|Fluorescence sensing of the influenza panhandle structure PNA-carrying thiazole orange base surrogate conjugated with a virus RNA promoter|
To detect the presence of the influenza A virus quickly, researchers created a fluorogenic probe that could bind to the promoter region. A fluorogenic probe employs fluorophores, which are tiny molecules that emit light when a specific target is present.
The influenza A virus, which causes seasonal flu outbreaks, is also the only influenza virus that has previously caused pandemic flu outbreaks. This makes influenza A an important research topic, as the seasonal flu kills between 290,000 and 650,000 people worldwide each year. The influenza A virus is difficult to detect, treat, and immunize against because it is constantly changing, or mutating. Researchers are looking for parts of the influenza virus that do not change when the virus mutates to solve this problem. The promoter region or promoter, a panhandle structure on the virus, has emerged as a potential target.
To detect the presence of the influenza A virus quickly, researchers created a fluorogenic probe that could bind to the influenza A virus RNA promoter region. A fluorogenic probe is made up of small molecules called fluorophores that emit light when they come into contact with a specific target. The fluorogenic probe developed in this study binds to a portion of the promoter region, which consists of a double-strand RNA structure carrying an internal loop, resulting in a significant light-up response that can detect the presence of influenza A.
The technique was described in a paper published in Analytical Chemistry on May 23.
"Because the sequences are not involved in gene variations related to pathogenesis (how the flu virus develops) and antiviral resistance, the promoter region of influenza A virus RNA has emerged as a new target for biochemical and therapeutic application," said Yusuke Sato, an associate professor at Tohoku University. "These findings represent the development of new molecular probes for influenza A research, with the goal of diagnosing influenza A infection and designing new antivirus drugs targeting the influenza A virus RNA promoter region."
The fluorogenic probe was created using a type of synthetic DNA known as peptide nucleic acid (PNA). The triplex-forming PNA can be developed specifically to target the double-stranded RNA in the panhandle structure of the influenza A virus RNA in a sequence-specific manner. The researchers then combined the triplex-forming PNA with a dye called thiazole orange with a small molecule that would bind to the RNA's internal loop structure.
This is referred to as a conjugate. To determine how effective the conjugate was, researchers first looked at how brightly it glowed when bound to the promoter region's target panhandle structure. It was more than 130 times brighter when it wasn't attached to anything. The combination of the PNA and the small molecules had a two-order-of-magnitude higher binding affinity than the small molecules alone. Because the promoter region remains stable regardless of influenza strain, this result demonstrates how promising this technique could be for the diagnosis of influenza A.
"The research group demonstrated the conjugate's selective fluorescence response for total RNA from influenza A virus H1N1-infected cells over mock-infected cells," Sato said. "In sharp contrast to the gold standard PCR method, this technique would serve as a promising candidate for the analysis of influenza A virus RNA based on direct sensing of the influenza A virus RNA promoter region."
As the world watches the ongoing COVID-19 pandemic, researchers are eager to find solutions for future influenza A outbreaks. This research could be used to develop more sensitive tests that can detect the influenza A virus more easily by discovering new ways to target specific parts of the influenza A virus that does not change when the virus mutates. In the future, this could be a promising target for antiviral drugs that treat influenza A infections.
Materials provided by Tohoku University. Note: Content may be edited for style and length.
Yusuke Sato, Hiromasa Miura, Takaaki Tanabe, Chioma Uche Okeke, Akiko Kikuchi, Seiichi Nishizawa. Fluorescence Sensing of the Panhandle Structure of the Influenza A Virus RNA Promoter by Thiazole Orange Base Surrogate-Carrying Peptide Nucleic Acid Conjugated with Small Molecule. Analytical Chemistry, 2022; DOI: 10.1021/acs.analchem.1c05488