Screening Internal Donor-Acceptor Biaryl Nucleobase Surrogates for Turn-On Fluorescence Affords an Aniline-Carboxythiophene Probe for Protein Detection by G-Quadruplex DNA.

Donor-acceptor biaryls serve as microenvironment fluorescent sensors with highly quenched intramolecular charge transfer (ICT) emission in polar protic solvents that turns on in aprotic media. In DNA, canonical donor-acceptor fluorescent base analogs can be prepared through on-strand Suzuki-Miyaura cross-coupling reactions involving 8-bromo-2'-deoxyguanosine (8-Br-dG) with an acceptor aryboronic acid. Herein, we demonstrate that replacement of 8-Br-dG with N-methyl-4-bromoaniline (4-Br-An) containing an acyclic N-glycol group can be employed in the on-strand Suzuki-Miyaura reaction to afford new donor-acceptor biaryl nucleobase surrogates with a 40-fold increase in emission intensity for fluorescent readout within single-strand oligonucleotides. Screening the best acceptor for turn-on fluorescence upon duplex formation afforded the carboxythiophene derivative [COOTh]An with a 7.4-fold emission intensity increase upon formation of a single-bulged duplex (-1) with the surrogate occupying a pyrimidine-flanked bulge. Insertion of the [COOTh]An surrogate into the lateral TT loops produced by the antiparallel G-quadruplex (GQ) of the thrombin binding aptamer (TBA) afforded a 4.1-fold increase in probe fluorescence that was accompanied by a 20 nm wavelength shift to the blue upon thrombin binding. The modified TBA afforded a limit of detection of 129 nM for thrombin and displayed virtually no emission response to off-target proteins. The fluorescence response of [COOTh]An to thrombin binding highlights the utility of the thienyl-aniline moiety for monitoring DNA-protein interactions.

Bis-Pyrene Photo-Switch Open- and Closed-Form Differently Bind to ds-DNA, ds-RNA and Serum Albumin and Reveal Light-Induced Bioactivity.

Newly designed and synthesized diarylethene (DAE) derivatives with aliphatic amine sidearms and one with two pyrenes, revealed excellent photo-switching property of central DAE core in MeOH and water. The only exception was bis-pyrene analogue, its DAE core very readily photochemically closed, but reversible opening completely hampered by aromatic stacking interaction of pyrene(s) with cyclic DAE. In this process, pyrene fluorescence showed to be a reliable monitoring method, an open form characterized by strong emission at 480 nm (typical for pyrene-aggregate), while closed form emitted weakly at 400 nm (typical for pyrene-DAE quenching). Only open DAE-bis-pyrene form interacted measurably with ds-DNA/RNA by flexible insertion in polynucleotide grooves, while self-stacked closed form did not bind to DNA/RNA. For the same steric reasons, flexible open DAE-bis-pyrene form was bound to at least three different binding sites at bovine serum albumin (BSA), while rigid, self-stacked closed form interacted dominantly with only one BSA site. Preliminary screening of antiproliferative activity against human lung carcinoma cell line A549 revealed that all DAE-derivatives are non-toxic. However, bis-pyrene analogue efficiently entered cells and located in the cytoplasm, whereby irradiation by light (315-400 nm) resulted in a strong, photo-induced cytotoxic effect, typical for pyrene-related singlet oxygen species production.

Structure-Property Relationships in CuII -Binding Tetramolecular G-Quadruplex DNA.

A series of artificial metal-base tetrads composed of a CuII cation coordinating to four pyridines, covalently attached to the ends of tetramolecular G-quadruplex DNA strands [LA-D d(G4 )]4 (LA-D =ligand derivatives), was systematically studied. Structurally, the square-planar [Cu(pyridine)4 ] complex behaves analogously to the canonical guanine quartet. Copper coordination to all studied ligand derivatives was found to increase G-quadruplex thermodynamic stability, tolerating a great variety of ligand linker lengths (1-5 atoms) and thus demonstrating the robustness of the chosen ligand design. Only at long linker lengths, the stabilizing effect of copper binding is compensated by the loss of conformational freedom. A previously reported ligand LE with chiral backbone enables incorporation at any oligonucleotide position. We show that ligand chirality distinctly steers CuII -induced G-quadruplex stabilization. 5'-End formation of two metal-base tetrads by tetramolecular G-quadruplex [LE2 d(G)4 ]4 shows that stabilization in the presence of CuII is not additive. All results are based on UV/Vis thermal denaturation, thermal difference, circular dichroism experiments and molecular dynamics simulations.

