Silver Nanoclusters Spot Single-Base Mutations In DNA

For genetic diseases, mutations in a gene don’t have to be large. Even a change of a single base can cause disease. Unfortunately, genetic tests that can spot single base mutations are expensive and time-consuming. A new inexpensive method based on fluorescent silver nanoclusters can quickly detect and identify base switches in a gene (J. Am. Chem. Soc., DOI: 10.1021/ja3024737).

If doctors know the sequence of a mutated gene, they not only can diagnose disease but also find the best medication to treat it, says James Werner of Los Alamos National Laboratory. Currently, detecting single-base mutations involves either multistep enzymatic reactions or DNA probes that bind specific gene sequences. While these probes can spot a gene with a mutation, they can’t tell which single base has replaced the original one.

Werner’s group wanted to do both. He, his postdoctoral fellow Hsin-Chih Yeh, and colleagues used silver nanoclusters that fluoresced, which they had previously reported designing (Nano Lett., DOI: 10.1021/nl101773c). The nanoclusters form when the researchers mix a silver nitrate solution with a piece of DNA containing a specific sequence. To make this group of between two and 30 silver atoms fluoresce, they add a piece of DNA containing what they call an enhancer sequence that binds to part of the cluster-forming sequence. The mechanism behind this fluorescence is unknown, Werner says.

In the new study, the scientists tuned the color of the glowing nanoclusters to reflect differing DNA sequences. When they added or deleted DNA bases surrounding the enhancer sequence, they changed how it binds to the cluster-forming sequence. As the binding changes, so does the light emitted by the nanocluster.

The researchers thought that they could use this color-changing phenomenon to spot single-base changes in disease-related genes. They designed the DNA sequences surrounding the cluster-forming and enhancer sequences to bind parts of a disease gene. One DNA probe contained the cluster-forming sequence and the section of the disease gene on one side of the possible mutation site. The other probe would consist of the enhancer sequence and the DNA sequence on the other side of the mutation site.

For the assay, the scientists first mix the probe containing the cluster-forming sequence with silver nitrate to produce nanoclusters. Then, when they mix those clusters with the enhancer sequence and the target gene, the DNAs form a T-shaped structure, with the gene of interest as the cross and the cluster-forming and enhancer sequences as the base. How the DNAs line up to form the “T” changes slightly based on the specific base at the mutation site. These subtle differences alter how the enhancer and cluster-forming sequences interact, leading to different colors depending on a single base.

To test the method, Werner’s team designed probe sets for six disease-related genes, including two associated with type-2 diabetes. Using a fluorometer, the scientists measured the wavelength of light produced by the silver nanoclusters after exciting them with ultraviolet light. The nanocluster glowed one of three reddish-orange colors depending on whether the mutation site contained a T, a G, or either an A or a C. The researchers also could spot the color changes by eye. They hope to develop probes that can distinguish between all four base possibilities with a single probe set.

Because the probes are inexpensive and easy to use, Jeffrey Petty of Furman University thinks that the approach could lead to more routine genetic testing. “This is a promising development,” he says, “that may contribute to a better understanding of the genetics of disease.”