Effect of divalent cations and cytosine protonation on thermodynamic properties of intermolecular DNA double and triple helices

The contribution of divalent cations and cytosine protonation to conformation and stability of duplex and triplex formation were intensively investigated and characterized by ultraviolet (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and electrophoresis mobility shift assay (EMSA). CD spectra showed that the divalent cations investigated would not significantly distort nucleotide geometry, while UV and DSC melting experiments revealed that the cation binding abilities to duplexes and triplexes were clearly dependent on the types of cations under near physiological conditions. The calorimetric enthalpies were generally underestimated relative to the corresponding van't Hoff enthalpies for Hoogsteen and Watson-Crick transitions, but free energy changes derived from the DSC measurements were in good agreement with those derived from the UV measurements. The adjacent placing of the C(+) x G.C triplets in triplexes lowered the stabilities of not only Hoogsteen base-pairing but also Watson-Crick base-pairing. The protonation contribution of the given cytosine residues might depend on the local and global structure of the protonated cytosine complex. A rigid structural targeted-strand would favor the protonation of cytosine residues. The apparent pK(a) values for parallel duplex and triplex investigated were determined to be 6.4 and 7.6, respectively, which are considerably heightened by 2.1 and 3.3 pH unit as compared to the intrinsic pK(a) value of the free cytosine residues.     

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices.

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.     

Thermodynamic and kinetic studies of the formation of triple helices between purine-rich deoxyribo-oligonucleotides and the promoter region of the human c-src proto-oncogene.

The thermodynamic and kinetic parameters of triplex formation between four purine-rich oligonucleotides and a 22 bp pyrimidine. purine tract in the promoter region of the c-src gene were determined by fluorescence polarization studies. Three of these four oligonucleotides were 11 nt in length, corresponding to the left, central or right portion of the tract, while the fourth was a 22mer covering the whole tract. Binding constants ( Ka) were measured as a function of Mg2+ concentration (0-10 mM) and temperature (0-41 degrees C). In 10 mM Mg2+, K a for the left, central and right 11mers were 0.26, 0.75 and 1.4 x 10(8)/M, respectively, while for the 22mer the value was 1.8 x 10(8)/M at 22 degrees C. Under the same conditions, Ka was estimated by an electrophoretic band shift technique. The agreement between the two methods was acceptable for the 22mer but not for the 11mers. Kinetic measurements demonstrated that the rate of dissociation of the 22mer from the triplex was significantly slower than that of the 11mers, providing an explanation for the observed discrepancy. The entropy and enthalpy of triplex formation were calculated from van't Hoff plots. In all cases the entropy was favourable, especially for the 22mer and for the 11mer with the lowest guanine content. The enthalpy was unfavourable for the 22mer and most favourable for the 11mer with the highest guanine content. These results provide a thermodynamic explanation for length and sequence effects on the formation of purine.pyrimidine.purine triplexes.     

Thermodynamics of oligonucleotide triple helices

Understanding of the cellular role of nucleic acid triple helices and utilization of triple helix forming oligonucleotides in biotechnology, diagnostics, and therapeutics depend on development of an understanding of triple helix formation as a function of the nucleic acid components and solution conditions. This article reviews developments in nucleic acid triple helix thermodynamics with emphasis on the thermal and thermodynamic stability and their dependence on solution conditions. Special emphasis is placed on the construction and interpretation of state diagrams as a means of characterizing the complex behavior of triple helix forming oligonucleotides. Recent developments, which strive to overcome some of the limitations imposed by the natural nucleotide bases and sugar-phosphate backbone, are reviewed from a thermodynamic perspective. © 1998 John Wiley & Sons, Inc. Biopoly 44: 241–256, 1997     

Stability of G,A triple helices.

