Overview

Our experimental approach focuses on developing the RADAR (RNA-Activated Detection And Response) biosensor system for detecting hepatocellular carcinoma biomarkers. This comprehensive methodology combines molecular cloning, cell culture, transfection protocols, and validation studies to create an effective detection system based on ADAR-mediated RNA editing.

The experimental design is structured around detailed standard protocols for reproducibility, comprehensive materials and methods documentation, and complete sequence information for all genetic components used in the biosensor development.

Standard Protocols

Restriction Cleavage

Media and materials needed:

• Thawed reaction buffer
• Thawed restriction enzymes of choice
• Suspended plasmid

1. Assemble the reaction mix on ice in PCR tubes.

2. Incubate at 37°C for 3-5 hours (using a PCR machine or heat block).
3. Heat-inactivate enzymes at 65°C for 20 minutes.
4. Store digested DNA at -20°C if not used immediately, or purify the backbone using a gel purification kit.

RADAR Transfection

Media and materials needed:

• Room Temperature or 37°C Opti-MEM medium
• L3000 and P3000 reagents
• Suspended plasmids

For transfection of the RADAR system, a typical amount of total plasmid DNA (in a volume less than or equal to 10µL) is 500ng (500ng/ml) plasmid DNA per well when using a 24 well format with 200 ng of the sensor plasmid, 250 ng of the trigger sequence, and 50 ng of the ADAR editor plasmid.

1. Cell confluency:
   1. One day before transfection, seed cells in appropriate plates so they are 70-90% confluent at the time of transfection.
2. Preparation of Transfection Mix:
   1. Dilute the required amount of plasmid DNA in 25µl of Opti-MEM medium, then add 1µl of P3000 reagent.
   2. In a separate tube, dilute 1 µl of L3000 reagent in 25 µl of Opti-MEM medium.
   3. Combine the two tubes and allow the mixture to incubate for 10-15 minutes to enable complex formation.
3. Transfection:
   1. Add the DNA-reagent complexes dropwise to the cells in the growth medium (antibiotic-free).
4. Incubation:
   1. Return the cells to the incubator (37°C, 5% CO₂) until data collection and analysis

Notes:

• Always include a control to monitor transfection efficiency (ex. GFP-only plasmid, untransfected well).
• Change medium 4-6 hours post-transfection if cytotoxicity is observed.

Cloning and In-Fusion Assembly

Media and materials needed:

• Thawed In-Fusion assembly master mix
• Suspended vector
• Suspended insert
• Nuclease-free H₂O
• SOC recovery medium
• Competent bacterial cells

All optimal amounts of vector and insert to use in the In-Fusion Cloning reaction were calculated via TakaRaBio molar ratio calculator using insert and vector base pair size inputs. Following receiving suggested ng amounts, volumetric amounts were calculated using the insert and vector concentrations.

1. Place all components into an eppendorf tube and mix the reaction components by pipetting up and down 5x.

2. Incubate the reaction mix at 50°C for 15 minutes using a heat block.
3. Place the samples on ice for 1-2 minutes.
4. Store samples in -20°C until ready for bacteria transformation to complete the ligation.
5. Bacterial transformation following assembly using Stellar Comp Cells (Takara):
   1. Thaw competent cells on ice. Mix gently to ensure even distribution.
   2. Transfer 50 µl of the competent cells into a Falcon tube.
   3. Add up to 2.5µl of the In-Fusion reaction mixture.
   4. Incubate on ice for 20-30 min.
   5. Heat shock cells at 42°C for 45 sec.
   6. Place tubes on ice for 1-2 min.
   7. Add SOC medium to bring the final volume to 500 µl.
   8. Incubate at 37°C with shaking (160-225 rpm) for 20-60 min depending on antibiotic selection.
   9. Plate 100-170 µl of the transformation mix onto selective LB agar plates and incubate plates overnight at 37°C.

Hep3B Cell Line Maintenance

Materials needed:

• Warm growth medium, PBS, and trypsin to 37°C using a water bath
• Check cells under a microscope to assess confluency. Cells should be around 70–80% confluent before repassaging

1. Using a sterile pipette, gently aspirate the spent growth medium from the culture dish.
2. Add 5 mL of PBS to wash off residual serum or medium. Swirl gently, then aspirate the PBS.
   1. Tip: When adding PBS, touch the pipette tip to the side of the dish to avoid disturbing the cells.
3. Add 1.5 mL of trypsin to the dish and incubate at 37°C for about 5 minutes to allow the cells to detach.
4. Check under a microscope to confirm cell detachment.
5. Add 3.5 mL of complete growth medium to neutralize the trypsin. Pipette up and down to break up any clumps and get a single-cell suspension.
6. Transfer an appropriate volume of this suspension into a new dish containing 10 mL of fresh DMEM complete growth medium.
   1. Example: For a 1:5 split, take 1 mL of the suspension and add it to 10 mL fresh medium. For smaller splits, adjust the transferred volume accordingly (ex. 200 µL into 10 mL for a 1:25 split).

