M. A. Livshits

Discrimination Between Perfect and Mismatched Duplexes with Oligonucleotide Gel Microchips: Role of Thermodynamic and Kinetic Effects During Hybridization

Abstract The efficiency of discrimination between perfect and mismatched duplexes during hybridization on microchips depends on the concentrations of target DNA in solution and immobilized probes, buffer composition, and temperature of hybridization and is determined by both thermodynamic relationships and hybridization kinetics. In this work, optimal conditions of discrimination were studied using hybridization of fluorescently labeled target DNA with custom-made gel-based oligonucleotide microchips. The higher the concentration of immobilized probes and the higher the association constant, the higher the concentration of the formed duplexes and the stronger the corresponding fluorescence signal, but, simultaneously, the longer the time needed to reach equilibrium. Since mismatched duplexes hybridize faster than their perfect counterparts, perfect-to-mismatch signal ratio is lower in transient regime, and short hybridization times may hamper the detection of mutations. The saturation time can be shortened by decreasing the probe concentration or augmenting the gel porosity. This improves the detection of mutations in transient regime. It is shown that the decrease in the initial concentration of oligonucleotide probes by an order of magnitude causes only 1.5–2.5-fold decrease of fluorescence signals after hybridization of perfect duplexes for 3–12 h. At the same time, these conditions improve the discrimination between perfect and mismatched duplexes more than two-fold. A similar improvement may be obtained using an optimized dissociation procedure.

Kinetics of Hybridization on Surface Oligonucleotide Microchips: Theory, Experiment, and Comparison with Hybridization on Gel-Based Microchips

Abstract The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5–1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5–20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2–4 times. These effects may be attributed to the better immobilization efficiency and to the higher thermodynamic association constants for duplex formation within gel pads.

Flexibility Difference Between Double-Stranded RNA and DNA as Revealed by Gel Electrophoresis

A systematic study of agarose gel electrophoresis of double-stranded RNA in the kilobase range of sizes was performed. The dsRNA to dsDNA relative mobility was found to depend on gel concentration: in low density gels RNA moves slower and in high density gels - faster than DNA of the same molecular size. The electrophoretic differences were interpreted within the reptation theory to be mainly due to the molecular stiffness differences. The dsRNA persistence length was roughly estimated to be about twice as great as that of DNA.

Kinetics of hybridization on the oligonucleotide microchips with gel pads.

Abstract The kinetics of hybridization on the oligonucleotide microchip with gel pads is studied both theoretically and experimentally. The monitoring of kinetics was performed with the measurements of fluorescence intensity produced by the labeled target oligonucleotides. As is shown, the hybridization time depends on the stability of the formed duplexes, the concentrations of target and probe oligonucleotides, and the diffusion of target oligonucleotides in solution and gel pad. The initial stage of hybridization is determined by the flow of target oligonucleotides from solution, then, followed by the diffusive propagation with approximately constant concentration of oligonucleotides at the boundary of gel pad and, finally, by the exponential saturation. The theoretical predictions of hybridization kinetics reveal a good correspondence with the experimental results and may be used for the choice of the optimal hybridization conditions. The possible applications of kinetic hybridization curves to the discrimination problems and assessment of diffusion coefficients in gel pads are briefly discussed. Finally, we discuss the relationships between the binding kinetics and the general functioning of biomolecular microchips.

Dissociation of duplexes formed by hybridization of DNA with gel-immobilized oligonucleotides.

