RNA Pull Down Assay Service

RNA-protein interactions play pivotal roles in various cellular processes, including mRNA assembly, cellular development, and disease pathogenesis. Understanding these interactions is essential for elucidating biological mechanisms and developing targeted therapies. At BOC Sciences, we offer cutting-edge RNA pull down assay services to facilitate the study of RNA-protein interactions with precision and reliability.

What is RNA Pull Down?

Currently, methods for studying RNA-protein interactions can be broadly categorized into two types. One focuses on the protein of interest and investigates the RNAs bound to it, such as RIP and CLIP. The other type centers on the RNA of interest and studies the proteins binding to it, including RNA pull down and ChIRP techniques. Among these, RNA pull down is a major method for detecting interactions between target RNAs and associated proteins, aiding in understanding RNA functionality and regulatory mechanisms, and deciphering the interaction networks between RNA and proteins. RNA pull down is a powerful technique used to study the interaction between RNA molecules and proteins. It allows researchers to isolate RNA binding proteins (RBPs) by capturing RNA-protein complexes formed in vitro. The interaction between RNA and its binding proteins (RBPs) is central to all gene expression processes, involving mRNA assembly, cellular development regulation, protein synthesis, and playing crucial roles in the onset and progression of various diseases. By using labeled RNA probes, researchers can specifically pull down target proteins bound to RNA molecules from complex cell lysates.

DNA Pull Down: A Comparison

While RNA Pull Down focuses on RNA-protein interactions, DNA pull down assays are designed to study DNA-protein interactions. DNA pull down typically involves immobilizing DNA fragments onto a solid support and incubating them with protein extracts to capture DNA-binding proteins. Both techniques offer valuable insights into gene regulation mechanisms but target different types of nucleic acid interactions.

Mechanism of RNA Pull Down

RNA pull down experiment typically involves incubating biotin-labeled RNA probes with cell/tissue protein extracts to form RNA-protein complexes. These complexes are then captured by streptavidin-coated magnetic beads, allowing the separation of RNA-protein complexes from other components in the incubation mixture through magnetic force. After elution, specific proteins interacting with RNA are detected through Western blot experiments, or mass spectrometry is employed to screen for unknown proteins binding to RNA.

Figure 1. The flowchart for the RNA pull down experiment includes the major steps of RNA probe preparation, RNA-protein binding, and protein identification. (Chopra, A.; et al, 2022)

BOC Sciences' RNA Pull Down Assay Service

BOC Sciences is committed to providing comprehensive RNA pull down assay services to global clients, catering to various diverse needs. With our extensive experience and professional expertise, particularly in the field of RNA-protein interaction analysis, we understand the significance of this technology. Therefore, our services are designed to ensure high sensitivity and reliable quality. Whether you are in the research field or pharmaceutical development, we customize RNA Pull Down services to meet your specific requirements. Our pricing strategy is competitive, allowing you to confidently use our services and focus on your research and projects. BOC Sciences looks forward to delivering outstanding services to you and collaborating with you to advance scientific research.

Service Process

Figure 2. Flowchart of BOC Sciences RNA pull down assay service.

a: In the process of constructing RNA overexpression plasmids, firstly, the RNA of interest is screened and identified through various methods, and the corresponding RNA nucleotide sequence is retrieved from databases. RACE amplification is then performed to confirm the presence of any unknown sequences. Subsequently, after confirming the sequence for RNA pull down, primers are designed to synthesize the entire target RNA sequence, which is then validated through sequencing to ensure accuracy. Finally, the overexpression plasmid is extracted and prepared for further use.

b: Using the constructed overexpression plasmid as a template and the designed synthetic primers, an in vitro transcription template is amplified. Gel electrophoresis is conducted to verify the amplification of PCR products, followed by excising and purifying the desired bands for subsequent use.

c: The purified PCR products are used as transcription templates for in vitro transcription, resulting in the generation of the target RNA.

d: Protein lysates are prepared in advance, and the RNA obtained from in vitro transcription is biotin-labeled and purified for later use. Additionally, the biotin-labeled RNA probe is incubated with streptavidin-coated magnetic beads to form complexes. These complexes, comprising the biotin-labeled RNA probe and streptavidin-coated magnetic beads, are used to enrich proteins.

e: Then, mass spectrometry identification is performed after silver staining.

