Helicase Activity Measurement of a Target Protein Using Biotin-Labeled RNA Duplexes

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.

For the MOV10 helicase activity assay, mix the reagents according to the manuscript directions, and add water for a final reaction volume of 20 microliters. Incubate the reaction at 37 degrees Celsius for 10 minutes, 30 minutes, and 60 minutes, and then, add 5X Stop Buffer to stop the reaction. Then, prepare a 20% native polyacrylamide gel and load 20 to 25 microliters of each sample into each well. Run the gel at 100 volts on an ice bath until the bromophenol blue marker has migrated to the bottom quarter of the gel.

Acrylamide is harmful and toxic. It is important to handle it with appropriate personal protective equipment.

Disassemble the gel plates and trim the gel by removing loading wells and unused lanes. Place the gel in 0.5X TBE buffer. Cut filter paper and nylon membrane to the size of the gel, pre-wet the paper and membrane, and assemble the stack for transfer.

Transfer the samples from the gel to the membrane in a semi-dry electrophoretic apparatus at 90 milliamperes for 20 minutes. Then, cross-link the samples by irradiating the membrane at 120 millijoules per centimeter squared for 45 to 60 seconds.

For chemiluminescence detection, begin by adding 20 milliliters of blocking buffer to the membrane and incubating it for 15 to 30 minutes while gently shaking. Carefully remove the blocking buffer, and replace it with conjugate blocking buffer. Incubate the membrane again for 15 minutes.

Wash the membrane four times while shaking for 5 minutes per wash. Then, add 30 milliliters of substrate equilibration buffer to the membrane, and incubate it for 5 minutes while shaking at 20 to 25 RPM.

Cover the entire surface of the membrane with substrate working solution and incubate for 5 minutes. After the incubation, scan the membrane in a chemiluminescent imaging system for 1 to 3 seconds.