AGT Activity Towards Intrastrand Crosslinked DNA is Modulated by the Alkylene Linker.

DNA oligomers containing dimethylene and trimethylene intrastrand crosslinks (IaCLs) between the O4 and O6 atoms of neighboring thymidine (T) and 2'-deoxyguanosine (dG) residues were prepared by solid-phase synthesis. UV thermal denaturation (Tm ) experiments revealed that these IaCLs had a destabilizing effect on the DNA duplex relative to the control. Circular dichroism spectroscopy suggested these IaCLs induced minimal structural distortions. Susceptibility to dealkylation by reaction with various O6 -alkylguanine DNA alkyltransferases (AGTs) from human and Escherichia coli was evaluated. It was revealed that only human AGT displayed activity towards the IaCL DNA, with reduced efficiency as the IaCL shortened (from four to two methylene linkages). Changing the site of attachment of the ethylene linkage at the 5'-end of the IaCL to the N3 atom of T had minimal influence on duplex stability and structure, and was refractory to AGT activity.

Triplex- and Duplex-Forming Abilities of Oligonucleotides Containing 2'-Deoxy-5-trifluoromethyluridine and 2'-Deoxy-5-trifluoromethylcytidine.

A facile synthesis of 2'-deoxy-5-trifluoromethyluridine and 2'-deoxy-5-trifluoromethylcytidine phosphoramidites from commercially available 2'-deoxyuridine and 2'-deoxycytidine was achieved, respectively. The obtained phosphoramidites were incorporated into oligonucleotides, and their binding affinity to double-stranded DNA (dsDNA) and single-stranded RNA (ssRNA) was evaluated by UV-melting experiments. The triplex-forming abilities of oligonucleotides including 5-trifluoromethylpyrimidine nucleobases with dsDNA were decreased. Especially, the stability of the triplex containing a trifluoromethylcytosine (CF3C)-GC base triplet was low, likely due to the low pKa of protonated CF3C by the electron-withdrawing trifluoromethyl group. A slight decrease in stability of the duplex formed with ssRNA by oligonucleotides including 5-trifluoromethylpyrimidine nucleobases was only observed, suggesting that they might be applicable to various ssRNA-targeted technologies using features of fluorine atoms.

Application of coamplification at lower denaturation temperature-PCR sequencing for early detection of antiviral drug resistance mutations of hepatitis B virus.

Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modified co-amplification at lower denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations.

Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target.

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine•pyrimidine (Pu•Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))•(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu•Py sequences may be pertinent to gene regulation.

Cleavage of oligonucleotides containing a P3'→N5' phosphoramidate linkage mediated by single-stranded oligonucleotide templates.

Double-stranded DNA (dsDNA) templates can hybridize to and accelerate cleavage of oligonucleotides containing a P3'→N5' phosphoramidate (P-N) linkage. This dsDNA-templated cleavage of P-N linkages could be due to conformational strain placed on the linkage upon triplex formation. To determine whether duplex formation also induced conformational strain, we examined the reactivity of the oligonucleotides with a P-N linkage in the presence of single-stranded templates, and compared these reactions to those with dsDNA templates. P-N oligonucleotides that are cleaved upon duplex formation could be used as probes to detect single-stranded nucleic acids.

CG base pair recognition within DNA triple helices by modified N-methylpyrrolo-dC nucleosides.

3-Aminophenyl-modified analogues of the bicyclic nucleoside N-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one were synthesised and incorporated directly into triplex-forming oligonucleotides in order to utilise their extended hydrogen bonding motif for recognition of the CG base pair. All analogues demonstrated strong binding affinity and very good selectivity for CG from pH 6.2 to 7.0; a marked improvement on previous modifications.

