Exosomes, small (40–100 nm) extracellular membranous vesicles, attract enormous research interest because they are carriers of disease markers and a prospective delivery system for therapeutic agents. Differential centrifugation, the prevalent method of exosome isolation, frequently produces dissimilar and improper results because of the faulty practice of using a common centrifugation protocol with different rotors. Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for “fixed-angle” rotors. For both types of rotors – “swinging bucket” and “fixed-angle” – we express the theoretically expected proportion of pelleted vesicles of a given size and the “cut-off” size of completely sedimented vesicles as dependent on the centrifugation force and duration and the sedimentation path-lengths. The proper centrifugation conditions can be selected using relatively simple theoretical estimates of the “cut-off” sizes of vesicles. Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements. Measured by the nanoparticle tracking analysis (NTA) technique, the concentration and size distribution of the vesicles after centrifugation agree with those theoretically expected. To simplify this “cut-off”-size-based adjustment of centrifugation protocol for any rotor, we developed a web-calculator.

A new method of DNA sequencing by hybridization using a microchip containing a set of immobilized oligonucleotides is being developed. A theoretical analysis is presented of the kinetics of DNA hybridization with deoxynucleotide molecules chemically tethered in a polyacrylamide gel layer. The analysis has shown that long-term evolution of the spatial distribution and of the amount of DNA bound in a hybridization cell is governed by "retarded diffusion," i.e., diffusion of the DNA interrupted by repeated association and dissociation with immobile oligonucleotide molecules. Retarded diffusion determines the characteristic time of establishing a final equilibrium state in a cell, i.e., the state with the maximum quantity and a uniform distribution of bound DNA. In the case of cells with the most stable, perfect duplexes, the characteristic time of retarded diffusion (which is proportional to the equilibrium binding constant and to the concentration of binding sites) can be longer than the duration of the real hybridization procedure. This conclusion is indirectly confirmed by the observation of nonuniform fluorescence of labeled DNA in perfect-match hybridization cells (brighter at the edges). For optimal discrimination of perfect duplexes from duplexes with mismatches the hybridization process should be brought to equilibrium under low-temperature nonsaturation conditions for all cells. The kinetic differences between perfect and nonperfect duplexes in the gel allow further improvement in the discrimination through additional washing at low temperature after hybridization.

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.

Short term storage of extracellular vesicle (EV) solutions at +4°C is a common practice, but the stability of EVs during this procedure has not been fully understood yet. Using nanoparticle tracking analysis, we have shown that EVs isolated from the conditioned medium of HT-29 cells exhibit a pronounced concentration decrease when stored in PBS in ordinary polypropylene tubes within the range of (0.5–2.1) × 1010 particles/ml. EV losses reach 51±3% for 0.5 ml of EVs in Eppendorf 2 ml tube at 48 hours of storage at +4°C. Around 2/3 of the observed losses have been attributed to the adsorption of vesicles onto tube walls. This result shows that the lower part (up to at least 2 × 1010 particles/ml) of the practically relevant concentration range for purified EVs is prone to adsorption losses at +4°C. Total particle losses could be reduced to 18–21% at 48 hours by using either Eppendorf Protein LoBind tubes or ordinary tubes with the surface blocked with bovine serum albumin or EVs. Reduction of losses to 15% has been shown for isolated EVs dissolved in the supernatant after 100 000 g centrifugation as a model of conditioned medium. Also, a previously unknown feature of diffusion-controlled adsorption was revealed for EVs. In addition to the decrease in particle count, this process causes the predominant losses of smaller particles.

Parallel-stranded (ps) DNAs with mixed AT/GC content comprising G.C pairs in a varying sequence context have been investigated. Oligonucleotides were devised consisting of two 10-nt strands complementary either in a parallel or in an antiparallel orientation and joined via nonnucleotide linkers so as to form 10-bp ps or aps hairpins. A predominance of intramolecular hairpins over intermolecular duplexes was achieved by choice of experimental conditions and verified by fluorescence determinations yielding estimations of rotational relaxation times and fractional base pairing. A multistate mode of ps hairpin melting was revealed by temperature gradient gel electrophoresis (TGGE). The thermal stability of the ps hairpins with mixed AT/GC content depends strongly on the specific sequence in a manner peculiar to the ps double helix. The thermodynamic effects of incorporating trans G.C base pairs into an AT sequence are context-dependent: an isolated G. C base pair destabilizes the duplex whereas a block of > or =2 consecutive G.C base pairs exerts a stabilizing effect. A multistate heterogeneous zipper model for the thermal denaturation of the hairpins was derived and used in a global minimization procedure to compute the thermodynamic parameters of the ps hairpins from experimental melting data. In 0.1 M LiCl at 3 degrees C, the formation of a trans G.C pair in a GG/CC sequence context is approximately 3 kJ mol(-)(1) more favorable than the formation of a trans A.T pair in an AT/TA sequence context. However, GC/AT contacts contribute a substantial unfavorable free energy difference of approximately 2 kJ mol(-)(1). As a consequence, the base composition and fractional distribution of isolated and clustered G.C base pairs determine the overall stability of ps-DNA with mixed AT/GC sequences. Thus, the stability of ps-DNA comprising successive > or =2 G.C base pairs is greater than that of ps-DNA with an alternating AT sequence, whereas increasing the number of AT/GC contacts by isolating G.C base pairs exerts a destabilizing effect on the ps duplex. Molecular modeling of the various helices by force field techniques provides insight into the structural basis for these distinctions.

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.

Structure-specific ligands are convenient tools for the recognition, targeting or probing of non-canonical DNA structures. Porphyrin derivatives exhibit a preference for interaction with G-quadruplex (G4) structures over canonical duplex DNA and are able to cause photoinducible damage to nucleic acids. Here, we show that Zn(II) 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (ZnP1) interacts with different conformations of the telomeric sequence d(TAGGG(TTAGGG)3) at submicromolar concentrations without any detectible disturbance of the particular fold. Among different folds, potassium (3+1) hybrid G4-structure. reveal the highest affinity to ZnP1. The pattern of guanine oxidation is specific for each telomeric DNA conformation and may serve as an additional tool for probing the G4 topology. The potassium (3+1) and parallel G4 conformations are more susceptible to light-induced oxidation than the sodium G4 conformation or double helix of the telomeric DNA. The major products of the guanine modifications are spiroiminodihydantoin (Sp) and 8-oxoguanine (8-oxoG). ZnP1-induced oxidation of guanines results in the structural rearrangement of parallel and (3+1) G4 conformations yielding an antiparallel-like G4 conformation. The mechanism of the observed light-induced conformational changes is discussed.

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.