Stabilization of short collagen-like triple helices by protein engineering

Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions.     

Stabilization of DNA double and triple helices by conjugation of minor groove binders to oligonucleotides

New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.     

Human telomerase inhibition and cytotoxicity of regioisomeric disubstituted amidoanthraquinones and aminoanthraquinones

Telomerase is an attractive target for the rational design of new anticancer drugs due to its central role in the control of cellular proliferation. A number of 1,4-disubstituted amidoanthraquinones and 1,5-disubstituted aminoanthraquinones that are related to mitoxantrone and ametantrone have previously been prepared. The present study details the effects on human telomerase of these new classes of 1,4- and 1,5-difunctionalized tricyclic anthraquinone compounds. We have used cytotoxicity assay, reporter SEAP assay to monitor the hTERT expression, and TRAP-G4 assay to measure the relative activity of these compounds, and have examined how the attached substituents affect their ability to influence telomerase. Cytotoxicity levels in human tumor cell lines were at comparable levels for several compounds. Structural and activity relationships indicated that the position of disubstituent side chains is important for its inhibitory effect. Moreover, a primary amine or tertiary amine on the substitution group appears to be required for the telomerase inhibitory effect. There is no significant correlation between telomerase activity and cytotoxicity. These symmetrical disubstituted anthraquinones may represent useful leads for the development of human telomerase inhibitors as potential anticancer agents, and the exact mode of intercalative binding is dictated by the positional placement of substituent side chains for effective telomerase inhibition.     

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.     

Structural heterogeneity in intramolecular DNA triple helices

Oligodeoxynucleotides designed to form intramolecular triple helices are widely used as model systems in thermodynamic and structural studies. We now report results from UV, Raman and NMR experiments demonstrating that the strand polarity, which also determines the orientation of the connecting loops, has a considerable impact on the formation and stability of pyr x pur x pyr triple helices. There are two types of monomolecular triplexes that can be defined by the location of their purine tract at either the 5'- or 3'-end of the sequence. We have examined four pairs of oligonucleotides with the same base composition but with reversed polarity that can fold into intramolecular triple helices with seven base triplets and two T4 loops under appropriate conditions. UV spectroscopic monitoring of thermal denaturation indicates a consistently higher thermal stability for the 5'-sequences at pH 5.0 in the absence of Mg2+ ions. Raman spectra provide evidence for the formation of triple helices at pH 5 for oligomers with purine tracts located at either the 5'- or 3'-end of the sequence. However, NMR measurements reveal considerable differences in the secondary structures formed by the two types of oligonucleotides. Thus, at acidic pH significant structural heterogeneity is observed for the 3'-sequences. Employing selectively 15N-labeled oligomers, NMR experiments indicate a folding pattern for the competing structures that at least partially changes both Hoogsteen and Watson-Crick base-base interactions.     

Symmetry and structure of RNA and DNA triple helices

Despite wide interest in nucleic acid triple helices, there has beenno stereochemically satisfactory structure of an RNA triple helixin atomic detail. An RNA triplex structure has previously been proposed based on fiber diffraction and molecular modeling [S. Arnott and P. J. Bond (1973) Nature New Biology, Vol. 244. pp. 99–101; S. Arnott. P. J. Bond. E. Seising, and P. J. C. Smith (1976) Nucleic Acids Research, Vol. 3. pp.2459–2470], but it has nonallowed close contacts at every triplet and is therefore not stereochemically acceptable. We propose here a new modelfor an RNA triple helix in which the three chains have identical backbone conformations and are symmetry related. There are no short contacts. The modeling employs a novel geometrical approach using the linked atom least squares [P. J. C. Smith and S. Arnott (1978) Acta Crystallographica, Vol. A34, pp. 3–11] program and is not based on energy minimization. In general, the method leads to a range of possible structures rather than a unique structure. In the present case, however, the constraints resulting from theintroduction of a third strand limit the possible structures to a very small range of conformation space. This method was used previously to obtain a model for DNA triple helices [G. Raghunathan, H. T. Miles, and V. Sasisekharan (1993) Biochemistry, Vol. 32, pp. 455–462], subsequently confirmed by fiber-type x-ray diffraction of oligomeric crystals [K. Liu. H. T. Miles. K. D. Parris, and V. Sasisekharan (1994) Nature Structural Biology, Vol. 1. pp. 11–12]. The above triple helices have Watson–Crick–Hoogsteen [K. Hoogsteen (1963) Acta Crystallographica, Vol. 16. pp. 907–916] pairing of the three bases. The same modeling method was used to investigate the feasibility of three-dimensional structures based on the three possible alternative hydrogen-bonding schemes: Watson–Crick–reverse Hoogsteen, Donogue [J. Donohue (1953) Proceeding of the national Academy of Science USA, Vol. 39, pp. 470–475] (reverse Watson–Crick)–Hoogsteen, and Donohue–reverse Hoogsteen. We found that none of these can occur in either RNA or DNA helices because they give rise only to structures with prohibitively short contacts between backbone and base atoms in the same chain. © 1995 John Wiley & Sons, Inc.     

Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase.

Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved.     

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.     

Stabilities of intrastrand pyrimidine motif DNA and RNA triple helices

Nucleic acid triple helices have provoked interest since their discovery more than 40 years ago, but it remains unknown whether such structures occur naturally in cells. To pursue this question, it is important to determine the stabilities of representative triple helices at physiological temperature and pH. Previous investigations have concluded that while both DNA and RNA can participate in the pyrimidine triplex motif under mildly acidic conditions, these structures are often relatively unstable at neutral pH. We are now explorin g the stability of intrastrand DNA and RNA pyrimidine motif triplexes at physiological temperature and pH. Duplex and triplex formation were monitored by thermal denaturation analysis, circular dichroism spectroscopy and gel shift experiments. Short intrastrand triplexes were observed to form in the pyrimidine motif in both DNA and RNA. In the presence of physiological concentrations of Mg²⁺ and at physiological pH, all detected triplexes were sufficiently stable to persist at physiological temperature. If sequences specifying such intrastrand triplexes are encoded in genomes, the potential exists for the formation of stable structures in RNA or DNA in vivo.     

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.     

Coralyne has a preference for intercalation between TA.T triples in intramolecular DNA triple helices.

Intercalating ligands may improve both the stability and sequence specificity of triple helices. Numerous intercalating drugs have been described, including coralyne, which preferentially binds triple helices, though its sequence specificity has been reported to be low [Lee,J.S., Latimer,L.J.P. and Hampel,K.J. (1993) Biochemistry , 32, 5591-5597]. In order to analyse the sequence preferences of coralyne we have used a combination of DNase I footprinting, UV melting, UV-visible spectrophotometry, circular dichroism and NMR spectroscopy to examine defined intermolecular triplexes and intramolecular triplexes linked either by hexaethylene glycol chains or by octandiol chains. DNase I footprinting demonstrated that coralyne has a moderate preference for triplexes over duplexes, but a substantial preference for TA.T triplets compared with CG. C+triplets. The drug was found to have essentially no effect on the melting temperatures of duplexes of the kind d(A)n.d(T)n or d(GA)n.d(TC)n. In contrast, it increased the T m for triplexes of the kind d(T)nd(A)n.dTn, but had little effect on the stability of d(TC)nd(GA).d(CT)n at either low or high pH. On binding to DNA triplexes, there is a large change in the absorption spectrum of coralyne and also a substantial fluorescence quenching that can be attributed to intercalation. The changes in the optical spectra have been used for direct titration with DNA. For triplexes d(T)6d(A)6.d(T)6, the Kd at 298 K was 0.5-0.8 microM. In contrast, the affinity for d(TC) nd(GA)n.d(CT)n triplexes was 6- to 10-fold lower and was characterized by smaller changes in the absorption and CD spectra. This indicates a preference for intercalation between TAT triples over CG.C+/TA.T triples. NMR studies confirmed interaction by intercalation. However, a single, secondary binding was observed at high concentrations of ligand to the triplex d(AGAAGA-L-TCTTCT-L-TCTTCT), presumably owing to the relatively low difference in affinity between the TA.T site and the competing, neighbouring sites.     

A database of 32 DNA triplets to study triple helices by molecular mechanics and dynamics

We present here a database of 32 deoxyribonucleotide triplets, that can be used as building blocks of triple helix forming deoxyribonucleotides on a computer. This database is made of all the pairing schemes of the triplets ATT, GCC⁺, ATA and GCG where the third base forms two hydrogen bonds with the purine of the first two Watson-Crick strands. The essential features of the known triple helices were preserved in the resulting structures. A triple helix can be easily built from any combination of these basic triplets. Four homogeneous and alternate triple helices thus obtained were studied by molecular mechanics and dynamics in vacuo. The results are in agreement with known experimental observations for ATT and suggest a possible structure for the GCG triple helix. In order to characterize the geometry of the structures obtained, the definitions of nucleic acid structure parameters (R.E. Dickerson et al., EMBO J. 8 (1989) 1–4) have been extended to triple helical polynucleotides.     

