Engineering circular RNA for potent and stable translation in eukaryotic cells

Abstract

Messenger RNA (mRNA) has broad potential for application in biological systems. However, one fundamental limitation to its use is its relatively short half-life in biological systems. Here we develop exogenous circular RNA (circRNA) to extend the duration of protein expression from full-length RNA messages. First, we engineer a self-splicing intron to efficiently circularize a wide range of RNAs up to 5 kb in length in vitro by rationally designing ubiquitous accessory sequences that aid in splicing. We maximize translation of functional protein from these circRNAs in eukaryotic cells, and we find that engineered circRNA purified by high performance liquid chromatography displays exceptional protein production qualities in terms of both quantity of protein produced and stability of production. This study pioneers the use of exogenous circRNA for robust and stable protein expression in eukaryotic cells and demonstrates that circRNA is a promising alternative to linear mRNA.

Fig. 1

Permuted intron-exon splicing and addition of homology arms. a Schematic diagram showing permuted intron-exon construct design and mechanism of splicing. The group I catalytic intron of the T4 phage Td gene is bisected in such a way to preserve structural elements critical for ribozyme folding. Exon fragment 2 is then ligated upstream of exon fragment 1, and a coding region roughly 1.1 kb in length is inserted between the exon-exon junction. During splicing, the 3′ hydroxyl group of a guanosine nucleotide engages in a transesterification reaction at the 5′ splice site. The 5′ intron half is excised, and the freed hydroxyl group at the end of the intermediate engages in a second transesterification at the 3′ splice site, resulting in circularization of the intervening region and excision of the 3′ intron. b RNAFold predictions of precursor RNA secondary structure for homology arm design. Colors denote base pairing probability, with red indicating higher probability. Without homology arms, no base pairing is predicted to occur between the ends of the precursor molecule. c Agarose gel demonstrating the effect of homology arms on splicing. Putative circRNA runs at a higher molecular weight than heavier precursor RNA, as indicated. (−): no homology arms. Weak: weak homology arms, 9 nt. Strong: strong homology arms, 19 nt. d Agarose gel confirmation of precursor RNA circularization. C: precursor RNA (with strong homology arms) subjected to circularization conditions. C + R: Lane C, digested with RNase R. C + R + H: Lane C + R, digested with oligonucleotide-guided RNase H. U: precursor RNA not subjected to circularization conditions. U + H: Lane U, digested with oligonucleotide-guided RNase H. e Sanger sequencing output of RT-PCR across the splice junction of the sample depicted in lane C + R from (d)

Fig. 2

Spacer design and splicing using the Anabaena autocatalytic intron. a RNAFold predictions of precursor RNA secondary structure in the context of designed spacers. Secondary structures potentially important for ribozyme function are identified by black arrows. b Agarose gel demonstrating the effect of spacers on splicing. (−): no spacer. D: disruptive spacer. P1: permissive spacer 1. P2: permissive spacer 2. c RNAFold predictions of precursor RNA secondary structure for internal homology region design. Lack of significant internal homology (Anabaena 1.0) and introduced internal homology (Anabaena 2.0) indicated by black arrows. Splicing bubble indicated as the region between homology arms and internal homology regions that contains the splicing ribozyme. d Agarose gel demonstrating the effect of internal homology on splicing. e Agarose gel comparing the optimized T4 phage splicing reaction to the optimized Anabaena splicing reaction. Anabaena intron halves are of roughly equal lengths, and are less likely to remain associated after splicing in comparison to the T4 phage intron halves despite stronger homology arms

