Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

1. Primer design strategy

1. Design primers to introduce genes of interest into pBiT1.1-C [TK/LgBiT], pBiT2.1-C [TK/SmBiT], pBiT1.1-N [TK/LgBiT] and pBiT2.1-N [TK/SmBiT] Vectors.
2. Select at least one of these three sites as one of the two unique restriction enzymes needed for directional cloning due to the presence of an in-frame stop codon that divides the multicloning site as shown in Figure 1¹¹.
3. Incorporate nucleotide sequence into the primers as shown in Table 1 to encode the linker residues shown in red in Table 2¹¹.
4. For pBiT1.1-C [TK/LgBiT] and pBiT2.1-C [TK/SmBiT] vectors, make sure that the 5 ́ primer contains an ATG codon and a potent Kozak consensus sequence (GCCGCCACC).
5. For pBiT1.1-N [TK/LgBiT] and pBiT2.1-N [TK/SmBiT] vectors, ensure that the 3 ́ primer contains a stop codon.
   NOTE: Each vector contains the HSV-TK promoter to minimize nonspecific association and reduce experimental artifacts, also each vector has an expression cassette for ampicillin resistance in bacteria.

2. PCR

1. Set up and run PCR reactions to amplify the insert DNA of the gene of interest using the primers designed from step 1. It is important to use a high-fidelity DNA polymerase to minimize mutations.
2. Use exactly the following order to prepare 50 µL of PCR reaction. Add 35.5 µL of distilled water, 5 µL of 10x Polymerase buffer, 5 µL of dNTP mixture (2.5 mM each), 1 µL of plasmid template (200 ng/µL), 1.25 µL of forward and reverse primers (10 μM) and 1 µL of high-fidelity polymerase (5 U/µL).
3. Using a thermocycler set up the following DNA amplification program.
   1. Denature at 95 °C for 5 min.
   2. Repeat 25 times the following thermal cycle: 95 °C for 30 s, 60 °C for 1 min, 72 °C for 2 min per 1 kbp to be amplified.
   3. Run a final extension at 72 °C for 10 min.
   4. Hold the samples at 4 °C within the thermocycler.
      NOTE: It is highly recommended to use a high-fidelity polymerase in order to minimize point mutations particularly those occurring during the amplification of long sequences. As the amplicon becomes longer, the degree of accuracy in the replication of the DNA decreases. For the PCR reaction, choose the annealing temperature based on the melting temperature of the region where the oligos directly hybridize with the DNA template and not with all the sequence of the primer.
4. PCR product purification
   1. Isolate the PCR product from the rest of the PCR reaction using a kit from a manufacturer of preference¹². The PCR product is now ready for restriction digestion.

3. DNA digestion

1. For the PCR product digestion prepare 50 μL of digestion reaction as follows.
   1. Using a 1.5 mL tube, add 12 μL of distilled water.
   2. Add 5 μL of 10x buffer with the best compatibility with both restriction enzymes.
   3. From step 2.4 add 30 μL of PCR product
   4. Finally, add 1.5 μL of each restriction enzyme.
   5. Briefly mix by vortex and incubate at 37.5 °C overnight.
2. For the recipient plasmid digestion prepare 50 μL of digestion reaction as follows:
   1. Using a 1.5 mL tube, add 23 μL of distilled water.
   2. Add 5 μL of 10x buffer with the best compatibility with both restriction enzymes.
   3. Add 15 μL of recipient plasmid (200 ng/μL).
   4. Add 1.5 μL of each restriction enzyme.
   5. Briefly mix by vortex and incubate at 37.5 °C overnight.
      NOTE: It is important to use 3 μg of recipient plasmid in order to obtain sufficient material after DNA agarose gel purification. It is also relevant to leave DNA digestions overnight using both enzymes to obtain high cloning efficiency.

4. DNA agarose gel purification and cloning

1. Prepare a 1% agarose gel to run the digested DNA plasmid and inserts and proceed to cut the corresponding bands. Once the corresponding vector and insert bands have been purified12, determine the DNA concentration using a spectrophotometer.
2. Perform DNA ligation to fuse the insert to the recipient plasmid.
3. Prepare ligation reactions of around 100 ng of total DNA including 50 ng of plasmid vector.
4. Set up recipient plasmid-insert ratio of approximately 1:3; it can be calculated using a vector-insert calculator¹³.
5. Set up negative controls in parallel. For instance, a ligation of the recipient plasmid DNA without any insert will provide information about how much background of undigested or self-ligating recipient plasmid is present.

