Considering the oligonucleotide secondary structures in thermodynamic and kinetic analysis of DNA duplex 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.     

Triplex formation of chemically modified homopyrimidine oligonucleotides: thermodynamic and kinetic studies.

We have investigated effects of chemical modifications of a third strand on the thermodynamic and kinetic properties of the triplex formation between a 23-bp duplex and each of four kinds of 15-mer chemically modified third strands using isothermal titration calorimetry and interaction analysis system. The chemical modifications of the third strand included one base modification, with replacement of thymine by uracil; two sugar moiety modifications, RNA and 2'-O-methyl-RNA; and one phosphate backbone modification, with replacement of phosphodiester by phosphorothioate backbone. The thermodynamic and kinetic parameters obtained were similar in magnitude at room temperature for the triplex formation with the base-modified and the sugar-modified third strands. By contrast, binding constant for the triplex formation with the third strand containing phosphorothioate backbone was much smaller by a factor of 10 than that for the other triplex formations. Kinetic analyses have also demonstrated that the third strand containing phosphorothioate backbone was much slower in the association step and much faster in the dissociation step than the other third strands, which resulted in the much smaller binding constant. The reason for the instability of the triplex with the third strand containing phosphorothioate backbone will be discussed. We conclude that, at least in the triplex formation with the chemically modified third strands studied in the present work, the modification of phosphate backbone of the third strand produces more significant effect on the triplex formation than the modifications of base and sugar moiety.     

Hybridization of the bridged oligonucleotides with DNA: thermodynamic and kinetic studies

Hybridization properties of oligonucleotides containing non-nucleotide inserts designed on the basis of synthetic abasic sites, oligomethylene diols or oligoethylene glycols have been characterized. The influence of the inserts which generate extrahelical anucleotidic bulges on thermodynamics, kinetics of hybridization of bridged oligonucleotide with DNA has been studied by UV-melting and stopped-flow techniques. Circular dichroism spectrometry data show that anucleotidic bulges in the middle of the duplex does not alter the B-form helix conformation. Nevertheless, the insert induces destabilization of the duplex structure, caused mostly by the considerable enhancement of the dissociation rates. Free energy increments for the extrahelical anucleotidic bulges can be described in the nearest-neighbor approximation. The thermodynamic effect of the insert lengthening obeys a simple Jacobson-Stockmayer entropy extrapolation. Independently of the insert type, the free energy term is directly proportional to the logarithm of the number of bonds between the oligonucleotide fragments. The behavior of hydrophobic inserts formed by 10-hydroxydecyl-1-phospate units is an exception to the rule.     

dC-dG alternating oligonucleotides: thermodynamic and kinetic aspects of the B-Z transformation

The alternating cytosine-guanine oligodeoxyribonucleotides (dCdG)n, (dGdC)n, (dCdG)ndC (n = 3,4), (dGdC)7 and dG(dCdG)3 have been studied by UV and CD spectroscopy at different temperatures and NaCl concentrations. The analysis of the melting data, assuming an all-or-none model, reveals that in the B-conformation the 5'G/C3' stacking interactions are enthalpically favoured with respect to the 5'C/G3' one. The CD investigation of the B-Z equilibrium shows that the Z-conformation is enthalpically stabilized, while the B-conformation is entropically favoured, in the range of NaCl concentration considered (1 to 5 M). The kinetic data for the B-Z transformation, obtained with a salt-jump technique for the hexamer (dCdG)3, support a mechanism by which the Watson-Crick hydrogen bonds are broken before the bases flip over separately and eventually stack, reforming the H-bonds, in the new helix.     

Hybridization of 2'-ribose modified mixed-sequence oligonucleotides: thermodynamic and kinetic studies

In this study, we characterize the thermodynamics of hybridization, binding kinetics and conformations of four ribose-modified (2′-fluoro, 2′-O-propyl, 2′-O-methoxyethyl and 2′-O-aminopropyl) decameric mixed-sequence oligonucleotides. Hybridization to the complementary non-modified DNA or RNA decamer was probed by fluorescence and circular-dichroism spectroscopy and compared to the same duplex formed between two non-modified strands. The thermal melting points of DNA–DNA duplexes were increased by 1.8, 2.2, 0.3 and 1.3°C for each propyl, methoxyethyl, aminopropyl and fluoro modification, respectively. In the case of DNA–RNA duplexes, the melting points were increased by 3.1, 4.1 and 1.0°C for each propyl, methoxyethyl and aminopropyl modification, respectively. The high stability of the duplexes formed with propyl-, methoxyethyl- and fluoro-modified oligonucleotides correlated with high preorganization in these single-strands. Despite higher thermodynamic duplex stability, hybridization kinetics to complementary DNA or RNA was slower for propyl- and methoxyethyl-modified oligonucleotides than for the non-modified control. In contrast, the positively-charged aminopropyl-modified oligonucleotide showed rapid binding to the complementary DNA or RNA.     