In this work we selected double-stranded DNA sequences capable of forming stable triplexes at 20 or 50 degrees C with corresponding 13mer purine oligonucleotides. This selection was obtained by a double aptamer approach where both the starting sequences of the oligonucleotides and the target DNA duplex were random. The results of selection were confirmed by a cold exchange method and the influence of the position of a 'mismatch' on the stability of the triplex was documented in several cases. The selected sequences obey two rules: (i) they have a high G content; (ii) for a given G content the stability of the resulting triplex is higher if the G residues lie in stretches. The computer simulation of the Mg2+, Na+and Cl-environment around three triplexes by a density scaled Monte Carlo method provides an interpretation of the experimental observations. The Mg2+cations are statistically close to the G N7 and relatively far from the A N7. The presence of an A repels the Mg2+from adjacent G residues. Therefore, the triplexes are stabilized when the Mg2+can form a continuous spine on G N7.     

Calorimetric and spectroscopic studies of aminoglycoside binding to AT-rich DNA triple helices

Calorimetric and fluorescence techniques were used to characterize the binding of aminoglycosides-neomycin, paromomycin, and ribostamycin, with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′ intramolecular DNA triplex (x = hexaethylene glycol) and poly(dA)·2poly(dT) triplex. Our results demonstrate the following features: (1) UV thermal analysis reveals that the T_m for triplex decreases with increasing pH value in the presence of neomycin, while the T_m for the duplex remains unchanged. (2) The binding affinity of neomycin decreases with increased pH, although there is an increase in observed binding enthalpy. (3) ITC studies conducted in two buffers (sodium cacodylate and MOPS) yield the number of protonated drug amino groups (Δn) as 0.29 and 0.40 for neomycin and paromomycin interaction with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′, respectively. (4) The specific heat capacity change (ΔC_p) determined by ITC studies is negative, with more negative values at lower salt concentrations. From 100 mM to 250 mM KCl, the ΔC_p ranges from −402 to −60 cal/(mol K) for neomycin. At pH 5.5, a more positive ΔC_p is observed, with a value of −98 cal/(mol K) at 100 mM KCl. ΔC_p is not significantly affected by ionic strength. (5) Salt dependence studies reveal that there are at least three amino groups of neomycin participating in the electrostatic interactions with the triplex. (6) FID studies using thiazole orange were used to derive the AC₅₀ (aminoglycoside concentration needed to displace 50% of the dye from the triplex) values. Neomycin shows a seven fold higher affinity than paromomycin and eleven fold higher affinity than ribostamycin at pH 6.8. (7) Modeling studies, consistent with UV and ITC results, show the importance of an additional positive charge in triplex recognition by neomycin. The modeling and thermodynamic studies indicate that neomycin binding to the DNA triplex depends upon significant contributions from charge as well as shape complementarity of the drug to the DNA triplex Watson–Hoogsteen groove.     

Kinetic and mechanistic studies of proline-mediated direct intermolecular aldol reactions

Reaction progress kinetic analysis of the proline-mediated intermolecular aldol reaction shows that the rate depends on the concentrations of both the donor ketone1 and the electrophilic aldehyde 2, implying that enamine formation cannot be rate-determining. The observed kinetics and deuterium isotope effects are consistent with formation of the product iminium species as the rate-determining step.     

Spectroscopic studies on the interaction of aristololactam-beta-D-glucoside with DNA and RNA double and triple helices: A comparative study.

The interaction of aristololactam-beta-D-glucoside (ADG), a DNA intercalating alkaloid, with the DNA triplexes, poly(dT). poly(dA)xpoly(dT) and poly(dC).poly(dG)xpoly(dC+), and the RNA triplex poly(rU).poly(rA)xpoly(rU) was investigated by circular dichroic, UV melting profile, spectrophotometric, and spectrofluorimetric techniques. Comparative interaction with the corresponding Watson-Crick duplexes has also been examined under identical experimental conditions. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature-dependent circular dichroic measurements. The binding of ADG to triplexes and duplexes is characterized by the typical hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield, an increase or decrease of thermal melting temperatures, and perturbation in the circular dichroic spectrum. Scatchard analysis indicates that ADG binds both to the triplexes and the duplexes in a noncooperative manner. Binding parameters obtained from spectrophotometric measurements are best fit by the neighbor exclusion model. The binding affinity of ADG to the DNA triplexes is substantially stronger than to the RNA triplex. Thermal melting study further indicates that ADG stabilizes the Hoogsteen base-paired third strand of the DNA triplexes whereas it destabilizes the same strand of RNA triplex but stabilizes its Watson-Crick strands. Comparative data reveal that ADG exhibits a stronger binding to the triple helical structures than to the respective double helical structures.     

