Phage display technology
Introduction by Doctor Waffle
Dr. Waffle introduces himself and explains his preferred nickname due to the complexity of his first name. He shares his background, including his experience working on viruses and his involvement in COVID-19 research.
Other students introduce themselves, sharing their names, origins, and programs of study.
Phage Display Technology
Molecular Display
When a molecule is presented on the surface of a cell, ribosome, or mRNA with a covalent link, it's considered a molecular display. This concept is essential for understanding phage display technology.
Phage Definition
Phage refers to viruses infecting only bacteria, which means they don't harm eukaryotic cells. Phages are manipulated to apply this technology for drug discovery.
If a foreign gene is inserted into the phage genome, it creates a fusion. When this genome is expressed (DNA transcription, translation), and the resulting protein is part of the minor coat protein of the virus, it is displayed. This is phage display technology.
This technology requires that the genome within the phage capsid contains the gene of the foreign protein, and the protein is displayed on the surface. This links the genotype (genome) with the phenotype (functional molecule).
Biology Definition
Biology refers to anything used to cure a disease, specifically biologic peptide-related compounds. If protein A binds to protein B and this interaction harms the cell, targeting this interaction becomes important. A peptide that prevents protein B from binding to A can serve as a biologic to reduce that interaction.
Mimicking Peptide
A mimicking peptide involves taking a peptide structure and finding any chemical that mimics it, using software. This chemical can then be used in place of the peptide. Such chemicals are called peptido-mimetics.
Nanobody
Nanobodies discovered during COVID-19 research are single-chain antibodies that block receptors. They are small enough to be displayed on the surface and can interact to prevent or stimulate pathways.
Tags, such as CMV, T7, and histidine tags, help purify specific proteins or signal their presence. Phage technology can also lead to biosensors that detect specific molecules, adding more tools to biology.
Limits of Using Peptide
Peptides are easily degradable and antigenic, meaning that they stimulate responses from the body's defenses, and they are degradable because the body's first defense is made of peptidase. To avoid peptide degradation, D isomers can be synthesized instead of L isomers (naturally accepted by the body).
Circularizing peptides is also more effective. Issues with peptides include degradation, antigenicity, and aggregation. Phage display applications can help overcome these problems.
Nobel Laureate George Smith
2018 Nobel Laureate George Smith's work with the DNA M13 phage is foundational to phage display technology. The presenter developed an RNA incubator for phage display. DNA replication systems have proofreading, while RNA phages lack this, leading to greater diversity.
Different Phages
Different phages were tentatively used. Cubit is even nanometer in diameter, whereas another is almost a micrometer. It can affect bacteria in animal power by killing them. Phages are part of the microbiome regulating the level of bacteria.
RNA and DNA Phages
Two phage systems are commonly used: RNA and DNA. DNA phages have a long structure, while RNA phages are equally sprayed on the surface. The key difference is that DNA has proofreading capabilities, while RNA does not.
DNA systems require a phage mid (plasmid with a structural gene) and a helper. The minor structural gene is fused with a foreign gene for display.
DNA system
In the DNA system, the foreign gene with a minor structural gene are getting the helper, and you will get your phages.
A library is a bunch of molecules with different positions. A mil library, it's like taking the amino acid 20 times those 20. Libraries are crucial for discovering biology because they combine nucleotides to create various amino acid sequences.
Using the library to discover biology
The first step is to immobilize the target to have a surface binding to your library. This is performed by expressing proteins with specific binding properties with the help of companies.
If the desired surface is the N-terminal, the C-terminal is fused with an FC, allowing the protein to be positioned on the plate. The library is then introduced, specific binding occurs, nonspecific elements are washed away.
The molecules are in acidic environment and neutralized after washing. The selected candidate is amplified, with cycles repeated up to six times to filter out any nonspecific sequences. Sequencing each candidate leads to enrichment, which is the process of amplifying specific sequences until they dominate the sample to make them very specific against Biotins.
Biopanning
This is known as penning or biopanning because it is biological at the level. Involves a library and will select what you want. The key technology for selecting a specific candidate from a library.
Cubita Phase (RNA)
The advantage of this system is that the cell doesn't have to be removed and amplified like M13 phage. The A1 with 12 binding affinity has high capacity. The cell is added on target with A1 binding allowing A2 to infect and make more protein and RNA, enabling the completion of many cycles in a day.
RNA
With the RNA phage, after washing the plate, the cells are added to perform the amplification directly without a harsh pH elution. It is composed of four genes, a node nine and the code protein and the A1, they have the same beginning. But here there is a read through one time over for a reason we don't know, even to this day, we always have it going through
All is the same: Library, Expression, Library of Phage, Library of DNA, Library of Phage, Selection, Amplification, Enrichment, Specific Target. Against one of the protein, right?
