Over the past decade, researchers have demonstrated that it is possible to expand the basic DNA alphabet—made up of the four basic nucleotide bases adenine (A), cytosine (C), guanine (G), and thymine (T)—with the addition of unnatural bases to aptamers, strands of oligonucleotides that specifically bind to target proteins much like antibodies.
Two years ago, Hirao and colleagues reported that the unnatural nucleotide 7-(2-thienyl) imidazo[4,5-b]pyridine, called Ds for short, could be successfully incorporated into and replicated in DNA. Taking that work one step further, they now report that aptamers containing Ds bind two human proteins, VEGF-165 and interferon-g, with 100 times the affinity of aptamers containing only natural base pairs in in vitro selection experiments. The findings were published in Nature Biotechnology (1).
“By adding this extra base into the nucleic acid, affinity and functionality increased,” says Hirao. He credits the increased affinity to Ds’ hydrophobic properties. Natural nucleic acids are relatively hydrophilic, limiting interactions with hydrophobic regions of proteins and thus resulting in low binding affinities. By adding a hydrophobic base to the aptamer, Hirao and colleagues significantly increased the aptamer’s affinity for the hydrophobic protein elements.
While these unnatural base pair aptamers may one day have therapeutic applications, Hirao is currently concentrating on how they might be used as a diagnostic tool. “Diagnostics are easier to do than therapeutics,” he says. “You can use an aptamer instead of a protein antibody to detect a target protein, for example.” While protein antibodies exhibit low sensitivity and low selectivity for target molecules, aptamers that incorporate unnatural base pairs can bind a wider variety of molecular targets within a protein and, as a result, have increased affinity for the target protein.
That’s not to say, however, that therapeutic applications are too far out of reach. Hirao and colleagues are already developing a pharmacological standardization method for therapeutic nucleic acids containing unnatural bases.
In the end, Hirao believes the recent study highlights the potential of his expanded genetic alphabet. “If we can add in new bases into DNA, we can make proteins containing more than twenty amino acids, we can perhaps apply it to protein synthesis with completely new amino acids, or even create new organisms with five or six different bases in the DNA. There are many, many possibilities.”
1. Kimoto, M., R. Yamashige, K.-i. Matsunaga, S. Yokoyama, and I. Hirao. 2013. Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Nat Biotech advance online publication(April).