The Research and Protocols Involved in Our Project
In the early stages of project definition, we conducted extensive literature reviews, consultations with multiple professors and researchers in related fields, and project planning to understand the feasibility and significance of our project. We also formulated a project plan.
During this period, we learned about the crucial role of the conservative Argonaute protein family in gene editing and regulation in organisms. They hold great potential to become the primary tool for gene editing. Predicting the Tm values of Ago proteins can assist experimentalists in designing experiments, such as setting protein annealing temperatures, significantly enhancing experimental efficiency. Furthermore, we also discovered that directed evolution is one of the core methods for protein modification in the field of life sciences, helping us obtain more stable Argonaute proteins under different temperature conditions.
Argonaute proteins are a large protein family and are the principal components of the RNA-induced silencing complex (RISC), a crucial entity in RNA interference (RNAi) technology. Some AGO proteins possess a PIWI domain, conferring endonuclease activity, playing a critical role in binding and catalysis during the RNA silencing process.
Therefore, studying Argonaute proteins is significant for the following reasons:
1) Unraveling the molecular mechanism of RNA silencing: Argonaute proteins are key players in RNA interference. By binding to small RNAs such as siRNA or miRNA, Argonaute can recognize, bind, and degrade target RNA molecules. In-depth research into this molecular mechanism helps us better understand how RNA silencing works and how precise control of gene expression can be achieved by regulating Argonaute proteins. This has important implications for biological research and gene therapy.
2) Development and application of downstream RNA interference techniques: The study of Argonaute proteins forms the foundation for the development and improvement of RNA interference techniques, including the applications of siRNA and miRNA for gene silencing, gene expression regulation, and gene function research. These techniques have wide-ranging potential in drug development, disease treatment, and biotechnology applications.
3) Potential in antiviral defense: Argonaute proteins have significant potential in antiviral defense. Compared to CRISPR-Cas9, Argonaute proteins more efficiently recognize and degrade viral RNA under relatively high-temperature conditions or in biological environments. This means that Argonaute proteins can be used to inhibit viral infections, offering new approaches to the development of antiviral immunotherapies. This has potential applications in pandemic control and the management of viral infections, especially when dealing with emerging pathogens.
KmAgo is a programmable universal Argonaute ribonuclease originating from mesophilic bacteria. Its position in the evolutionary tree is shown in the figure below. KmAgo can effectively cleave most types of nucleic acids, as indicated by some of its cleavage sites and corresponding GC contents in the figure. KmAgo exhibits relatively good thermal stability, making it an ideal choice for applications requiring heat resistance, as compared to CRISPR.
Our team conducted in-depth research to understand the fundamental principles of directed evolution and its applications in protein modification. Directed evolution is a commonly used method in protein and synthetic biology design, simulating the evolutionary process at the molecular level in the laboratory. Through random mutations and recombination, a large number of mutant variants can be artificially generated. By applying selective pressure, protein variants with desired features can be screened, simulating molecular-level evolution.
The process includes:
1) Mutation: Introduction of random mutations into the gene to be modified, for example, through error-prone PCR, DNA shuffling, etc.
2) Expression: Transfer of the modified gene into host cells for expression.
3) Selection: Typically achieved by rapidly evaluating optical properties such as turbidity, color, fluorescence, or chemiluminescence. Traditional selection methods are often laborious and susceptible to environmental factors. Therefore, computer-assisted screening is required. This project uses a thermal stability prediction model as a screening tool, enabling faster and more convenient selection.
After extensive research, we summarized existing data and clarified our project's objectives, including the following three steps:
1.1 Train a high-precision thermal stability prediction model and fine-tune it for AGO proteins and mutant strains.
1.2 Develop an online platform for thermal stability prediction, allowing users to submit sequence data and receive Tm values along with ranking.
1.3 Employ the DARWINS model for assisted directed evolution of KmAgo to obtain a more stable and active KmAgo at various temperatures.
The detailed design of this approach ensures the concretization and feasibility of our project.