Triple Helix Stability - Introduction

Introduction to Triple Helix Stability

Triple-Helix Forming Oligonucleotides (TFO) are a DNA sequence-specific tool that binds to a polypurine:polypyrimidine region of a DNA duplex, resulting in a triple-helix structure at that location. Their sequence-specificity enables them to be used for directing cleaving/cross-linking reagents, transcription factors, nucleases to specific regions of a DNA duplex target (1). They also can be used as tools for altering or controlling gene expression via site-directed mutagenesis or DNA recombination (2), or as part of biochemical assays, e.g. monitoring topoisomerase activity or protein translocation (3,4).

TFOs have several natural limitations associated with them which must be addressed by the researcher in order for TFOs to be effective in in vitro or in vivo contexts. First, TFOs bind in the major groove of the duplex via Hoogsteen or reverse Hoogsteen hydrogen bonds, which are weaker than the Watson-Crick hydrogen bonds between the two strands of duplex itself. In addition, because all three strands of a triple helix are negatively charged under physiological conditions, strand repulsion is significantly and destabilizing. Moreover, in the cell, there are additional concerns: the need for the TFO to be nuclease-resistant, be able to form a triple-helix at physiological pH (7.2), not be locked in a stable secondary structure, and form a triple-helix stable enough to successfully interfere with the targeted biological process working on DNA (1).

Overcoming these limitations involves incorporating different kinds of modifications, depending on the particular TFO application. Modifications can be in the base, the ribose sugar, or the phosphodiester backbone (1).