Letter pubs.acs.org/synthbio
Artificial OFF-Riboswitches That Downregulate Internal Ribosome Entry without Hybridization Switches in a Eukaryotic Cell-Free Translation System Atsushi Ogawa,* Hiroki Masuoka, and Tsubasa Ota Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan S Supporting Information *
ABSTRACT: We constructed novel artificial riboswitches that function in a eukaryotic translation system (wheat germ extract), by rationally implanting an in vitro-selected aptamer into the intergenic internal ribosome entry site (IRES) of Plautia stali intestine virus. These eukaryotic OFF-riboswitches (OFF-eRSs) ligand-dose-dependently downregulate IRES-mediated translation without hybridization switches, which typical riboswitches utilize for gene regulation. The hybridization-switch-free mechanism not only allows for easy design but also requires less energy for regulation, resulting in a higher switching efficiency than hybridization-switch-based OFF-eRSs provide. In addition, even a small ligand such as theophylline can induce satisfactory repression, in contrast to other types of OFF-eRSs that modulate the 5′ cap-dependent canonical translation. Because our proposed hybridization-switch-free OFF-eRSs are based on a versatile IRES that functions well in many types of eukaryotic translation systems, they would be widely usable elements for synthetic gene circuits in both cell-free and cell-based synthetic biology. KEYWORDS: aptamer, cell-free translation, IRES, riboswitch
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type of OFF-eRS has a strong advantage in that it is easily designed without the need for complicated, ligand-responsive hybridization switches, which general riboswitches require for gene regulation. In fact, due partly to their facile design, various OFF-eRSs based on this mechanism have been synthetically constructed one after another.9−13 However, this design strategy has an issue in that an aptamer with the AUG triplet is not in principle available, because this triplet should manage to function as the start codon instead of the codon of the downstream gene. In addition, depending on the aptamer to be implanted, there is another problem, in that one aptamer in the 5′ UTR is not sufficient to prevent ribosome progression. For example, in the case of a theophylline aptamer,14 which has been widely used in both bacterial and eukaryotic artificial riboswitches,15−25 as many as three aptamers in tandem are needed to satisfactorily suppress the canonical translation,11 though it is difficult to prepare a DNA template with relatively long repeating sequences at narrow intervals.
riboswitch is an allosteric regulatory element with a ligand-binding domain, called an aptamer, in an untranslated region (UTR) of mRNA.1 The specific binding between the aptamer domain and its ligand induces conformational change of mRNA, which turns gene expression ON or OFF. Although several types of natural riboswitches have been identified thus far, mainly in bacteria,1 a riboswitch can be also artificially constructed with an in vitro-selected aptamer.2 Because an aptamer can potentially be selected against any ligand of interest through a method called SELEX,3 there has recently been much interest in artificial riboswitches as useful elements of synthetic gene circuits, not only in cell-based systems4,5 but also cell-free systems.6−8 The first reported instance of artificial riboswitches was a eukaryotic OFF-riboswitch (OFF-eRS) that was constructed merely by inserting an in vitro-selected aptamer into the 5′ UTR.9 Since the eukaryotic ribosome is loaded onto the 5′ terminus of mRNA and then scans the mRNA for the start codon (in the canonical translation system), a rigid structure of an aptamer-ligand complex in the 5′ UTR inhibits ribosome loading or scanning, thus downregulating gene expression. This © XXXX American Chemical Society
Received: April 18, 2017
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DOI: 10.1021/acssynbio.7b00124 ACS Synth. Biol. XXXX, XXX, XXX−XXX
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Figure 1. Schematic illustration (above) and a partial sequence (below) of the control mRNA 2′ encoding the FLuc gene downstream of the PSIV IRES.17 The domain 3 is composed of the SL-VI and the PK-I. The numbers under mRNA and large dots represent nucleotide positions in the PSIV genome and base-pair interactions, respectively. 2′-CrNCP was constructed by replacing the N-CP region in 2′ with that of the CrPV to improve the translation efficiency.
