型聚(N-异丙基丙烯酰胺-co-N-乙烯基吡咯烷酮)共聚物,通过弹性光散射(elastic light scattering,ELS)、荧光光谱与动态光散射研究了共聚物水溶液分子链可逆聚集的温度和时间依赖性.研究表明,升温时,ELS强度增加,分子链聚集;降温时,ELS强度降低,聚集的分子链解离.荧...     

Synthesis, cytotoxicity and spectroscopy studies of a new copper (II) complex: calf thymus DNA and T47D as targets

A water-soluble Cu (II) complex [(dien)Cu(??-1,6-DAH)Cu(dien) (NO₃)₂](NO₃)₂ has been synthesized and its effect on the carrier model DNA structure and cancer cell line proliferation was investigated. In this regard, calf thymus DNA (CT-DNA) and human breast cancer cell line, T47D, were the targets. The effect of the complex on DNA structure was investigated by means of UV/vis, fluorescence and circular dichroism (CD) spectroscopic techniques as well as dynamic light scattering (DLS), zeta potential analysis and docking assay for more analysis. The UV?Cvis absorption spectra of complex with DNA showed a slight red shift and hypochromic effect, which indicated the intercalation and electrostatic effect of complex with CT-DNA. Using ethidium bromide (EB) as a probe in fluorescence studies revealed that complex can quench the EB?CDNA fluorescence emission at different temperatures. Besides, the far UV?CCD studies displayed that the complex induces changes in the secondary structure of CT-DNA and can increase the melting temperature of DNA up to 14?°C. The DLS and zeta potential measurements confirmed the electrostatic interaction of complex with the negatively charged DNA and subsequent DNA condensation. Besides, computational studies reflect that major and minor groove binding are two modes of interaction between complex and DNA. On the other hand, growth inhibition of the complex toward T47D cell line was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, which showed no cytotoxic properties.     

Kinetic and thermodynamic control of g-quadruplex folding

A matter of speed: When allowed to fold in a K(+) /poly(ethylene glycol) solution, the guanine?(G)-rich strand of vertebrate telomere DNA forms a parallel/antiparallel G-quadruplex, which is a (3+1) hybrid, within microseconds before slowly transforming into the parallel one within hours. Thus, the conformation that a G-quadruplex initially adopts under physiological conditions may not be the one it adopts at the equilibrium state.     

Label-free detection of sequence-specific DNA with multiwalled carbon nanotubes and their light scattering signals

A detection method for DNA sequence-specificity in a homogeneous medium is presented with multiwalled carbon nanotubes (MWCNTs) as optical probes on the basis of the measurements of light scattering signals. ssDNA can prevent MWCNTs from coagulation in electrolyte solution while dsDNA cannot, displaying different light scattering signals. With the light scattering signals, target DNA in the range of 8.6-86.4 nM could be detected and one base pair mismatch could be discriminated. The sequence specificity for the present method has been identified with PCR products.     

An abnormal resonance light scattering arising from ionic-liquid/DNA/ethidium interactions

In the aqueous phase, ethidium bromide (EB) intercalates into the double helix structure of dsDNA (ds=double-stranded) with a notable enhancement in fluorescence and resonance light scattering (RLS). However, when dsDNA was extracted into an ionic liquid (IL), 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF(6)), an abnormal RLS arising from the interactions of IL-DNA-EB was observed, with a substantial decrease of the recorded RLS. The cationic Bmim(+) groups of BmimPF(6) intercalate into the DNA helix structure, in which they interact with the P-O bonds of phosphate groups in DNA strands and result in a reduction of the base-pair interstice along with transformation of DNA conformations that consequently prohibits the intercalation of EB with DNA. Thus, in the IL phase, the interactions between ethidium and DNA were dominated by electrostatic interactions and hydrogen bonding, leading to a congregation of EB entities around the DNA strands that results in an increase of absorption by ethidium, and consequently the inner filter effect leads to a reduction of the RLS. The present observation has been applied to the direct quantification of DNA in an ionic-liquid phase after DNA from human whole blood was extracted into BmimPF(6).     

Cationic helicenes as selective G4 DNA binders and optical probes for cellular imaging

The important role that G-quadruplex DNA (G4 DNA) structures play in regulating biological processes is becoming widely recognised. These structures have also been proposed to be attractive drug targets. Therefore, there has been significant interest in developing small molecules that can selectively bind to G4 DNA over other topologies. In this paper we investigate the interaction between DNA and helical compounds (helicenes) based on a central carbocation trisubstituted with aromatic rings. We show that the non-planar structure of these helicenes results in a significantly reduced affinity for dsDNA when compared to their planar analogues, whilst maintaining a high affinity for G4 DNA. Additionally, the right- and left-handed enantiomers of one of these helicenes recognise the chiral DNA environments of G4 and dsDNA differently. We show that upon DNA binding the helicenes display a fluorescence switch-on effect, which we have successfully used for cellular imaging in live and fixed U2OS cells, staining mitochondria and the nucleus, respectively.