Terahertz Wave Accelerates DNA Unwinding: A Molecular Dynamics Simulation Study.

Unwinding the double helix of the DNA molecule is the basis of gene duplication and gene editing, and the acceleration of this unwinding process is crucial to the rapid detection of genetic information. Based on the unwinding of six-base-pair DNA duplexes, we demonstrate that a terahertz stimulus at a characteristic frequency (44.0 THz) can serve as an efficient, nonthermal, and long-range method to accelerate the unwinding process of DNA duplexes. The average speed of the unwinding process increased by 20 times at least, and its temperature was significantly reduced. The mechanism was revealed to be the resonance between the terahertz stimulus and the vibration of purine connected by the weak hydrogen bond and the consequent break in hydrogen bond connections between these base pairs. Our findings potentially provide a promising application of terahertz technology for the rapid detection of nucleic acids, biomedicine, and therapy.

The effect of microwave irradiation on DNA hybridization.

The effect of microwave irradiation on DNA/DNA hybridization has been studied under controlled power and temperature conditions. It was discovered that microwave irradiation led to the melting of double-stranded deoxyoligonucleotides well below their thermal melting temperature and independent of the length of the deoxyoligonucleotides. These observations indicate a specific interaction of microwaves with DNA, and have important implications in the chemical or enzymatic processing of DNA under microwave heating.

Low-power laser irradiation inhibiting Abeta25-35-induced PC12 cell apoptosis via PKC activation.

Apoptosis is a contributing pathophysiological mechanism of Alzheimer's disease (AD). Recently, low-power laser irradiation (LPLI) has been applied to moderate AD, but the underlying mechanism remains unknown. In this study, the techniques of fluorescence resonance energy transfer (FRET) and real-time quantitative RT-PCR were used to investigate the anti-apoptotic mechanism of LPLI. Rat pheochromocytoma (PC12) cells were treated with amyloid beta 25-35 (Abeta(25-35)) for induction of apoptosis before LPLI treatment. The cell viability assays and morphological examinations show that low fluence of LPLI (0.156 J/cm(2)-0.624 J/cm(2)) could inhibit the cells apoptosis. An increase of PKC activation was dynamically monitored in the cells treated with PMA (specific activator of PKC), LPLI only or Abeta(25-35) followed by 5 min LPLI treatment, respectively. However, the effect of LPLI activating PKC could be inhibited by Go 6983 (specific inhibitor of PKC). Similar results were obtained by using Western blot analysis. Furthermore, LPLI involved an increase in mRNA of the cell survival member bcl-xl and a decrease in the up-regulation of cell death member bax mRNA caused by Abeta(25-35). Further data show that low fluence of LPLI could reverse the increased level of bax/bcl-xl mRNA ratio caused by Abeta(25-35) treatment. In addition, Go 6983 could inhibit the decreased level of bax/bcl-xl mRNA ratio. Taken together, these data clearly indicate that LPLI inhibited Abeta(25-35)-induced PC12 cell apoptosis via PKC-mediated regulation of bax/bcl-xl mRNA ratio.

Hydration regulates the thermodynamic stability of DNA structures under molecular crowding conditions.

Although water is an integral part of DNA structures, the effects of water molecules on various DNA structures which are formed by not only Watson-Crick but also Hoogsteen base pairs are still unclear. Here, we studied quantitatively the effects of molecular crowding on the thermodynamics of a parallel G-quadruplex formation of [d(TG 4)2]4 with Hoogsteen base pairs. It was demonstrated that molecular crowding conditions stabilized the parallel G-quadruplex. Moreover, the plot of stability of the parallel G-quadruplex structure versus water activity suggested that water molecules were released through the G-quadruplex formation. The stabilization of the DNA structures consisting of Hoogsteen base pairs under cell-like conditions may lead to a structural polymorphism of various DNA sequences regulated by water molecules.

Temperature dependence of unwinding forces between complementary oligonucleotides.