this IRES to function well in many types of eukaryotic translation systems,26,30−32 including wheat germ extract (WGE). We used WGE here as a eukaryotic expression system because it is useful for designing eukaryotic riboswitches.33,34 We previously determined a sufficient region of the PSIV IRES to effectively express the downstream gene in WGE: a 6004− 6204 segment also covering 12 nucleotides that encode the Nterminal region of the capsid protein precursor (N-CP).25 In the present study, we integrated this segment into the firefly luciferase (FLuc) gene lacking the first AUG codon to prepare a control mRNA 2′ (Figure 1). Unlike the original mRNA 2 that we previously constructed,25 the first codon was removed to completely inhibit initiator tRNA-dependent translation of the FLuc gene.31 In addition, this control mRNA 2′ has a 5′ stem-loop structure beginning with a G triplet (5′-SL) to prevent canonical translation,35 because the ribosome can be loaded on mRNA without a 5′ cap (at least in WGE).36 We then replaced the N-CP region with that of cricket paralysis virus (CrPV), which belongs to the same class in the same family (Dicistroviridae) as PSIV, to improve the translation initiation efficiency (2′-CrNCP, Figure 1), by reference to a report describing that the GCU codon is suitable for the start codon in Dicistroviridae intergenic IRES-mediated translation.37 In fact, 2′-CrNCP exhibited 1.6-fold higher translation efficiency, comparable to that of the canonical translation of IRES-free mRNA with the most effective translational enhancer, E0138 (c-mRNA in the ref 23). The PSIV IRES is known to form a higher-order structure with four stem loops (SL-III, SL-IV, SL-V, and SL-VI) and three pseudoknots (PK-I, PK-II, and PK-III) so that it can directly recruit the ribosome with no need for eukaryotic initiation factors (Figure 1).29 Moreover, a recent study revealed that the 3′ terminal domain involving the SL-VI and the PK-I in a Dicistroviridae intergenic IRES (called domain 3) mimics the tRNA-mRNA interaction in the P site of the
Aside from these classic OFF-eRSs (i.e., canonical translationregulating ones), we recently reported another type of synthetic OFF-eRS that ligand-dose-dependently downregulates internal ribosome entry site (IRES)-mediated translation in such a way that an aptamer-ligand complex distorts the IRES structure.22 In this type of OFF-eRS, an aptamer is placed upstream of the IRES (i.e., a ribosome loading site), so that there is much less limitation of its sequence. Moreover, even when we implant a theophylline aptamer, which is ill-suited to classic OFF-eRSs as described above, one aptamer per riboswitch is sufficient to achieve moderate switching efficiency: the repression ratio of the optimized OFF-eRS with one theophylline aptamer (∼6fold at 1 mM theophylline) was comparable to that of a classic OFF-eRS with three continuous aptamers (∼7-fold at 1 mM theophylline).11 This is probably because the aptamer-ligand complex functions as an IRES-distortion inducer, not just as a ribosome blocker as in classic OFF-eRSs. Nonetheless, this type of IRES-based OFF-eRS exploits complicated, ligand-responsive hybridization switches, thus requiring much more design effort and thermodynamic energy to change its conformations in response to the ligand compared to hybridization-switch-free ones.23,24 We herein report a novel type of efficient, IRES-based OFF-eRS that has an aptamer within an IRES and requires no hybridization switches to regulate downstream gene expression.
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RESULTS AND DISCUSSION We chose the Plautia stali intestine virus (PSIV) intergenic IRES,26 which was previously used to create hybridizationswitch-based OFF-eRSs,22 in which to embed an aptamer. This IRES has been well studied: its tertiary structure and translation initiation mechanism have already been elucidated.27−29 Surprisingly, it allows for translation initiation from any sense codon right after its core structure by using the corresponding elongator tRNA, without any help from initiation factors or an initiator tRNA (vide inf ra).30 This unique mechanism allows B
DOI: 10.1021/acssynbio.7b00124 ACS Synth. Biol. XXXX, XXX, XXX−XXX
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Figure 2. Design strategy for PSIV IRES-based OFF-eRSs that use no hybridization switches: the wild-type IL in the SL-VI is replaced with a split aptamer, an aptamer-IL (left to middle). Because a ligand-free aptamer is generally flexible, this mutant is expected to be folded into a moderately active IRES structure (middle, ON state). In contrast, when the ligand binds to the embedded aptamer, the SL-VI is likely to be extended and/or distorted and thus unable to form the PK-I, which should repress the IRES-mediated translation (right, OFF state).