A kinetic approach to the prediction of RNA secondary structures

A new approach to the prediction of secondary RNA structures based on the analysis of the kinetics of molecular self-organisation is proposed herein. The Markov process is used to describe structural reconstructions during secondary structure formation. This process is modelled by a Monte-Carlo method. Examples of the calculation by this method of the secondary structures kinetic ensemble are given. Distribution of time-dependent probabilities within the ensembles is obtained. An effective method for search for the equilibrium ensemble is also suggested. This method is based on the construction of a tree of all possible secondary structures of RNA. By ascribing a probability for each structure (according to its free energy) the Boltzmann equilibrium ensemble can be obtained.     

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.     

A theoretical analysis of structural restructuring during formation of secondary RNA structures

An improved method for predicting the RNA secondary structure is proposed. The process of self-organization of structure is considered as a Markov chain. The kinetic of secondary structure is analysed by the Monte-Carlo method. The topological compatibility of helices is discussed. On the base of analysis it follows that the dynamical process of secondary structure formation is so that it is impossible to define a static set of complementary pairs. The method was used for predicting the mRNA secondary structure of a series of recombinant plasmids, containing the cro gene. The observed variation in expression can be explained by secondary structure.     

Physico-chemical and biological properties of antisense phosphodiester oligonucleotides with various secondary structures.

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.     

Effects of abasic sites on structural,thermodynamic and kinetic properties of quadruplex structures

Abasic sites represent the most frequent lesion in DNA. Since several events generating abasic sites concern guanines, this damage is particularly important in quadruplex forming G-rich sequences, many of which are believed to be involved in several biological roles. However, the effects of abasic sites in sequences forming quadruplexes have been poorly studied. Here, we investigated the effects of abasic site mimics on structural, thermodynamic and kinetic properties of parallel quadruplexes. Investigation concerned five oligodeoxynucleotides based on the sequence d(TGGGGGT), in which all guanines have been replaced, one at a time, by an abasic site mimic (dS). All sequences preserve their ability to form quadruplexes; however, both spectroscopic and kinetic experiments point to sequence-dependent different effects on the structural flexibility and stability. Sequences d(TSGGGGT) and d(TGGGGST) form quite stable quadruplexes; however, for the other sequences, the introduction of the dS in proximity of the 3′-end decreases the stability more considerably than the 5′-end. Noteworthy, sequence d(TGSGGGT) forms a quadruplex where dS does not hamper the stacking between the G-tetrads adjacent to it. These results strongly argue for the central role of apurinic/apyrimidinic site damages and they encourage the production of further studies to better delineate the consequences of their presence in the biological relevant regions of the genome.     

Structural, kinetic, and thermodynamic analysis of glucoimidazole-derived glycosidase inhibitors

Inhibition of glycosidases has great potential in the quest for highly potent and specific drugs to treat diseases such as diabetes, cancer, and viral infections. One of the most effective ways of designing such compounds is by mimicking the transition state. Here we describe the structural, kinetic, and thermodynamic dissection of binding of two glucoimidazole-derived compounds, which are among the most potent glycosidase inhibitors reported to date, with two family 1 beta-glycosidases. Provocatively, while inclusion of the phenethyl moiety improves binding by a factor of 20-80-fold, this does not appear to result from better noncovalent interactions with the enzyme; instead, improved affinity may be derived from significantly better entropic contributions to binding displayed by the phenethyl-substituted imidazole compound.     

Prediction of alternative RNA secondary structures based on fluctuating thermodynamic parameters.

In this paper we present a new method for predicting a set of RNA secondary structures that are thermodynamically favored in RNA folding simulations. This method uses a large number of 'simulated energy rules' (SER) generated by perturbing the free energy parameters derived experimentally within the range of the experimental errors. The structure with the lowest free energy is computed for each SER. Structural comparisons are used to avoid multiple generation of similar structures. Computed structures are evaluated using the energy distribution of the lowest free energy structures derived in the simulation. Predicted be graphically displayed with their occurring frequencies in the simulation by dot-plot representations. On average, about 90% of phylogenetic helixes in the known models of tRNA, Group I self-splicing intron, and Escherichia coli 16 S rRNA, were predicted using the method.     