The method of DNA sequencing by hybridization with oligonucleotides matrix (SHOM) developed in this laboratory (1.2) uses the matrix of oligonucleotides immobilized within polyacrylamide gel. The particular feature of this matrix is that the apparent thermostability of the duplexes depends on the concentration of gel-immobilized oligonucleotides. This dependence is specific for oligonucleotides immobilized in the gel volume (3-D-immobilization) rather than on a flat surface of a filter or glass (2-D-immobilization). The theory has been developed that provides a quantitative description of temperature-dependent duplex dissociation within gel. The theory takes into account that the diffusion of dissociated DNA out of the gel is retarded by multiple acts of association-dissociation of DNA with immobilized oligonucleotides. It allows to calculate the apparent dissociation temperature of duplexes and describes quantitatively its growth upon increase in the enthalpy of duplex dissociation, concentration of immobilized oligonucleotides, gel thickness and decrease of dissociation entropy and washing time. Concentration of gel-immobilized oligonucleotides can be calculated for a normalized matrix in which GC-rich and AT-rich duplexes exhibit the same apparent thermostabilities and are washed off at the same temperature. This simplifies identification of perfect duplexes formed on the matrix which can be carried out for all duplexes at the same temperature. The gel-immobilized oligonucleotide matrix provides also a higher capacity for immobilization and therefore a higher sensitivity of measurements, resulting in a higher discrimination power for identification of perfect duplexes as compared with matrixes of oligonucleotides immobilized on a surface.

Disordering of human telomeric G-quadruplex with novel antiproliferative anthrathiophenedione.

Linear heteroareneanthracenediones have been shown to interfere with DNA functions, thereby causing death of human tumor cells and their drug resistant counterparts. Here we report the interaction of our novel antiproliferative agent 4,11-bis[(2-{[acetimido]amino}ethyl)amino]anthra[2,3-b]thiophene-5,10-dione with telomeric DNA structures studied by isothermal titration calorimetry, circular dichroism and UV absorption spectroscopy. New compound demonstrated a high affinity (Kass∼106 M−1) for human telomeric antiparallel quadruplex d(TTAGGG)4 and duplex d(TTAGGG)4∶d(CCCTAA)4. Importantly, a ∼100-fold higher affinity was determined for the ligand binding to an unordered oligonucleotide d(TTAGGG TTAGAG TTAGGG TTAGGG unable to form quadruplex structures. Moreover, in the presence of Na+ the compound caused dramatic conformational perturbation of the telomeric G-quadruplex, namely, almost complete disordering of G-quartets. Disorganization of a portion of G-quartets in the presence of K+ was also detected. Molecular dynamics simulations were performed to illustrate how the binding of one molecule of the ligand might disrupt the G-quartet adjacent to the diagonal loop of telomeric G-quadruplex. Our results provide evidence for a non-trivial mode of alteration of G-quadruplex structure by tentative antiproliferative drugs.

Analysis of Perfect and Mismatched DNA Duplexes with a Generic Hexanucleotide Microchip

Thermodynamic analysis was performed for the duplexes formed by fluorescently labeled oligonucleotide targets on a generic hexanucleotide microchip. All 4096 different hexanucleotide chains were immobilized as probes in individual gel pads of the microchip. To strengthen the hybridization, each hexamer was extended at both ends by one nucleotide from the equimolar mixture of all four nucleotides to serve as nonselective linkers. It has been shown that the melting curves for oligonucleotide duplexes formed on the microchip and in a solution are quite similar. The influence of ionic surrounding has been studied in terms of the hybridization efficiency and discrimination between the mismatched and perfect duplexes. Different approaches have been tested to compensate the dependence of duplex stability on the GC content. It has been demonstrated that the use of chaotropic agents, addition of nonlabeled GC-rich competitor oligonucleotides, as well as creation of a temperature gradient along the microchip reproducing the distribution of melting temperatures, efficiently level out the AT/GC differences. The use of tetramethylammonium chloride for the same purpose was accompanied by weakening to some extent the discrimination between the mismatched duplexes and the perfect ones.

