Elsevier

Biochimie

Volume 90, Issue 8, August 2008, Pages 1117-1130
Biochimie

Review
DNA triple helices: Biological consequences and therapeutic potential

https://doi.org/10.1016/j.biochi.2008.02.011Get rights and content

Abstract

DNA structure is a critical element in determining its function. The DNA molecule is capable of adopting a variety of non-canonical structures, including three-stranded (i.e. triplex) structures, which will be the focus of this review. The ability to selectively modulate the activity of genes is a long-standing goal in molecular medicine. DNA triplex structures, either intermolecular triplexes formed by binding of an exogenously applied oligonucleotide to a target duplex sequence, or naturally occurring intramolecular triplexes (H-DNA) formed at endogenous mirror repeat sequences, present exploitable features that permit site-specific alteration of the genome. These structures can induce transcriptional repression and site-specific mutagenesis or recombination. Triplex-forming oligonucleotides (TFOs) can bind to duplex DNA in a sequence-specific fashion with high affinity, and can be used to direct DNA-modifying agents to selected sequences. H-DNA plays important roles in vivo and is inherently mutagenic and recombinogenic, such that elements of the H-DNA structure may be pharmacologically exploitable. In this review we discuss the biological consequences and therapeutic potential of triple helical DNA structures. We anticipate that the information provided will stimulate further investigations aimed toward improving DNA triplex-related gene targeting strategies for biotechnological and potential clinical applications.

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).

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