G-quadruplex DNA (G4 DNA) structures are selectively recognised by helical optical probes.     

Inducement and stabilization of G-quadruplex DNA by a thiophene-containing dinuclear ruthenium(II) complex

G-quadruplex structures are attractive targets for the development of anticancer drugs, as their formation in human telomere could impair telomerase activity, thus inducing apoptosis in cancer cells. In this work, a thiophene-containing dinuclear ruthenium(II) complex, [Ru₂(bpy)₄(H₂bipt)]⁴⁺ {bpy = 2,2′-bipyridine, H₂bipt = 2,5-bis[1,10]phenanthrolin[4,5-f]-(imidazol-2-yl)thiophene}, was prepared and the interaction between the complex and human telomeric DNA oligomers 5′-G₃(T₂AG₃)₃-3′ (HTG21) has been investigated by UV-Vis, fluorescence and circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay, fluorescent intercalator displacement (FID) titrations, Job plot and color reaction studies. The results indicate that the complex can well induce and stabilize the formation of antiparallel G-quadruplex of telomeric DNA in the presence or absence of metal cations, and the ΔT_m value of the G-quadruplex DNA treated with the complex was obtained to be 12.8 °C even at levels of 50-fold molar of duplex DNA (calf-thymus DNA), suggesting that the complex exhibits higher G-quadruplex DNA selectivity over duplex DNA. The complex shows high interaction ability with G-quadruplex DNA at (1.17 ± 0.12) × 10⁷ M^?1 binding affinity using a 2:1 [complex]/[quadruplex] binding mode ratio. A novel visual method has been developed here for making a distinction between G-quadruplex DNA and duplex DNA by our ruthenium complex binding hemin to form the hemin-G-quadruplex DNAzyme.     

Characterization of single- and double-stranded DNA on gold surfaces

Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.     

A fluorescence aptasensor based on DNA charge transport for sensitive protein detection in serum

A novel fluorescence aptasensor based on DNA charge transport for sensitive protein detection has been developed. A 15nt DNA aptamer against thrombin was used as a model system. The aptamer was integrated into a double strand DNA (dsDNA) that was labeled with a hole injector, naphthalimide (NI), and a fluorophore, Alexa532, at its two ends. After irradiation by UV light, the fluorescence of Alexa532 was bleached due to the oxidization of Alexa532 by the positive charge transported from naphthalimide through the dsDNA. In the presence of thrombin, the binding of thrombin to the aptamer resulted in the unwinding of the dsDNA into ssDNA, which led to the blocking of charge transfer and the strong fluorescence emission of Alexa532. By monitoring the fluorescence signal change, we were able to detect thrombin in homogeneous solutions with high selectivity and high sensitivity down to 1.2 pM. Moreover, as DNA charge transfer is resistant to interferences from biological contexts, the aptasensor can be used directly in undiluted serum with similar sensitivity as that in buffer. This new sensing strategy is expected to promote the exploitation of aptamer-based biosensors for protein assays in complex biological matrixes.     

Human telomeric DNA forms parallel-stranded intramolecular G-quadruplex in K+ solution under molecular crowding condition

The G-rich strand of human telomeric DNA can fold into a four-stranded structure called G-quadruplex and inhibit telomerase activity that is expressed in 85-90% tumor cells. For this reason, telomere quadruplex is emerging as a potential therapeutic target for cancer. Information on the structure of the quadruplex in the physiological environment is important for structure-based drug design targeting the quadruplex. Recent studies have raised significant controversy regarding the exact structure of the quadruplex formed by human telomeric DNA in a physiological relevant environment. Studies on the crystal prepared in K+ solution revealed a distinct propeller-shaped parallel-stranded conformation. However, many later works failed to confirm such structure in physiological K+ solution but rather led to the identification of a different hybrid-type mixed parallel/antiparallel quadruplex. Here we demonstrate that human telomere DNA adopts a parallel-stranded conformation in physiological K+ solution under molecular crowding conditions created by PEG. At the concentration of 40% (w/v), PEG induced complete structural conversion to a parallel-stranded G-quadruplex. We also show that the quadruplex formed under such a condition has unusual stability and significant negative impact on telomerase processivity. Since the environment inside cells is molecularly crowded, our results obtained under the cell mimicking condition suggest that the parallel-stranded quadruplex may be the more favored structure under physiological conditions, and drug design targeting the human telomeric quadruplex should take this into consideration.