6 June 2005: A third strand for DNA

The DNA double helix can under certain conditions accommodate a third strand in its major groove. Researchers in the UK have now presented a complete set of four variant nucleotides that makes it possible to use this phenomenon in gene regulation and mutagenesis.

Natural DNA will only form a triplex if the targeted strand is rich in purines -   guanine (G) and adenine (A) - which in addition to the bonds of the Watson-Crick base pairing can form two further hydrogen bonds, and the ’third strand’ oligonucleotide has the matching sequence of pyrimidines - cytosine (C) and thymine (T). Any Cs or Ts in the target strand of the duplex will only bind very weakly, as they contribute just one hydrogen bond. Moreover, the recognition of G requires the C in the probe strand to be protonated, such that the triplex formation will only work at low pH. 

To overcome all these problems, the groups of Tom Brown and Keith Fox at the University of Southampton have developed modified building blocks, and have now completed a set of four new nucleotides, each of which will bind to one DNA nucleotide from the major groove of the double helix.¹

They tested the binding of a 19-mer of these designer nucleotides to a double helix target sequence in comparison with the corresponding triplex-forming oligonucleotide made from natural DNA bases. Using fluorescence-monitored thermal melting and DNase I footprinting, the researchers showed that their construct forms stable triplex even at neutral pH. 

Tests with mutated versions of the target sequence showed that three of the novel nucleotides are highly selective for their target base pair, while the ’S’ nucleotide, designed to bind to T, also tolerates C. 

In principle, triplex formation has already been demonstrated as a way of inducing mutations in cell cultures and animal experiments.² The new set of building blocks ’opens the way to the exploitation of the triplex approach in many areas of biology,’ says Brown. Michael Gross

Image: A triplex forming oligonucleotide in green bound to the major groove of a DNA duplex