Thermodynamics of Interaction of Phthalocyanine‐Oligonucleotide Conjugates with Single‐ and Double‐Stranded DNA

Thermodynamics of interaction of phthalocyanine‐oligonucleotide conjugates with single‐ and double‐stranded DNA resulting in formation of duplexes and triplexes was measured by UV melting method. It was shown that a phthalocyanine moiety of conjugates stabilized the formation of duplexes and triplexes.     

Spectroscopic and thermodynamic studies on the binding of sanguinarine and berberine to triple and double helical DNA and RNA structures

A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T.AxT and U.AxU) and duplexes (A.T and A.U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C.GxC(+) triplex and G.C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent duplexes.     

Evidence from CD spectra and melting temperatures for stable Hoogsteen-paired oligomer duplexes derived from DNA and hybrid triplexes.

The pyr*pur.pyr type of nucleic acid triplex has a purine strand that is Hoogsteen-paired with a parallel pyrimidine strand (pyr*pur pair) and that is Watson-Crick-paired with an antiparallel pyrimidine strand (pur.pyr pair). In most cases, the Watson-Crick pair is more stable than the Hoogsteen pair, although stable formation of DNA Hoogsteen-paired duplexes has been reported. Using oligomer triplexes of repeating d(AG)12 and d(CT)12 or r(CU)12 sequences that were 24 nt long, we found that hybrid RNA*DNA as well as DNA*DNA Hoogsteen-paired strands of triplexes can be more stable than the Watson-Crick-paired strands at low pH. The structures and relative stabilities of these duplexes and triplexes were evaluated by circular dichroism (CD) spectroscopy and UV absorption melting studies of triplexes as a function of pH. The CD contributions of Hoogsteen-paired RNA*DNA and DNA*DNA duplexes were found to dominate the CD spectra of the corresponding pyr*pur.pyr triplexes.     

Vacuum UV CD spectra of homopolymer duplexes and triplexes containing A.T or A.U base pairs.

Vacuum UV circular dichroism (CD) spectra were measured down to 174 nm for five homopolymers, five duplexes, and four triplexes containing adenine, uracil, and thymine. Near 190 nm, the CD bands of poly[d(A)] and poly[r(A)] were larger than the CD bands of the polypyrimidines, poly[d(T)], poly[d(U)], and poly[r(U)]. Little change was observed in the 190 nm region upon formation of the duplexes (poly[d(A).d(T)], poly[d(A).d(U)], poly[r(A).d(T)], poly[r(A).d(U)], and poly[r(A).r(U)]) or upon formation of two of the triplexes (poly[d(T).d(A).d(T)] and poly[d(U).d(A).d(U)]). This showed that the purine strand had the same or a similar structure in these duplexes and triplexes as when free in solution. Both A.U and A.T base pairing induced positive bands at 177 and 202 nm. For three triplexes containing poly[d(A)], the formation of a triplex from a duplex and a free pyrimidine strand induced a negative band centered between 210 and 215 nm. The induction of a band between 210 and 215 nm indicated that these triplexes had aspects of the A conformation.     

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.     

Development of a simple coarse-grained DNA model for analysis of oligonucleotide complex formation

In this paper, we develop a coarse-grained nucleotide model for the purpose of simulating large-scale aptamer-based hydrogel network formation in future research. In the model, each nucleotide is represented by a single interaction site containing sugar, phosphate and base. Discontinuous molecular dynamics (DMD) simulations are performed to simulate formation and denaturation of oligonucleotide duplexes as a function of temperature. The simulated melting temperatures of oligonucleotide duplexes are calculated in simulations of systems with different sequences, lengths and concentrations of oligonucleotides, and compared to data from the OligoAnalyzer tool. The denaturation of oligonucleotide triplexes containing a hybridised structure of three different oligonucleotides is analysed using both simulations and experiments. The nucleotide model is found to be a good predictor of the oligonucleotide’s hybridised state for both duplexes and triplexes. This coarse-grained model has wide ranging applications in the development or optimisation of DNA-based technologies including DNA origami, DNA-enabled hydrogels and DNA-based biosensors.     

Metalloregulation of Triple Helix Formation by Control of the Loop Conformation

The flexible polypyridine ligand, 2,2′:6′,2^″-terpyridine (terpy), was built into the backbone of oligonucleotides to form DNA conjugates. The terpy unit functioned as a good loop when the conjugates formed the bimolecular triplexes with complementary oligopurine. The triplex structure was destabilized by the specific interaction with divalent transition metal ions (Cu²⁺, Zn²⁺, and Fe²⁺), in particular Cu²⁺ ions. This ion destabilized one of the triplexes by 4.2 kcalmol^?1 or made the triplex formation constant less than 1/10³ at 298 K. This result is attributed to the substantial turbulence of the terminal structure of the triplexes.     

Recognition of base pair inversions in duplex by chimeric (alpha,beta) triplex-forming oligonucleotides

DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes.     

Metalloregulation of triple helix formation by control of the loop conformation

The flexible polypyridine ligand, 2,2':6',2(')-terpyridine (terpy), was built into the backbone of oligonucleotides to form DNA conjugates. The terpy unit functioned as a good loop when the conjugates formed the bimolecular triplexes with complementary oligopurine. The triplex structure was destabilized by the specific interaction with divalent transition metal ions (Cu(2+), Zn(2+), and Fe(2+)), in particular Cu(2+) ions. This ion destabilized one of the triplexes by 4.2 kcalmol(-1) or made the triplex formation constant less than 1/10(3) at 298 K. This result is attributed to the substantial turbulence of the terminal structure of the triplexes.     

