Triple Helix Structures: Sequence Dependence,Flexibility and Mismatch Effects

Abstract

By means of molecular modelling, electrostatic interactions are shown to play an important role in the sequence-dependent structure of triple helices formed by a homopyrimidine oligonucleotide bound to a homopurine, homopyrimidine sequence on DNA. This is caused by the presence of positive charges due to the protonation of cytosines in the Hoogsteen-bonded strand, required in order to form C.GxC+ triplets. Energetic and conformational characteristics of triple helices with different sequences are analyzed and discussed. The effects of duplex mismatches on the triple helix stability are investigated via thermal dissociation using UV absorption.     

Sequence-specific recognition of the major groove of DNA by oligodeoxynucleotides via triple helix formation. Footprinting studies.

Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. The oligonucleotide is bound parallel to the homopurine strand of the duplex. This binding can be revealed by a footprinting technique using copper-phenanthroline as a cleaving reagent. Oligonucleotide binding in the major groove prevents cleavage by copper-phenanthroline. The cleavage patterns on opposite strands of the duplex at the boundaries of the triple helix are asymmetric. They are shifted to the 3'-side, indicating that the copper-phenanthroline chelate binds in the minor groove of the duplex structure. Binding of the chelate at the junction between the triple and the double helix is not perturbed on the 5'-side of the bound homopyrimidine oligonucleotide. In contrast, a strong enhancement of cleavage is observed on the purine-containing strand at the triplex-duplex junction on the 3'-side of the homopyrimidine oligonucleotide.     

Stable triple helices are formed upon binding of RNA oligonucleotides and their 2'-O-methyl derivatives to double-helical DNA.

Pyrimidine oligoribonucleotides bind to the major groove of double-helical DNA at homopurine.homopyrimidine sequences. They recognize Watson-Crick base pairs by forming T.A x U and C.G x C base triplets via Hoogsteen hydrogen bonding. The stability of these triple helices is much higher than that of triple helices formed by oligodeoxyribonucleotides as shown by an increase of the temperature at which half-dissociation of the third strand occurs. When the 2'-hydroxyl group of ribose moieties is replaced by 2'-O-methyl substituent, triple helix stability is further increased.     

Extension of the range of recognition sequences for triple helix formation by oligonucleotides containing guanines and thymines.

Oligodeoxynucleotides containing G and T can bind to homopurine.homopyrimidine sequences on double-stranded DNA by forming C.G x G and T.A x T base triplets. The orientation of the third strand in such triple helices depends on the number of GpT and TpG steps. Therefore a single oligonucleotide can be designed to bind to two consecutive homopurine.homopyrimidine sequences where the two homopurine stretches alternate on the two strands of DNA. The oligonucleotide switches from one homopurine strand to the other at the junction between the two sequences. This result shows that it is possible to extend the range of DNA sequences that can be recognized by a single oligonucleotide.     

Towards mixed sequence recognition by triple helix formation

The formation of intermolecular DNA triple helices offers the possibility of designing compounds with extensive sequence recognition properties which may be useful as antigene agents or tools in molecular biology. One major limitation of this approach is that these structures are generally restricted to homo-purine. homopyrimidine target sites. This review describes the strategies that have been employed to overcome this drawback and outlines the potential for triplex formation at mixed sequence DNA targets.     

Strand orientation of [alpha]-oligodeoxynucleotides in triple helix structures: dependence on nucleotide sequence.

The aims of the present theoretical study of the conformations of [alpha]-oligodeoxynucleotides forming triple helices with DNA duplexes are to understand the structural and energetic factors involved in [alpha]-triple helix formation by means of energy minimization, and to explain the experimentally observed dependence of strand orientation on the nucleotide sequence. It is found that the energetically preferred orientation of the [alpha]-oligonucleotide with respect to the homopurine strand depends on the sequence of the homopurine.homopyrimidine tracts. This is a consequence of the structural heteromorphism of base triplets in the intrinsically more stable reverse Hoogsteen hydrogen bonding configuration. Practical rules are proposed for determining the orientation of the nuclease-resistant [alpha]-oligodeoxynucleotide strand which will form the most stable triple helix.     

COMBO-FISH: specific labeling of nondenatured chromatin targets by computer-selected DNA oligonucleotide probe combinations

Here we present the principle of fluorescence in situ hybridization (FISH) with combinatorial oligonucleotide (COMBO) probes as a new approach for the specific labeling of genomic sites. COMBO-FISH takes advantage of homopurine/homopyrimidine oligonucleotides that form triple helices with intact duplex genomic DNA, without the need for prior denaturation of the target sequence that is usually applied for probe binding in standard FISH protocols. An analysis of human genome databases has shown that homopurine/homopyrimidine sequences longer than 14 bp are nearly homogeneously distributed over the genome, and they represent from 1% to 2% of the entire genome. Because the observation volume in a confocal laser-scanning microscope equipped with a high numerical aperture lens typically corresponds to an approximate 250-kb chromatin domain in a normal mammalian cell nucleus, this volume should contain 150-200 homopurine/homopyrimidine stretches. Using DNA database information, one can configure a set of distinct, uniformly labeled oligonucleotide probes from these stretches that is expected to exclusively co-localize within a 250-kb chromatin domain. Due to the diffraction-limited resolution of a microscope, the fluorescence signals of the configured oligonucleotide probe set merge into a typical, nearly homogenous FISH spot. Using a set of 32 homopyrimidine probes, we performed experiments in the Abelson murine leukemia region of human chromosome 9 as some of the very first proofs-of-principle of COMBO-FISH. Although the experimental protocol currently contains several steps that are incompatible with living cell conditions, the theoretical approach may be the first methodological advance toward the long-term but still elusive goal of carrying out specific FISH in high-resolution fluorescence microscopy of vital cells.     