Qb Virus Cycle
In the Qb virus cycle, A1 is the protein binding the receptor and A2 is the bacteria used to attach. Through A2 they inject the rna from the tail to the cytosol. The A1 is the one binding the tail of the bacteria and it attaches through the A2 allowing the phage to inject its genetic material. The cycle of Q-beta is to minutes.
Library Construction
The RNA phage is copied into cDNA, and any foreign sequence is inserted. The cDNA is in a stable plasmid, and there is no need for a helper. When inserted into the minor cord, the phage is released because the plasmid has transduplication allowing the plasmids to transcribe.
The first library used Randomized five amino acids as a proof of concept. If the five mer is added, it checks if some epitope are just four amino acids.
Determining Motifs
Phage display can be used to identify motifs. Large protein can be chopped into overlapping fragments, fused with phages, and selected against antibodies or receptors.
Library Design Considerations
When designing libraries, it is essential to assess the insertion site of the minor code protein and predict how the structure will change using AlphaFold to guarantee phage function. When we design is to be able to get the A1 like this and see what that structure would do using AlphaFold. If I insert something and you have a different portion of insertion. So
The secondary structure of RNA is another consideration. Also confirming phage particles with plaque assays confirms their presence post-transfection or transformation.
Conclusion
During the COVID-19 pandemic, the spike protein was targeted for vaccine design. However, there are portions of the virus that binds to receptors. This technology can identify key epitopes. It can then be used to identify a biotin or other protein that will be appropriate for detecting specific sequence.
Dr. Waffle introduces himself, expressing his preference for the nickname 'Waffle' to simplify interactions due to the complexity of his first name. He shares insights into his academic and professional background, highlighting his extensive experience in virology, particularly with respect to viruses that affect human health. His involvement in critical research during the COVID-19 pandemic aimed at understanding virus behavior and developing effective countermeasures showcases his commitment to advancing public health through science.
Other students in the room also introduce themselves. They share their names, origins, and programs of study, illustrating a diverse group of individuals from various academic and cultural backgrounds, bringing different perspectives and experiences to the learning environment.
Phage Display Technology
Molecular Display
Molecular display refers to the presentation of a specific molecule on the surface of a cell, ribosome, or mRNA, achieved through a covalent link. This is a crucial concept in the study of phage display technology, which relies on successful interaction between the displayed molecules and target entities to facilitate drug discovery and development.
Phage Definition
Phages, or bacteriophages, are viruses that exclusively infect bacteria without harming eukaryotic cells, making them ideal tools for various biotechnological applications. The manipulation of phages to apply this technology in drug discovery, diagnostics, and therapeutics underscores their importance in modern biomedical research.
If a foreign gene is inserted into the phage genome, it creates a fusion protein. Upon expression of this genome (which includes processes like DNA transcription and translation), the resulting protein, which is often integrated into the minor coat protein of the virus, will be displayed on the phage surface. This aspect of phage display technology enables researchers to link the genotype (the genetic code within the phage) with the phenotype (produced functional molecule).
Biology Definition
In a broad context, biology encompasses various biocompounds used to cure diseases, specifically focusing on biologic peptide-related compounds. For instance, if protein A binds to protein B and this interaction has deleterious effects on a cell, it becomes critical to target this interaction. A synthetic peptide designed to inhibit the binding of protein B to A can serve as a biologic agent to mitigate these unwanted interactions.
Mimicking Peptide
A mimicking peptide embodies the structural characteristics of a naturally occurring peptide. Identifying chemical compounds that can mimic these peptides, often through computational software, allows for the development of novel drugs. These compounds, referred to as peptido-mimetics, can serve as substitutes in therapeutic applications.
Nanobody
Nanobodies, discovered during research pursuits surrounding COVID-19, represent a class of single-chain antibodies that uniquely block receptor interactions. Their small size enables them to be effectively displayed on the surface of phages, thus allowing them to interact with target pathways to either inhibit or enhance biological responses.
Tags—including CMV, T7, and histidine tags—facilitate the purification of specific proteins or signal their presence in various experimental contexts. Furthermore, advancements in phage technology may contribute to the development of biosensors capable of detecting specific molecules, which adds profound versatility to the field of biology.
Limits of Using Peptide
Despite their advantages, peptides exhibit significant challenges such as susceptibility to degradation by peptidases and potential antigenic responses that can elicit immune reactions. To overcome these challenges, D-amino acid isomers can be utilized instead of the more common L-isomers. Additionally, employing circularization of peptides is a method to enhance their stability. Issues relating to peptide degradation, antigenicity, and aggregation are prevalent, but phage display technology provides a promising avenue to address and mitigate these complications effectively.
Nobel Laureate George Smith
2018 Nobel Laureate George Smith's pioneering work with the DNA M13 phage serves as a foundational pillar of phage display technology. His contributions significantly improved our understanding of phage biology. The development of an RNA incubator for phage display represents a significant leap forward, particularly as DNA phage systems possess proofreading capabilities that RNA phages lack, resulting in increased variability in the phage population.