5. Transformation of clones

1. Place a tube of DH5α competent cells from the freezer at -80 °C and immediately transfer it on ice for 20 min.
2. After that time, take 55 µL of DH5α competent cells and add 4 µL of ligation reaction and mix by flicking the tube and store on ice for 45 min.
3. Place the tube in a water bath previously warmed at 42 °C for exactly 48 s and immediately get the tubes back on ice for another 3 min.
4. Add 600 µL of Luria Broth (LB) medium previously warmed at 37.5 °C and incubate with shaking for 1 hour at 200 rpm.
5. Transfer 200 µL into an agar plate containing ampicillin 100 μg/mL and gently spread over the surface with the liquid is mostly absorbed.
6. Incubate the plates overnight to see the colonies next morning. The recipient plasmid on the insert plate should have significantly more colonies than the recipient plasmid alone plate.

6. Isolation of the finished plasmid

1. Pick 3-10 individual bacterial colonies and transfer into 1 mL of LB medium containing ampicillin (100 μg/mL) and incubate for 6 h.
2. Take 200 µL of bacterial suspension and transfer to 5 mL of LB medium containing the same concentration of ampicillin as in step 6.1 and incubate overnight at 37.5 °C with shaking at 200 rpm.
3. Using a miniprep DNA kit purification, perform miniprep DNA purifications using 5 mL of LB grown overnight following the manufacturer instructions¹⁴.
4. To identify successful ligations, set up PCR reactions in the same way as in section 2 using the DNA obtained from step 6.3 as a template with the same primers as in section 2 during the first PCR. Positive clones will produce PCR products with the corresponding size.
5. After large prep DNA purification of the positive clones, conduct a diagnostic restriction digestion of 500 ng of purified DNA with the enzymes used during the cloning step and run the digested products on an 1% agarose gel. There should be two bands: one the size of the vector and one the size of the new insert.
6. Verify the construct sequence by sequencing using the following primers: Forward 5’- aaggtgacgcgtgtggcctcgaac-3’ and reverse 5’-gcatttttttcactgcattctagtt-3’.
   NOTE: When DNA is replicated using PCR, there is always the possibility of errors during the amplification even when using a high-fidelity polymerase, therefore is very important to sequence the final constructs.

7. Transfection and protein expression

1. In a previously poly-L-lysine-coated white 96 well plate, perform cell seeding one day before transfection at 2.5 x 10⁴ cells per well using Dulbecco’s modified Eagle’s medium supplemented with 10% of Fetal Bovine Serum, 100 U/mL penicillin G, and 100 μg/mL streptomycin.
2. Use only the 60 inner wells to minimize the potential for thermal gradients across the plate and edge effects from evaporation. Add 200 μL of sterile distilled water to the 36 outside wells and 150 μL in the spaces between wells and incubate overnight at 37.5 °C and 5% of CO₂.
3. The following morning perform transfection using 100 ng of total DNA (50 ng each construct).
4. Set up four different plasmid combinations (receptor:β-arrestin) according to Figure 2b.
5. For each plasmid combination use 20 µL of modified Eagle's Minimum Essential Media buffered with HEPES using 0.3 µL of lipidic transfection reagent per well.
6. Add 20 μL of lipidic transfection reagent-DNA mixture to each well and mix the plate in circles for 10 s.
7. Change fresh medium after 6 hr incubation at 37.5 °C and 5% CO₂.
8. Incubate the plate for 24 h at 37.5 °C and 5% CO₂.

8. Monitoring receptor-β-arrestin1/2 interactions in HEK293 cells

1. Aspirate medium and add 100 μL of modified Eagle's Minimum Essential Media buffered with HEPES to each well and let the plate stabilize at RT for 10 min.
2. Prepare the furimazine substrate by combining 1 volume of 100x substrate with 19 volumes of LCS Dilution Buffer (a 20-fold dilution)¹¹, creating a 5x stock to mix with cell culture medium.
3. Add 25 μL of 5x furimazine to each well and gently mix in circles for 10 s.
4. Measure luminescence for 10 min for signal stabilization at RT.
   NOTE: By using this baseline signal to normalize the response of each well it will help to reduce variability caused by differences in the number of cells plated per well, also differences in transfection efficiency, etc. Once calculated, average the normalized response from replicate wells for a given drug treatment.
5. Prepare 13.5x ligand solution in modified Eagle's Minimum Essential Media buffered with HEPES.
6. For experiments at room temperature with 10 µL of 13.5x ligand addition, add the compounds using injectors or a multichannel pipette and mix the plate by hand or using an orbital shaker (20 s at 200 rpm).
7. For experiments at 37.5 °C with 10 µL of 13.5x ligand addition, use injectors to dispense compounds and mix by using the instrument orbital shaker. In case of not using injectors, remove the plate from the luminometer, add the ligands and mix the plate by hand or using an orbital shaker (20 s at 200 rpm).
   NOTE: Use injectors and a shaker within the detection instrument to minimize temperature fluctuations associated with removing the plate from the luminometer. Standard benchtop luminometers can be used for this assay. Use an integration time of 0.25–2 s.