RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degradation. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Additionally, an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.
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We thank J. D. Moon and H. Kim of the Jaffrey laboratory for comments and suggestions. This work was supported by NIH grant no. R01NS064516 (to S.R.J.) and by NIH fellowship no. F31AI134100 (to J.L.L.). Fluorescence-assisted cell-sorting experiments reported in this publication were supported by the Office of the Director of the National Institutes of Health under award no. S10OD019986 to Hospital for Special Surgery.
S.R.J, J.L.L. and Weill Cornell Medicine have filed a patent application covering aspects of this technology. S.R.J. is a co-founder of Lucerna Technologies and has equity in this company. Lucerna has licensed commercialization of technology related to Spinach and other RNA–fluorophore complexes.
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Integrated supplementary information
Supplementary Figure 1 tRNA intronic circular RNA (tricRNA) processing and its use for expression of circular Broccoli.
(a) We previously described an expression system to generate circular RNA aptamers based on aptamer insertion into a tRNA intron (RNA, Lu, et. al., 2014). Endogenous processing of tricRNA is illustrated. tRNA splicing endonuclease (TSEN) cleaves both strands of a stem in the transcribed primary tricRNA (pri-tricRNA), containing the tRNA exon (orange) and tRNA intron (black). Each cleaved 5' end has a 5'-hydroxyl modification and each 3' end has a 2',3'-cyclic phosphate modification. RNA stems containing these unique termini are substrates for the ubiquitous endogenous RNA ligase, RtcB, which ligates ends to make a new stem loop. Exon ligation forms a mature tRNA, while intron ligation forms a circular RNA. In the “tricY” system, the aptamer is expressed within the intron sequence. (b) We derived a way to encode aptamers, here with Broccoli (green), so that they would contain the exon-associated RtcB substrate as in a. Design of this system required a circular permutation of the tricRNA approach (cp-tricY). (c) When Broccoli is encoded within the intron of the tRNA in the tricY system, we observe weak expression of circular Broccoli. After 6 h of actinomycin D (ActD) treatment, linear RNAs are degraded while circular RNAs are stable. The cp-tricY system generated circular RNA more abundantly than tricY; however, it also generates a relatively abundant linear Broccoli species.
Supplementary Figure 2 Engineered sequence of a Broccoli-containing pri-racRNA and the corresponding cleaved pre-racRNA.
This transcript contains a 5' P3 Twister U2A ribozyme (blue) and a 3' P1 Twister ribozyme (cyan) that flank the stem-forming sequences (orange). For each construct, the 3′ fragment of the 5′ ribozyme and the 5′ fragment of the 3′ ribozyme (not shown) were replaced with either of the stem-forming sequences (orange). This was done so that the pre-racRNA that results from ribozyme cleavage (sites of cleavage denoted by black arrow heads) can form a stem that presents the tRNA substrate of RtcB. Mutations were made to the ribozyme sequences so that the ribozyme’s preferred secondary structure would form. Regions where these compensatory mutations were made is marked. These positions do not show high sequence conservation through mutational analysis (Angew. Chemie, Kobori & Kobayashi, 2016), consistent with the idea that the structure needs to be maintained, rather than the sequence25 (Nat. Chem. Biol., Roth, et. al., 2014). Additionally, in the 5′ P3 Twister, replacing this ribozyme’s 3′ fragment with the 5′ stem-forming sequence introduces a uridine where an adenosine is highly conserved among different ribozyme orthologs25 (Nat. Chem. Biol., Roth, et. al., 2014). This adenosine is also near the catalytic site of the ribozyme31 (Proc. Natl. Acad. Sci. U.S.A., Eiler, Wang & Steitz, 2015) (Nat. Chem. Biol., Liu, et. al., 2014). Thus, we tested two versions of the 5′ P3 Twister ribozyme. The first contained the same stem-forming sequence as all other 5′ ribozymes, and the second contained the mutation of this uridine to adenosine (U2A) that we predicted would help promote cleavage. The 5′ U6+27 and 3′ U6 terminator stems of the pri-racRNA are not shown but would be present at this stage of circular RNA expression in mammalian cells.
