Novel method to isolate and identify specific RNA/DNA binding proteins
By Venu Gurram
Owing to their role in establishing cell asymmetry and their role in pathological conditions such as neurodegenerative diseases, it is very important to focus on eukaryotic RNA-binding proteins(RBPs) binding to a specific transcript(s). Although the biotin-conjugated method is currently in use to isolate RBPs, it has certain limitations such as background interference. Here, I am proposing a new idea which is simple, yet effective.
To isolate RBPs binding to specific RNA transcript, we can introduce a prokaryotic RBP binding site in the transcript of our interest while simultaneously co-expressing the corresponding prokaryotic RBP. Followed by RNA-Immunoprecipitation and mass spectrometry, we can identify RBPs binding to a specific transcript. This method, which is first of its kind, could have many advantages like identifying RBPs of prokaryotic-site linked endogenous transcripts(using genetically modified animals) while still retaining the native secondary structure of RNA.
Similarly, to isolate DNA binding proteins that bind to a specific DNA sequence(Promoter), we can introduce a prokaryotic DNA sequence in the DNA segment of our interest and co-express the corresponding prokaryotic DNA binding protein. This can later be followed by Chromatin Immunoprecipitation and mass spectrometry.