Figure 3. Theophylline-responsive OFF-eRSs that downregulate the IRES-mediated translation without hybridization switches. (A) and (B) The sequences of domain 3 (SL-VI, PK-I, and VLR) in theoAn (n = 0, 1, 2, 3, or 4) and theoA3-rS, respectively. A box named An and the suffix n represent the inserted adenosines and the number thereof, respectively. (C) Relative activities of FLuc translated from mRNAs with theophyllineresponsive OFF-eRSs, compared to that from 2′, in the absence (ON) or presence (OFF) of 1 mM theophylline. Average ON/OFF ratios of FLuc activities are shown below the graph. (D) The ON/OFF ratios of theoA3-rS, 2′-CrNCP and m-theoA3-rS at various concentrations of theophylline. (E) Chemiluminescence images of FLuc translated from theoA3-rS (left) and m-theoA3-rS (right) in the absence or presence of 1 mM theophylline (theo) or caffeine (caf). (F) Fluorescence images of YPet translated from theoA3-rS-YPet without or with 1 mM theophylline.
SL-VI with an essential stem loop in the 3′ side,29,37 we considered that this IL in the PSIV IRES might be replaceable with a larger, flexible split aptamer (Figure 2):23,24 the mutated IRES with an aptamer-IL in the SL-VI would maintain a certain level of translation efficiency (Figure 2, middle: ON state). If
ribosome to facilitate the elongator tRNA-dependent translation initiation from the A site.28 We herein focused on this domain 3, especially the SL-VI, which has a small internal loop (IL). In light of the fact that Taura syndrome virus (TSV), another class-member of the Dicistroviridae family, has a larger C
DOI: 10.1021/acssynbio.7b00124 ACS Synth. Biol. XXXX, XXX, XXX−XXX
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translation, though the stabilized stem set contributes much more. Although it was difficult to reduce the adverse effect derived from the third possibility since we could not shorten the wellminimized aptamer, the translation efficiency in the ON state of theoA3-rS (16% of that by 2′) was high enough to be comparable to that of previously reported, hybridization-switchbased OFF-eRSs with the wild-type PSIV IRES.22 This suggests that the P site somewhat tolerates the occupancy of the mutated domain 3 with a larger but flexible IL. In contrast, the IRES-mediated translation of theoA3-rS was considerably repressed in the presence of 1 mM ligand, theophylline (OFF state, 1.1% of that by 2′), indicating that domain 3 with a rigid aptamer-ligand complex cannot access the P site, probably due to the distortion and/or extension of the SL-VI (vide inf ra). Consequently, the ON/OFF ratio at 1 mM theophylline was 14 ± 1 (Figure 3C), which is more than twice that of the optimized hybridization-switch-based OFF-eRS that is responsive to theophylline (5.8 at 1 mM theophylline).22 This remarkable difference can be attributed largely to the hybridization-switch-free mechanism of the present riboswitch.23,24 theoA3-rS also does not require a ligand-responsive formation of stems in both ends of the embedded aptamer, since these stems already form in the absence of the ligand, in contrast to previously reported, hybridization-switch-free ONeRSs.23,24 This means that much less energy is required for ligand-responsive conformational change in theoA3-rS, though small energy may be required to disrupt the weak PK-I (a stronger PK-I is harder to disrupt (theoA3-rS-rPKI, Figure S1)). We then carried out several experiments to confirm that the specific aptamer-ligand interaction actually repressed the IRESmediated translation. As shown in Figure 3D, the ON/OFF ratio of theoA3-rS increased theophylline-dose-dependently in a broad range of concentrations (from 5 μM to 3 mM), while 2′-CrNCP, devoid of the theophylline aptamer in the SL-VI, showed no dependence on theophylline. The lower limit (5 μM with the ON/OFF ratio of 1.3) is much lower than that of the previously reported, hybridization-switch-based OFF-eRS22 and is comparable to detection limits of theophylline aptamer-based fluorescent biosensors that are composed of much shorter RNA.25 This indicates that the context of the IRES has little effect on the aptamer binding. In contrast to its sensitive responsiveness to theophylline, theoA3-rS did not respond to caffeine, whose structure is identical to that of theophylline except for the N7 methyl group (Figure 3E, left), indicating its high specificity. In addition, when a mutated aptamer with C22A and U24A (m-theo, Figure 3B)40 was used instead so as not to bind to theophylline (m-theoA3-rS), the theophylline