Application of the weighted histogram method for calculating the thermodynamic parameters of the formation of oligodeoxyribonucleotide duplexes.

To date, many derivatives and analogs of nucleic acids (NAs) have been developed. Some of them have found uses in scientific research and biomedical applications. Their effective use is based on the data about their properties. Some of the most important physicochemical properties of oligonucleotides are thermodynamic parameters of the formation of their duplexes with DNA and RNA. These parameters can be calculated only for a few NA derivatives: locked NAs, bridged oligonucleotides, and peptide NAs. Existing predictive approaches are based on an analysis of experimental data and the consequent construction of predictive models. The ongoing pilot studies aimed at devising methods for predicting the properties of NAs by computational modeling techniques are based only on knowledge about the structure of oligonucleotides. In this work, we studied the applicability of the weighted histogram analysis method (WHAM) in combination with umbrella sampling to the calculation of thermodynamic parameters of DNA duplex formation (changes in enthalpy ΔH°, entropy ΔS°, and Gibbs free energy ΔG° 37). A procedure was designed involving WHAM for calculating the hybridization properties of oligodeoxyribonucleotides. Optimal parameters for modeling and calculation of thermodynamic parameters were determined. The feasibility of calculation of ΔH°, ΔS°, and ΔG° 37 was demonstrated using a representative sample of 21 oligonucleotides 4-16 nucleotides long with a GC content of 14-100 %. Error of the calculation of the thermodynamic parameters was 11.4, 12.9, and 11.8 % for ΔH°, ΔS°, and ΔG° 37, respectively, and the melting temperature was predicted with an average error of 5.5 °C. Such high accuracy of computations is comparable with the accuracy of the experimental approach and of other methods for calculating the energy of NA duplex formation. In this paper, the use of WHAM for computation of the energy of DNA duplex formation was systematically investigated for the first time. Our results show that a reliable calculation of the hybridization parameters of new NA derivatives is possible, including derivatives not yet synthesized. This work opens up new horizons for a rational design of constructs based on NAs for solving problems in biomedicine and biotechnology.

Application of W-band 19F electron nuclear double resonance (ENDOR) spectroscopy to distance measurement using a trityl spin probe and a fluorine label.

Recently, Marina Bennati and coworkers (M. Bennati et al., Angew. Chem., Int. Ed., 2020, 59, 373-379., M. Bennati et al., J. Magn. Reson., 2021, 333, 107091) proposed to use electron nuclear double resonance (ENDOR) spectroscopy in the W-band for a pair of labels, nitroxide and 19F, for measurements of short (0.5-1.0 nm) distances in biomolecules. In our paper, we investigated the suitability of high-field ENDOR spectroscopy in the W-band for pairs of triarylmethyl and fluorine labels using five newly synthesized model compounds. It is shown that the application of strong magnetic fields allows distinguishing nuclear frequencies of 19F and protons with sufficient resolution. On the one hand, in contrast to nitroxides, for triarylmethyl radicals, it is not necessary to obtain spectra in different orientations owing to low g-factor anisotropic and long electron spin relaxation times of triarylmethyls. On the other hand, the size of the triarylmethyl radical is substantially larger than that of nitroxide and comparable with measured distances. We theoretically analyzed the suitability of the dipole-dipole approach for triarylmethyl to be used in a 19F ENDOR experiment and determined limitations of this approach. Finally, for comparison, we performed paramagnetic relaxation enhancement (PRE) NMR on the same compounds. In addition, we applied this approach to study the process of a thiol exchange between molecules of triarylmethyl-labeled and 19F-labeled human serum albumin (HSA).

Flow-Seq Method: Features and Application in Bacterial Translation Studies.

The Flow-seq method is based on using reporter construct libraries, where a certain element regulating the gene expression of fluorescent reporter proteins is represented in many thousands of variants. Reporter construct libraries are introduced into cells, sorted according to their fluorescence level, and then subjected to next-generation sequencing. Therefore, it turns out to be possible to identify patterns that determine the expression efficiency, based on tens and hundreds of thousands of reporter constructs in one experiment. This method has become common in evaluating the efficiency of protein synthesis simultaneously by multiple mRNA variants. However, its potential is not confined to this area. In the presented review, a comparative analysis of the Flow-seq method and other alternative approaches used for translation efficiency evaluation of mRNA was carried out; the features of its application and the results obtained by Flow-seq were also considered.

