The ability to regulate gene expression in vivo is of major importance to experimenters working with cells. It gives the researcher incredible power in determining the nature of the cell's activity. Current in vivo tools for experimental gene regulation generally produce permanent changes in the cell. Knock-outs, transformants, and in situ immunoprecipitation all modify the gene or gene product such that it can no longer perform its normal function. Unfortunately, the processes also permanently change the phenotype of the cell or cause other irreparable changes. It thus becomes impossible to see how the cell would react to a temporary loss of gene function, or to return the cell to the ground state to gauge overall effects of the experiment.
Antisense RNA has provided limited amounts of transient experimental in vivo gene regulation (Dash et al 1987). However, its efficiency is questionable and its use not always possible. Triplex DNA technology provides an alternative method to regulating in vivo gene expression on demand. Previous work has shown that oligonucleotides designed for third-strand binding can change levels of gene expression in cells (Cooney et al 1994) or affect cellular processes (Orson et al 1991; Strobel et al 1991). Therefore, if an appropriate third-strand binding site can be found in a gene of interest, it might be possible to modulate its expression levels. While the ability to turn off a gene and reestablish its expression is not new, certain properties of triplex DNA make it a promising new technology for this kind of work.
To begin with, third-strand probes do not cause gross distortions in the structure of the existing DNA double-helix nor do they create insertions or deletions in the target nucleotide sequence. Furthermore, the original Watson-Crick hydrogen bonding motifs remain undisturbed as the helix does not need to be denatured for third-strand binding. As a result, gene expression can readily be turned on again by allowing the third-strand to disassociate from the DNA double helix.
Also, third-strands target the DNA template and not the RNA message. Most genes are present only once in the genome, but over 1000 copies of the transcribed mRNA counterpart can often be found in the cell. In order to achieve useful reductions in cellular mRNA levels, the antisense probe concentration must be several orders of magnitude higher than the DNA target. With third-strand "anticode" probes, approximately ten times as much probe as target should suffice for most assays or tests.
Sensitivity to mispairing events and low third-strand binding affinity are potential restrictions to third-strand binding applications. Because of the rigidity of the current third-strand binding code, it has been difficult to fully exploit the possible applications. For this reason, work is currently ongoing to create base analogs that can bind to double stranded DNA with much better affinity regardless of sequence. As noted earlier, the addition of certain small hydrophobic groups to third-strand bases can improve binding strength. More dramatic modifications to the Hoogsteen hydrogen-binding face of the bases have met with less success (P Miller, personal communication).
Nucleoside methylphosphonates are a nuclease resistant backbone analog that has been used successfully in binding assays (Kean et al 1994). Peptide nucleic acids (PNA) are another type backbone analog that are stable within the cell and have a high binding affinity (Demidov et al 1993). Finally, White et al have designed de novo ligands using pyrrole and imidazole rings on a polyamide chain (1998). Through a hairpin mechanism, they have made a stable, non-degenerate, unrestricted synthetic ligand that can recognize any base pair in the minor groove.