Transcription of pairwise 5′ and 3’ ribozyme combinations generate fully cleaved pre-racRNA in combinations involving Twister, Twister Sister, and/or Pistol. RNAs are transcribed for 2 h and then in vitro transcription is halted with urea before resolving the RNA on a denaturing 10% polyacrylamide gel and visualizing with DFHBI-1T (See Methods). All constructs contain Broccoli as an effector, and the Broccoli-containing bands pertaining to full-length, 3'- and 5'-cleaved, and fully cleaved pre-racRNA are identified according to expected RNA size. Constructs generating fully cleaved RNA require a P1 Twister on the 3' end and generate the most fully cleaved product when containing a Twister Sister 4, P3 Twister U2A, or Pistol on the 5' end. Other constructs generate much lower amounts of the fully cleaved pre-racRNA. Overexposure of fluorescent band detection is represented in red.
Supplementary Figure 4 Twister-cleaved pre-racRNA is ligated, forming racRNA, which is highly stable.
(a) We tested whether the RNA generated by transcription of the construct containing P1 Twister and P3 Twister U2A (see Fig. 2a,b) and then treated with RtcB resists exoribonucleolytic cleavage. Similarly, we tested whether the T4-PNK-treated RNA in Fig. 2b would degrade upon RNase R treatment. RNase R degrades RNA from the 3′ end. As expected, the T4 PNK-treated RNA is completely degraded while the RtcB-treated RNA is not. This suggests that the RtcB-treated RNA is a circular RNA. (b) Twister is thought to fold somewhat poorly in vitro25 (Nat. Chem. Biol., Roth, et. al., 2014). Thus, we compared the completely Twister-based Tornado with a similar system that uses the reliably-folded but slower wild-type hepatitis delta virus ribozyme instead of Twister as a 3’ ribozyme (RNA, Chadalavada, Cerrone-Szakal & Bevilacqua, 2007). This system has cleavage sites arranged so that the same pre-racRNA is generated by cleavage. Surprisingly, replacing the 3′ Twister with HDV generates an RNA of the same size; however it generates 30% less than Tornado. These data imply that Twister may fold better than expected in cells. (c) We tested whether the Broccoli fluorescent band in Fig. 2c is resistant to exoribonucleolytic cleavage. The band was excised and purified, then treated with RNase R. The Broccoli fluorescent band does not degrade. This suggests that the RNA lacks a 3′ end, consistent with being a circular RNA. (d) Expression of high levels of circular RNA using the Tornado expression system is dependent on strong promoters. Broccoli was expressed as a racRNA in HEK293T cells using the Pol II CMV promoter or the Pol III U6+27 promoter. After separating total RNAs on a 10% PAGE gel, DFHBI-1T staining identifies the Broccoli racRNA only when the RNA was expressed using the Pol III promoter.
Supplementary Figure 5 RNA expression by Tornado is circular and contains homogenous sequences at the ligation site.
(a) The stable Broccoli-containing band expressed by Tornado migrates at an unusual rate that depends on the gel percentage. In a 6% polyacrylamide gel, it runs faster than tRNA, but in a 10% polyacrylamide gel, it runs more slowly than tRNA. This characteristic is known for circular RNAs33,36,37 (Nucleic Acids Res., Zaug & Cech, 1982) (Cell, Ruskin et. al., 1984) (Nucleic Acids Res., Tabak, 1988), suggesting that the Broccoli-containing band is indeed a circular RNA. Gels were first imaged with DFHBI-1T to identify the Broccoli-fluorescent band and subsequently stained with SYBR Gold and imaged, which is shown. The 10% polyacrylamide gel image has been elongated to better compare migration of bands. (b) RT-PCR of two racRNAs produces a ladder pattern of PCR products. Two RNAs were expressed by Tornado in HEK293T cells; Broccoli as in Fig. 2c and Broccoli with an additional unstructured region as in Fig. 2d. Each RNA was purified and excised by gel electrophoresis, and then amplified by PCR after reverse transcription. PCR products migrated in an agarose gel as a ladder of equally-spaced bands. The smallest band was the length of the distance between the two primers, and each longer band increased in length according to the full-length size of the circular RNA (96 nt and 236 nt). The second bottom-most rungs (marked with arrows) were excised and purified. Gel images have been gamma-corrected to highlight dimmer bands. (c) Purified DNA bands from b were sequenced (see Methods). In all clones of Broccoli racRNA (top), the sequence of the stem-forming sequences was homogenous after endogenous ligation. This same sequence of a racRNA containing Broccoli with unstructured RNA (bottom) was nearly homogenous.
