New Fluorogenic Probe Senses Non-Mutating Section of Influenza A

Researchers in Japan have developed a new probe that attaches to the region of influenza A that does not mutate—a breakthrough with the potential to speed diagnosis of the virus and one day identify treatments that would not lose effectiveness due to frequent mutations.

The influenza A virus is responsible for seasonal flu outbreaks that cause between 290,000 and 650,000 deaths per year globally. Because influenza A is constantly mutating, it can be difficult to detect, treat, and inoculate against. To solve this problem, researchers are looking for parts of the influenza virus that do not change when the virus mutates. A panhandle structure on the virus known as the promoter region or promoter has emerged as a potential target.

"The promoter region of influenza A virus RNA has emerged as a new target for biochemical and therapeutic application because the sequences are not involved in the gene variations related to pathogenesis (how the flu virus develops) and antiviral resistance," said Yusuke Sato, an associate professor at Tohoku University. "These results represent the development of new molecular probes for influenza A research, with a view toward the diagnosis of influenza A infection, as well as the design of new antivirus drugs targeting the influenza A virus RNA promoter region."

To create the fluorogenic probe, the researchers used a type of synthetic DNA called peptide nucleic acid (PNA). The triplex-forming PNA can be specifically developed to target the double-stranded RNA found in the panhandle structure of the influenza A virus RNA in a sequence-selective manner.

Researchers then combined the triplex forming PNA having a type of dye called thiazole orange with a small molecule that would bind with the internal loop structure of the RNA. This combination is called a conjugate; to determine how effective the conjugate was, researchers first analyzed how brightly the conjugate glowed when it was bound to the target panhandle structure of the promoter region. It was more than 130-fold brighter than when it was not bound to anything. Compared to the small molecules alone, the combination of the PNA and the small molecules had a stronger binding affinity by two orders of magnitude. The team also demonstrated the selective fluorescence response of the conjugate for total RNA from influenza A virus H1N1-infected cells over that from mock-infected ones.

"This technique would serve as a promising candidate for the analysis of influenza A virus RNA based on the direct sensing of the influenza A virus RNA promoter region, in sharp contrast to the gold standard PCR method,” Sato adds.

By finding new ways to target specific parts of the influenza A virus that do not change when the virus mutates, this research could be used to create more sensitive tests that can detect the influenza A virus more easily. In the future, this could even be a promising target for antiviral drugs that could treat infections of influenza A.

The findings were published recently in Analytic Chemistry.