Positive: 3,5,6
Negative: 1,2,4,7,8

1. To detect the sequence of the circular mRNA in the cDNA derived from the RNA after RNaseA processing
2. To detect the sequence of the linear mRNA in the cDNA derived from the RNA after RNaseA processing
3. To detect the sequence of the circular mRNA in the cDNA derived from the RNA after RNaseR processing
4. To detect the sequence of the linear mRNA in the cDNA derived from the RNA after RNaseR processing
M. Marker
5. To detect the sequence of the circular mRNA in the cDNA derived from the non-treated RNA
6. To detect the sequence of the linear mRNA in the cDNA derived from the non-treated RNA
7. To detect the sequence of the circular mRNA in the non-treated RNA
8. To detect the sequence of the linear mRNA in the non-treated RNA

See the lane 7,8. → RNA is not detected by the electrophoresis, namely, the matter detected is cDNA.
See the lane 5,6,7,8. → The factor involved in the existence of cDNA is the ribonuclease processing.
See the lane 1,2,5,6. → The endoribonuclease decomposes the all RNA.
See the lane 3,4,5,6. → There is the RNA decomposed by the exoribonuclease.
Therefore, the RNA that is decomposed by the endoribonuclease but is not decomposed by the exoribonuclease exists. We think this RNA is the circular mRNA!

Elucidation of the cyclization mechanism

After having reverse-transcripted it, I amplified a joining part between the intron by PCR and read sequence. I show below the sequence for the junction of 3'intron parts and 5'intron parts.

As a result of having read Sequence, I understood that U of 5'intron was combined with C of 3'intron and mRNA became a loop.

The existence of a long-chain protein

RFP -1- SDS-PAGE

1. RFP from linear RNA (with stop codon)
2. RFP from circular RNA (with stop codon)
3. RFP from circular RNA (without stop codon)
4. RFP from circular RNA (with the stop codon of mRNA circular device)
S. supernatant
P. precipitation
M. marker

There is a long-chain protein near a band that indicates 250 kDa. The molecular weight of a monomeric RFP is 25423.7(→ BBa_E1010), so we guess that the protein is not less than decameric RFP. In addition, long chain protein looked like a ladder. This reason is because it dissociates while ribosome continues translating it. There will be three reasons that ribosome leaves.

1. It is caused by a distortion to occur to the cyclic mRNA.
2. When other ribosome was binded, the ribosome which turned around was pushed outside of the circular mRNA.
3. Originally there may be little tRNA like a rare codon.

RFP -2- Western blotting

We separated “long-chain RFP” from cell bodies by 12%, 10%, 7% of SDS-PAGE and transferred PVDF membrane. After that we performed Western blot by using peroxidase-labeled RFP antibody (rabbit) and color coupler (DAB). We showed below the result.

As a result of having dyed gel of SDS-PAGE in CBB after the membrane transfer.

As a result of dyed gel after the membrane transcription. The protein more than 100KDa was not transferred and stayed in gel of 12%. The protein more than 250KDa was not transferred and stayed in gel of 10% Most of the long chain protein more than 250KDa was transferred on PVDF membrane.

The result of having performed Western blot of membrane.

A band was detected and was able to confirm that an antibody was connected, and it was developed a pigment by DAB. A band of RFP (27KDa) was detected clearly when we watched the membrane which transferred from 12% and 10% gel after color development. However, if gel density is high, it is hard to elute the long chain protein. So it is hard for us to show that long chain protein was detected from this date.

The determination of long-chain RFP

We aimed to find how much long-chain RFP would be synthesized per cell body of E. coli. RFP combines with Histag. So it ought to have gained monomer RFP and long-chain RFP by conducting affinity chromatography with Ni-NTA. However, the long-chain RFP was insolubilized and produced precipitation because it is polymer compound. And it could not combine with the Ni-NTA column, flowing out in flow-through. So it was difficult to refine the long-chain RFP, which has different size. On the other hand, the monomer RFP combined with the Ni-NTA column, being able to refine them using imidazole. By using these monomer RFP, we tried to calculate protein mass of the long-chain RFP.