Triplex formation by morpholino oligodeoxyribonucleotides in the HER-2/neu promoter requires the pyrimidine motif

Triplex-forming oligonucleotides (TFOs) are good candidates to be used as site-specific DNA-binding agents. Two obstacles encountered with TFOs are susceptibility to nuclease activity and a requirement for magnesium for triplex formation. Morpholino oligonucleotides were shown in one study to form triplexes in the absence of magnesium. In the current study, we have compared phosphodiester and morpholino oligonucleotides targeting a homopurine–homopyrimidine region in the human HER2/neu promoter. Using gel mobility shift analysis, our data demonstrate that triplex formation by phosphodiester oligonucleotides at the HER-2/neu promoter target is possible with pyrimidine-parallel, purine-antiparallel and mixed sequence (GT)-antiparallel motifs. Only the pyrimidine-parallel motif morpholino TFO was capable of efficient triple helix formation, which required low pH. Triplex formation with the morpholino TFO was efficient in low or no magnesium. The pyrimidine motif TFOs with either a phosphodiester or morpholino backbone were able to form triple helices in the presence of potassium ions, but required low pH. We have rationalized the experimental observations with detailed molecular modeling studies. These data demonstrate the potential for the development of TFOs based on the morpholino backbone modification and demonstrate that the pyrimidine motif is the preferred motif for triple helix formation by morpholino oligonucleotides.     

Sequence specificity in triple-helix formation: experimental and theoretical studies of the effect of mismatches on triplex stability

The specificity of a homopyrimidine oligonucleotide binding to a homopurine-homopyrimidine sequence on double-stranded DNA was investigated by both molecular modeling and thermal dissociation experiments. The presence of a single mismatched triplet at the center of the triplex was shown to destabilize the triple helix, leading to a lower melting temperature and a less favorable energy of interaction. A terminal mismatch was less destabilizing than a central mismatch. The extent of destabilization was shown to be dependent on the nature of the mismatch. Both single base-pair substitution and deletion in the duplex DNA target were investigated. When a homopurine stretch was interrupted by one thymine, guanine was the least destabilizing base on the third strand. However, G in the third strand did not discriminate between a C.G and an A.T base pair. If the stretch of purines was interrupted by a cytosine, the presence of pyrimidines (C or T) in the third strand yielded a less destabilizing effect than purines. This study shows that oligonucleotides forming triple helices can discriminate between duplex DNA sequences that differ by one base pair. It provides a basis for the choice of antigene oligonucleotide sequences targeted to selected sequences on duplex DNA.     

Design of oligonucleotides for sequence-specific triple-helix formation. Properties of oligonucleotides containing 2'-deoxyxanthosine.

Homopyrimidine-homopurine sequences are targeted by homopyrimidine oligodeoxynucleotides to form triple helix structure. Xanthine was introduced to the third strand of pyrimidine-rich 15-mer oligodeoxy-nucleotides to recognize a cytosine in the purine strand in a homopyrimidine-homopurine duplex target.     

Site-specific oligodeoxynucleotide binding to maize Adh1 gene promoter represses Adh1-GUS gene expression in vivo

There is a 36 bp tract of extreme homopurine/homopyrimidine (PuPy) asymmetry in the maize Adh1 gene promoter (from –44 to –79) that is S1-hypersensitive in plasmids under supercoil tension. Oligodeoxynucleotides corresponding to the PuPy tract were designed to examine the secondary structure of the region and address the possible role of the tract in gene regulation. On the basis of oligodeoxynucleotide band-shift and DNase I footprinting analyses, it was concluded that the homopyrimidine oligodeoxynucleotide can form a triple helix with the duplex PuPy tract in vitro. Transient assays in protoplasts, suspension cells, and seedling roots show that the homopyrimidine oligodeoxynucleotide is also capable of repressing Adh1-GUS gene expression during co-transformation, presumably by the formation of a triple helix with the PuPy tract in vivo. The complementary homopurine oligodeoxynucleotide would not form a triple helix in vitro, nor would it repress Adh1-GUS in vivo. We propose that triple helix formation is a potential regulatory phenomenon in vivo, and that an intraregion triple helix could occur within the Adh1 promoter via the formation of H-DNA.     

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.     