Rupture Event Scanning (REVS) was used to study oligonucleotide unwinding under mechanical load. Oligonucleotide melting temperature was successfully estimated using this method. To estimate the enthalpy of reaction, we represented denaturation process as a unimolecular reaction. This gave us the possibility to recover the force profile from the experimental data obtained in force measurements at different scanning time (reaction time) for different temperatures.

Volumetric contributions of loop regions of G-quadruplex DNA to the formation of the tertiary structure.

DNA guanine-quadruplexes (G-quadruplexes) are unique DNA structures formed by guanine-rich sequences. The loop regions of G-quadruplexes play key roles in stability and topology of G-quadruplexes. Here, we investigated volumetric changes induced by pressure in the folding of the G-quadruplex formed by the thrombin binding aptamer (TBA) with mutations within the loop regions. The change of partial molar volume in the transition from coil to G-quadruplex, ∆Vtr, of TBA with a mutation from T to A in the 5' most loop (TBA T3A) was 75.5cm3mol-1, which was larger than that of TBA (54.6cm3mol-1). TBA with a G to T mutation in the central loop (TBA G8T) had thermal stability similar to TBA T3A but a smaller ∆Vtr of 41.1cm3mol-1. In the presence of poly(ethylene)glycol 200 (PEG200), ∆Vtr values were 14.7cm3mol-1 for TBA T3A and 13.2cm3mol-1 for TBA G8T. These results suggest that the two mutations destabilize the G-quadruplex structure differently. Thus, volumetric data obtained using pressure-based thermodynamic analyses provides information about the dynamics of the loop regions and the roles of loops in the stabilities and folding of G-quadruplex structures.

The role of loops and cation on the volume of unfolding of G-quadruplexes related to HTel.

In aqueous solutions containing sodium or potassium cations, oligodeoxyribonucleotides (ODNs) rich in guanine form four-stranded DNA structures called G-quadruplexes (G4s). These structures are destabilized by elevated hydrostatic pressure. Here, we use pressure to investigate the volumetric changes arising from the formation of G4 structures. G4s display a great deal of structural heterogeneity that depends on the stabilizing cation as well as the oligonucleotide sequence. Using UV thermal unfolding at different pressures, we have investigated the volume change of the helix-coil equilibrium of a series of ODNs whose sequences are related to the G-rich ODN HTel (d[A(GGGTTA)3GGG]), which contains four repeats of the human telomeric sequence. The experiments are conducted in aqueous buffers containing either 100mM NaCl or KCl at pH7.4. The G4s stabilized by Na+ are less sensitive to pressure perturbation than those stabilized by K+. The overall molar volume changes (ΔVtot) of the unfolding transition for all of the G4s are large and negative. A large fraction of the measured ΔVtot value arises from the re-hydration of the cations released from the interior of the folded structure. However, the differences in the measured ΔVtot values demonstrate that variations in the structure of G4s formed by each ODN, arising from differences in the sequence of the loops, contribute significantly to ΔVtot and presumably the hydration of the folded structures. Depending on the sequence of the loops, the magnitude of the measured ΔVtot can be larger or smaller than that of HTel in solutions containing sodium. However, the magnitude of ΔVtot is smaller than HTel for the unfolding of all G4s that are stabilized by potassium ions.

Biochemical and biophysical properties of positively supercoiled DNA.

In this paper we successfully developed a procedure to generate the (+) supercoiled (sc) plasmid DNA template pZXX6 in the milligram range. With the availability of the (+) sc DNA, we are able to characterize and compare certain biochemical and biophysical properties of (+) sc, (-) sc, and relaxed (rx) DNA molecules using different techniques, such as UV melting, circular dichroism, and fluorescence spectrometry. Our results show that (+) sc, (-) sc, and rx DNA templates can only be partially melted due to the fact that these DNA templates are closed circular DNA molecules and the two DNA strands cannot be completely separated upon denaturation at high temperatures. We also find that the fluorescence intensity of a DNA-binding dye SYTO12 upon binding to the (-) sc DNA is significantly higher than that of its binding to the (+) sc DNA. This unique property may be used to differentiate the (-) sc DNA from the (+) sc DNA. Additionally, we demonstrate that E. coli topoisomerase I cannot relax the (+) sc DNA. In contrast, E. coli DNA gyrase can efficiently convert the (+) sc DNA to the (-) sc DNA. Furthermore, our dialysis competition assays show that DNA intercalators prefer binding to the (-) sc DNA.