Supplementary Figure 6 Circular Broccoli accumulates in the cytoplasm and accumulates more highly with Tornado than with other expression systems.
(a) Circular Broccoli racRNA fluorescence is predominantly localized to the cytoplasm. Three days after transfection, HEK293T cells expressing Broccoli using Tornado generate significant fluorescence in the cytoplasm. In some cells, bright signal localizes to nuclear puncta that may correspond to Cajal bodies due to U6 promoter expression. Scale bar is 25 μm. (b) Flow cytometry analysis of Broccoli signal in different expression systems at the population level. We wanted to compare the population-level fluorescence of HEK293Ts expressing Broccoli using expression systems that were investigated in Fig. 3a (see lower table). By flow cytometry, we collected the Broccoli fluorescence of individual cells (488 [530_30]-A) and plotted signal against forward scattering area (FSC-A), which is related to cell size. We subset the cells that had fluorescence higher than untransfected cells (within black diagonal gate) and plotted a histogram of the number of cells at different fluorescence levels. The largest population of fluorescent cells expressed from Tornado is approximately 50 times brighter than that of the cells expressing Broccoli with tricY, and 200 times brighter than that of the linear Broccoli expressing cells.
HEK293T cells were transfected with plasmids encoding the Corn aptamer with a tRNA scaffold (tCorn). We sought to express tCorn as a circular RNA (circ-tCorn) using our circularization strategy. To demonstrate that the RNA bands with Corn fluorescence are circular, cells were treated with actinomycin D (ActD) for 6 h hours prior to harvesting the cellular RNA. The aptamer is expressed as a linear RNA or as a circle using either the tricY31 (RNA, Lu, et. al., 2014) or Tornado circular RNA expression systems (see Fig. 3d). We observed a very faint band after linear expression of tCorn using this exposure time (5 sec). This band is no longer detected when the cell is treated with ActD, indicating this RNA is unstable. On the other hand, the RNA expressed using the Tornado expression system is readily detected as a bright, fluorescent RNA that is insensitive to ActD treatment. The tricY expression system does not appear to generate any Corn fluorescent bands at this exposure time. We can also identify the circ-tCorn bands generated by Tornado expression when staining total cellular RNA using SYBR Gold. Here we also observed that the circular Corn RNA exhibits stability even after application of ActD. Overall these data show that the Tornado expression system can generate circular RNA containing the Corn aptamer.
Supplementary Figure 8 Expression of circular Broccoli by Tornado is not cytotoxic by various measures of normal cell function.
(a) We sought to determine if Tornado expression of circular Broccoli triggers apoptosis. HeLa cells were transfected with a mCherry-expressing plasmid or a plasmid encoding Broccoli for Tornado-based expression. Mock transfected cells can be induced into apoptosis with doxorubicin (1 mg/mL, 24 h), which can be detected from elevated levels of cleaved poly (ADP-ribose) polymerase (cleaved PARP). The cells expressing mCherry and circular Broccoli using Tornado both showed very low levels of cleaved PARP. No increase in toxicity is detected when expressing a circular RNA. (b) We compared cellular proliferation rates of HEK293T cells transfected with plasmids for mCherry expression or Tornado-based expression of Broccoli to see if Tornado expression affects cell proliferation. Non-fluorescent cells were present in each culture that were untransfected. We imaged cells from each culture and counted the number of non-fluorescent and fluorescent cells in each. To accurately count cells, cell nuclei were stained with Hoechst 33342. The rate of increase in non-fluorescent and fluorescent cells from three days post transfection until five days post transfection was calculated using the formula for exponential growth. Using the computed rates, we derived a doubling time for each population of cells, shown adjacent to each set of data in hours. Fluorescent cells proliferate somewhat slower than non-fluorescent cells – an effect largely attributed to transfection. Thus, expression of circular RNAs by Tornado has a relatively minimal effect on cellular proliferation rate. Data represents the mean number of cells (n = 4 independent fields of independent cells).