[Calculation of Energy for RNA/RNA and DNA/RNA Duplex Formation by Molecular Dynamics Simulation].

The development of approaches for predictive calculation of hybridization properties of various nucleic acid (NA) derivatives is the basis for the rational design of the NA-based constructs. Modern advances in computer modeling methods provide the feasibility of these calculations. We have analyzed the possibility of calculating the energy of DNA/RNA and RNA/RNA duplex formation using representative sets of complexes (65 and 75 complexes, respectively). We used the classical molecular dynamics (MD) method, the MMPBSA or MMGBSA approaches to calculate the enthalpy (ΔH°) component, and the quasi-harmonic approximation (Q-Harm) or the normal mode analysis (NMA) methods to calculate the entropy (ΔS°) contribution to the Gibbs energy (ΔG°37) of the NA complex formation. We have found that the MMGBSA method in the analysis of the MD trajectory of only the NA duplex and the empirical linear approximation allow calculation of the enthalpy of formation of the DNA, RNA, and hybrid duplexes of various lengths and GC content with an accuracy of 8.6%. Within each type of complex, the combination of rather efficient MMGBSA and Q-Harm approaches being applied to the trajectory of only the bimolecular complex makes it possible to calculate the ΔG°37 of the duplex formation with an error value of 10%. The high accuracy of predictive calculation for different types of natural complexes (DNA/RNA, DNA/RNA, and RNA/RNA) indicates the possibility of extending the considered approach to analogs and derivatives of nucleic acids, which gives a fundamental opportunity in the future to perform rational design of new types of NA-targeted sequence-specific compounds.

A QCM-based rupture event scanning technique as a simple and reliable approach to study the kinetics of DNA duplex dissociation.

Rupture Event Scanning (REVS) is applied for the first time within an approach based on dynamic force spectroscopy. Using model DNA duplexes containing 20 pairs of oligonucleotides including those containing single mismatches, we demonstrated the possibility of reliable determination of the kinetic parameters of dissociation of biomolecular complexes: barrier positions, the rate constants of dissociation, and the lifetime of complexes. Within this approach, mechanical dissociation of DNA duplexes occurs according to a mechanism similar to unzipping. It is shown that this process takes place by overcoming a single energy barrier. In the case where a mismatch is located at the farthest duplex end from the QCM surface, a substantial decrease in the position of the barrier between the bound and unbound states is observed. We suppose that this is due to the formation of an initiation complex containing 3-4 pairs of bases, and this is sufficient for starting duplex unzipping.

Peptide-oligonucleotide conjugates exhibiting pyrimidine-X cleavage specificity efficiently silence miRNA target acting synergistically with RNase H.

Taking into account the important role of miRNA in carcinogenesis, oncogenic miRNAs are attractive molecules for gene-targeted therapy. Here, we developed a novel series of peptide-oligonucleotide conjugates exhibiting ribonuclease activity targeted to highly oncogenic miRNAs miR-21 and miR-17. When designing the conjugates, we enhanced both nuclease resistance of the targeted oligodeoxyribonucleotide by introducing at its 3'-end mini-hairpin structure displaying high thermostability and robustness against nuclease digestion and the efficiency of its functioning by attachment of the catalytic construction (amide)NH2-Gly(ArgLeu)4-TCAA displaying ribonuclease activity to its 5'-end. Designed miRNases efficiently cleaved miRNA targets, exhibiting Pyr-X specificity, and cleavage specificity had strong dependence on the miRNA sequence in the site of peptide location. In vitro, designed miRNases do not prevent cleavage of miRNA bound with the conjugate by RNase H, and more than an 11-fold enhancement of miRNA cleavage by the conjugate is observed in the presence of RNase H. In murine melanoma cells, miRNase silences mmu-miR-17 with very high efficiency as a result of miR-17 cleavage by miRNase and by recruited RNase H. Thus, miRNases provide a system of double attack of the miRNA molecules, significantly increasing the efficiency of miRNA downregulation in the cells in comparison with antisense oligonucleotide.