Sample Requirements

• Sample Type: Cell or tissue lysates
• Sample Size: Cells (> 5×10⁸) or Tissue (> 500 mg)
• Sample Preservation: Cell samples or fresh tissue stored in liquid nitrogen and at -80°C

BOC Sciences' Advantages in Offering RNA Pull Down Assay Service

• Expertise: Our team of experienced scientists ensures the highest quality and reliability of results.
• Advanced Technology: We utilize cutting-edge equipment and methodologies to optimize RNA-protein interaction studies.
• Customization: We tailor our services to meet your specific research objectives, providing personalized solutions for every project.
• Comprehensive Analysis: In addition to capturing RNA-protein complexes, we offer comprehensive analysis services, including western blotting and mass spectrometry identification.

Application of RNA Pull Down

The interaction between RNA and proteins is crucial for maintaining cellular homeostasis. Disruption of this interaction can lead to cellular dysfunction and the occurrence of related diseases. Currently, apart from a few RNAs that function independently as enzymes, most RNAs exert their effects by forming RNA-protein complexes through binding to proteins. On one hand, RNA binding proteins (RBPs) participate in multiple post-transcriptional processes such as RNA synthesis, alternative splicing, modification, transport, translation, and intracellular localization. They regulate various biological processes, including mRNA stability, lncRNA activity, and miRNA silencing complex formation. On the other hand, RNA also modulates the activity, localization, or interaction of these proteins, thereby influencing various cellular events mediated by RBPs. Therefore, RNA pull down, as a common and important tool for analyzing the interaction between RNA and proteins, has wide applications in biological research and drug development.

• Functional Characterization: Investigating the role of specific RNA molecules in regulating protein function and cellular processes.
• Disease Mechanisms: Studying the dysregulation of RNA-protein interactions in disease states, including cancer, neurological disorders, and viral infections.
• Drug Discovery: Identifying potential therapeutic targets and developing novel RNA-targeted therapies for various diseases.

Case Study

Case study 1 Evaluate the binding affinity between His-Nab3 RRM (329–419) and biotinylated snR47 RNA probe.

Figure 3. Graph of the results of chemiluminescent detection of recombinant proteins and RNA probes incorporating His tags using HRP-conjugated HisProbe. (Chopra, A.; et al, 2022)

This case study focuses on investigating protein-RNA interactions using RNA pull-down. The objective is to assess the binding affinity between His-Nab3 RRM (329–419) and a biotinylated snR47 RNA probe. Recombinant His-tagged proteins and RNA probes are incubated for in vitro interactions, followed by RNA pull-down using streptavidin-coated agarose beads. Chemiluminescence assays are then employed to measure binding affinity, with dose-response curves determining KD values. Additionally, a comparison between wild-type and mutant proteins sheds light on the specificity of the binding interaction. Overall, this approach provides a concise method for studying protein-RNA interactions and offers insights into molecular mechanisms underlying cellular processes.

FAQ

1. How do proteins interact with RNA?

Proteins interact with RNA through specific binding sites, where amino acids of the protein directly contact nucleotides of the RNA molecule. Some proteins have domains or motifs that recognize certain RNA sequences or structures, facilitating binding. These interactions are crucial for gene expression, RNA processing, and other biological processes.

2. What does an RNA binding protein do?

RNA binding proteins (RBPs) regulate RNA metabolism by binding to RNA molecules, influencing processes like splicing, stability, localization, and translation. They help form RNA-protein complexes, modulate gene expression, and contribute to cellular functions and homeostasis.

3. How do you Biotinylate RNA?

Biotinylation of RNA can be achieved enzymatically using T4 RNA ligase or E. coli biotin ligase (BirA), or chemically using biotinylated nucleotides or reactive biotin derivatives. These methods attach biotin to RNA for various applications like affinity purification or detection assays.

4. What is the difference between clip-seq and rip-seq?

CLIP-seq cross-links RNA to proteins before sequencing, providing high-resolution mapping of direct interactions. RIP-seq immunoprecipitates RNA-protein complexes without cross-linking, capturing both direct and indirect interactions but with lower resolution.

5. How does RNA bind to protein?

RNA binds to proteins through specific interactions between amino acids in the protein and nucleotides in the RNA. This binding is facilitated by factors like hydrogen bonding and electrostatic interactions. RNA-binding proteins often have specialized domains that recognize and bind to specific RNA sequences or structures, allowing them to regulate RNA metabolism.

Reference

1. Chopra, A.; et al. Assessing the in vitro Binding Affinity of Protein-RNA Interactions Using an RNA Pull-down Technique. Bio Protoc. 2022, 12(23): e4560.