Custom Circular RNAs Synthesis Service
With our well-established platforms and techniques in the field of circular RNAs (circRNAs), outstanding and well-trained scientists at our Creative Biolabs have capable of fulfilling the various needs of circRNAs synthesis with comprehensive, one-stop services for our worldwide clients, such as sequence design and optimization, in vitro transcriptional, quality control and expression verification.
Transcription of CircRNAs
For in vitro synthesis of circRNAs, a T7 transcriptional system-contained plasmid is usually used. In this system, the well-designed sequence is cloned and flanked by the T7 promoter and terminator, and the target sequence can be correctly transcribed with the help of T7 RNA polymerase in vitro.
Fig.1 The map of circRNA-expression plasmid.
The plasmid can be divided into three parts.
- Part A contains the essential cassettes of a plasmid for amplification in bacteria. It also contains a selection gene when delivered into cells.
- Part B contains the T7-associated elements used for in vitro transcription (IVT). When IVT primers are used, the PCR-amplified DNA fragment can be used for the template of IVT. When Part B is cloned into a lentiviral backbone (selection marker can be contained if necessary), lentivirus-based stable integration of circRNA for overexpression can be achieved. The CMV promoter makes it possible to transcription of precursor RNA in cells.
- Part C involves the important portions for the circulation of circRNAs in vitro or in vivo. Usually, T4 ligase-based circulation of precursor RNA can only occur out of experimental cells, whereas specific sequence-induced looping can happen in experimental cells or in vivo such as permuted intron-exon (PIE) splicing strategy. The former won't introduce any extra bases in the circRNAs, while the latter will contain part of the additional sequence of the PIE system, which causes no or less influence on the function of circRNAs. Other strategies based on chemical, RNA-binding protein, or tRNA can also be used for the production of circRNAs.
Verification of CircRNAs
When the circulation of circRNAs is finished, the products will undergo a series of verification.
- Resistance to RNase R
RNase R can degrade the linear RNA and circRNAs are resistant to digestion. Based on these, when the products of the circulation reaction are loaded onto the agarose gel, the linear part in RNase R-treated sample (C + R) will be digested, and the circRNAs will be kept compared with the untreated one (C).
Fig.2 The result of RNase R treatment to the products of the circulation reaction.¹
- Concentration & Circulation efficiency & Purity of circRNAs
The concentration of circRNAs can be determined by the UV absorbance at 260 nm, and the circulation efficiency can be roughly estimated by the ratio of sample concentration after RNase R treatment to the concentration before RNase R treatment. The purification of circRNAs can be achieved by column purification, gel extraction, or HPLC purification. Among these, circRNAs purified from HPLC have the highest purity (> 95%) and best performance when transfected into cells.
- Sequencing of circRNAs
We use FBXW7 as an example. Both linear mRNA of FBXW7 and circ-FBXW7 can be transcribed in vivo. The convergent primer pair (blue) and divergent primer pair (red) are designed for PCR after the extraction of total RNA or genomic DNA. The PCR products are analyzed by electrophoresis. Both primer pairs can amplify the corresponding bands when using the cDNA produced by reverse transcription. If the total RNA is treated with RNase R, the convergent primer pair (blue) fails to form the bands near 100 bp. When the genomic DNA is used as the template, the divergent primer pair (red) can't amplify the bands above 100 bp. When sequencing the amplifications in the red box, the circRNAs can be verified.
Fig.3 The PCR and sequencing of circRNAs.²
Being a reputed biotech business, Creative Biolabs specializes in circRNAs and builds up a skilled team of educated specialists, and establishes well-defined approaches in this field. Please feel free to contact us for more details about your project involving synthetic circRNAs.
References
- Wesselhoeft, R. Alexander, Piotr S. Kowalski, and Daniel G. Anderson. "Engineering circular RNA for potent and stable translation in eukaryotic cells." Nature communications 9.1 (2018): 2629.
- Yang, Yibing, et al. "Novel role of FBXW7 circular RNA in repressing glioma tumorigenesis." JNCI: Journal of the National Cancer Institute 110.3 (2018): 304-315.
