Creative Biolabs is dedicated to providing yeast three-hybrid (Y3H) services including library construction and screening, which are introduced to assay RNA-protein interactions and devised to the selection of RNA binding protein with modifications to use a small RNA as bait. In 1996, the Wickens and Kuhl labs developed the yeast three-hybrid system independently. This system also makes it possible to identify those regions of an RNA or protein that are required for a known interaction and to test the combinations of RNA and protein to confirm whether they interact in vivo.

The Functions of Y3H

It is well-known that Y3H methods can be applied in a variety of research fields:

a. Finding protein partners of a known RNA sequence from cDNA libraries
b. Searching a natural RNA partner or ligand for a known RNA binding protein via RNA libraries
c. Testing suspected RNA-protein interactors
d. Mutational analysis of interacting RNA and protein
e. Discovery of multiprotein-RNA complexes

In addition, Y3H is still a sensitive method to screen for the interactions between small molecule drugs and their protein targets.

The Principle of Y3H

Yeast three-hybrid system is a derivative of yeast two-hybrid (Y2H). It’s a kind of powerful tool to dissert RNA-protein interactions of interest and that typically consists of three chimeric components.

The first hybrid protein is made up of an RNA binding protein (RBD) fused to a DNA binding domain (DBD). The second fusion protein molecule contains a second RNA binding protein fused to the transcriptional activation domain (AD). The third hybrid part is an RNA molecule which bridges above two fusion proteins by providing two specific RNA targets for the RNA binding proteins. When this tripartite constituent forms at a promoter, the reporter gene is turned on, even transiently. And the expressed reporter products can be recognized by simple biochemical or phenotypic assays.

Fig.1 General strategy of Y3H system.

Key Benefits of Y3H

In an RNA Y3H method, there are lots of great advantages presented on Creative Biolabs’ platforms:

• Fast and sensitive approach to identify and characterize RNA-protein interaction
• Applicable to a wide range of RNA-protein interactions
• Detection of both very strong and very weak interactions of RNA and proteins
• Assay results monitored easily by cell growth, colony color, or the levels of a specific enzyme
• The species of yeast is an ideal model organism in eukaryotic genetics
• Technique improved to reduce the selection of false-positive clones

RNA-protein interactions are essential for the proper execution and regulation of each step in the life of eukaryotic mRNAs, such as splicing, translational control, mRNA processing, and infection of RNA viruses. With this, Creative Biolabs provides a potentially effective technique by which to examine RNA-protein interactions towards a variety of purposes. The final submitted deliverables include an exhaustive report, a full list of identified protein partners, and interaction domain on each protein, together with scientific assistance whenever clients or their projects need for the better outcome.

Other optional protein-nucleic acid interaction (PNI) assay services:

• Yeast One-Hybrid (Y1H) Service
• Magic™ CARRY Two-Hybrid Service

Published Data

• Study of Interaction between 3c and Receptors by Yeast Three-hybrid Screening

Fig. 2 Binding mode of compound 3c within acetylcholinesterase (A) and butyrylcholinesterase (B). (Kamila Czarnecka, 2020)

Here, the researchers designed and synthesized a series of novel tetrahydroacridine and 3,5-dichlorobenzoic acid hybrids with different spacers and evaluated their ability to inhibit two kinds of cholinesterase. These four compounds—3a, 3b, 3f, and 3G—showed selective inhibition of butyrylcholinesterase. Among them, compound 3b had the highest activity, and 3c showed double cholinesterase inhibitory activity and had the highest acetylcholinesterase (AChE) activity. They also tested the ability of the active compound 3c to inhibit A β aggregation and studied the catalytic active site (CAS) and peripheral anion site (PAS) of 3c targeting AChE. In addition, 3c seems to have neuroprotective activity and can be regarded as a free radical scavenger. It also shows little inhibitory activity on hyaluronidase (HYAL), which may indicate that it has anti-inflammatory properties. Finally, the researchers screened the new in vivo interaction between 3c and known receptors using yeast three-hybrid (Y3H) screening.

FAQ

1. What is a Yeast Three-Hybrid (Y3H) Assay?

   The Yeast Three-Hybrid (Y3H) assay is an extension of the Yeast Two-Hybrid (Y2H) system, designed to study protein interactions with various molecules, typically RNA or small molecules. This method involves three components: a DNA-binding domain fused to a protein of interest, an activation domain fused to another protein or molecule, and a reporter gene that is activated only when all components come together in the yeast nucleus. This assay is particularly useful for identifying and characterizing the interaction between a protein and a third molecule, providing insights into complex biological processes and potential therapeutic targets.