responsiveness was lost (Figure 3C, 3D and 3E, right). Taken together, these results clearly show that the aptamer-ligand complex formation switches off the IRES-mediated translation of theoA3-rS. This is also supported by the fact that the theophylline dependency remained high even after the FLuc gene was changed to another one encoding a yellow fluorescent protein, YPet41 (theoA3-rS-YPet, Figure 3F), which means that the riboswitch functions independently from the regulated gene. To next investigate how the stem extension in the SL-VI affects translation efficiency, we altered the 5′ or 3′ side of the split theophylline aptamer in theoA3-rS into a sequence complementary to the opposite side in order to form a long stem (3S-theoA3-rS or 5S-theoA3-rS, respectively, Figure
so, a ligand specific to the embedded aptamer was expected to repress the IRES-mediated translation through a distortion and/or extension of the SL-VI that would be induced by forming a rigid, ligand-aptamer complex therein (Figure 2, right: OFF state). We thus substituted a split theophylline aptamer for the IL in the SL-IV of 2′-CrNCP in such a way that the 3′ side is longer (theoA0, Figure 3A). However, this replacement considerably attenuated the translation efficiency (0.48% and 0.30% of that by 2′ and 2′-CrNCP, respectively), contrary to our expectation, though it showed a slight riboswitch activity (the repression ratio, i.e., the ON/OFF ratio, was 2.2 with 1 mM theophylline) (Figure 3C). There were three possible explanations for this low efficiency, all of which can be attributed to the longer IL composed of the aptamer: (1) the variable loop region (VLR) intervening between the SL-VI and the PK-I was too short for the lengthened SL-VI to precisely form the PK-I; (2) the two stems at both ends of the embedded aptamer did not reliably form duplexes (upper and lower stems, Figure 3A); (3) the mutated domain 3 with the longer IL (especially in the 5′ side) was not suitable for occupying the P site in the ribosome due to the space limitation, even though the aptamer was sufficiently flexible. To initially address the first possibility, we inserted one to four adenosines (As) into the 3′ terminus of the VLR (theoA1−4, Figure 3A), on the basis of the fact that CrPV IRES activity tolerated base insertion of up to four nucleotides into this region.39 These VLR extensions except for theoA4 increased the translation efficiency in the absence of theophylline (ON state) by 3−4 fold, which led to higher ON/OFF ratios (Figure 3C). While theoA2 with two extra As exhibited the highest translation efficiency in the ON state, its riboswitch activity was slightly lower than that of theoA3 with three extra As. The ON/OFF ratio of theoA3 (4.4 at 1 mM theophylline) was comparable to those of previously reported theophyllineresponsive OFF-eRSs.11,22 Nonetheless, the translation efficiency in the ON state was still relatively low (1.4% of that by 2′). We thus next sought to strengthen the two stems in the SLVI of theoA3 to deal with the second possibility. When we altered all A−U pairs in both stems to stronger G−C pairs (theoA3-rS, Figure 3B), the IRES-mediated translation (in the ON state) was approximately 11.5 times more efficient than that by theoA3 (Figure 3C). This result indicates that the wildtype base pairs in the SL-VI are not stable enough to form helical stems with a longer IL (in theoA0−3), while stronger ones allow the mutated IRES to be more easily folded into an active structure. However, when we inserted an additional G−C pair into the lower stem of theoA3-rS to further enhance stem stability, the translation efficiency was decreased (theoA3-rlS, Figure S1). This is probably because the stem-stabilization effect was overcompensated by a stem-extension effect, which inhibits IRES-mediated translation (vide inf ra). Therefore, we concluded that the upper and lower stem set in theoA3-rS was the most appropriate for efficient translation mediated by the muted IRES with a long aptamer-IL. Incidentally, although we tried to make the domain 3 more rigid by altering A−U pairs in the PK-I to G−C pairs (theoA3-rS-rPKI), these alterations adversely affected the translation efficiency (Figure S1). In addition, when we removed the extra As in the VLR to confirm their effect again, the translation efficiency was slightly attenuated (theoA0-rS, theoA1-rS, and theoA2-rS, Figure S1), meaning that they partially contribute to IRES-mediated D
DOI: 10.1021/acssynbio.7b00124 ACS Synth. Biol. XXXX, XXX, XXX−XXX