[Modified Oligonucleotides for Guiding RNA Cleavage Using Bacterial RNase P].

The ability of a series of novel modified external guide sequences (EGS oligonucleotides) to induce the hydrolysis of target RNA with bacterial ribonuclease P has been studied; the most efficient modification variants have been selected. We have found patterns of the oligonucleotide sugar-phosphate backbone modi-fications that enhance oligonucleotide stability in the biological environment and do not violate the ability to interact with the enzyme and induce the RNA hydrolysis. It has been shown that analogues of EGS oligonucleotides selectively modified at 2'-position (2'-O-methyl and 2'-fluoro) or at internucleotide phosphates (phosphoryl guanidines) can be used for the addressed cleavage of a model RNA target by bacterial RNase P. The ability of new phosphoryl guanidine analogues of oligodeoxyribonucleotides that are stable in biological media to induce the hydrolysis of target RNA with bacterial ribonuclease P has been shown for the first time. The modified EGS oligonucleotides with an optimal balance between functional activity and stability in biological media can be considered as potential antibacterial agents.

Phosphoryl guanidines: a new type of nucleic Acid analogues.

A new type of nucleic acid analogues with a phosphoryl guanidine group is described. Oxidation of polymer-supported dinucleoside 2-cyanoethyl phosphite by iodine in the presence of 1,1,3,3-tetramethyl guanidine yields a dinucleotide with an internucleoside tetramethyl phosphoryl guanidine (Tmg) group as the main product. The Tmg group is stable under conditions of solid-phase DNA synthesis and subsequent cleavage and deprotection with ammonia. Oligonucleotides with one or more Tmg groups bind their complementary DNA or RNA with affinity similar to that of natural oligodeoxyribonucleotides.

A simple approach to prepare molecularly imprinted polymers from nylon-6.

A convenient and simple approach for the preparation of molecularly imprinted polymers (MIPs) based on polyamide (nylon-6) was developed. The polymer matrix formation occurred during the transition of nylon from dissolved to solid state in the presence of template molecules in the initial solution. 2,2,2-Trifluoroethanol (TFE) was chosen as a main solvent for the polyamide. It provides a high solubility of nylon and does not significantly change the structure of biopolymers. The alteration of the polymer matrix structure after the addition of different types of porogens in the nylon/TFE solution was investigated. The structured polymers in the form of films and microparticles were prepared in the chosen optimal conditions. Different model biomolecular templates (of low- and high-molecular weight) were used for the preparation of MIPs, which were shown to specifically recognize these molecules upon binding experiments. The binding of the template molecules to MIPs was monitored using spectrophotometric, radioisotopic, or fluorometric detection. The selectivity coefficients of the MIPs were estimated to be 1.4-4.6 depending on the type of templates and conditions of the polymer matrix formation.

[Synthesis and properties of methylene carboxamide mimetics of nucleic acids based on morpholine nucleosides].

Uracyl and adenine containing oligocarboxamide mimetics of nucleic acids based on morpholine nucleosides (MorGly) are synthesized using peptide chemistry methods. Conditions for an analysis of homogeneity of protonated at physiological pH oligomers using a capillary electrophoresis are proposed. Studies of thermostability of complementary complexes formed by MorGly oligomers revealed that melting temperature dramatically depends on heterocyclic base composition (uracyl or adenine). Cooperative interactions realized at junctions in tandem complexes give more contribution to the thermostability in the case of complexes formed by modified oligomers than native oligodeoxyriboadenilates. Adenine containing MorGly oligomers form more stable complexes with poly(U) than native oligodeoxyriboadenilate of the same length. Complexes formed by modified oligomers with polyribonucleotides are more stable in compare with polydeoxyribonucleotide.

[Considering the oligonucleotides secondary structures at thermodynamic and kinetic analysis of the DNA-duplexes formation].

