To do this, Limozins group, in collaboration with coworkers from Marseille and Paris, combined two existing technologies: acoustic force spectroscopy, which allows lots of molecular sets to be tested simultaneously, and DNA scaffolds, which make it possible for the very same bonds to be evaluated repeatedly.
During acoustic force spectroscopy, sets of bonded proteins are tested inside a liquid-filled chamber. The proteins are limited by DNA scaffolding such that a person hair of DNA connects the very first protein to the bottom of the chamber, while another hair attaches the 2nd protein to a little silica bead. When the scientists blast the chamber with a soundwave, the waves force pulls the silicon bead– and the protein its connected to– far from the bottom of the chamber. If the force is strong enough, this pulling action ruptures the bond between the two proteins. Nevertheless, in this new method, a third strand of DNA functions as a leash to keep the proteins close together after their bond is ruptured.
” The originality of our method is that in addition to these two hairs on each side, in the center you have this leash that connects the 2 strands and keeps the proteins together upon rupture,” states Limozin. “Without this leash, the detachment would be irreversible, but this allows you to repeat the measurement nearly as often times as you wish.
As a proof of concept, the research study team utilized the method to identify two single-molecule interactions of biomedical interest– the bond between proteins and rapamycin, an immunosuppressive drug, and the bond in between a single-domain antibody and an HIV-1 antigen.
The researchers observed these cycles of bonding and rupturing utilizing a microscope. Having the ability to evaluate the exact same protein-protein bond multiple times is essential for exploring variation in between molecularly similar pairs. It also allows researchers to examine how these interactions change as the particles age, which could be crucial for determining the half-life of drugs or antibodies.
” With this tool, we have a way to go deeper and really probe experimentally concepts about molecular heterogeneity and molecular aging,” says Limozin. “We, and others, believe that characterizing these properties will be really beneficial for creating future rehabs that will need to operate in situations where mechanical forces are included.”
Reference: “Combining DNA scaffolds and acoustic force spectroscopy to define individual protein bonds” by Yong Jian Wang, Claire Valotteau, Adrien Aimard, Lorenzo Villanueva, Dorota Kostrz, Maryne Follenfant, Terence Strick, Patrick Chames, Felix Rico, Charlie Gosse and Laurent Limozin, 7 June 2023, Biophysical Journal.DOI: 10.1016/ j.bpj.2023.05.004.
The study was moneyed by AMIDEX Emergence Innovation, the Plan Cancer PhysCancer program, the European Research Council, the Human Frontier Science Program, and PSL University.
Illustration showing a DNA scaffold holding 2 proteins close together while their bond is defined using acoustic force spectroscopy. Credit: Vladimir Kunetki
We rely on temporary protein-protein bonds for essential processes including enzymatic reactions, antibody binding, and reaction to medication. The capability to exactly identify these interactions is essential for assessing the effectiveness of possible treatments. However, the existing methodologies for this function are restricted in their abilities, either in terms of producing detailed insights at the level of private interactions or in examining huge quantities of these interactions.
In a study just recently released in the Biophysical Journal, scientists unveiled an enhanced method for assessing the toughness and effectiveness of protein-protein bonds under conditions that simulate those within our bodies. The technique uses sound waves to pull bonded proteins apart and DNA leashes to keep the 2 proteins close together so that they can re-bond after their connection is burst. This innovation permits the exact same protein bonds to be re-tested as much as 100 times, offering valuable information about how bond strength modifications as molecules age.
” We wanted to propose a method that is adequately modular to use to various types of bonds, that has a sensible throughput, and that reaches high molecular precision that is currently only available with really fine-tuned strategies, like magnetic or optical tweezers, that are frequently hard to grasp for non-specialists,” says senior author Laurent Limozin, a biophysicist at the Centre National de la Recherche Scientifique (CNRS).
The approach utilizes sound waves to pull bonded proteins apart and DNA leashes to keep the two proteins close together so that they can re-bond after their connection is burst. Throughout acoustic force spectroscopy, pairs of bonded proteins are tested inside a liquid-filled chamber. The proteins are limited by DNA scaffolding such that one hair of DNA attaches the very first protein to the bottom of the chamber, while another hair attaches the 2nd protein to a small silica bead. If the force is strong enough, this pulling action ruptures the bond between the two proteins.
