ReviewDNA triple helices: Biological consequences and therapeutic potential
Introduction
The DNA of a single cell contains all of the genetic information necessary for life's processes. Friedrich Miescher discovered DNA in 1868, yet it took more than 70 years to demonstrate that it is the molecule that carries genetic information [1]. Once this was realized, tremendous effort has been made to better understand both the structure and function of DNA. Not only does the DNA primary nucleic acid sequence define the genetic code, its secondary structure plays important roles in regulating gene expression such that the formation of multi-stranded DNA structures at specific sites in the genome can influence many cellular functions. DNA can form multi-stranded helices through either folding of one of the two strands or association of two, three, or four strands of DNA. A well-established multi-stranded DNA structure, triple helical DNA (triplex DNA), both naturally occurring intramolecular H-DNA structures, and triplex-forming oligonucleotide (TFO)-targeted intermolecular triplexes will be the focus of this review.
Triple-helical nucleic acids were first described in 1957 by Felsenfeld and Rich [2], who demonstrated that polyuridylic acid and polyadenylic acids strands in a 2:1 ratio were capable of forming a stable complex. In 1986, it was demonstrated that a short (15-mer) mixed-sequence triplex-forming oligonucleotide (TFO) formed a stable specific triple helical DNA complex [3]. The third strand of DNA in the triplex structure (i.e. the TFO) follows a path through the major groove of the duplex DNA. The specificity and stability of the triplex structure is afforded via Hoogsteen hydrogen bonds [4], which are different from those formed in classical Watson–Crick base pairing in duplex DNA. Because purines contain potential hydrogen bonds with incoming third strand bases, the binding of the third strand is to the purine-rich strand of the DNA duplex [5], [6].
Section snippets
Classification of DNA triple helices
Since the original discovery of triple helical nucleic acids, a number of triplex DNA structures that form under various conditions in vitro and/or in vivo have been identified (reviewed in [7], [8], [9]). These include intermolecular triplexes (with a pyrimidine third strand “Y:RY”, a purine strand or mixed pyrimidine/purine third strand “R:RY”), and intramolecular triplexes (H-DNA).
Targeting genes as an approach to molecular-targeted therapeutics
The ability to target specific genes to modulate their structure and/or function in the genome has far-reaching implications in biology, biotechnology, and medicine. TFOs represent near-ideal molecules for this purpose because of their ability to bind duplex DNA with high affinity and specificity. Facile chemistries for TFO modification are also available, allowing the covalent attachment of DNA damaging agents, for example, to target damage to specific sites in a genome. Oligonucleotides have
Approaches to improve the efficacy of TFOs in biological systems
As discussed above (Section 3.3), there are many factors that can limit the efficacy of triplex technology in cellular systems. Improvements in TFO chemistries are under active investigation, as these modifications could considerably increase the efficacy of antigene oligonucleotide therapeutics.
H-DNA conformation and its occurrence in genomic DNA
Eukaryotic genomes contain many S1 nuclease sensitive sites with a common feature being runs of polypurine–polypyrimidine sequences. These types of sequences are capable of adopting non-canonical DNA structures. For example, H-DNA, or intramolecular triplex DNA is a structure in which half of the pyrimidine tract swivels its backbone parallel to the purine strand in the underlying duplex, or the purine strand (in *H-DNA) binds to the purine strand of the underlying duplex in an antiparallel
H-DNA induces genetic instability
Bacolla et al. found that genes carrying long polypurine–polypyrimidine sequences are more susceptible to chromosomal translocations [103]. Certain “fragile site” or “hotspot” regions of the genome are mapped in or near sequences that have the potential to adopt non-B DNA conformations. For example, a segment in the promoter of the human c-MYC gene capable of adopting H-DNA [112], overlaps with the one of major breakage hotspots found in c-MYC-induced lymphomas and leukemias [113], [114], [115]
H-DNA is implicated in transcription regulation
Sequences that are capable of forming H-DNA are found in promoter regions of genes more frequently than expected by random distribution of bases in eukaryotic genomes, suggesting that they may be involved in the regulation of gene expression [139]. There are many published reports that H-DNA can either up-regulate or down-regulate gene expression, depending on a number of factors, including the location of H-DNA in a gene, and the adjacent sequences and elements. In bacteria, when an
Modulating H-DNA structure as a potential gene targeting strategy
Anticancer agents that target DNA are among the most effective agents in cancer therapeutics, but are often extremely toxic due to lack of specificity for the tumor cells. Although the mechanisms by which H-DNA influences DNA metabolism are not well understood, it is clear that it plays important roles in a variety of DNA processes, and the unique structure of H-DNA provides a potential target for a the development of a new class of more selective DNA-based therapeutics.
Concluding remarks
The formation of triplex DNA, either in an intramolecular fashion from the same DNA molecule, or in an intermolecular fashion by delivery of a TFO into cells, has very attractive application potential. Naturally occurring intramolecular triplexes play important roles in regulating DNA metabolism and gene function, and are inherently mutagenic and recombinogenic. Regulating H-DNA conformation or specifically interfering with H-DNA-related interactions using small molecules or oligonucleotides
Acknowledgments
We thank Dr. Rick A. Finch for critical reading of the manuscript. Support was provided by an NIH/NCI grant to K.M.V. (CA93729).
References: (162)
- et al.
Studies on the formation of two- and three-stranded polyribonucleotides
Biochim. Biophys. Acta
(1957) - et al.
Stabilities of double- and triple-strand helical nucleic acids
Prog. Biophys. Mol. Biol.
(1992) - et al.
