In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.
Characterization of protein binding to RNA in vivo remains a major challenge due to the complex nature of RNA and the many factors affecting its interaction with proteins. RNA binding protein or RBP-RNA affinity is measured inside live bacterial cells. Since cellular environment affects both the structure of RNA and resultant RBP-RNA binding, measuring the affinity in vivo is crucial for understanding such interactions in natural systems.
The secret to success is in the design of the plasmids. The design should use several waves of delta and avoid insertion of stop codons and frameshift mutations. Begin this protocol by designing the binding site cassette.
Each mini gene contains an EagI restriction site, 40 bases from the five prime end of the kanamycin resistance gene, the pLac/Ara promoter, the ribosome binding site, AUG of the mCherry gene, a spacer, an RBP binding site, 80 bases from the five prime end of the mCherry gene, and an ApaLI restriction site. To increase the success rate of the assay, design three binding site cassettes for each binding site with spacers consisting of at least one, two and three bases. Double digest both the mini genes and the target vector with EagI HF and ApaLI before performing column purification of the digests.
Ligate the digested mini genes to the binding site backbone containing the rest of the mCherry reporter gene, terminator and a kanamycin resistance gene. Then transform the ligation solution into Escherichia coli top 10 cells. After identifying positive transformants via Sanger sequencing, store purified plasmids in the 96 well format at minus 20 degrees Celsius.
Also store the bacterial strains as glycerol stocks in the 96 well format at minus 80 degrees Celsius. Custom order the required RBP sequence lacking a stop codon as a double-stranded DNA mini gene with restriction sites at the ends. Transform the prepared plasmids into chemically competent bacterial cells already containing an RBP mCerulean plasmid.
To save time, plate the cells using an eight channel pipetter on eight lane plates containing Luria-Bertani agar with relevant antibiotics. Colonies should appear in 16 hours at 37 degrees Celsius. Select a single colony for each double transformant and transfer to 48 well plates containing 1.5 milliliters of LB with appropriate antibiotics.
Grow the cells over a period of 18 hours at 37 degrees Celsius shaking at 250 RPM. Store the overnights as glycerol stocks at minus 80 degrees Celsius in 96 well plates. In the morning, program the robot to warm 180 microliters of semi-pore medium or SPM in 96 well plates.
Program the robot to prepare the inducer plate. In a clean 96 well plate, prepare wells with SPM consisting of 95%BA and 5%LB. The number of wells corresponds to the desired number of inducer concentrations.
Add C4-HSL to the wells in the inducer plate that will contain the highest inducer concentration. Next, program the robot to serially dilute medium from each of the highest concentration wells into 23 lower concentrations ranging from zero to 218 nanomolar. The volume of each inducer dilution should be sufficient for all strains.
To dilute the overnight strains, program the robot to dilute by a factor of 10 by mixing 20 microliters of bacteria with 180 microliters of SPM in 48 well plates. Then dilute again by a factor of 10 by taking 20 microliters from the diluted solution into the pre-prepared strain plate suitable for fluorescent measurement. Now program the robot to add the diluted inducer from the inducer plate to the 96 well plates with the diluted strains according to the final concentrations.
Shake the 96 well plates at 37 degrees Celsius for six hours. During that time, take measurements of the optical density or OD at 595 nanometers as well as mCherry and mCerulean fluorescence via a plate reader every 30 minutes. For normalization purposes, measure growth of SPM with no cells added.
Shown here are three-dimensional plots for a positive strain. The plots depict raw OD levels, mCerulean fluorescence, and mCherry fluorescence as a function of time and inducer concentration. mCerulean steady-state expression levels were computed for each inducer concentration for both the positive and negative strains.
The mCherry production rate was also computed for each inducer concentration for both positive and negative strains. mCherry production rate was plotted as a function of mean mCerulean fluorescence averaged over two biological duplicates for two strains. The fit KRBP using the fitting formula is shown for the positive strain exhibiting a specific binding response.
For the negative strain, no KRBP value was extracted. Normalized dose response curves were determined for 30 different strains based on two RBPs as 10 binding sites at different locations. Three types of responses are observed, high affinity, low affinity, and no affinity.
Shown here are quantitative KRBP results for two RBPs, MS2 coat protein and PP7 coat protein with 10 different binding site cassettes. Dose response curves for MS2 coat protein with a mutant binding site at four different locations are shown here to demonstrate the effect of the mutant spacing on mCherry production. The sequence of the tested mutated binding site is shown.
It is important to use a structural technique such as shape seek to chemically probe the structure of the protein-RNA complex and to visualize which mRNA regions are affected by RNA binding. Recently, we used this technique in a high throughput manner to simultaneously measure the affinity of the RBPs to 10, 000 binding sites.