Different Phages
Various phages show a range of structural and functional versatility. For example, the Cubit phage, which measures approximately nanometers in diameter, stands in contrast to larger phages, some reaching almost a micrometer in size. These entities play a pivotal role in the microbiome, directly regulating bacterial populations and influencing health outcomes.
RNA and DNA Phages
The two primary classes of phages utilized in research and industry are RNA and DNA phages. DNA phages are often characterized by their elongated structure and sophisticated genome. The distinctive ability of DNA to undergo proofreading significantly impacts the fidelity of genetic transactions within the phage. In notable contrast, RNA phages lack this proofreading mechanism, which results in a higher mutation rate.
In constructing DNA systems, one requires a phage mid, often a plasmid carrying a structural gene, along with a helper mechanism to facilitate successful genome incorporation of a foreign gene, typically fused with a minor structural gene for optimal display.
DNA System
In a typical DNA system, the integration of the foreign gene to the minor structural gene is facilitated through the helper phage to produce usable phage particles for experimental work. The design of a molecular library, often comprising a plethora of molecules with diverse positions, is fundamental to discovering biological interactions. For example, a million library represents the exhaustive combination of amino acids across numerous sequences, critical in the study of molecular biology.
Using the Library to Discover Biology
The initial step in using a molecular library for discovery purposes entails immobilizing the target molecule to provide a surface for binding with the library constituents. This can typically be achieved by utilizing proteins known for their specific binding properties, often obtained through collaborative work with specialized companies.
When targeting specific surface properties, fusion techniques can be employed where a protein's N-terminal is linked to an FC tag at the C-terminal, thereby ensuring the correct orientation for binding during experimental setups. Following the introduction of the library, effective binding occurs, while nonspecific interactions are systematically washed away.
Post washing, the bound molecules are placed in an acidic environment and neutralized, allowing the selected candidates to undergo amplification through repeated cycles that can extend to six iterations. This process of sequencing each candidate ultimately leads to enrichment, optimizing specific sequences until they predominate the sample, enhancing specificity against biotin or other key targets.
Biopanning
The process of biopanning, also known in some contexts as penning, is essential for the biological selection of desired candidates from a molecular library. This fundamental technology enables researchers to isolate specific candidates based on their interactions within the library context.
Cubita Phase (RNA)
The Cubita phase presents significant advantages, allowing for the amplification of phage particles within a living cell context, eliminating the need for external removal processes needed with M13 phage. The A1 subtype exhibits exceptional binding affinity, facilitating high-capacity responses. Adding a target cell allows for enhanced protein and RNA production through infection cycles, greatly increasing efficiency.
RNA
In utilizing RNA phages, following the binding and washing phases, the cells can be introduced directly for amplification without harsh elution conditions. The structural composition includes four essential genes, leading to dynamic protein interaction and expression cycles. The potential variation in the gene output remains an intriguing field of investigation, with numerous unknowns still prevalent in RNA phage functioning.
Qb Virus Cycle
In the Qb virus cycle, A1 refers to the critical protein responsible for receptor binding, while A2 designates the specific bacteria utilized for attachment. The phage employs A2 to inject its RNA genome into the bacterial cytosol, taking advantage of specific receptor interactions to ensure successful genetic material transfer. The complete cycle duration of the Qb virus ranges between to minutes, indicating a rapid replication and infection process, which is a hallmark of phage biology.
Library Construction
Construction of an RNA phage library involves translating RNA into complementary DNA (cDNA), where any desired foreign sequence is integrated. The resultant cDNA can be stabilized within plasmid formats, negating the reliance on helper systems. Once incorporated into the minor coat structure, the phage is released, leveraging the transduplication capabilities of the plasmid for further transcription processes.
The initial library design employed randomized five-amino-acid sequences as proof of concept, facilitating checks on epitope formations within the full-length chains, providing rich data for identifying key protein interactions.
Determining Motifs
Phage display methodologies extend to motif identification wherein larger proteins can be fragmented into overlapping segments, specifically fused with phages for targeted selection against antibodies or receptors, showcasing the versatility of this technology in proteomic studies.
Library Design Considerations
Strategic considerations arise when designing molecular libraries, including assessing optimal insertion sites on the minor coat protein and predicting subsequent structural alterations using computational tools like AlphaFold. The structural integrity of designed libraries is critical to maintaining phage functionality, alongside confirming the presence of phage particles through plaque assays to verify success rates post-transfection or transformation.
Conclusion
Amidst the global COVID-19 pandemic, significant focus was directed towards the spike protein in vaccine development strategies. However, other viral components interacting with cell receptors also warrant attention. By employing phage display technology, it becomes feasible to identify pivotal epitopes which, in turn, can direct the discovery of suitable biotin or complementing proteins, aiding in the effective detection of specific viral sequences and ultimately contributing to enhanced public health responses.