Prediction of antisense oligonucleotides using structural and thermodynamic motifs

Specific gene expression regulation strategy using antisense oligonucleotides occupy significant space in recent clinical trials. The therapeutical potential of oligos lies in the identification and prediction of accurate oligonucleotides against specific target mRNA. In this work we present a computational method that is built on Artificial Neural Network (ANN) which could recognize and predict oligonucleotides effectively. In this study first we identified 11 major parameters associated with oligo:mRNA duplex linkage. A feed forward multilayer perceptron ANN classifier is trained with a set of experimentally proven feature vectors. The classifier gives an exact prediction of the input sequences under 2 classes – oligo or non-oligo. On validation, our tool showed comparatively significant accuracy of 92.48% with 91.7% sensitivity and 92.09% specificity. This study was also able to reveal the relative impact of individual parameters we considered on antisense oligonucleotide predictions.     

Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding

The equilibrium folding of a series of self-complementary RNA duplexes and the unmodified yeast tRNA(Phe) is studied as a function of urea and Mg(2+) concentration with optical spectroscopies and chemical modification under isothermal conditions. Via application of standard methodologies from protein folding, the folding free energy and its dependence on urea concentration, the m value, are determined. The free energies of the RNA duplexes obtained from the urea titrations are in good agreement with those calculated from thermal melting studies [Freier, S. I., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373]. The m value correlates with the length of the RNA duplex and is not sensitive to ionic conditions and temperature. The folding of the unmodified yeast tRNA(Phe) can be described by two Mg(2+)-dependent transitions, the second of which corresponds to the formation of the native tertiary structure as confirmed by hydroxyl radical protection and partial nuclease digestion. Both transitions are sensitive to urea and have m values of 0.94 and 1.70 kcal mol(-)(1) M(-)(1), respectively. Although the precise chemical basis of urea denaturation of RNA is uncertain, the m values for the duplexes and tRNA(Phe) are proportional to the amount of the surface area buried in the folding transition. This proportionality, 0.099 cal mol(-)(1) M(-)(1) A(-)(2), is very similar to that observed for proteins, 0.11 cal mol(-)(1) M(-)(1) A(-)(2) [Myers, J., Pace, N., and Scholtz, M. (1995) Protein Sci. 4, 2138]. These results indicate that urea titration can be used to measure both the free energy and the magnitude of an RNA folding transition.     

A thermodynamic and structural analysis of DNA minor-groove complex formation

As part of an effort to develop a better understanding of the structural and thermodynamic principles of DNA minor groove recognition, we have investigated complexes of three diphenylfuran dications with the d(CGCGAATTCGCG)(2) duplex. The parent compound, furamidine (DB75), has two amidine substituents while DB244 has cyclopentyl amidine substituents and DB226 has 3-pentyl amidines. The structure for the DB244-DNA complex is reported here and is compared to the structure of the DB75 complex. Crystals were not obtained with DB226 but information from the DB75 and DB244 structures as well as previous NMR results on DB226 indicate that all three compounds bind in the minor groove at the AATT site of the duplex. DB244 and DB75 penetrate to the floor of the groove and form hydrogen bonds with T8 on one strand and T20 on the opposite strand while DB226 forms a complex with fewer interactions. Binding studies by surface plasmon resonance (SPR) yield -delta G degrees values in the order DB244>DB75>DB226 that are relatively constant with temperature. The equilibrium binding constants for DB244 are 10-20 times greater than that for DB226. Isothermal titration calorimetric (ITC) experiments indicate that, in contrast to delta G degrees, delta H degrees varies considerably with temperature to yield large negative delta Cp degrees values. The thermodynamic results, analyzed in terms of structures of the DNA complexes, provide an explanation of why DB244 binds more strongly to DNA than DB75, while DB266 binds more weakly. All three compounds have a major contribution to binding from hydrophobic interactions but the hydrophobic term is most favorable for DB244. DB244 also has strong contributions from molecular interactions in its DNA complex and all of these factors combine to give it the largest-delta G degrees for binding. Although the factors that influence the energetics of minor groove interactions are varied and complex, results from the literature coupled with those on the furan derivatives indicate that there are some common characteristics for minor groove recognition by unfused heterocyclic cations that can be used in molecular design.     