Sequence-dependent stability of intramolecular DNA triple helices

A set of 21 oligodeoxynucleotides were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. UV melting experiments on the triplexes which only differ in the number and distribution of third strand cytosines reveal the influence of sequence and pH on triplex stability and can be summarized as follows: (1) increasing the cytosine content in the third strand results in a higher thermal stability of the triplex at acidic pH but lowers the triplex to duplex melting temperature at neutral pH; (2) cytosines at terminal positions destabilize the triple helical structure as compared to non-terminal positions; (3) contiguous cytosines lead to a pH dependent destabilization of the triplex, the destabilizing effect being more pronounced at higher pH. Analysis of these effects in terms of the various interactions within a triple helical complex indicate that the sequence-dependent stabilities are largely determined by the extent of protonation for individual third strand cytosines.     

Kinetic studies on activation of peptide bond formation by hyaluronic acid.

1. In this study, a cell-free system derived from Escherichia coli has been used in order to examine in detail the effect of hyaluronic acid on peptide bond formation with the aid of puromycin reaction. 2. This reaction is activated by hyaluronic acid. 3. The degree of activation of peptide bond formation depends on the molecular size of hyaluronic acid. 4. The kinetic analysis revealed that the hyaluronic acid acts as a mixed-type nonessential activator. 5. The presence of hyaluronic acid improves about 9-fold the activity status of ternary complex as it can be calculated by k3/k5 ratio.     

Intracellular location of triple helix formation of collagen. Enzyme probe studies.

Primary cultures of chick embryo fibroblasts were used to study ribosomal events in the processing of procollagen. Polyribosomes from radiolabeled cells were subjected to enzyme probe analysis using collagenase and pepsin digestion to assess both the amount of procollagen present on the polyribosomes and the conformation of the molecule. The peptides rendered dialyzable by each enzyme treatment were analyzed for radioactive proline and hydroxyproline. Approximately 30% of the nascent proteins were collagenous. Although some hydroxyproline was dialyzable in the pepsin-treated material, a low ratio of hydroxyproline to proline (0.04) indicated that considerable amounts of noncollagenous proteins were digested. Polyribosomal material, previously treated with pepsin, was digested with purified collagenase. Similarly, collagenase-digested polyribosomes were treated with pepsin. The pepsin pretreatment released noncollagenous protein and served to purify the remaining ribosomally bound pepsin-resistant collagenous protein. Collagenase treatment of the pepsin-resistant ribosomally bound peptides released peptides with a hydroxyproline to proline ratio of 0.65, indicating that considerable hydroxylation of proline occurs on nascent ribosomally bound procollagen. This finding combined with the well documented stabilizing effect of hydroxyproline on the collagen triple helix and the demonstrated resistance of ribosomally bound procollagen to pepsin digestion indicates that the collagen triple helix may well form on the polyribosome.     

Thermodynamic and kinetic stability of intermolecular triple helices containing different proportions of C+*GC and T*AT triplets

We have used oligonucleotides containing appropriately placed fluorophores and quenchers to measure the stability of 15mer intermolecular triplexes with third strands consisting of repeats of TTT, TTC, TCC and TCTC. In the presence of 200 mM sodium (pH 5.0) triplexes that contain only T·AT triplets are unstable and melt below 30°C. In contrast, triplets with repeats of TTC, TCC and CTCT melt at 67, 72 and 76°C, respectively. The most stable complex is generated by the sequence containing alternating C⁺·GC and T·AT triplets. All four triplexes are stabilised by increasing the ionic strength or by the addition of magnesium, although triplexes with a higher proportion of C⁺·GC triplets are much less sensitive to changes in the ionic conditions. The enthalpies of formation of these triplexes were estimated by examining the concentration dependence of the melting profiles and show that, in the presence of 200 mM sodium at pH 5.0, each C⁺·GC triplet contributes about 30 kJ mol^–1, while each T·AT contributes only 11 kJ mol^–1. Kinetic experiments with these oligonucleotides show that in 200 mM sodium (pH 5.0) repeats of TCC and TTC have half-lives of ~20 min, while the triplex with alternating C⁺·GC and T·AT triplets has a half-life of ~3 days. In contrast, the dissociation kinetics of the triplex containing only T·AT are too fast to measure.     

