Riboswitch module and method for controlling eukaryotic protein translation

Abstract

It provides a platform for regulating the translation of any protein of interest within eukaryotic cells. [Solution] This disclosure provides a gene construct comprising a recombinant internal ribosome entry site (IRES) that can be used as a riboswitch to functionally regulate the translation of an mRNA sequence encoding a protein of interest. In other aspects, this disclosure provides recombinant cells, methods, kits and systems that utilize it to provide a platform for regulating the expression of essentially any protein of interest in eukaryotic cells, for example.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 038,536, filed June 12, 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing, which has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on June 10, 2021, is named 002806-190120WOPT_SL.txt and has a size of 42,737 bytes.

[0003] Parties in a joint research agreement The claimed invention was made by, on behalf of, and / or in connection with, one or more of the following parties to the joint research agreement, namely the President and Fellows of Harvard College and BASF Corporation. The joint research agreement was in effect prior to the effective filing date of the claimed invention, and the claimed invention was made as a result of activities carried out within the scope of the joint research agreement.

[0004] Technical field This disclosure provides constructs and methods for regulating protein expression in eukaryotic cells using recombinant group 1 internal ribosome entry site (IRES) elements derived from viral IRES elements. [Background technology]

[0005] Background of this disclosure The regulation of gene expression is crucial for growth and development, as well as for maintaining proper homeostasis in the face of changing environmental conditions. Therefore, cells utilize various mechanisms to increase or decrease the production of specific gene products (e.g., proteins or RNA). Expression levels can be regulated, for example, to trigger developmental pathways, in response to environmental stimuli, or to adapt to new food sources. Gene expression can be regulated at the transcriptional level, for example, by increasing or decreasing the rate of transcription initiation or the phase of RNA processing. Post-translational modifications of proteins (e.g., by increasing or decreasing their degradation rates) can also be controlled. Numerous different mechanisms exist in nature for regulating gene expression, and these mechanisms are typically interconnected to form complex regulatory networks. The use of different mechanisms and triggers allows cells to express specific subsets of genes or adjust the levels of specific gene products as needed. Doing so allows cells to respond more quickly to environmental stimuli while conserving energy and resources. For example, bacteria and eukaryotic cells often regulate the expression of enzymes used in synthetic or metabolic pathways based on the availability of required substrates or end products. Similarly, many cell types induce the synthesis of protective molecules (e.g., heat shock proteins) in response to environmental stress.

[0006] Several methods have been developed to artificially control the level of gene expression, many of which are modeled after naturally occurring regulatory systems. Generally, gene expression can be controlled at the level of RNA transcription, or post-transcriptionally, for example, by controlling the processing or degradation of mRNA molecules, or by controlling their translation. For example, gene expression can be regulated by administering small molecule activators or inhibitors (e.g., to increase or decrease the activity of transcription factors), or by administering nucleic acids designed to inactivate or degrade mRNA (e.g., using ribozymes, antisense DNA / RNA, and RNA interference techniques). While these methods have proven useful in many applications, their usefulness can be limited by certain drawbacks. For example, ribozyme, antisense DNA / RNA, and RNAi-based methods typically require sequence-specific techniques (e.g., small interfering RNAs used in RNAi and antisense DNA / RNA must be specifically designed for each target). Furthermore, the use of small molecule activators and inhibitors to regulate transcription is also not ideal, as such methods typically have slow response times.

[0007] Research efforts to address these shortcomings have led to the development of a prokaryotic RNA sensing module called a "toehold switch," which relies on trigger-based unfolding of the ribosome-binding site (RBS). See, for example, U.S. Patent No. 10,208,312 (Patent Document 1), the entirety of which is incorporated herein by reference. The toehold switch selectively represses the translation of a target transcript by concealing the RBS in the absence of a distinct trigger RNA ("trRNA"), and reveals the RBS in the presence of trRNA, initiating the translation of a functionally linked sequence encoding a protein of interest. The prokaryotic toehold switch partially addresses the shortcomings of other prior art methods by providing an efficient mechanism for regulating translation in prokaryotes. However, this toehold switch mechanism is generally incompatible with eukaryotic systems that rely on a more complex set of epigenetic signals for initiating and regulating translation. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 10,208,312 [Overview of the Initiative]

[0009] Summary of Exemplary Aspects of This Disclosure This disclosure addresses various needs in the art by providing novel gene constructs and methods for regulating protein translation. These constructs can be used, for example, as platforms for regulating the translation of any protein of interest in eukaryotic cells without requiring sequence-specific redesign. Furthermore, the systems described herein enable the artificial control of intracellular gene expression in response to external stimuli.

[0010] In particular, the present disclosure describes gene constructs, recombinant cells, methods, kits, and systems that provide, for example, a platform for regulating the expression of essentially any protein of interest within a eukaryotic cell.