Transcription factor decoy molecules based on a peptide nucleic acid (PNA)-DNA chimera mimicking Sp1 binding sites
J. Biol. Chem.
(2003) - et al.
Triplex-induced recombination in human cell-free extracts. Dependence on XPA and HsRad51
J. Biol. Chem.
(2001) - et al.
Psoralen adducts induced by triplex-forming oligonucleotides are refractory to repair in HeLa cells
J. Mol. Biol.
(2000) - et al.
Triplex-directed modification of genes and gene activity
Trends Biochem. Sci.
(1998) - et al.
Chemical modification of pyrimidine TFOs: effect on i-motif and triple helix formation
Arch. Biochem. Biophys.
(2000) - et al.
Chromosome targeting at short polypurine sites by cationic triplex-forming oligonucleotides
J. Biol. Chem.
(2001) - et al.
Electron microscopy visualization of oligonucleotide binding to duplex DNA via triplex formation
J. Mol. Biol.
(1993) - et al.
Strand displacement recognition of mixed adenine-cytosine sequences in double stranded DNA by thymine-guanine PNA (peptide nucleic acid)
Bioorg. Med. Chem.
(2001)
Branched oligonucleotides containing bicyclic nucleotides as branching points and DNA or LNA as triplex forming branch
Bioorg. Med. Chem. Lett.
Exploring cellular activity of locked nucleic acid-modified triplex-forming oligonucleotides and defining its molecular basis
J. Biol. Chem.
Targeted gene knockout by 2′-O-aminoethyl modified triplex forming oligonucleotides
J. Biol. Chem.
Stabilization of purine motif DNA triplex by a tetrapeptide from the binding domain of HMGBI protein
Biochimie
Formation of a combined H-DNA/open TATA box structure in the promoter sequence of the human Na,K-ATPase alpha2 gene
J. Biol. Chem.
Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III
J. Exp. Med.
Design of sequence-specific DNA-binding molecules
Science
The structure of crystals containing a hydrogen-bonded complex of 1-methylthymine and 9-methyladenine
Acta Cryst.
Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro
Science
Second structural motif for recognition of DNA by oligonucleotide-directed triple-helix formation
Science
Triplex DNA structures
Annu. Rev. Biochem.
Triplex-forming oligonucleotides: principles and applications
Q. Rev. Biophys.
Triplex DNA and human disease
Front. Biosci.
Specificity in formation of triple-stranded nucleic acid helical complexes: studies with agarose-linked polyribonucleotide affinity columns
Biochemistry
Binding of triple helix forming oligonucleotides to sites in gene promoters
Biochemistry
Complexes formed by (pyrimidine)n. (purine)n DNAs on lowering the pH are three-stranded
Nucleic Acids Res.
DNA triplex formed by d-A-(G-A)7-G and d-mC-(T-mC)7-T in aqueous solution at neutral pH
Anticancer Drug Des.
Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide-directed triple helix formation at single DNA sites
Biochemistry
Monovalent cation effects on intermolecular purine-purine-pyrimidine triple-helix formation
Nucleic Acids Res.
Triple helix formation: binding avidity of acridine-conjugated AG motif third strands containing natural, modified and surrogate bases opposed to pyrimidine interruptions in a polypurine target
Nucleic Acids Res.
Oligodeoxyribonucleotide length and sequence effects on intermolecular purine-purine-pyrimidine triple-helix formation
Nucleic Acids Res.
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
Nucleic Acids Res.
DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines
Nucleic Acids Res.
High-affinity triple helix formation by synthetic oligonucleotides at a site within a selectable mammalian gene
Biochemistry
A directional nucleation-zipping mechanism for triple helix formation
Nucleic Acids Res.
Thermodynamic and kinetic stability of intermolecular triple helices containing different proportions of C+*GC and T*AT triplets
Nucleic Acids Res.
Kinetic studies on the formation of DNA triplexes containing the nucleoside analogue 2′-O-(2-aminoethyl)-5-(3-amino-1-propynyl)uridine
Org. Biomol. Chem.
Kinetic studies on the formation of intermolecular triple helices
Nucleic Acids Res.
A web-based search engine for triplex-forming oligonucleotide target sequences
Oligonucleotides
High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes
Mol. Carcinog.
Overcoming potassium-mediated triplex inhibition
Nucleic Acids Res.
Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells
EMBO Rep.
RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions
Proc. Natl. Acad. Sci. U.S.A.
Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair
Science
Targeting oncogenes to improve breast cancer chemotherapy
Cancer Res.
Proton NMR and optical spectroscopic studies on the DNA triplex formed by d-A-(G-A)7-G and d-C-(T-C)7-T
J. Biomol. Struct. Dyn.
The vacuum UV CD spectra of G.G.C triplexes
Nucleic Acids Res.
Sequence-specific cleavage of double helical DNA by triple helix formation
Science
High-efficiency triple-helix-mediated photo-cross-linking at a targeted site within a selectable mammalian gene
Biochemistry
Triplex targets in the human rhodopsin gene
Biochemistry
Cited by (229)
Examining DNA structures with in-droplet hydrogen/deuterium exchange mass spectrometry
2024, International Journal of Mass SpectrometryBiophysical evaluation of antiparallel triplexes for biosensing and biomedical applications
2024, International Journal of Biological MacromoleculesUnexplored power of CRISPR-Cas9 in neuroscience, a multi-OMICs review
2024, International Journal of Biological MacromoleculesNon-B DNA structures as a booster of genome instability
2023, Biochimie
- 1
These authors contributed equally to the study.