MemDX™ CARRY Two-Hybrid Service for Unraveling RNA-Protein Interaction

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Creative Biolabs, with over two decades of expertise in molecular biology, proudly introduces our state-of-the-art MemDX™ CARRY Two-Hybrid Service, a revolutionary platform designed to explore this complex interactome with unparalleled sensitivity and reliability. Harnessing the power of the CRISPR-assisted RNA/RBP yeast (CARRY) two-hybrid system, our MemDX™ platform provides a robust, in vivo solution for identifying and characterizing RNA-RBP interactions on a genome-wide scale. Move beyond the limitations of traditional methods and unlock a deeper understanding of the post-transcriptional regulatory networks that drive your research forward.

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Introducing the Advanced CARRY Two-hybrid System

The CARRY two-hybrid system represents a significant leap forward from conventional yeast three-hybrid (Y3H) and in vitro assays. By ingeniously integrating CRISPR-dCas9 technology into a yeast two-hybrid framework, the CARRY system overcomes common issues of low sensitivity and high background noise.

How It Works:

The system is based on tethering a specific RNA "bait" to a reporter gene's promoter region.

RNA Bait Delivery: The RNA of interest is fused to a guide RNA (gRNA), which is then expressed in yeast.
CRISPR-dCas9 Tethering: A catalytically inactive Cas9 (dCas9) protein is fused to the LexA DNA-binding domain (DBD). The dCas9-LexA fusion is directed by the gRNA to bind specifically to a LexA operator sequence upstream of a reporter gene (e.g., HIS3, LacZ). This anchors the RNA bait to the promoter.
RBP Prey Screening: A cDNA library of potential RBPs is expressed as fusions to a transcription activation domain (AD).
Interaction & Activation: If an RBP "prey" from the library binds to the RNA "bait," the AD is brought into proximity with the promoter, activating the reporter gene and allowing yeast cells to grow on selective media. This positive selection makes identifying genuine interactors highly efficient.

This innovative CRISPR-assisted RNA/RBP yeast (CARRY) two-hybrid system forms the technological backbone of our MemDX™ platform, ensuring the highest quality data for your projects.

Fig. 1 Mechanisms of CRISPR/Cas9-mediated genome editing and epigenome modulation. (OA Literature). Fig. 1 Epigenome and RNA editing via CRISPR.¹

Workflow of the MemDX™ CARRY Two-Hybrid Service

We have streamlined the entire process into a transparent and efficient workflow, ensuring you are informed at every stage.

Step 1: Project Consultation & Design

Our PhD-level scientists consult with you to understand your research goals.
We assist in designing the optimal RNA bait construct and selecting the appropriate prey library.

Step 2: Bait & Prey Construct Generation

Synthesis and cloning of the gRNA-RNA bait construct.
Verification of dCas9-LexA expression and prey library quality.

Step 3: CARRY Two-Hybrid System Screening

Transformation of yeast reporter strains with bait and prey constructs.
High-throughput screening on selective media to isolate positive colonies.

Step 4: Hit Confirmation and Validation

Plasmids from positive clones are isolated and sequenced to identify the interacting RBP.
Re-transformation and one-on-one interaction assays are performed to eliminate false positives.

Step 5: Data Analysis & Reporting

Comprehensive bioinformatics analysis of the identified protein hits.
A detailed final report is delivered, including raw data, validated interactor lists, and expert interpretation.

Comparative Analysis of RNA-RBP Interaction Screening Methods

To highlight the superiority of our platform, here is a comparison of the CARRY Two-Hybrid system against other common methodologies.

