High-stringency method for the isolation and proteomic identification of RNA binding proteins

Unmet Need

RNA binding proteins (RBPs) play a critical role in maintaining stable gene expression, and they are involved in several related functions throughout eukaryotic cells, including splicing and translation. RBPs interact with RNA in intricate ways to form RNA-protein complexes. The role of RBPs in disease, in particular as correlative markers, causative agents, or responsive mediators, is an active area of research. Additionally, researchers are considering RBPs as therapeutic targets for previously difficult-to-treat diseases like certain cancers. While there are several current methods for studying RBPs and their interactions with RNA, their usability and scalability is limited due to their low-throughput nature, low recovery of RNA-protein complexes, high cost, and the limited availability of the antibodies that these methods rely on. There is a need for a low-cost, scalable method to isolate RNA-protein complexes with speed and precision.

Technology

Duke inventors have developed a high-stringency method to purify RNA-protein complexes. This is intended to be used by researchers at any type of organization studying RBP interactions with RNA that require a cost-effective, high throughput methodology to study RBPs. This technology aligns with common, existing assays like mass spectrometry and Western blot. This technology could also be assembled into a kit. Specifically, this method uses AGPC to extract RBPs and a unique method to isolate RBPs. The approach involves selectively precipitating and extracting RNA-bound proteins, assessing RBP enrichment and RNA binding activity, and validating these interactions in vivo. This method allows researchers to validate endogenous RNA-protein interactions in a matter of hours with nearly 100% recovery of materials at a low cost. This has been demonstrated in bench top tests with pancreatic beta cell lines and metabolic syndrome models.

Advantages

• Competitively low-cost relative to current solutions
• High throughput and high efficiency, resulting in robust yields of RNA-protein complex in approximately 2 hours and recovery of RNA and associated RBPs
• Can potentially be turned into a kit