Supplementary Figure 9 Expression of circular RNA by Tornado does not activate the innate immune response.
(a) RIG-I levels reflect activation of the innate immune response. RIG-I normally triggers the innate immune response by recognizing RNAs that have a triphosphate on their 5' end. Transfection of HeLa cells with triphosphate-containing linear Broccoli RNA generates a readily detected innate immune response compared to mock transfected cells. In contrast, the innate immune response elicited by the expression of circular Broccoli using the Tornado expression system is minimal. The Tornado-expressed Broccoli level of innate immune response is similar to that induced by expressed of mCherry or a linear Broccoli aptamer. (b) NF-κB activation is not induced by expression of circular RNA. We detected NF-κB activation in HEK293 cells expressing luciferase driven by an NF-κB promoter. Treatment with IL-1β generates robust activation of NF-κB signaling, whereas expression of an RNA encoding mCherry or the Broccoli aptamer generates negligible levels of luminescence. We observed no difference in NF-κB activation when comparing linear expression of Broccoli with Tornado-based expression. Data in this panel represent the mean (n = 5 stimulation- and assay-independent samples).
Supplementary Figure 10 Levels of endogenous pre-tRNA tRNA are unperturbed by Tornado expression of RNA aptamers.
We wanted to determine if expressing circular RNA using the Tornado expression system perturbs endogenous tRNA processing, since the Tornado-expressed RNAs might sequester endogenous RtcB. We measured levels of pre-tRNA, which contains an intron, and mature tRNA by northern blotting in HEK293T cells expressing a variety of RNAs. The northern blot was probed using a biotin-conjugated DNA oligonucleotide that is complementary to the 5’ end of the human pre-tRNATyr, a region which precedes the intron. In cells expressing linear Broccoli and cells expressing circular Broccoli using Tornado, the bands corresponding to pre-tRNA and mature tRNA have the same intensity as in cells that were mock transfected. This suggests that expressing circular RNA does not perturb endogenous tRNA processing. The tricY system overexpresses pre-tRNATyr with an aptamer in its intron. In this blot, this exogenous pre-tRNA and tRNA generate the two strongest bands. Additionally, the endogenous pre-tRNA and tRNA are present at elevated levels in the cells expressing Broccoli using tricY. Collectively, this data demonstrates that Tornado expression of circular RNAs does not affect levels of endogenous tRNA. Full scan of blot is shown.
Supplementary Figure 11 Circular NF-κB aptamer expressed by Tornado inhibits NF-κB pathway activation.
(a) HEK293 cells containing the NF-κB luciferase reporter (see Methods) were transfected with a plasmid expressing a fusion (see Fig. 4a) of Broccoli with the indicated variant of the NF-κB aptamer (1-5) using the Tornado expression system. An aptamer fusion that lacks any NF-κB aptamer (null) is also separately expressed in this cell line. Each construct generates Broccoli fluorescent RNA that is resistant to degradation by actinomycin D treatment, suggesting that each RNA is circular. Images have been gamma-corrected to improve band visibility. (b) We tested whether expression of these racRNAs affects NF-κB pathway activation (left). NF-κB aptamer 1 was omitted due to high rates of cell death observed when this aptamer was expressed. We stimulated cells with 50 ng/mL IL-1β for 2.5 h and detected NF-κB pathway activation by an increase in the luminescence signal. Activation is completely blocked by a known inhibitor (BAY 11-7082) of IκB kinase, a critical kinase in this pathway. Each of the NF-κB aptamers led to significant inhibition of pathway activation compared to expression of the Broccoli aptamer (‘null’). For NF-κB aptamer 5, we determined whether inhibition was caused specifically by the aptamer binding (right). When circular Broccoli is expressed, there is a lack of inhibition, comparable to mock transfected cells. When a circular RNA comprising Broccoli and NF-κB aptamer 5 is expressed, inhibition is robust. When aptamer 5 is replaced with a mutant of the aptamer that has weaker binding (Kd ~ 1.1 μM)45 (RNA, Wurster & Maher, 2008), inhibition is reduced. Since circular RNA concentration in this cell line is similar to the Kd, the protein is expected to be ~50% inhibited. All luminescence values were normalized by the number of cells counted in each condition at the time of assay, and also to the stimulated signal for the null aptamer condition without chemical inhibitor (left) or to the mock-transfected cells (right). Data in this panel are presented as the mean (n = 4 stimulation- and assay-independent samples).