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Figure 4. Two other ligand-responsive riboswitches. (A) The sequence of SL-VI in FMN-A3-rS with a split FMN aptamer. (B) FMN dependency of FMN-A3-rS and 2′-CrNCP. (C) The sequence of SL-VI in TMR-A3-rS with a split TMR aptamer. (D) TMR dependency of TMR-A3-rS and 2′CrNCP. It should be noted that the concentration is up to 500 μM for FMN and 1 mM for TMR because higher concentrations of these ligands adversely affect the PSIV IRES-mediated translation.25
S1A). As a result, 3S-theoA3-rS with a stem totalling 17 bp significantly suppressed translation efficiency, to 24-fold lower than that by theoA3-rS in the ON state (i.e., 1.7-fold lower than in the OFF state (at 1 mM theophylline)), while 5S-theoA3-rS with a 14-bp stem exhibited an incomplete suppression, specifically a 3-fold lower translation efficiency (Figure S1B). Given the fact that the actual length of the aptamertheophylline complex is closer to the latter stem,40 these results indicate that the theophylline-dependent translational repression in theoA3-rS can be attributed to not only stem extension but also stem distortion in the SL-VI, both of which would be induced by an aptamer-ligand binding. Finally, we checked the modularity of the present hybridization-switch-free, IRES-based OFF-eRSs. Specifically, the split theophylline aptamer in the SL-VI of theoA3-rS was replaced with a split FMN aptamer42 or a split 5-carboxy-tetramethylrhodamine (TMR) aptamer,43 both of which have similar affinities to their small ligands, in order to prepare FMN-A3-rS or TMR-A3-rS, respectively (Figure 4A or 4C, respectively). We also constructed i-theoA3-rS by inverting each side of the theophylline aptamer (Figure S1A). As expected, all derivatives repressed the IRES-mediated translation well in response to each ligand, though two riboswitches with a different aptamer (i.e., FMN-A3-rS and TMR-A3-rS) each exhibited a slightly lower ligand dependency than theoA3-rS (Figure 4B, 4D, and S1B). These slightly lower activity levels are probably due to the smaller stem-distortion effect, as estimated from the smaller difference in length between the sides of these split aptamers. This indicates that it is more favorable to embed an aptamer that can induce stem distortion (and stem extension) to a large extent by forming a complex with the ligand. In terms of itheoA3-rS, although the ON/OFF ratio was comparable to that of theoA3-rS, the translation efficiency in the ON state was 4-fold lower (Figure S1B). This is probably because the longer sequence in the 5′ side is less favorable for the IRES-mediated translation, due to the space limitation of the P site. In fact, the translation efficiency in the ON state of TMR-A3-rS, which has a similar sequence length in the 5′ side, was as low as that of itheoA3-rS. Nonetheless, this preference would hardly affect the riboswitch activity, because it remains also in the OFF state. In any case, the fact that it is very straightforward to construct efficient OFF-eRSs merely by replacing the aptamer part in a theophylline-responsive one (theoA3-rS) clearly shows how
free from hybridization switches these riboswitches are and thus how versatile their design strategy is. In conclusion, we rationally constructed a novel type of OFFeRS that ligand-dose-dependently downregulates the IRESmediated translation with no hybridization switches. By virtue of its hybridization-switch-free mechanism, this artificial riboswitch requires much less energy to achieve conformational change,23,24 resulting in a more sensitive response to the ligand, compared to previous hybridization-switch-based OFF-eRSs.22 It also requires for downregulation only a minor extension and/ or distortion around an aptamer embedded in the local core IRES structure, so that even a small ligand such as theophylline can induce a satisfactory repression of translation with one aptamer-ligand complex per mRNA. This is largely in contrast to the classic type of OFF-eRS that regulates the canonical translation, in which the aptamer-ligand complex functions just as an obstacle to the ribosome.11 In addition to these advantages in the activity of the present OFF-eRSs, their design is very facile: one need only replace the aptamer in the theophylline-responsive riboswitch (theoA3-rS) with a wellminimized split aptamer (preferably, a shorter one that can induce a lot of stem extension and stem distortion) that is specific to a ligand of interest, as in the cases of FMN and TMR. It is possible to obtain such a