Combined thermodynamic and kinetic analysis of GroEL interacting with CXCR4 transmembrane peptides

GroEL along with ATP and its co-chaperonin GroES has been demonstrated to significantly enhance the folding of newly translated G-protein-coupled receptors (GPCRs). This work extends the previous studies to explore the guest capture and release processes in GroEL-assisted folding of GPCRs, by the reduced approach of employing CXCR4 transmembrane peptides as model substrates. Each of the CXCR4-derived peptides exhibited high affinity for GroEL with a binding stoichiometry near seven. It is found that the peptides interact with the paired α helices in the apical domain of the chaperonin which are similar with the binding sites of SBP (strongly binding peptide: SWMTTPWGFLHP). Complementary binding study with a single-ring version of GroEL indicates that each of the two chaperonin rings is competent for accommodating all the seven CXCR4 peptides bound to GroEL under saturation condition. Meanwhile, the binding kinetics of CXCR4 peptides with GroEL was also examined; ATP alone, or in combination of GroES evidently promoted the release of the peptide substrates from the chaperonin. The results obtained would be beneficial to understand the thermodynamic and kinetic nature of GroEL-GPCRs interaction which is the central molecular event in the assisted folding process.     

Lipase-catalyzed remote kinetic resolution of citalopram intermediate by asymmetric alcoholysis and thermodynamic analysis

Lipase-catalyzed remote resolution of the tertiary alcohol, citalopram intermediate (diol acetate), has been achieved. The chiral discrimination was obtained by the Novozym435-catalyzed alcoholysis of the primary hydroxyl ester which was four bonds away from the center. The influence of acyl acceptor structure and the organic solvents on the reaction rate and enantioselectivity were investigated. Based on the thermodynamic analysis, the difference of activation free energy between the two enantiomers which dominated the enantioselectivity was significantly affected by the organic solvents, while the acyl acceptor showed less effect. In addition, the enantiomer discrimination was driven by both the difference of activation enthalpy and activation entropy. The thermodynamic analysis provides further insights into the prediction and optimization of enantioselectivity and reaction rate in remote resolution.     

CALB-catalyzed kinetic resolution of (RS)-3-benzoylthio-2-methylpropyl azolides: kinetic and thermodynamic analysis

Abstract

Zofenopril as an ACE inhibitor expired recently was found to have a favourable safety profile in comparison with other ACE inhibitors in treating high blood pressure, congestive heart failure, and acute myocardial infarction. It can be synthesised from the key building blocks of (S)-3-benzoylthio-2-methylpropanoic acid and (4S)-phenylthio-L-proline. In this report, an efficient hydrolytic resolution via Candida antarctic lipase B (CALB) for preparing the former block in isopropyl ether (IPE) containing (RS)-3-benzoylthio-2-methylpropyl pyrazolide (1) and water was developed. Quantitative improvements of the enzyme activity and enantioselectivity in terms of k_2SK_mS^?1?=?5.726?L h^?1 g^?1 and E?=?217 at 45?°C were found from the kinetic analysis. Insights into the CALB performance via thermodynamic analysis were then addressed and compared with those by using (RS)-3-benzoylthio-2-methylpropyl 1,2,4-triazolide (2) as the substrate. A putative thermodynamic model was moreover hypothesised for elucidating the more enthalpy reduction of 68.92-70.86?kJ mol^?1 from the acyl part of (S)-1 and (S)-2 as well as that of 23.69-25.63?kJ mol^?1 from the triad imidazolium to Ser105 and leaving 1,2,4-triazole moiety of (R)-2 and (S)-2 on stabilising the corresponding transition states.     

Promotion of triplex formation by a fixed N-form sugar puckering: thermodynamic and kinetic studies.

We analyzed the effect of a fixed N-form sugar puckering of TFO (triplex-forming oligonucleotide) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. Both thermodynamic and kinetic analyses revealed that the binding constant of the pyrimidine motif triplex formation at pH 6.8 with modified TFO containing the fixed N-form sugar puckering was about 20-times larger than that observed with unmodified TFO. Kinetic data also demonstrated that the observed increase in the binding constant at neutral pH by the fixed N-form sugar puckering resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the fixed N-form sugar puckering of TFO could be a key modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.