Notes on Hep 3B culture:

• When thawed, Hep 3B cells were initially cultured in DMEM complete growth medium with 20% FBS.
• After the first passage, cells were maintained in DMEM complete growth medium with 10% FBS.

Cell Seeding

Materials needed:

• TC-treated cell culture plates
• DMEM Growth Media warmed to room temperature
• Trypan Blue dye

1. Gather all the cells in the original dish in a 10 ml centrifuge tube and spin in a centrifuge at 1200 rpm for about 3 minutes. Following the spin down, you should see a pellet at the bottom of a tube.
2. Aspirate the supernatant containing any medium and trypsin.
3. Rack the tube to facilitate resuspension of the cell pellet.
4. Use about 5ml of DMEM to resuspend the cells.
5. Use the cell counter to inspect the number of live cells:
   1. 10µl cell suspension + same amount of trypan blue into the cell counter slide
6. Calculate the total volume of the cell suspension needed for the wells you are seeding.
7. Move the volume of cells needed for a concentration of 1.5×10⁵ cell/ml to another tube and fill the rest of that tube with the remaining volume of DMEM to make 24 ml of master mix
8. Fill each well with 1 ml of the master mix.
9. Label the transfection plate with the date, your experimental conditions, and which wells will receive the cells.
10. Place the plate in the 37°C incubator with 5% CO₂ for 16-18 hours before transfection.

Data Collection and Analysis

All data was collected using fluorescent microscopy and flow cytometry. Fluorescent microscopy was used for initial verification of sensor and trigger presence as well as GFP production, while flow cytometry was used to quantify fluorescence intensity values. Fluorescent images were processed using ImageJ for brightness and contrast values, with no major modifications made. For flow cytometry, around 10k cells were recorded per sample with the FSC/SSC gating strategy used to exclude debris and capture only fluorescing cells. For each experiment, at least three replicates were made.

Materials and Methods

Plasmid Construction

All plasmids were constructed using In-Fusion Assembly reactions using inserts designed by the team and synthesized by Twist Bioscience and vectors digested from previously validated sensor plasmids. To initially clone the glypican-3 (GPC3), hepatitis B x antigen (HBx), and aldo-keto reductase family 1 member B10 (AKR1B10) sensor plasmids, the Kaseniit et al., 2022 sensor plasmid (plasmid EK0698) was digested to remove the sensor sequence for later replacement with the appropriate synthesized sensor fragment. Reactions were completed in a 30 µl volume consisting of 3µg of the appropriate plasmid, 1 µl of each restriction enzyme (MluI-HF and XhoI), 3µl of 10X rCutSmart Buffer, and the remaining volume of Nuclease-free water. Digests were performed using a thermocycler, incubating the samples at 37°C for 5 hours followed by heat inactivation at 65°C for 20 minutes. The PCR products were then run on a 0.8% agarose gel in 1X TAE buffer for 45 minutes at 120 volts. Following the gel, appropriate bands respective to the vector backbone length were removed from the gel using a blade and gel purified as per the Zymoclean Gel DNA Recovery Kit for later plasmid construction.

Following sensor validation, to alter the output, digestions were run on GPC3 and HBx sensor plasmids, enabling isolation and removal of the GFP output initially used to test the sensors. Restriction enzymes used for the GPC3 and HBx sensor digestions were ApaI and XhoI to frame the GFP output fragment. This reaction was performed according to the description above using ApaI, XhoI, and 10X FastDigest Buffer.

All In-Fusion Assembly reactions were performed in 10 µl volumes at a molar ratio of 2:1 insert:vector and a total DNA mass of 200ng. To assemble the GPC3 and AKR1B10 sensor fragments, around 178 ng of the vector, 24 ng of the insert, 2 µl of In-Fusion Master Mix, and the volume to 10µl of Nuclease-free water was used. To assemble split GFP output fragments, for the GPC3 sensor around 162 ng of vector and 38 ng of GFP 1-10 insert were used with the aforementioned amounts of In-Fusion Master Mix and Nuclease-free water, while the HBx sensor reaction required 191 ng vector and 39 ng GFP 11 insert were used with the aforementioned amounts of In-Fusion Master Mix and Nuclease-free water.

Cell Culture Conditions

Hep 3B and HEK 293T cells were maintained at 37°C with 5% CO₂ in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin–Streptomycin. Hep 3B cells were thawed into DMEM containing 20% FBS for the first passage and subsequently cultured in 10% FBS medium. Both cell lines were passaged at 70–80% confluency using 0.05% trypsin–EDTA, and split at ratios between 1:3 and 1:10 depending on growth rate and experimental needs.

Sequences