In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

This protocol offers a biotin-labeled platform for the study of protein-nucleic acid interactions that proves to be robust, reliable, efficient, and affordable. The same batch of the biotin-labeled nucleic acids can be used over a long period of time, maintaining reproducibility of experiments. Finally, all biotin-labeled assays described here can be performed within a day and do not require special equipment.

Demonstrating the procedure will be Lina Yu, a postgraduate from our laboratory. Prepare MEIOB and MOV10 constructs, and transform them into the appropriate bacteria. To extract MEIOB, pellet the cells via centrifugation and resuspend them in 20 milliliters of ice-cold Dulbecco's phosphate buffered saline, or DPBS, buffer.

Sonicate the bacterial suspension on ice. Then, centrifuge the lysate at 12, 000 times g and four degrees Celsius for 15 minutes, and transfer the supernatant to a fresh tube. Pre-wash glutathione-Sepharose beads, and incubate them with the lysate at four degrees Celsius for two hours.

Centrifuge the mixture at 750 times g and four degrees Celsius for one minute to pellet the beads. Rinse the beads with 10 milliliters of ice-cold PBS eight times. Then, add one milliliter of elution buffer, incubate the beads at four degrees Celsius for 10 minutes, and elute the beads via centrifugation.

Repeat the elution six times, and pool together the six fractions. Transfer the eluted proteins into a centrifugal filter, and concentrate by centrifugation at 7, 500 times g for a final volume of 100 to 200 microliters. To extract MOV10, express the protein in HEK293T cells and pellet the cells according to the manuscript directions.

Resuspend the pellet in three milliliters of cell lysis buffer with complete EDTA-free protease inhibitor cocktail, and incubate for 30 minutes on ice. Then, centrifuge the lysate at four degrees Celsius. Prepare anti-FLAG magnetic beads by washing them twice with K150 buffer.

After the wash, resuspend the beads in one milliliter of ice-cold K150 buffer and incubate them for two minutes at four degrees Celsius with gentle rotation. Collect the beads on a magnet, and remove the supernatant. Add the beads to the cell lysate, and incubate at four degrees Celsius for two hours.

Wash the beads with K150 buffer according to the manuscript directions, and resuspend the beads in 300 microliters of FLAG elution buffer. Place the beads on a magnet, collect the supernatant with the MOV10 proteins, and determine the protein concentration. Prepare the RNA oligonucleotides by diluting each oligo to 20 micromolar and annealing them to form RNA duplexes according to the manuscript directions.

For the MEIOB electrophoretic mobility shift assay, or EMSA, mix the reagents according to the manuscript directions. Add water for a final reaction volume of 20 microliters. Incubate the reaction at room temperature for 30 minutes, and then add 5x stop buffer.

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, crosslink 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 five minutes per wash.

Then, add 30 milliliters of substrate equilibration buffer to the membrane, and incubate it for five minutes while shaking at 20 to 25 rpm. Cover the entire surface of the membrane with substrate working solution, and incubate for five minutes. After the incubation, scan the membrane in a chemiluminescent imaging system for one to three seconds.

Purification of the MEIOB proteins A, C, and E can be verified with Coomassie blue staining and western blot analysis. The red arrows indicate the positions of the purified MEIOB proteins. Interactions of MEIOB and single-stranded DNA can be visualized using EMSA.

Concentration-dependent binding and cleavage are observed. As expected, only the wild-type MEIOB-A interacts with the DNA while the mutant MEIOB-E and MEIOB-C do not. Interactions of MEIOB and single-stranded RNA can also be observed.

Wild-type MEIOB shows concentration-dependent binding and exonuclease activity on single-stranded RNA. However, quantitative comparison shows that MEIOB binds DNA with higher efficiency than RNA. Purification of MOV10 was verified with Coomassie blue staining, and the protein's helicase activity was measured on an RNA duplex with a five-prime overhang.

As reaction time increases, MOV10 progressively unwinds the double-stranded RNA. To reduce the cost of the assay, the chemiluminescence detection can be performed with either a two-fold dilution of the detection kit reagents or with self-made reagents. The protocol described here successfully used biotin-labeled nucleic acids as substrates for in vitro biochemical assays such as EMSA and for enzymatic reactions in which biotin has not been commonly used.