In vivo persistence of DNA triple helices containing psoralen-conjugated oligodeoxyribonucleotides.

Triple helices represent an attractive method for modulating specific gene expression. In particular, cross-linking between a triplex-forming oligonucleotide (TFO) and its duplex DNA target, typically through the formation of psoralen photoadducts, allows efficient blocking of elongation by RNA polymerases in vitro. However, in vivo, this approach is limited by DNA repair of the photoadduct. Here we describe the use of an oligodeoxyribonucleotide 19mer psoralen-modified TFO to form covalent linkages between an oligonucleotide and both strands of the targeted duplex DNA, thereby efficiently blocking expression of a luciferase reporter gene. Most importantly, we demonstrate that both the psoralen cross-link and the purine-motif triplex remained intact for at least 72 h post-transfection, indicating that such species can persist for an extended period of time in vivo. These findings support the feasibility of an antigene approach for the therapeutic regulation of specific gene expression.     

Thermodynamics of the melting of PNA(2)/DNA triple helices.

Equilibrium melting curves were obtained for triplexes, formed by single stranded DNA containing an A10 target with bis-PNA consisting of two T10 decamers. Thermodynamic parameters of melting were determined for Na(+) concentrations 50, 200 and 600mM by two methods. The melting enthalpy Delta H degrees was evaluated from the width of the differential melting curves and from the concentration dependence of the melting temperature. The latter method allowed also evaluating the melting entropy Delta S degrees. The following results were obtained: Delta H degrees = - 137 kcal/M, Delta S degrees = - 368 cal/M.K, Delta G degrees (298)= - 27 kcal/M. No dependence of Delta H degrees, Delta S degrees and Delta G degrees (298) was observed upon ionic strength within the accuracy of the experiment (+/- 10%). The absolute values of Delta H degrees, Delta S degrees and Delta G degrees(298) are 2 to 3 times higher than the published values of corresponding melting parameters for decameric PNA/DNA duplexes of various nucleic base sequences. The origin of the extremely high stability of the triplexes is discussed.     

Molecular stability of chemically modified collagen triple helices

Ionic residues influence the stability of collagen triple helices and play a relevant role in the spontaneous aggregation of fibrillar collagens. Collagen types I and II and some of their CNBr peptides were chemically modified in mild conditions with two different protocols. Primary amino groups of Lys and Hyl were N-methylated by formaldehyde in reducing conditions or N-acetylated by sulfosuccinimidyl acetate. The positive charge of amino groups at physiological pH was maintained after the former modification, whereas it was lost after the latter. These chemical derivatizations did not significantly alter the stability of the triple helical conformation of peptide trimeric species. Also the enthalpic change on denaturation was largely unaffected by derivatizations. This implies that no significant variation of weak bonds, either in number or overall strength, and of entropy occur on modification. These properties can probably be explained by the fact that chemically modified groups maintain the ability to form hydrogen bonds.     

Conformational stability of triazolyl functionalized collagen triple helices

Functionalized collagen is attractive for the development of synthetic biomaterials. Herein we present the functionalization of azidoproline containing collagen model peptides with various alkynes using click chemistry. The influence on the stability of the collagen triple helix of the stereochemistry of the introduced triazolyl prolines (4R or 4S), the position of their incorporation (Xaa or Yaa) and the substituents attached to them are shown. The results provide a useful guide for the optimal functionalization of collagen using click chemistry.     

Thermodynamic and computational studies of DNA triple helices containing a nucleotide or a non-nucleotide linker in the third strand.

