dC-dG Alternating Oligonucleotides: Thermodynamic and Kinetic Aspects of the B-Z Transformation

Abstract

The alternating cytosine-guanine oligodeoxyribonucleotides (dCdG)_n, (dGdC)_n, (dCdG)_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 5M). The kinetic data for the B-Z transformation, obtained with a salt-jump technique for the hexamer (dCdG)₃, support a mechanism by which (he 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 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.     

B-Z conformational transition and hydration of poly (dC-dG).poly (dC-dG) in fibres.

The B-Z transition of poly(dC-dG).poly(dC-dG) has been studied by fibre X-ray diffraction and measurement of fibre dimensions. The polymorphism of the Z form is well observed as a function of variations of the r.h. (relative humidity). The Z to B transition is obtained at very high r.h. values. The cooperative transition from B to Z is associated with a disorganization of the fibre. Details about the hydration of the polynucleotide during conformational transitions are presented and it is shown that a nucleotide in Z form can be associated with up to 16 water molecules and up to 22 when in the B form.     

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.     

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.     

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.     

Conformation and dynamics of short DNA duplexes: (dC-dG)3 and (dC-dG)4

Natural abundance 13C NMR spectra of duplexed (dC-dG)3 and (dC-dG)4 exhibit resolved resonances for most of the carbons at 0.1M NaCl in aqueous solution. Large transitions in chemical shift for many of the hexamer carbons (up to 1.8 ppm) are observed in variable temperature measurements. Determination of spin-lattice relaxation times and nuclear Overhauser enhancements in 0.1M NaCl indicate that the duplexes tumble almost isotropically, with overall correlation times near 5 nsec; the sugar carbons experience more rapid local motions than do the base carbons. The relaxation data are also consistent with the most rapid local motions occurring at the chain-terminal residues, especially in the Cyd(1) sugar. 4M NaCl causes changes in the 13C chemical shifts of most of the guanine base carbons, and rearrangements in the deoxyribose carbon shifts; this is consistent with changes predicted by a salt-induced B to Z transition, viz. conversion of the guanylates from the anti to syn range about the glycosyl bond, and from the S to N pseudorotational state of the deoxyribose ring.     

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

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.     

Conformation and Dynamics of Short DNA Duplexes: (dC-dG)3 and (dC-dG)4

Abstract

Natural abundance ¹³C NMR spectra of duplexed (dC-dG)₃ and (dC-dG)₄ exhibit resolved resonances for most of the carbons at 0.1M NaCl in aqueous solution. Large transitions in chemical shift for many of the hexamer carbons (up to 1.8 ppm) are observed in variable temperature measurements. Determination of spin-lattice relaxation times and nuclear Overhauser enhancements in 0.1M NaCl indicate that the duplexes tumble almost isotropically, with overall correlation times near 5 nsec; the sugar carbons experience more rapid local motions than do the base carbons. The relaxation data are also consistent with the most rapid local motions occurring at the chain-terminal residues, especially in the Cyd(l) sugar. 4M NaCl causes changes in the ¹³C chemical shifts of most of the guanine base carbons, and rearrangements in the deoxyribose carbon shifts; this is consistent with changes predicted by a salt-induced B to Z transition, viz. conversion of the guanylates from the and to syn range about the glycosyl bond, and from the S to N pseudorotational state of the deoxyribose ring.     

Bovine skeletal muscle adenosine deaminase: kinetic and thermodynamic studies

Adenosine deaminase from bovine skeletal muscle catalyzes the hydrolytic deamination of adenosine to inosine and ammonia via an ordered Uni-Bi mechanism, if water is not considered as a true second substrate, as deduced from the inhibition pattern products. The inhibition constants (Ki) obtained for inosine and ammonia were 316 mumol/l and 2 mol/l, respectively. The activation energy of the reaction has been calculated as 10 kcal/mol, delta H* and delta F* as 7.9 and 15.6 kcal/mol, respectively, and delta S* as -23 cal/mol/degrees K.     

Influence of 5-N-carboxamide modifications on the thermodynamic stability of oligonucleotides

We have recently shown that the incorporation of modified nucleotides such as 5-N-carboxamide-deoxyuridines into random nucleic acid libraries improves success rates in SELEX experiments and facilitates the identification of ligands with slow off-rates. Here we report the impact of these modifications on the thermodynamic stability of both duplexes and intramolecular ‘single-stranded’ structures. Within duplexes, large, hydrophobic naphthyl groups were destabilizing relative to the all natural DNA duplex, while the hydrophilic groups exhibited somewhat improved duplex stability. All of the significant changes in stability were driven by opposing contributions from the enthalpic and entropic terms. In contrast, both benzyl and naphthyl modifications stabilized intramolecular single-stranded structures relative to their natural DNA analogs, consistent with the notion that intramolecular folding allows formation of novel, stabilizing hydrophobic interactions. Imino proton NMR data provided evidence that elements of the folded structure form at temperatures well below the T_m, with a melting transition that is distinctly less cooperative when compared to duplex DNA. Although there are no data to suggest that the unmodified DNA sequences fold into structures similar to their modified analogs, this still represents clear evidence that these modifications impart thermodynamic stability to the folded structure not achievable with unmodified DNA.     

Lipase-catalyzed synthesis of propyl-phenyl acetate: a kinetic and thermodynamic study

Bioprocess and Biosystems Engineering - This study focuses on the synthesis of propyl-phenyl acetate via esterification reaction in the presence of immobilized Candida antartica...     

Radioimmunoassay of epitestosterone: methodology, thermodynamic aspects and applications

A radioimmunoassay system for 17 alpha-hydroxy-4-androstene-3-one (epitestosterone) was developed and evaluated using rabbit antisera against 17 alpha-hydroxy-4-androstene-3-one-3-(O-carboxymethyl)-oxime-bovine serum albumin conjugate, and radioiodinated homologous histaminyl derivative of epitestosterone as a tracer. Two antisera with different specificity were used for radioimmunological determination of epitestosterone in hydrolyzed urine and plasma, respectively. Apparent intrinsic association constants and derived thermodynamic variables for ligand-antibody interaction were measured under various conditions in order to set up an optimal assay protocol. The mean plasma epitestosterone levels for healthy subjects were 0.44 +/- 0.15 nmol.l-1 (means +/- SD) in males and 0.092 +/- 0.056 nmol.l-1 in females. The corresponding concentrations in urine were 320 +/- 70 nmol.l-1 (males) and 273 +/- 100 nmol.l-1 (females).