Helicase Activity Measurement of a Target Protein Using Biotin-Labeled RNA Duplexes
Transcrição
Proteins with RNA helicase activity can help unwind and separate individual RNA molecules in an RNA duplex.
To determine a protein's helicase activity, take tubes containing a mixture of biotin-labeled RNA duplexes with a 5'-overhang, ATPs, and RNA traps — unlabeled strands complementary to the duplexes' biotinylated strands.
Add the protein with helicase activity to each tube and incubate for different durations. The protein binds to the duplexes' 5'-overhang.
Using ATP molecules, it breaks hydrogen bonds between two duplex strands, resulting in their unwinding and releasing the biotinylated single-stranded RNA. The RNA traps interact with the biotin-labeled strands, preventing reannealing with the separated strands.
Terminate the reaction using a termination buffer.
Load the samples in different wells of a native polyacrylamide gel. Run electrophoresis. Low-molecular-weight, single-stranded RNA moves faster than high-molecular-weight duplexes. This mobility shift generates two distinct bands.
Blot the gel on a nylon membrane, transferring the RNAs to the membrane. Post-blotting, expose the membrane to UV light, crosslinking the RNAs onto the membrane.
Add chemiluminescent enzyme-conjugated streptavidin, which interacts with the biotin on the duplexes and single-stranded strands. Use chemiluminescent substrates to produce chemiluminescence products.
Upon imaging, the low-molecular-weight band's intensity increases over time, correlating with the target protein's helicase activity.
