Part:BBa_K1777008

RNA accelerating cyclization

This RNA can bind to the flanking region of exon4 of hsACTB in HEK293T cells and facilitate the back-splicing mechanism. This part can be used to cyclize certain RNA exon.
This part has the similar function with BBa_K1777009[1]

Function

This RNA is a short(~180nt) linear RNA composed of two binding region and a short linker. The principle of start cyclization is the same as “3C” strategy. We use the two binding region on acRNA to bind the double ends of pre-circle RNA through complementary base-pairing and then the acRNA will pull the two flanking region of the exon to be cyclized close to each other, forming a ‘U’ structure.

We design this RNA to promote the cyclization of hsACTB in HEK293T cells.

Results

Forty-eight hours after transfection with pZW1-sno-ACTB into HEK 293T cells, we use the specific circular exon-4 primers to test the circular RNA.

Figure 1. Circular RNA form with acRNA (pZW1-sno-ACTB).
HEK-293T cells were seeded into a 24-well plate. Forty-eight hours post-transfection, the total RNAs are extracted. Use circular RNA specific primers (have been BLAST on UCSC-In Silico, proved to be specific) doing PCR analysis, Unexpectedly, we amplify other circular RNAs. Extract non-specific gel and sequence it.Both vector without insert pZW1-sno and plasmid with acRNA pZW1-sno-ACTB appear non-specific electrophoretic band.
After sequencing, we found 200bp: circular RNA consists of exon4-exon3-exon4, 500bp: circular RNA onsists of exon4-exon5-exon6-exon2-exon3-exon4 while 700bp: circular RNA consists of exon4-exon5-exon3-exon4.

Progress

We are still trying to make specific circular RNA through acRNA.However, the amount of RNA is far less than that of protein, even with the most efficient promoter, the number of acRNA molecular wouldn’t overpass that of pre-circle RNA too much. Most importantly, the total number of any specific RNA is quite small(Global quantification of mammalian gene expression control), which means it’s hard for acRNA and pre-circle RNA crashing into each other in cell nucleus. Therefore, acRNA cannot efficiently pull ends close like designed proteins, which will greatly lower cyclization efficiency.