Feature	MemDX™ CARRY Two-Hybrid	Yeast Three-Hybrid (Y3H)	RIP-Seq/CLIP-Seq	*EMSA / In Vitro* Assays**
Environment	In vivo (Yeast)	In vivo (Yeast)	In vivo (Cell culture)	In vitro
Principle	CRISPR-dCas9 tethering	Hybrid RNA bridge	Immunoprecipitation	Gel mobility shift
Sensitivity	Very High	Moderate to Low	Moderate	Low (for discovery)
False Positives	Very Low	High	Moderate	N/A (not for screening)
Bait Requirement	Any RNA sequence	Requires specific hybrid RNA design	Requires high-quality antibody	Purified RNA and protein
Format	High-throughput screening	Screening is possible but cumbersome	Screening is indirect	Low-throughput
Primary Use	Discovery of novel interactions	Validation / limited screening	Mapping binding sites of a known RBP	Validation of a single interaction

Applications of MemDX™ CARRY Two-Hybrid System

The insights generated by our MemDX™ CARRY Two-Hybrid Service can accelerate a wide range of research areas:

Functional Genomics: Elucidating the function of novel lncRNAs by identifying their protein partners.
Drug Discovery: Identifying RBPs that interact with viral RNA genomes, providing novel targets for antiviral therapeutics.
Disease Mechanism Research: Characterizing how mutations in RNA or RBPs disrupt critical interactions in genetic diseases.
Systems Biology: Mapping comprehensive RNA-protein interactome networks to understand cellular regulation on a global scale.
Biomarker Discovery: Identifying RNP complexes that are unique to diseased states.

Advantages of Our MemDX™ CARRY Two-Hybrid Service

Choosing Creative Biolabs means partnering with a leader in molecular interaction analysis. Our service is distinguished by:

Unmatched Sensitivity: The CRISPR-based tethering mechanism significantly boosts the signal-to-noise ratio, enabling the detection of weak or transient interactions missed by other systems.
Exceptional Specificity: The precise targeting of dCas9 minimizes off-target effects and dramatically reduces the rate of false positives, saving you valuable time in downstream validation.
Versatile Bait Design: Our platform accommodates a wide range of RNA baits, including long non-coding RNAs (lncRNAs), mRNAs, viral RNAs, and specific RNA fragments or structural motifs.
Comprehensive Library Screening: We offer both pre-made and custom cDNA library screening services to identify interacting partners from any species or tissue type.
Full-Spectrum Service: From initial consultation and construct design to library screening, hit confirmation, and detailed bioinformatics analysis, our expert team manages the entire workflow.
Rapid Turnaround: Our optimized protocols and dedicated project managers ensure you receive actionable data in a timely manner.

Explore Our Related Services

Creative Biolabs offers a comprehensive suite of services to complement your RNA-RBP interaction studies. Explore our other platforms to take your research to the next level. Discover our advanced solutions for PNA analysis: Protein-Nucleic Acid Interaction (PNI) Assay Services

Propel your research into the intricate world of RNA-RBP interactions. Contact us today to discuss your project with one of our experts and receive a customized quote for our MemDX™ CARRY Two-Hybrid Service.

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Frequently Asked Questions (FAQs)

Q: What kind of RNA can be used as bait in the CARRY two-hybrid system?

A: The system is highly versatile. You can use full-length mRNAs, lncRNAs, viral RNAs, or specific sub-fragments and structural domains. Our team can help you design the optimal bait for your target.

Q: How does the CARRY system compare to CLIP-Seq?

A: They are complementary. The CARRY two-hybrid system is a discovery tool used to identify what proteins bind to a specific RNA. CLIP-Seq is a mapping tool used to identify the specific binding sites across the entire transcriptome for a known RBP. Using CARRY first to find the RBP and then CLIP to map its sites is a powerful strategy.

Q: Can you screen a custom library?

A: Absolutely. In addition to our high-quality pre-made libraries, we can screen a custom cDNA library provided by you or constructed by us from your specific cell line, tissue, or organism of interest.

Reference

Fadul, Shaima M., Aleeza Arshad, and Rashid Mehmood. "CRISPR-based epigenome editing: mechanisms and applications." Epigenomics 15.21 (2023): 1137-1155. Distributed under Open Access license CC BY 4.0, without modification. https://doi.org/10.2217/epi-2023-0281

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