split aptamer through in vitro selection with an appropriate initial pool followed by minimization (i.e., deleting extra bases that are not required for binding to its ligand).34 Alternatively, one could probably construct an OFF-eRS with the same design strategy by using another Class I intergenic IRES (e.g., the CrPV IRES) instead of the PSIV IRES.37 Because the present OFF-eRSs and our previously reported ON-eRSs25 modulate internal ribosome entry in contrast to typical artificial riboswitches, more than two of them can be embedded into one polycistronic mRNA to regulate expression of various genes on it: to create a larger single-mRNA gene circuit.44 Although their activities were verified only in the WGE optimized for cell-free translation, they may be further improved by altering the translation conditions. In addition, given the fact that these riboswitches are based on the versatile PSIV IRES,26,30−32,37,45 they are expected to function well in many types of eukaryotic translation systems. Therefore, they are promising as widely usable elements for synthetic gene circuits in both cell-free and cell-based synthetic biology.4−8 E
DOI: 10.1021/acssynbio.7b00124 ACS Synth. Biol. XXXX, XXX, XXX−XXX
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METHODS Plasmids, Primers, and Enzymes for Preparing DNA Templates. A pUC57-based plasmid named pPSIV-IRES, encoding the PSIV IRES sequence, was artificially synthesized by GENEWIZ (Saitama, Japan). The whole sequence of this plasmid is described in Supporting Information. Those of pE01-Luc encoding the FLuc gene and pHis-SRY-YPet encoding the YPet gene were reported elsewhere.46 Synthetic primers were purchased from Life Technologies (Tokyo, Japan) or Eurofins Genomics (Tokyo, Japan). The primer sequences are also presented in Supporting Information. All PCRs were performed using PrimeSTAR MAX DNA Polymerase, which has extremely high fidelity, from Takara Bio (Ohtsu, Japan). The restriction enzyme SpeI was also purchased from Takara Bio. The ligation reactions were carried out with a Ligation high Ver.2 kit from TOYOBO (Osaka, Japan). Preparation of DNA Templates. DNA templates were prepared by SpeI ligation (and the subsequent final PCR) between the 5′ segment (the T7 promoter and an IRES part) and the 3′ segment (an open reading frame and the 3′ UTR), which were PCR-amplified from pPSIV-IRES and pE01-Luc (or pHis-SRY-YPet), respectively, by using a primer with the SpeI site.33 The former 5′ segment was constructed using sequential PCRs in which the PCR product was agarose-gel-purified and then used as a template in the next PCR. Preparation of mRNA Templates. mRNA templates were constructed by in vitro runoff transcription of the DNA templates with a T7-Scribe Standard RNA IVT Kit (CellScript, Madison, WI, USA) according to the manufacturer’s protocol. The transcribed mRNAs were purified with an RNeasy MinElute Cleanup Kit (QIAGEN, Tokyo, Japan) or a QIAquick Nucleotide Removal Kit (QIAGEN) and quantified by absorbance at 260 nm. Cell-Free Translation of mRNA Templates. Cell-free translation of mRNA templates in WGE was carried out as previously described.25 Evaluation of Translation Efficiency. The chemiluminescence intensities of translated FLuc (with luciferin) and the fluorescence intensities of translated YPet were measured as previously described.25,46
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ABBREVIATIONS UTR, untranslated region; eRS, eukaryotic riboswitch; IRES, internal ribosome entry site; PSIV, Plautia stali intestine virus; WGE, wheat germ extract; N-CP, N-terminal region of the capsid protein precursor; SL, stem-loop; PK, pseudoknot; CrPV, cricket paralysis virus; IL, internal loop; TSV, Taura syndrome virus; VLR, variable loop region; m-theo, mutated theophylline aptamer; TMR, 5-carboxy-tetramethylrhodamine
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REFERENCES
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00124.
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Letter
DNA sequences of pPSIV-IRES and primers, translation activities of theoA3-rS derivatives (Figure S1) (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Atsushi Ogawa: 0000-0001-5240-3395 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partially supported by JSPS KAKENHI Grant Number 16K05846. F
DOI: 10.1021/acssynbio.7b00124 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Letter
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DOI: 10.1021/acssynbio.7b00124 ACS Synth. Biol. XXXX, XXX, XXX−XXX