In this paper we report a thermodynamic characterisation of stability and melting behaviour of four different triple helices at pH 6.0. The target duplex consists of 16 base pairs in alternate sequence of the type 5'-(purine)(m)(pyrimidine)(m)-3'. The four triplexes are formed by targeting the 16-mer duplex with an all pyrimidine 16-mer or 15-mer or 14-mer third strand. The 16-mer oligonucleotide contains a 3'-3' phosphodiester junction and corresponding triplex was named 16-mer P. The 14-mer oligonucleotide contains a non-nucleotide linker, the 1,2,3 propanetriol residue and the corresponding triplex was named 14-mer PT. For the 15-mer oligonucleotide both junctions were alternatively used and the relative triplexes were named 15-mer P and 15-mer PT, respectively. These linkers introduce the appropriate polarity inversion and let the third strand switch from one oligopurine strand of the duplex to the other. Thermal denaturation profiles indicate the initial loss of the third strand followed by the dissociation of the target duplex. Transition enthalpies, entropies and free energies were derived from differential scanning calorimetric measurements. The comparison of Gibbs energies reveals that a more stable triplex is obtained when in the third strand there is the lack of one nucleotide in the junction region and a propanetriol residue as linker was used. The thermodynamic data were discussed in light of molecular mechanics and dynamics calculations.     

Different conformational families of pyrimidine.purine.pyrimidine triple helices depending on backbone composition.

Different helical conformations of DNA (D), RNA (R), and DNA.RNA (DR) hybrid double and triple helices have been detected using affinity cleavage analysis. Synthetic methods were developed to attach EDTA.Fe to a single nucleotide on RNA as well as DNA oligonucleotides. Cleavage patterns generated by a localized diffusible oxidant in the major groove on the pyrimidine strand of four purine.pyrimidine double helices consisting of all DNA, all RNA, and the corresponding hybrids reveal that the relative cleavage intensity shifts to the 5' end of the purine strand increasingly in the order: DD < DR < RD < RR. These results are consistent with models derived from structural studies. In six pyrimidine.purine.pyrimidine triple helices, the altered cleavage patterns of the Watson-Crick pyrimidine strands reveal at least two conformational families: (i) D + DD, R + DD, D + DR, and R + DR and (ii) R + RD and R + RR.     

Underhydroxylated minor cartilage collagen precursors cannot form stable triple helices.

Matrix-free cells from chick-embryo sterna were incubated with various concentrations of 2,2'-bipyridyl, an iron chelator that inhibits prolyl hydroxylase and lysyl hydroxylase. At concentrations in the region of 0.1 mM, significant effects on cartilage collagen hydroxylation and secretion were observed. When the underhydroxylated collagens were subsequently digested with chymotrypsin or chymotrypsin plus trypsin at 4 degrees C for 15 min, the minor cartilage collagen precursors (namely types IX and XI) were extensively degraded; type II procollagen was only partially susceptible and was converted into underhydroxylated collagen. The results demonstrate that there were significant differences in triple-helix stability among cartilage collagens such that the underhydroxylated minor collagen precursors were unable to attain a native structure under conditions where type II procollagen was successful.     

Resolution of parallel and antiparallel oligonucleotide triple helices formation and melting processes by multivariate curve resolution

A procedure is described for the complete resolution of concentration profiles of oligonucleotide triplexes as a function of pH and temperature. The pH and temperature ranges at which triplexes are present and the relative concentrations of all the species involved in acid-base and conformational equilibria are successfully estimated from Multivariate Curve Resolution analysis of UV absorbance spectra recorded along acid-base titrations and melting experiments of single stranded, hairpin and their mixtures. The dependence of formation constants upon pH was successfully estimated. The hairpin h26 (5'-GAAGGAGGAGA-TTTT-TCTCCTCCTTC-3'), and the single stranded oligonucleotides s11CT (5'-CTTCCTCCTCT-3'), s11AG (5'-AGAGGAGGAAG-3') and s11TG (5'-TGTGGTGGTTG-3') were synthesized and their protonation and conformational equilibria were studied in detail. The procedure was shown to be especially useful for the study of triplexes with a low hypochromism upon formation.