Fig. 3

Evaluation of circularization efficacy and translation for a range of protein-coding circRNAs generated from de-novo engineered precursor RNA. a Schematic diagram showing elements of the engineered self-splicing precursor RNA design. b Agarose gel of precursor RNA containing an EMCV IRES and a variable insert including Gaussia luciferase (GLuc), human erythropoietin (hEpo), EGFP, Firefly luciferase (FLuc), or Cas9 coding regions after circularization and recircularization (R−). CircRNA was enriched by RNase R degradation (R+). c Approximate circRNA yields from treatment of 20 µg of splicing reaction with RNase R, as assessed by spectrophotometry (n = 3). d Luminescence in the supernatant of HEK293 cells 24 h after transfection with circRNA coding for GLuc (n = 4). e Expression of human erythropoietin in the supernatant of HEK293 cells 24 h after transfection with circRNA coding for hEpo as assessed by solid-phase sandwich ELISA (n = 4). f Luminescence in the lysate of HEK293 cells 24 h after transfection with circRNA coding for FLuc (n = 4). g GFP fluorescence in HEK293 cells 24 h after transfection with circRNA coding for EGFP (scale bar: 400 µm). h FACS analysis demonstrating GFP ablation in HEK293-EF1a-GFP cells 4 days after transfection with sgGFP alone (circCas9−) or co-transfected with circRNA coding for Cas9 (circCas9+), indicated by the appearance of a GFP-negative cell population (all data presented as mean + SD, *p < 0.05 (Welch’s t-test))

Fig. 4

IRES efficacy in varying cell and sequence contexts. a Luminescence in the supernatant of HEK293 cells 24 h after transfection with circRNA containing a panel of viral 5′ UTR IRES sequences in GLuc (left axis, dark purple with black outline) and FLuc (right axis, light purple with gray outline) contexts. b Luminescence in the supernatant of HEK293 cells 24 h after transfection with circRNA containing an added polyA(30) or polyAC(30) spacer sequence separating the IRES from the splice junction. (−): no spacer. pAC: 30nt spacer consisting of adenosines and cytosines. pA: 30nt spacer consisting of adenosines. c Luminescence in the supernatant of HEK293 (left axis, dark purple with black outline) and HeLa (right axis, light purple with gray outline) cells 24 h after transfection with the most effective circRNAs by IRES in (b). d Luminescence in the supernatant of A549 (left axis, dark purple with black outline) and Min6 (right axis, light purple with gray outline) cells 24 h after transfection with the most effective circRNAs by IRES in (b) (all data presented as mean + SD, n = 4, *p < 0.05 (Welch’s t-test))

Fig. 5

HPLC purification of circRNA from splicing reactions. a HPLC chromatogram of linear GLuc RNA (top) and a CVB3-GLuc-pAC splicing reaction (middle). Agarose gel of collected fractions (bottom). b Agarose gel of CVB3-GLuc-pAC purified by different methods. C: splicing reaction. +R: splicing reaction treated with RNase R. +G,R: splicing reaction gel extracted, and then treated with RNase R. +H,R: splicing reaction HPLC purified, and then treated with RNase R. c Luminescence in the supernatant of HEK293 cells 24 h after transfection with the CVB3-GLuc-pAC splicing reactions purified by different methods as noted in (b) (data presented as mean + SD, n = 4, *p < 0.05 (Welch’s t-test))

Fig. 6

Translation efficacy of circRNA compared to linear mRNA. a Luminescence in the supernatant of HEK293 (left, black outline) and HeLa (right, gray outline) cells 24 h after transfection with CVB3-GLuc-pAC circRNA or modified or unmodified linear GLuc mRNA (n = 4 HEK293, n = 3 HeLa). b Luminescence in the supernatant of HEK293 cells starting 24 h after transfection with CVB3-GLuc-pAC circRNA or modified or unmodified linear GLuc mRNA and continuing for 6 days (n = 4). c Relative cumulative luminescence produced over 6 days by HEK293 (left, black outline) and HeLa (right, gray outline) cells transfected with CVB3-GLuc-pAC circRNA or modified or unmodified linear GLuc mRNA (n = 4 HEK293, n = 3 HeLa). d Luminescence in the supernatant of HeLa cells starting 24 h after transfection with CVB3-GLuc-pAC circRNA or modified or unmodified linear GLuc mRNA and continuing for 6 days (n = 3). (all data presented as mean + SD, *p < 0.05 (Welch’s t-test))