Biori Development of CircRNA Preparation Process

01. Why Choose circRNA?

Using a typical eukaryotic organism as an example, the degradation of mRNA in yeast cells involves the following steps:

Initiation of deadenylation—shortening of the 3' end polyA tail—mediated by the Pan2/Pan3 complex and the Ccr4/Pop2/Not complex.

Degradation pathway 1: Degradation occurs from the 3' end to the 5' end through the action of exonucleases.

Degradation pathway 2: The decapping enzyme (Dcp1/Dcp2) removes the cap structure, allowing the mRNA to be degraded from the 5' end to the 3' end by Xrn1. When protein translation is prematurely terminated (premature termination of translation), a special mRNA degradation mechanism in yeast is triggered. mRNAs undergo decapping independent of deadenylation or rapid 3'-5' degradation, or they are cleaved by endonucleases.

Previous strategies to enhance the stability of linear mRNA included the use of UTRs (such as the 5' UTR of beta-globin mRNA), cap analogs (methylguanosine cap analogs), nucleotide modifications, and codon optimization.

02. Design Principles of circRNA

CircRNA lacks free ends, meaning it has neither a 3' polyA tail nor a 5' cap structure, allowing it to escape deadenylation and decapping reactions. Comparing the half-life of circRNA and its corresponding linear mRNA transcribed from the same gene shows that the half-life of circRNA (18.8–23.7 hours) is at least 2.5 times longer than that of linear mRNA (4.0–7.4 hours).

The cyclization of RNA can be achieved using a self-splicing group I intron sequence, which only requires the addition of Mg²+ and GTP for the process. However, the natural group I intron has a limited distance between the two splice sites (300nt-500nt), restricting the cyclization efficiency for long sequences.

Next, 9-base-pair and 19-base-pair homologous arms were added at the 5' and 3' ends, respectively, to improve cyclization efficiency. Results showed that adding these homologous arms significantly increased the cyclization rate.

Although adding homologous arms improved cyclization efficiency, it was still insufficient. Since both the IRES sequence and the 3′ PIE splice site are highly structured, it was hypothesized that the IRES sequence might interfere with the folding of the splicing ribozyme. Therefore, a series of spacer sequences were designed between the 3′ PIE splice site and the IRES sequence to either block or allow splicing and cyclization. The permissive spacers retained the secondary structure within the intron sequence (essential for ribozyme activity), while disruptive spacers broke the secondary structure, especially within the 5' end intron sequence. The results showed that adding permissive spacers could double the cyclization efficiency, whereas disruptive spacers completely blocked splicing and cyclization. By adding homologous arms and permissive spacers, it was possible to cyclize RNA sequences up to 5kb in length.

pacer design should prioritize the following:

a. No homologous sequences or structured sequences (no secondary structures) should exist between the IRES sequence and adjacent intron sequences.

b. Spacer sequences should separate the intron and IRES sequences, ensuring that each element can fold and function independently.

c. Spacer sequences should have some complementary regions between them to promote the formation of a splicing bubble, which contains the catalytic intron sequences.

03. Biori GFP-circRNA Development Case Study

Circ GFP Template Preparation

The circ GFP plasmid was digested using EcoRI. The digestion system was as follows:

Digestion at 37°C for 1 hour. After digestion, 1/10 volume of 3M sodium acetate, 1/20 volume of 0.5M EDTA, and 2 volumes of anhydrous ethanol were added. The mixture was incubated at -20°C for 1 hour to precipitate the digested products. The specific steps are as follows:

1）Centrifuge at 16000rpm for 20min at 4°C, and remove the supernatant.

2）Resuspend the pellet with anhydrous ethanol, centrifuge at 16000rpm for 20min at 4°C, and discard the supernatant.

3）Centrifuge at 16000rpm for 3min at 4°C, and remove the supernatant.

4）Once the ethanol has evaporated, dissolve the pellet in DEPC water.

Circ GFP Plasmid Digestion Recovery Electrophoresis Results

M: DL10000 marker

1.     Circ GPF before digestion

2.     Circ GPF after digestion

In Vitro Synthesis of Linear RNA

Linear RNA was synthesized in vitro using T7 RNA polymerase under the following conditions: 1µg of linear DNA template, reacted at 37°C for 2 hours to produce RNA. After transcription, DNase I was added to remove the DNA template at 37°C for 15min. The transcription product can be purified using column purification, phenol/chloroform extraction, magnetic bead purification, or lithium chloride purification.

The circ GFP transcription yield was around 220μg, and the integrity of the transcription product was confirmed by 2% agarose gel electrophoresis, showing no degradation.

