Tracking the structural changes that proteins undergo as they perform various functions can be difficult when these changes are especially subtle, and may require complicated tasks such as inserting fluorophores at several specific sites on the protein. A technique that simplifies the real-time monitoring of protein conformation and function could aid in a better understanding of biological processes and diseases, as well as in the development of new pharmaceutical drugs. Researchers at the Université de Montréal have now developed fluorescent nanoantennas made from DNA that can report protein conformational changes and be used in conjunction with any conventional spectrofluorometer.
Over the years, DNA has proven to be a useful material for building different nanostructures and nanomachines due to its “Lego-like” structure and very small size, said Alexis Vallée-Bélisle, the senior author on the study. The researchers leveraged these properties to build nanoantennas about 5 nm in length with a fluorescent dye at one end and biotin on the other end. The biotin end binds to a streptavidin platform while dye-protein interactions cause the fluorescent signal to change in response to the target protein’s behavior. Thus, the signal transmitted by the DNA nanoantenna can be received and analyzed to determine how the protein changes and functions in real time.
The nanoantennas were first tested to detect the activity of calf intestinal alkaline phosphatase (AP), an enzyme of medical interest that is difficult to directly monitor in real time through conventional means. Various nanoantenna lengths and fluorescent dyes were tested to determine the ideal configuration for monitoring AP, with an L12 linker length and fluorescein (FAM) dye showing some of the best results. The nanoantennas were able to monitor AP-mediated hydrolysis of several substrates and biomolecules as well as detect different conformational states of AP, including states induced by vanadate and tungstate binding. The composition, length and dyes used in the antennas could be further tuned for studying different proteins of interest, the researchers wrote. This study was published in Nature Methods.
“In addition to helping us understand how natural nanomachines function and malfunction, consequently leading to disease, this new method can also help chemists identify promising new drugs as well as guide nanoengineers to develop improved nanomachines,” said Dominic Lauzon, a co-author of the study.
The researchers are now forming a start-up company to commercialize the nanoantenna technology and make it available to other researchers, according to Vallée-Bélisle.
“Perhaps what we are most excited by is the realization that many labs around the world, equipped with a conventional spectrofluorometer, could readily employ these nanoantennas to study their favorite protein, such as to identify new drugs or to develop new nanotechnologies,” Vallée-Bélisle said.
Photo: Like a two-way radio that can both receive and transmit radio waves, the fluorescent nanoantenna designed by Alexis Vallée-Bélisle and his team receives light in one color and depending on the protein movement it senses, then transmits light back in another color, which can be detected. One of the main innovations of these nanoantennas is that the receiver part of the antenna (bright green) is also employed to sense the molecular surface of the protein studied via molecular interaction. Credit: Caitlin Monney