Interactions of cryptolepine and neocryptolepine with unusual DNA structures

Cryptolepine, the main alkaloid present in the roots of Cryptolepis sanguinolenta, presents a large spectrum of biological properties. It has been reported to behave like a DNA intercalator with a preference for GC-rich sequences. In this study, dialysis competition assay and mass spectrometry experiments were used to determine the affinity of cryptolepine and neocryptolepine for DNA structures among duplexes, triplexes, quadruplexes and single strands. Our data confirm that cryptolepine and neocryptolepine prefer GC over AT-rich duplex sequences, but also recognize triplex and quadruplex structures. These compounds are weak telomerase inhibitors and exhibit a significant preference for triplexes over quadruplexes or duplexes.     

PROPYNYL GROUPS IN DUPLEX,HAIRPIN AND TRIPLEX DNA: 7-DEAZAPURINES AND 9-DEAZAPURINES

Oligonucleotides containing 7-deazapurines or 9-deazapurines with propynyl groups at the 7- or 9-position were prepared. The stabilizing effect of the propynyl group was studied on DNA duplexes, hairpins and triplexes.     

Nucleosides and nucleotides. 208. Alternate-strand triple-helix formation by the 3'-3'-linked oligodeoxynucleotides with the anthraquinonyl group at the junction point.

The synthesis of 3'-3'-linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point is described. The ODNs were synthesized on a DNA synthesizer using a controlled pore glass (CPG) carrying pentaerythritol that has an intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3'-3'-linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer 10. It was found that the ODNs 12 and 13 carrying the anthraquinonyl groups can form thermally stable triplexes by skipping two or three extra base pairs between two binding domains of the target duplexes. The ability of the 3'-3'-linked ODNs to inhibit cleavage of the target DNA 22 by the restriction enzyme Hind III was tested. It was found that the 3'-3'-linked ODN 16 with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer 14 and the 3'-3'-linked ODN 15 without the intercalator.     

Intercalating Nucleic Acids: The Influence of Linker Length and Intercalator Type on Their Duplex Stabilities

Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes.     

A study of H-bonding of 3- and 5-substituted 6-aminouracils in duplex and triplex structures

All possible dimers of the title modified bases with native nucleobases [10 dimers from 3-methylated 6-aminouracils (3sau) and 20 from 5-methyled 6-aminouracils (5sau), respectively have been calculated by ab initio method (Hartree-Fock method, 3 21G basis set). We have found two potential duplexes of 5sau and three possible duplexes of 3sau. Altogether seven dimers containing one or two bifurcating H-bonds have been found. Later on, five triplexes from ten possible calculated dimers have been found. In two of them the amino group of 6-aminouracil moiety takes part in H-bonding and there are H-bonds, too, between the first and third base of the triplexes causing an extra stabilization.     

Propynyl groups in duplex, hairpin and triplex DNA: 7-deazapurines and 9-deazapurines

Oligonucleotides containing 7-deazapurines or 9-deazapurines with propynyl groups at the 7- or 9-position were prepared. The stabilizing effect of the propynyl group was studied on DNA duplexes, hairpins and triplexes.     

Intercalating nucleic acids: the influence of linker length and intercalator type on their duplex stabilities

Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes.     

DNA triplex stabilization by a delta-carboline derivative tethered to third strand oligonucleotides

A delta-carboline derivative was covalently coupled to a 7 mer oligonucleotide at its 5'- or 3'-end. The stability of triplexes formed from the conjugates and a double-helical target was studied by UV melting experiments. Compared to the unmodified control triple helices, triplexes with the conjugate exhibit a significantly higher stability. However, the degree of stabilization depends on the particular triplex structure formed.     

Triplex formation at physiological pH: comparative studies on DNA triplexes containing 5-Me-dC tethered at N4 with spermine and tetraethyleneoxyamine.

Oligodeoxynucleotides with spermine conjugation at C4 of 5-Me-dC ( sp -ODN) exhibit triple helix formation with complementary Watson-Crick duplexes, and were optimally stable at physiological pH 7.3 and low salt concentration. This was attributed to a favored reassociation of the polycationic third strand with the anionic DNA duplex. To gain further insights into the factors that contribute to the enhancement of triplex stability and for engineering improved triplex systems, the spermine appendage at C4 of 5-Me-dC was replaced with 1,11-diamino-3,6,9-trioxaundecane to create teg -ODNs. From the triple helix forming abilities of these modified ODNs studied by hysteresis behaviour and the effect of salts on triplex stability, it is demonstrated here that teg- ODNs stabilise triplexes through hydrophobic desolvation while sp -ODNs stabilise triplexes by charge effects. The results imply that factors in addition to base stacking effects and interstrand hydrogen bonds are significantly involved in modulation of triplex stability by base modified oligonucleotides.     

Relative specificities in binding of Watson-Crick base pairs by third strand residues in a DNA pyrimidine triplex motif.

The specificity of binding of Watson-Crick base pairs by third strand nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The host duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 duplexes with the Watson-Crick base pairs (XY) and 12 duplexes with mismatch pairs (XZ), all of whose stabilities were compared. Two Z:XY combinations confirm the primary binding of AT and GC target pairs in homopurine.homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic strength and pH, all of which were previously untested. The conclusions now drawn also differ substantially from those in previous studies. Thus, by evaluating the depression in Tm due to base triplet mismatches strictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combinations that destabilize qualify for inclusion in the third strand binding code for the pyrimidine triplex motif. Hence, none of the mismatch triplets afford a general way of circumventing the requirement for homopurine.homopyrimidine targets when third strands are predominated by pyrimidines, as others have suggested. At the same time, the applicability of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than are Watson-Crick duplexes to base pair mismatches.