The influence of secondary structures of DNA oligonucleotides on thermodynamics and kinetics at the formation of their bimolecular complexes (duplexes) has been studied. The models considering inherent secondary structures of duplex components and their influence on quantitative thermodynamic and kinetic characteristics of the duplexes have been developed. The values of thermodynamic impacts given by individual structural elements of the double helix have been shown to depend on hairpin structuring of the duplex components. The "concentration" method to consider oligonucleotides intramolecular structure with thermodynamic parameters of bimolecular duplex formation has been proposed. According to stop-flow measurements, the observed values of association and dissociation constants are influenced by the presence of inherent structures in duplex components. The influence observed is increased with the lowering of the sample temperature. The analysis of experimental data involving the developed models provides the possibility to determine "proper" kinetic constants for the helix-to-coil transition. The difference between observed and calculated rate constants can amount up to two or more orders of magnitude.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insert].

Non-nucleotide phosporamidites were synthetized, having branched backbone with different position of functional groups. Obtained phosphoramidite monomers contain intercalator moiety--6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with tert-butyldimethylsilyl group for post-synthetic modification. Synthesized oligothymidilates contain one or more modified units in different positions of sequence. Melting temperature and thermodynamic parameters of formation of complementary duplexes formed by modified oligonucleotides was defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA.

[A convenient approach to the synthesis of the phosphoramidite non-nucleotide monomers for the preparation of functionalized oligonucleotide derivatives].

A simple approach to the synthesis of phosphoramidite synthons for non-nucleotide inserts using 4-(2-(4,4'-dimethoxytrityloxy)ethyl)morpholine-2,3-dione as a key precursor is suggested. Using the method developed various inserts have been introduced into oligonucleotides to obtain intercalator-containing (acridine) as well as branched oligonucleotides.

Interaction of poly(ADP-ribose) polymerase 1 with apurinic/apyrimidinic sites within clustered DNA damage.

To study the interaction of poly(ADP-ribose) polymerase 1 (PARP1) with apurinic/apyrimidinic sites (AP sites) within clustered damages, DNA duplexes were created that contained an AP site in one strand and one of its analogs situated opposite the AP site in the complementary strand. Residues of 3-hydroxy-2-hydroxymethyltetrahydrofuran (THF), diethylene glycol (DEG), and decane-1,10-diol (DD) were used. It is shown for the first time that apurinic/apyrimidinic endonuclease 1 (APE1) cleaves the DNA strands at the positions of DEG and DD residues, and this suggests these groups as AP site analogs. Insertion of DEG and DD residues opposite an AP site decreased the rate of AP site hydrolysis by APE1 similarly to the effect of the THF residue, which is a well-known analog of the AP site, and this allowed us to use such AP DNAs to imitate DNA with particular types of clustered damages. PARP1, isolated and in cell extracts, efficiently interacted with AP DNA with analogs of AP sites producing a Schiff base. PARP1 competes with APE1 upon interaction with AP DNAs, decreasing the level of its cross-linking with AP DNA, and inhibits hydrolysis of AP sites within AP DNAs containing DEG and THF residues. Using glutaraldehyde as a linking agent, APE1 is shown to considerably decrease the amount of AP DNA-bound PARP1 dimer, which is the catalytically active form of this enzyme. Autopoly(ADP-ribosyl)ation of PARP1 decreased its inhibitory effect. The possible involvement of PARP1 and its automodification in the regulation of AP site processing within particular clustered damages is discussed.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon].

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DNA.

The analysis of DNA nucleotide polymorphisms is one of the main goals of DNA diagnostics. DNA-dependent enzymes (DNA polymerases and DNA ligases) are widely used to enhance the sensitivity and reliability of systems intended for the detection of point mutations in genetic material. In this article, we have summarized the data on the selectiveness of DNA-dependent enzymes and on the structural factors in enzymes and DNA which influence the effectiveness of mismatch discrimination during enzymatic conversion of oligonucleotide probes on a DNA template. The data presented characterize the sensitivity of a series of DNA-dependent enzymes that are widely used in the detection of noncomplementary base pairs in nucleic acid substrate complexes. We have analyzed the spatial properties of the enzyme-substrate complexes. These properties are vital for the enzymatic reaction and the recognition of perfect DNA-substrates. We also discuss relevant approaches to increasing the selectivity of enzyme-dependent reactions. These approaches involve the use of modified oligonucleotide probes which "disturb" the native structure of the DNA-substrate complexes.

