Orders of magnitude faster
Scientists have invented a fast method of "directed evolution" of molecules
The technique, dubbed VEGAS, works in mammalian cells and can create useful new molecules within days, providing scientists with a powerful new research tool and a potential path to better treatment of various diseases, according to a press release from the University of North Carolina Scientists Invent Fast Method for ‘Directed Evolution’ of Molecules.
Article by English et al. VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells is published in the journal Cell – VM.
Scientists demonstrated this technique by developing several proteins to perform specific new tasks, each time doing it for several days. The existing methods of directed evolution are more labor-intensive, time-consuming, and, as a rule, are used in bacterial cells, which limits the usefulness of this technology for the evolution of proteins for use in human cells.
Directed evolution is an artificial, accelerated version of the evolutionary process in nature. The idea is to focus the evolutionary process on a single DNA sequence so that it performs a specific task. Directed evolution can be used to create new therapeutic agents that work powerfully to stop diseases and have few or no side effects. The initial scientific work on directed evolution received The Nobel Prize in Chemistry in 2018.
"What we have developed is the most reliable system for directed evolution in mammalian cells," said lead study author Justin English, PhD, a research associate in the Department of Pharmacology at the University of North Carolina School of Medicine.
"The scientific community has long needed such a tool," said senior study author Brian L. Roth, MD, Michael Hooker Distinguished Professor in the Department of Pharmacology at the University of North Carolina School of Medicine. "We believe that our technique will accelerate research and ultimately lead to improved therapy for people suffering from many diseases for which we need much more effective treatment."
The broad concept of directed evolution is not new. Researchers have used it for centuries in the selection and breeding of animal and plant variants that have the desired characteristics, such as varieties of crops with larger fruits. Biologists in recent decades have also used directed evolution at the molecular level in the laboratory, for example, by randomly mutating a gene until a variant that has the desired property appears. But in general, the methods of directed evolution of biological molecules were difficult to use and limited in their application.
The new method developed by Roth, English and colleagues is relatively fast, simple and versatile. It uses the Sindbis virus as a carrier of the gene to be modified. A virus with its genetic cargo can infect cells in a cup and mutate quite quickly. Researchers have created conditions for only mutant genes to flourish – those that encode proteins capable of performing a desired function in cells, such as activating a certain receptor or turning on certain genes. Since the system works in mammalian cells, it can be used to generate new proteins in humans, mice, or other mammals that would be burdensome or impossible to generate using traditional bacterial cell-based methods.
English and his colleagues call the new system VEGAS (Viral Evolution of Genetically Actuating Sequences) – a system for the viral evolution of genetically activated sequences. In an initial demonstration, Roth's lab modified a protein called a tetracycline transactivator (tTA), which works as a gene activation switch and is a standard tool used in biological experiments. tTA usually stops working if it meets the antibiotic tetracycline or closely related doxycycline, but researchers have developed a new version with 22 mutations that allows tTA to continue working despite very high levels of doxycycline. The process took only seven days.
"To understand how effective this is, keep in mind that previously the mammalian directed evolution method applied to the tetracycline transactivator required four months to obtain only two mutations that confer only partial insensitivity to doxycycline," English said.
The scientists then applied VEGAS to a commonly accepted type of cellular receptors called G-protein-coupled receptors (G-protein-coupled receptors). There are hundreds of different GPCRs on human cells, and many of them are targets of modern medicines for the treatment of a wide range of diseases. How exactly this GPCR changes shape when it switches from an inactive state to an active one is of great interest to researchers trying to develop more accurate therapies. English and his colleagues used VEGAS to rapidly mutate a little-studied GPCR called MRGPRX2 so that it would always remain active.
"Identifying the mutations that occurred during this rapid evolution helps us understand for the first time the key regions in the receptor protein involved in the transition to the active state," English said.
The team demonstrated the potential of VEGAS to lead drug development more directly. They used VEGAS to rapidly develop small biological molecules called nanotels that can activate various GPCRs, including serotonin and dopamine receptors, which are found in brain cells and are a target for many psychiatric drugs.
The team is currently using VEGAS to develop highly effective gene editing tools, potentially for the treatment of genetic diseases, as well as to create nanotells that can neutralize cancer-causing genes.
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