Supplementary Figure 12 Comparison of linear and circular NF-κB aptamer folding through measurement of Bmax
Electrophoretic mobility shift assays were performed to quantify binding of the NF-κB aptamer to its protein target, p65 (left). Shown is a representative shift in RNA mobility due to p65 binding to either the linear (top) or circular aptamers (bottom). RNA sequences containing Broccoli and NF-κB aptamer fusions as in Fig. 4a were in vitro transcribed, and, in the case of the circular aptamer, were ligated by RtcB. Samples containing these linear or circular RNAs were titrated with recombinant p65 protein at concentrations ranging from 50 nM to 750 nM and separated on a gel under native conditions (see Methods). After transfer to a membrane, the shifted RNA was detected by Northern blot. In these experiments we detected the RNA using a 32P-labeled anti-Broccoli DNA probe. At right, the fraction of the aptamer that is folded was calculated by quantifying Bmax using a Scatchard plot of the binding data. The x-intercept of the linear aptamer data series indicates a Bmax of ~0.24 and the circular aptamer shows a Bmax of ~0.68. Thus, we observed that circularizing the NF-κB aptamer improves its folding by ~2.5-fold compared to the linear form of the aptamer.
Four variants of the circular SAM biosensor were generated, each with different transducers identified in Fig. 5b. Each biosensor was expressed using the Tornado expression system in HEK293T cells, and total RNA was separated on a denaturing gel. Using DFHBI-1T staining in the absence of SAM, low background signal was observed for variant 1 relative to variants 2-4, a favorable property for a biosensor. All variants were circular RNAs, since they were not degraded by actinomycin D (ActD) treatment. We observed that linear Broccoli degrades following this treatment. We were also able to identify these Broccoli-fluorescent RNAs by SYBR Gold staining. As can be seen in the lower image, roughly similar levels of each biosensor variant are expressed, but markedly reduced Broccoli fluorescence is seen with transducer variant 1, further supporting the idea that it has relatively low background.
Supplementary Figure 14 The circular SAM biosensor is specific and rapid, and accumulates to detectable levels in mammalian cells.
(a) S-adenosyl-methionine (SAM) activates the fluorescence of the SAM biosensor in vitro, whereas SAM analogs are much less effective. RNA was transcribed to contain the same transducer sequence as that which is expressed as circular RNA by Tornado. The biosensor RNA is present at 1 μM, and 100 μM of the indicated SAM analog was added. As can be seen, the biosensor shows the most efficient activation with SAM. For these experiments, the stem loop that is normally formed during circular RNA maturation is replaced by an extended double-stranded stem without a loop. (b) Kinetic measurement of fluorescence activation of biosensor upon addition of SAM. The fluorescence signal reachs 75% of total fluorescence within 5 min. In these experiments, the biosensor RNA is present at 1 μM, while SAM is added to 100 μM. As in a, the stem loop is replaced by an extended double-stranded stem to simulate the circular RNA. (c) The SAM biosensor containing transducer variant 1 is expressed more abundantly using Tornado than when expressed as a linear RNA. Treatment with actinomycin D (ActD) reveals that the linear biosensor is unstable, as expected. In contrast, the circular biosensor remains highly expressed even when new transcription is inhibited by this treatment. Scale bar is 25 μm. (d) HEK293T cells expressing the linear form of the SAM biosensor do not generate detectible fluorescence. In contrast, SAM biosensor fluorescence is readily observed when the biosensor is expressed as a circular RNA using the Tornado expression system. In each case, transcription was driven by the same U6 promoter.
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Litke, J.L., Jaffrey, S.R. Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nat Biotechnol 37, 667–675 (2019). https://doi.org/10.1038/s41587-019-0090-6
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