Parallel-Stranded DNA with Natural Base Sequences

Noncanonical parallel-stranded DNA double helices (ps-DNA) of natural nucleotide sequences are usually less stable than the canonical antiparallel-stranded DNA structures, which ensures reliable cell functioning. However, recent data indicate a possible role of ps-DNA in DNA loops or in regions of trinucleotide repeats connected with neurodegenerative diseases. The review surveys recent studies on the effect of nucleotide sequence on preference of one or other type of DNA duplex. (1) Ps-DNA of mixed AT/GC composition was found to have conformational and thermodynamic properties drastically different from those of a Watson–Crick double helix. Its stability depends strongly on the specific sequence in a manner peculiar to the ps double helix, because of the energy disadvantage of the AT/GC contacts. The AT/GC boundary facilitated flipping of A and T out of the ps double helix. Proton acceptor groups of bases are exposed into both grooves of the ps-DNA and are accessible to solvent and ligands, including proteins. (2) DNA regions containing natural minor bases isoguanine and isomethylisocytosine were shown to form ps-DNA with transAT-, trans isoGC, and transiso5meCG pairs exceeding in stability a related canonical duplex. (3) Nucleotide sequence dG(GT)4G from yeast telomeres and microsatellites was demonstrated to form novel ps-DNA with GG and TT base pairing. Unlike d(GT)n- and d(GnTm) sequences able to form quadruplexes, the dG(GT)4G sequence formed no alternative double- or multistranded structures in a wide range of experimental conditions, thus suggesting that the nucleotide context governs the observed structural polymorphism of the d(GT)n sequence. The possible biological role of ps-DNA and the prospects of its study are discussed.

Novel multi-targeting anthra[2,3-b]thiophene-5,10-diones with guanidine-containing side chains: Interaction with telomeric G-quadruplex, inhibition of telomerase and topoisomerase I and cytotoxic properties

Novel generations of antitumor anthraquinones are expected to be advantageous over the conventional chemotherapeutic agents. Previous structure-activity relationship studies demonstrated an importance of the positively charged side chains conjugated to anthra[2,3-b]thiophene-5,10-dione scaffolds. Exploring a role of individual side chain moieties in binding to the duplex and G-quadruplex DNA, modulation of telomerase and topoisomerase I activities, intracellular accumulation and cytostatic potency, we herein analyzed a series of reported and newly synthesized guanidine-containing derivatives of anthra[2,3-b]thiophene-5,10-dione. We found that the number of cationic side chains (namely, two) is critical for a tight interaction with human telomeric G-quadruplex (TelQ). Along with a larger drug-TelQ association constant, the telomerase attenuation by anthrathiophenediones with two basic groups in the side chains was more pronounced than by the analogs bearing one basic group. For mono-guanidinated compounds the substituent with the amino group in the side chain provided better TelQ affinity than the methylamine residue. The intracellular uptake of the mono-guanidino derivative with two side chains was >2-fold higher than the respective value for the bis(guanidino) derivative. This difference can explain a lower antiproliferative potency of bis(guanidine) containing compounds. Thus, the modifications of side chains of anthra[2,3-b]thiophene-5,10-dione differently modulated drug-target interactions and cellular effects. Nevertheless, the selected compound 11-(3-aminopropylamino)-4-(2-guanidinoethylamino)anthra[2,3-b]thiophene-5,10-dione 13 demonstrated a high affinity to TelQ and the ability to stabilize the quadruplex structure. These properties were paralleled by reasonable potency of 13 as a telomerase/topoisomerase I inhibitor and an antiproliferative agent. These results indicate that the structural elements of anthra[2,3-b]thiophene-5,10-dione derivatives can be balanced to yield a candidate for further preclinical study.

Effects of External Transport on Discrimination between Perfect and Mismatch Duplexes on Gel-Based Oligonucleotide Microchips

Abstract Using hydrogel-based oligonucleotide microchips developed previously for the choice of drugs during leukemia treatment and the other diseases, it is shown that the acceleration of external transport by mixing buffer solution with peristaltic pump not only enhances the observable fluorescence signals, but also improves significantly the discrimination between perfect and mismatch duplexes at the intermediate stage of hybridization on the oligonucleotide microchips. The discrimination efficiency for a given hybridization time grows monotonously with the frequency of flow pulsations. The mixing with frequency 10 Hz accelerates the hybridization rate approximately thrice and improves the discrimination efficiency 1.5–2.5 times higher for overnight hybridization. To study these effects, we have developed the special peristaltic pump mixing solution in a hybridization chamber of 35 μl in volume (area ~1 × 1 cm2 and height 0.3 mm). We present also the brief theoretical summary for the interpretation and assessment of the observed experimental features.