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.     

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.     

Stability of triple helices containing RNA and DNA strands: experimental and molecular modeling studies.

UV-absorption spectrophotometry and molecular modeling have been used to study the influence of the chemical nature of sugars (ribose or deoxyribose) on triple helix stability. For the Pyrimidine.purine* Pyrimidine motif, all eight combinations were tested with each of the three strands composed of either DNA or RNA. The chemical nature of sugars has a dramatic influence on triple helix stability. For each double helix composition, a more stable triple helix was formed when the third strand was RNA rather than DNA. No stable triple helix was detected when the polypurine sequence was made of RNA with a third strand made of DNA. Energy minimization studies using the JUMNA program suggested that interactions between the 2'-hydroxyl group of the third strand and the phosphates of the polypurine strand play an important role in determining the relative stabilities of triple-helical structures in which the polypyrimidine third strand is oriented parallel to the polypurine sequence. These interactions are not allowed when the third strand adopts an antiparallel orientation with respect to the target polypurine sequence, as observed when the third strand contains G and A or G and T/U. We show by footprinting and gel retardation experiments that an oligoribonucleotide containing G and A or G and U fails to bind double helical DNA, while the corresponding DNA oligomers form stable triple-helical complexes.     

Stabilization of short helices by intramolecular cluster formation

The intramolecular formation of multiple clusters of interacting helices has been characterized in a homopolymer. The configuration partition function permits the formation of clusters in which the number of interacting helices may be as large as the greatest integer in n/2, where n denotes the number of amino acid residues in the chain. The theoretical formulation has its origin in a recent [Mattice, W. L. & Scheraga, H. A. (1984) Biopolymers 23 , 1701–1724], tractable matrix expression for the configuration partition function for intramolecular antiparallel β-sheet formation. Reassignment of the expression for one of the n(n+3)/2 elements in the sparse statistical weight matrix, along with a simple change in notation, converts that treatment into a matrix formulation of the configuration partition function for a chain containing multiple clusters of interacting antiparallel helices. The five statistical weights used are δ, f_l, w, and the Zimm-Bragg σ and s. Each tight bend that connects two interacting helices contributes a factor of δ, f_l is used in the weight for larger loops between interacting helices, and w arises from helix–helix interaction. The influence of the helix–helix interaction is well illustrated by two helix–coil transitions in a chain with n = 156 and σ = 0.001. In the absence of helix–helix interaction, the transition occurs by the nucleation and subsequent elongation of a small number of helices. When helix–helix interaction is attractive, the transition can occur by a different mechanism. Formation of a single pair of interacting helices is followed by addition of new helices to the initial cluster. In the latter process, individual helices experience relatively little growth after they are formed.     

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     

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.     

Energetics of strand-displacement reactions in triple helices: a spectroscopic study.

DNA triple helices offer exciting new perspectives toward oligonucleotide-directed inhibition of gene expression. Purine and GT triplexes appear to be the most promising motifs for stable binding under physiological conditions compared to the pyrimidine motif, which forms at relatively low pH. There are, however, very little data available for comparison of the relative stabilities of the different classes of triplexes under identical conditions. We, therefore, designed a model system which allowed us to set up a competition between the oligonucleotides of the purine and pyrimidine motifs targeting the same Watson-Crick duplex. Several conclusions may be drawn: (i) a weak hypochromism at 260 nm is associated with purine triplex formation; (ii) delta H degree of GA, GT and TC triplex formation (at pH 7.0) was calculated as -0.1, -2.5 and -6.1 kcal/mol per base triplet, respectively. This unexpectedly low delta H degree for the purine triple helix formation implies that its delta G degree is nearly temperature-independent and it explains why these triplexes may still be observed at high temperatures. In contrast, the pyrimidine triplex is strongly favoured at lower temperatures; (iii) as a consequence, in a system where two third-strands compete for triplex formation, displacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This original purine-to-pyrimidine triplex conversion shows a significant hypochromism at 260 nm and a hyperchromism at 295 nm which is similar to the duplex-to-triplex conversion in the pyrimidine motif. Further evidence for this triplex-to-triplex conversion is provided by mung bean-nuclease foot-printing assay.