In Vitro Synthesis of Circular RNA

The synthesized linear RNA was heated to 70°C for 5min, then immediately placed on ice for 3min to relax the secondary structures. The RNA was then added to the cyclization reaction system and incubated at 55°C for 15min to obtain circular RNA.

The product showed no degradation, but a minor by-product band was observed, which was likely the intron splicing product. RNase R can be used to remove impurities.

Electrophoresis Results for BR-circ GFP Cyclization Product

RNase R Treatment of Circular RNA

The RNase R digestion system was as follows: Circular RNA and linear precursor RNA were digested with RNase R at 37°C for 15min in a 20μL reaction system (scalable based on RNA quantity). The purified circular RNA product was then analyzed.

Purification of Circular RNA Products After RNase R Treatment (LiCl Purification)

The specific procedure is as follows:

1）Add 2× RNase-free H₂O and 2× LiCl to the RNase R digestion reaction system.

2）Mix thoroughly, place at -20°C for 1 hour, then centrifuge at 15,000 rpm for 15 minutes at 4°C. Collect the precipitate.

3）Carefully remove the supernatant, wash the precipitate with 500 μL of 70% ethanol, and centrifuge at 15,000 rpm for 15 minutes at 4°C.

4）Carefully remove the 70% ethanol. Depending on the needs of subsequent experiments, resuspend the RNA in the appropriate solution or buffer, and perform quality control on the circular RNA (circRNA). This includes measuring the circRNA concentration and checking the RNA 260/280 and 260/230 ratios. When the 260/280 ratio is in the range of 1.8 to 2.1 and the 260/230 ratio is greater than 2.0, the circRNA is considered qualified. Store qualified samples at -80°C.

Electrophoresis Results of Circular RNA Recovered After RNase R Treatment

The electrophoresis results show that after RNase R treatment of the circ GFP circular RNA product, there was no obvious degradation of circular RNA, and the recovered product showed a single band. Validation by 2% agarose gel electrophoresis for 60 minutes confirmed no degradation, indicating that the prepared circular RNA can be used for subsequent cell transfection.

BR-circ GFP circular RNA product RR-treated electrophoresis (8 minutes)

M: DL5000marker

1: circ RNA GFP Circular RNA product

2-6: circ RNA GFP Circular RNA product + RR

Circ GFP circular RNA product RR-treated electrophoresis (60 minutes)

M: DL5000 marker

1: BR-circ GFP Transcription Product

2: BR-circ GFP Circularized Product

3: BR-circ GFP Transcription Product + RR

Circular RNA Transfection into Cells (Circular RNA Recovered After RNase R Digestion)

293T Cells in 6-Well Plate Experiment

1) Discard the culture supernatant and wash the cells twice with 3 mL of sterile PBS.

2) Add 300 μL of trypsin to digest the cells, ensuring the digestion solution covers all the cells. Place the culture flask in a 37°C incubator for 1 minute and observe cell digestion under a microscope. Once most cells become rounded and detached, quickly return to the workbench and add 2 mL of 20% complete medium to stop the digestion.

3) Add 4 mL of medium to the culture flask, gently pipette to mix, then transfer the mixture to a sterile 15 mL centrifuge tube. Centrifuge at 1,000 rpm for 5 minutes and discard the supernatant. Add 6 mL of medium and mix well.

4) Add 2 mL of medium to each well of the 6-well plate, then add 500 μL of the cell suspension to the wells.

5) Transfer the remaining cell suspension to a new 25 mm² culture flask, add 20% complete medium up to 7 mL, observe the cells under a microscope, and incubate the flask.

Circular RNA Cell Transfection

1) Wash the 6-well plate with 2 mL of PBS once, then add 2 mL of serum-free 1640 medium.

2) Take different amounts of circRNA-GFP: 1 μg, 3 μg, and 5 μg of RNA, dissolve in 250 μL of serum-free medium, gently mix, and leave at room temperature for 5 minutes (to determine the optimal amount of circRNA-GFP for transfection).

3) Dissolve 3 μL of LipoHigh transfection reagent in 250 μL of serum-free medium, gently mix, and leave at room temperature for 5 minutes.

4) Add the LipoHigh-containing medium to the RNA-containing medium, gently mix, and leave at room temperature for 20 minutes.

5) Gradually add the transfection mixture to the pre-seeded cells, gently mix, and incubate at 37°C in a 5% CO₂ incubator.

6) After 8 hours, observe fluorescence and replace with fresh 20% serum complete medium for continued incubation.

Circ GFP RNA Expression in Cells