RNA Compete

RNA-binding proteins (RBPs) play important roles in regulating post-transcriptional processes such as splicing, polyadenylation, capping, modification, mRNA stabilization, and mRNA localization. Lifeasible is a leading biotechnology company. We have developed an advanced platform for the rapid characterization of the binding specificity of RBPs — RNA compete.

RNA compete is an in vitro protein-centric method used to analyze RNA-protein interactions. RNA compete allows for an estimation of relative preference for a large number of individual sequences using a single binding reaction. Moreover, RNA compete also can be used to design arrays that focus on variants of a specific type of sequence and structure. RNA compete involves in four basic steps (Figure 1):

• RNA pool generation

RNA compete relies on an RNA pool comprising a library of RNA sequences and structures. An RNA library is designed to include all possible 8-base sequences represented at least 12 times in unstructured RNAs and all possible 6-base and 7-base loop sequences (and 60% of 8-base loops) in RNA hairpins containing unique 10-base pair stems. ssDNA is generated using these sequences designed as a template and is then converted to dsDNA using PCR. Finally, an RNA pool is generated by in vitro transcription from the dsDNA and comprises specific structures.

• RNA pull down assay

Following the generation of the RNA library, transcripts are incubated with the protein of interest immobilized on an affinity matrix (e.g., streptavidin-biotin tag system). The RNAs bound to a tagged RBP of interest can be separated by the pull down assay. 

• Microarray analysis

The bound RNA sequences are subsequently fluorescently labeled and hybridized to a microarray of the same format as was used to generate the RNA library

• RNA compete data analysis

Computational analysis is used to assess the relative enrichment of bound RNAs relative to a sample from the starting library.

With years of experience in RNA-protein interaction, we are dedicated to providing high quality tailored one-stop services for RNA compete assay from RNA pool generation, RNA pull down assay, to microarray and computational analysis. We could develop novel solutions to meet the specific requirement of customers. Please feel free to contact us for further information.