Protocol

1. E. coli, which can synthesize long-chain protein, was cultivated for 3 hours, after that, IPTG, which is an inducing substance, was added, and it was cultivated for further 10 hours. 5mL of culture solution that reached the stationary phase was taken, it was centrifuged, the supernatant was removed, and 300µL of 1×PBS was added. The cell body was crushed, it was centrifuged, and supernatant was separated from precipitation. The supernatant was refined with Ni-NTA, creating almost pure monomer RFP solution. The solution was refined in 1, 2, 4, 8, 16, and 32 times, measuring Abs (280nm) of each solution by using an absorbance meter. And protein concentration of each solution from the molecular absorbance coefficient and the molecular weight of RBS was calculated.
2. 1mL of culture solution that reached the stationary phase was taken, it was centrifuged, the supernatant was removed, and 1mL of 1×PBS was added. Using this solution as a sample, an undiluted solution and a 10 times diluted solution are prepared.
3. 4µL of each sample solution and each monomer RFP solution of different dilution rate were applied on the gel. Each of them was separated by the 10% SDS-PAGE, dyeing by CBB. After that, using the image processing software (ImageJ), analytical curve based on “the total sum of its brightness and area of dyeing” and “the already known concentrate of monomer solution” was made. Adapting the former, the concentration of long-chain RFP was calculated.
4. On the other hand, to calculate the number of cultivated E. coli easily, OD¬600 of culture solution was measured. Using the conversion formula of E. coli (the number of cell bodies), the number of cell bodies from the value of OD600 was calculated.
5. From the number of cell bodies and the concentration of long-chain RFP, the mass of long-chain RFP per cell body was calculated.
6. And also, if silk fabrics are made of 900g raw silk by this E. coli, the number of cell bodies that is needed for it was estimated. We assumed that the fibers of same length from the same weight of raw silk and the long-chain RFP.

Result

The following table is the concentration of each monomer RFP solution determined from the absorbance.

We calculated the sum of the stained area with the chromaticity from the picture. We made a calibration curve from “the sum of the stained area with the chromaticity of the gel” and “known concentration of the monomer solution”. The result is shown in the following table.

Following table shows the number of bacteria which synthesizes long-chain protein calculated by OD600. And we calculate the amount of the proteins which one bacterial cell (E. coli) synthesized from concentration of the protein (the above).

In order to create 900 grams of polymer RFP to making silk fabric, we need many bacterial cell as below.

examination

It showed that Circular mRNA / liner mRNA=0.026 in Modeling. It can not be said that a high ratio. However, the ratio of polymer RFP / monomer RFP=0.71, we found that long-chain protein is synthesized in relatively large amounts. This shows Circular RNA is better in the ability to synthesize protein than linear RNA.

There is a problem that the way to synthesize of long-chain proteins using circular RNA. It can not adapt to all kind of proteins, Efficiency of RNA cyclization is low, and so on. However, if you improve that problems or devise a way to use, the method of　synthesizing protein using circular mRNA will be useful enough basis.

Activation of a long-chain protein

The determination of long-chain RFP

1.RFP from linear RNA (with stop codon)
2.RFP from circular RNA (with stop codon)
3.RFP from circular RNA (without stop codon)：using this device
4.RFP from circular RNA (with the stop codon of mRNA circular device)

The RFP (+histidine tag) polymer didn’t show the fluorescence.
Possible factor
1.The RFP polymer is too huge, so it becomes an inclusion body.
2.The repetitive amino acid sequences are too near, so the conformation of the RFP polymer is in disorder.

On the other hand, we understand that long chain protein was detected than 150KDa on the membrane which transferred from 7% gel. Because we used RFP antibody derived from a rabbit, this date shows that detected long chain protein is protein derived from RFP.

Synthesizing long-chain SmtA (Metallothionein)

Confirmation of the synthesis

I was able to confirm protein more than 250KDa in SmtA. Because the metallothionine is the simple　structure protein which only adsorbs metal such as Zn, the Cd, even if it becomes a long chain, it's not lose activity

Future Works

Our future work is the improvement of a functionality of a long-chain protein. For example, SmtA (Metallothionein) can catch heavy metal ions such as Zn²⁺. We think that a protein sheet made of long-chain SmtA (Metallothionein) prevents heavy metal from leak from factories.

If the improvement of a long-chain protein is achieved, we can gain practical achievements in many directions.

Conclusions

The existence of circular mRNA

Yes! ← RNase processing

The existence of a long-chain protein

Yes! ← SDS-PAGE

Yes! ← Western blotting

Activation of a long-chain protein

RFP

No… ← Comparing RFPs derived from RNAs in various states

SmtA

The experiment is now underway.

References

1. Rederick K. chu, Gladys F. Maley, and Frank Maley(1998) “RNA splicing in the T-even bacteriophage” Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201, USA
2. M. Puttaraju and Michael D. Been(1996) “Circularizing ribozymes and decoy-competitors by autocatalytic splicing in vitro and in vivo” SAAS Bull Biochem Biotechnol
3. R. Perriman and M. Ares, Jr: (1998) Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo.
4. So Umekage et al. (2012) “In Vivo Circular RNA Expression by the Permuted Intron-Exon Method” Innovations in Biotechnology