Probing DNA triple helix structure by chemical ligation

A series of oligomeric double and triple helical DNAs with irregular sequences of homopurine and homopyrimidine strands were prepared. DNA triplexes were identified by CD spectroscopy and thermal denaturation profiles (biphasic helix-coil transition). Condensation of oligonucleotides on single and double-stranded DNA templates was performed using water-soluble carbodiimide, phosphodiester and pyrophosphate internucleotide bonds being newly formed. Such chemical ligation proved to be a sensitive monitor of changes in the sugar-phosphate backbone resulting from conversion of double to triple helix and of third-strand binding.     

A computational and experimental study of the bending induced at a double-triple helix junction.

We have studied the conformation of a 17 base-pair homopyrimidine.homopurine triple helix formed on a fragment of duplex DNA derived from Simian Virus SV40. Gel retardation assays indicate that an 80 base-pair fragment has an altered conformation when the triple helix is formed, which is most likely to result from an induced bend in the DNA. Investigation of the detailed conformation of the double helix-triple helix junctions has been performed by means of molecular modelling. Bending on the 5' and 3' sides of the third strand oligonucleotide are not located at equivalent positions with respect to the junctions, which is explained in terms of base stacking. The junction effects on DNA structure, induced by the requirement for cytosine protonation in the Hoogsteen-bonded strand to form CGC+ base triplets, are also discussed.     

Regulation of DNA replication by homopurine/homopyrimidine sequences

The simple repeating homopurine/homopyrimidine sequences dispersed throughout many eukaryotic genomes are known to form triple helical structures comprising three-stranded and single-stranded DNA. Several lines of evidence suggest that these structures influence DNA replication in cells. Homopurine/homopyrimidine sequences cloned into simian virus 40 (SV40) or SV40 origin-containing plasmids caused a reduced rate of DNA synthesis due to the pausing of replication forks. More prominent arrests were observed in in vitro experiments using single-stranded and double-stranded DNA with triplex-forming sequences. Nucleotides unable to form triplexes when present in the template DNA or when incorporated into the nascent strand prevented termination. Similarly, mutations destroying the triplex potential did not cause arrest while compensatory mutations restoring triplex potential restored it. These and other observations from a number of laboratories indicating that homopurine/homopyrimidine sequences act as arrest signals in vitro and as pause sites in vivo during replication fork movement suggest that these naturally occurring sequences play a regulatory role in DNA replication and gene amplification.     

Collagens as multidomain proteins

The number of proteins known to contain collagen-like triple helical domains is rapidly increasing. The functions of these domains are to provide molecular rods that separate spatially non-triple helical domains with varied properties and structures and to permit lateral interactions between molecules. Two-thirds of the amino acids of the triple helical domains have their side-chains at the surface of the protein. The triple helix is also a structure that is easily predictable from the primary structure. The structure of several recently discovered collagens are discussed in terms of domains and functions. The triple helical domains have sizes varying from 33 to over 1,000 amino acid residues. The longest uninterrupted triple helices are involved in the formation of the classical quarter-staggered fibrils. Other triple helical domains permit varied molecular aggregates. A very broad spectrum of non-triple helical or globular domains are interspersed by triple helices. Only those located at the extremities of the molecules are large in size, sometimes several hundred kDa, while the domains separating 2 triple helices are small (less than 50 amino acids) and provide the molecules with hinges, proteolytic cleavage sites or other specialized functions like a glycosaminoglycan attachment site. If the assembly of the 3 chains required for the triple helix formation can be controlled in vitro, collagen-like molecules offer an as yet unexploited potential for protein engineering.     

Theoretical calculations, synthesis and base pairing properties of oligonucleotides containing 8-amino-2'-deoxyadenosine

Theoretical calculations on double and triple helices containing 8-amino-2'-deoxyadenosine were made to analyze the possible differences in base pairing properties between 8-aminoadenine and adenine. These calculations indicate a strong preferential stabilization of the triplex over the duplex when adenine is replaced by 8-aminoadenine. In addition, a protected phosphoramidite derivative of 8-amino-2'-deoxyadenosine was prepared for the introduction of 8-aminoadenine into synthetic oligonucleotides using the phosphite-triester approach. DNA triple helical structures are normally observed at acidic pH. However, when oligonucleotides carrying 8-aminoadenine are used, very stable triple helical structures can be observed even at neutral pH. Biological applications of triple helices could benefit from the use of 8-aminoadenine derivatives.     

The influence of oligodeoxyribonucleotide phosphorothioate pyrimidine strands on triplex stability.

The analogues of the homopyrimidine oligonucleotide dT15 has been synthesized. The analogues, contains phosphorothioate bonds of a mixture of diastereoisomers or one of the two stereoisomer (either Rp or Sp). The analogues were mixed under conditions conductive to the formation of triple stranded assemblies. The mixtures were characterized by their thermal stabilities (Tm values) and CD spectra.     

DNA triple helix stabilization by aminoglycoside antibiotics

The stabilization of the poly(dA) x 2poly(dT) triple helix by neomycin is reported. Preliminary results indicate that neomycin stabilizes DNA triple helices and the double helical structures composed of poly(dA) x poly(dT) are virtually unaffected. This is the first report of the interaction of aminoglycoside antibiotics with DNA triple helices.