Length and pH-dependent energetics of (CCG)n and (CGG)n trinucleotide repeats.

Trinucleotide repeats are involved in a number of debilitating diseases such as fragile-X syndrome and myotonic dystrophy. Eighteen to 75 base-long (CCG)(n) and (CGG)(n) oligodeoxynucleotides were analysed using a combination of biophysical (UV-absorbance, differential scanning calorimetry) and biochemical methods (non-denaturing gel electrophoresis, enzymatic footprinting). All oligomers formed stable intramolecular structures under near physiological conditions with a melting temperature which was only weakly dependent on oligomer length. Thermodynamic analysis of the denaturation process by UV-melting and calorimetric experiments revealed a length-dependent discrepancy between the enthalpy values deduced from model-dependent (UV-melting) and model-independent experiments (calorimetry), as recently shown for CTG and CAG trinucleotides (Nucleic Acids Res. 33 (2005) 4065). Evidence for non-zero molar heat capacity changes was also derived from the analysis of the Arrhenius plots. Such behaviour is analysed in the framework of an intramolecular "branched" or "broken" hairpin model, in which long oligomers do not fold into a simple long hairpin-stem intramolecular structure, but allow the formation of several independent folding units of unequal stability. These results suggest that this observation may be extended to various trinucleotide repeats-containing sequences.

Energetics of echinomycin binding to DNA.

Differential scanning calorimetry and UV thermal denaturation have been used to determine a complete thermodynamic profile for the bis-intercalative interaction of the peptide antibiotic echinomycin with DNA. The new calorimetric data are consistent with all previously published binding data, and afford the most rigorous and direct determination of the binding enthalpy possible. For the association of echinomycin with DNA, we found DeltaG degrees = -7.6 kcal mol(-1), DeltaH = +3.8 kcal mol(-1) and DeltaS = +38.9 cal mol(-1) K(-1) at 20 degrees C. The binding reaction is clearly entropically driven, a hallmark of a process that is predominantly stabilized by hydrophobic interactions, though a deeper analysis of the free energy contributions suggests that direct molecular recognition between echinomycin and DNA, mediated by hydrogen bonding and van der Waals contacts, also plays an important role in stabilizing the complex.

Detection of guanine-adenine mismatches by surface plasmon resonance sensor carrying naphthyridine-azaquinolone hybrid on the surface.

We have discovered a new molecule naphthyridine-azaquinolone hybrid (Npt-Azq) that strongly stabilized the guanine-adenine (G-A) mismatch in duplex DNA. In the presence of Npt-Azq, the melting temperature (T(m)) of 5'-d(CTA ACG GAA TG)-3'/3'-d(GAT TGA CTT AC)-5' containing a single G-A mismatch increased by 15.4 degrees C, whereas fully matched duplex increased its T(m) only by 2.2 degrees C. Npt-Azq was immobilized on the sensor surface for the surface plasmon resonance (SPR) assay to examine SPR detection of duplexes containing a G-A mismatch. Distinct SPR signals were observed when 27mer DNA containing a G-A mismatch was analyzed by the Npt-Azq immobilized sensor surfaces, whereas the signal of the fully matched duplex was approximately 6-fold weaker in intensity. The SPR signals for the G-A mismatch were proportional to the concentration of DNA in a range up to 1 microM, confirming that the SPR signal is in fact due to the binding of the G-A mismatch to Npt-Azq immobilized on the surface. Examination of all 16 G-A mismatches regarding the flanking sequence revealed that the sensor surface reported here is applicable to eight flanking sequences, covering 50% of all possible G-A mismatches.