Main

DNA trinucleotide repeat expansions are known to be associated with several hereditary neurodegenerative disorders, including Huntington disease, Fragile X syndrome and spinocerebellar ataxia. Unfortunately, because little is known about the mechanisms of pathogenesis, there are no effective therapies to treat these disabling diseases.

The presence of a (CAG)n repeat results in the inclusion of long tracts of polyglutamine in certain proteins, such as in the protein huntingtin, responsible for Huntington disease. The mutant huntingtin is thought to take on a new function that leads to cell death and debilitating symptoms. The causes of trinucleotide repeat expansions are not well-understood, but it has been proposed that these repeats are a result of DNA strand slippage mediated by the formation of a hairpin structure during DNA synthesis. The stability of the hairpin increases as the length of the repeat increases, as does the severity of the disease.

Recently, a collaborative effort from several institutions in Japan resulted in development of a small-molecule biosensor capable of determining (CAG)n hairpin repeat length. Formation of the (CAG)n repeat hairpin results in C–G pairs and A-A mismatches (Fig. 1, left). The researchers discovered a small-molecule ligand, naphthyridine-azaquinolone (NA), which recognizes and binds to the CAG-CAG triad. The 2-amino-1,8-naphthyridine moiety was designed to present hydrogen bonds complementary to guanine, and 8-azaquinolone to present hydrogen bonds complementary to adenine; the two groups are connected by a short, flexible linker. Solution of the NMR structure of the CAG-CAG triad revealed that two molecules of NA intercalate into the DNA helix, presenting hydrogen bonds to correct the A-A mismatch yet disrupting the C–G pair, causing the two cytidine nucleotides to 'flip' to the outside of the helix (Fig. 1, center and right).

Figure 1: Binding of NA to the CAG-CAG triad.
figure 1

The CAG-CAG triad results in an A-A mismatch (left). The small molecule naphthyridine-azaquinolone (NA) intercalates into the DNA helix, with the naphthyridine moiety (red) presenting complementary hydrogen bonds to guanine, and azaquinolone (blue) presenting complementary hydrogen bonds to adenine (center). Binding of two NA molecules causes extrusion of the two cytidines. The NMR structure of the NA-CAG-CAG complex with cytidines highlighted in red (right). Reprinted with permission from Nature Chemical Biology.

Full size image

The researchers used NA to create a surface plasmon resonance (SPR) biosensor that was sensitive to (CAG)n repeat length. “A longer (CAG)n repeat sequence bound to immobilized NA on the sensor more efficiently than the shorter (CAG)n repeat, and so we could see the difference in binding affinity by the SPR signal intensity,” reports Kazuhiko Nakatani, principal investigator of this study. The NA-SPR sensor could be calibrated and used to determine (CAG)n repeat lengths, and therefore the severity of the disease state.

In addition to diagnosing (CAG)n repeat disorders, the discovery of NA may be an important step toward understanding the mechanism of trinucleotide repeat pathogenesis, and potentially, a cure. “One therapy may be to use NA to stop the translation of the (CAG)n repeat to the protein,” says Nakatani. Nakatani and other researchers in this area are certainly hopeful that the discovery of new small-molecule ligands may one day open the door to effective therapies for this and other trinucleotide repeat expansion disorders.