Bridged oligonucleotides as molecular probes for investigation of enzyme-substrate interaction and allele-specific analysis of DNA.

The efficiency of enzymatic conversion of DNA complexes containing non-nucleotide inserts has been studied. T4 DNA ligase and Taq DNA polymerase have been included in the study as examples of widely used DNA-dependent enzymes. A series of substrate DNA complexes have been formed using native oligonucleotides and bridged ones bearing non-nucleotide inserts based on phosphodiesters of di-, tetra-, or hexaethylene glycol, 1,5-pentanediol, 1,10-decanediol, and 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran. The perturbation in DNA located far from the site of the enzyme action had almost no influence on the substrate properties of the complex, while insertion near this site significantly deteriorated them. The use of a series of modified duplexes allows one to locate the position of the enzyme-binding site on DNA substrate with the accuracy of 1-2 nucleotides. The presence of a non-nucleotide insert in the complex has been also shown to enhance the efficiency of single mismatch discrimination upon both template-directed ligation and extension of oligonucleotides.

[Thermodynamic description of oligonucleotide self-association in DNA concatamer structures].

A scheme of the formation of concatamer structures consisting of two different oligonucleotides has been considered. It was shown that, in the general case, the dependence of the concentration of oligonucleotide components on temperature cannot be found in the analytic form. Therefore, it is impossible to find the thermodynamic parameters of the formation of concatamer complexes (deltaH0, deltaS0) and melting temperature by analyzing the profiles of thermal denaturation of oligonucleotide complexes. An algorithm of the numerical solution of implicit dependences has been developed. A number of approaches have been considered that simplify the analysis of thermal denaturation curves of concatamer complexes. It was shown that the analytical dependence of the efficiency of the concatamer formation on temperature can be described when duplex fragments have close stability and there is no cooperativity at the helix-helix interface. In this case, the dependence of melting temperature on thermodynamic parameters and oligonucleotide concentration has the same form as in the case of the duplex structure formed by a pair of noncomplementary oligonucleotides. The capacity of various model approaches to describe the experimental curves of thermal denaturation of concatamer structures has been evaluated. For the case of concatamer structures used as signal amplifiers in DNA hybridization analysis, a function was introduced that shows a relative contribution of a concatamer of a fixed length to the magnitude of signal amplification. The dependence of the maximum of this function on the concentration of oligonucleotides, the thermodynamic characteristics of their complexes, and temperature has been determined. It was shown by the gel shift assay that the function of the length distribution of concatamers qualitatively correlates with the experimental dependences.

[Bent dsDNA with defined geometric characteristics in terms of complexes of bridged oligonucleotides].

An opportunity of designing nontypical double-stranded DNA structures containing nonnatural inserts in a regular nucleotide DNA sequence has been investigated. The looped nucleotide inserts on the basis of adenylates and thymidilates, and nonnucleotide inserts on the basis of phosphodiesters of diethyleneglycol, 1,10-decanediol, and 3-hydroxy-2-hudroxymethyltetrahydrofuran were introduced into the backbone of a 32-mer native DNA duplex. These inserts formed the internal loops in the modified double-stranded DNA fragments which were shown to lead to bending of the linear duplex structure by 16 to 83 degrees. The dependencies of the bend angle of dsDNA on the composition and the length of the looped regions were determined. It was established that the bend of the irregular region of dsDNA depended on the electrostatic interaction of the phosphate residues. The tension in the complex structure could be reduced by the introduction of additional nucleotide units opposite the loop, which led to some relaxation of the bent helix. The resulting parameters of the bend values were shown to be in a good agreement with the published data obtained by NMR spectroscopy. It was demonstrated that the variation of the nature or the length of the insert allowed one to regulate the level of the local perturbation of the duplex structure and, thereby, influence both the bend level of the double helix and the destabilization of the modified complex.