2. How is the Yeast Three-Hybrid (Y3H) Assay used in research?

   Researchers use the Y3H assay to explore interactions that involve not just two proteins, but also an additional molecule such as RNA, small peptides, or other small molecules. This capability is invaluable for understanding molecular mechanisms that involve complex formations beyond simple protein-protein interactions. For example, it can be used to identify RNA sequences that bind to a specific protein, or to screen for small molecules that can disrupt or enhance interactions between known binding partners. The versatility of the Y3H system makes it a powerful tool in drug discovery, genetic research, and the study of disease mechanisms.

3. What are the advantages of using the Yeast Three-Hybrid (Y3H) Assay?

   First, it allows the identification of interactions between proteins and other types of molecules, broadening the scope beyond protein-protein interactions. This is particularly useful for studying RNA-protein interactions or the influence of small molecules on protein complexes. Secondly, it provides a cost-effective and relatively straightforward method that can be performed in a basic laboratory setting without the need for expensive equipment. Additionally, the Y3H system is highly scalable, making it suitable for high-throughput screening applications.

4. What are the limitations of the Yeast Three-Hybrid (Y3H) Assay?

   The artificial nature of the yeast cellular environment can affect the relevance of the interactions observed to human cells or other biological systems. There may be differences in post-translational modifications and other cellular contexts that could influence the interaction dynamics. Also, the assay might not detect weak or transient interactions effectively due to the stability requirements of the three-component complex. Moreover, the potential for false positives or negatives necessitates careful control experiments and validation of results through additional methods.

5. What kind of molecules can be studied using the Yeast Three-Hybrid (Y3H) Assay?

   While traditionally used for RNA-protein interactions, it has been adapted to explore interactions with small peptides, DNA segments, and even small organic or inorganic compounds. This adaptability makes the Y3H assay a valuable tool in fields such as pharmacology, where it can be used to screen for potential drug molecules that affect protein interactions, or in genetic research to explore how certain molecules influence gene expression or protein function.

6. How are the results of a Yeast Three-Hybrid (Y3H) Assay interpreted?
   • Reporter Gene Activation: The primary readout is the activation of the reporter gene, which indicates that the three components are interacting within the yeast cells.
   • Quantitative vs. Qualitative Data: The assay can provide qualitative data (whether or not an interaction occurs) and quantitative data (the strength of the interaction based on the level of reporter gene expression).
   • Validation of Interactions: Due to potential false positives/negatives, interactions identified in Y3H assays often require further validation using alternative biochemical or biophysical methods, such as co-immunoprecipitation or fluorescence resonance energy transfer (FRET).
   • Contextual Factors: Results need to be interpreted in the context of the yeast cellular environment, recognizing that some interactions may not occur similarly in more complex or different cellular contexts.
7. How can the specificity of interactions in a Yeast Three-Hybrid (Y3H) Assay be enhanced?
   • Vector and Host Optimization: Using yeast strains and vectors specifically designed for Y3H systems can reduce background activation of the reporter gene, leading to clearer interaction signals.
   • Use of High-Fidelity Enzymes: Employing high-fidelity enzymes in the preparation of DNA constructs can decrease the likelihood of mutations that might lead to non-specific interactions.
   • Titration of Components: Adjusting the concentrations of the hybrid proteins and the bridging molecule can help establish conditions where only specific interactions lead to reporter activation, reducing false positives due to overexpression artifacts.
8. What advancements have been made in Yeast Three-Hybrid (Y3H) technology?
   • Automated High-Throughput Screening: Developments in automation have facilitated high-throughput screening capabilities, allowing for the rapid testing of thousands of interactions across varied conditions.
   • Integration with Next-Generation Sequencing (NGS): Some protocols now combine Y3H with NGS to rapidly identify and characterize interaction partners, significantly speeding up the discovery process and increasing the assay's throughput.
   • Enhanced Reporter Systems: Newer reporter genes that offer more sensitive and quantitative readouts of interactions have been developed, providing clearer and more actionable data for researchers studying complex molecular interactions.

Resources

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