[0011] In a first general aspect, the present disclosure provides recombinant IRES modules engineered to reduce or prevent the translation of a functionally linked mRNA sequence encoding a protein of interest. These recombinant IRES modules are further engineered to fold into an active form in the presence of a specific trRNA. When activated, translation of the functionally linked mRNA sequence proceeds. The trRNA can be an artificially introduced sequence into the cell (e.g., by plasmid, or chemically mediated transfection), or a sequence found in a naturally occurring mRNA (e.g., viral mRNA). Thus, these recombinant IRES modules can be used to regulate the translation of a protein of interest for therapeutic or industrial applications, and can also be used as sensors for detecting exogenous stimuli such as viral infection.

[0012] In another general aspect, the disclosure provides a recombinant nucleic acid molecule comprising: (a) a first segment encoding a Group 1 Dicistroviridae internal ribosome entry site (IRES) modified to incorporate an exogenous nucleotide sequence into a first site and a second site; and (b) a second segment encoding a protein, downstream of and operably linked to the first segment, such that translation of the protein is suppressed when the IRES is in an inactivated state, wherein the first site comprises a first nucleotide sequence and the second site comprises a second nucleotide sequence that is at least a partial reverse complement of the first nucleotide sequence. In some aspects, the nucleic acid molecule is mRNA. In some aspects, the second nucleotide sequence is substantially the entire reverse complement of the first nucleotide sequence. In some aspects, the Group 1 Dicistroviridae IRES is a Cricket paralysis virus (CrPV) IRES, a Kashmir bee virus (KBV) IRES, or an Acute bee paralysis virus (ABPV) IRES.

[0013] In some aspects, the Group 1 Dicistroviridae IRES is modified to incorporate an exogenous nucleotide sequence into a first site and a second site, and the first and second sites are each independently selected from any of Site 1, Site 2, Site 3, Site 4, Site 5, Site 6, Site 7, and Site 8 (Sites 1-8 are defined below and shown in the schematic provided as Figure 2). In some aspects, the first and second sites comprise Site 1 and Site 2, Site 1 and Site 4, Site 1 and Site 5, Site 1 and Site 6, Site 1 and Site 7, Site 1 and Site 8, Site 2 and Site 6, Site 2 and Site 7, Site 4 and Site 6, Site 5 and Site 6, Site 5 and Site 7, Site 6 and Site 7, Site 8 and Site 2, Site 8 and Site 6, or Site 8 and Site 7, respectively.

[0014] In some cases, the first nucleotide sequence is 25–80 nt long. In other cases, the first nucleotide sequence may have a length within a subrange (e.g., a length of 30–40 nt, 40–50 nt, 50–60 nt, or a length within a subrange defined by any pair of integer values ​​in the range of 25–80 nt). In some cases, the second nucleotide sequence is 8–25 nt long. In other cases, the second nucleotide sequence may have a length within a subrange (e.g., a length of 10–15 nt, 15–25 nt, or a length within a subrange defined by any pair of integer values ​​in the range of 8–25 nt).

[0015] In some cases, the first and second nucleotide sequences hybridize when expressed in eukaryotic cells under in vivo or in vitro conditions, allowing group 1 disistroviridae IRESs to fold into an inactive state.

[0016] In a further development, the group 1 dicistroviridae IRES is configured to fold into an activated state in the presence of a trigger RNA molecule containing a third nucleotide sequence, which is the reverse complementary strand of the first nucleotide sequence. When the first nucleotide sequence is expressed in eukaryotic cells under in vivo or in vitro conditions, it may hybridize to the third nucleotide sequence, causing the group 1 dicistroviridae IRES to fold into an activated state.

[0017] In another general context, this disclosure provides plasmids and eukaryotic cells encoding any of the recombinant nucleic acid molecules described herein (e.g., any recombinant IRES). With respect to eukaryotic cells, such recombinant nucleic acid molecules are intended to be incorporated into the cell's genomic DNA or plasmid DNA. In some contexts, eukaryotic cells are animal cells (e.g., human cells or primate cells). In some contexts, eukaryotic cells are not plant cells.

[0018] In another general context, the Disclosure provides systems and kits that may be used to regulate gene expression in eukaryotic cells. For example, the Disclosure provides a system for regulating gene expression comprising (a) a recombinant nucleic acid molecule in any aspect described herein and (b) a trigger RNA molecule comprising a third nucleotide sequence which is the inverse complement of the first nucleotide sequence of the recombinant nucleic acid molecule. Similarly, the Disclosure provides a kit comprising (a) a plasmid encoding any of the recombinant nucleic acid molecules described herein and (b) a trigger RNA molecule comprising a third nucleotide sequence which is the inverse complement of the first nucleotide sequence of the recombinant nucleic acid molecule.

[0019] In another general context, the present disclosure provides a recombinant mRNA molecule comprising (a) a first segment encoding a first protein, (b) a second segment downstream of the first segment encoding a group 1 dicistroviridae IRES modified to incorporate exogenous nucleotide sequences at the first and second sites, and (c) a third segment downstream of the second segment and functionally linked to the second segment encoding a second protein, wherein the translation of the second protein is suppressed when the IRES is in an inactivated state, and the transcription of the recombinant mRNA molecule is dependent on RNA polymerase II, the first site comprising a first nucleotide sequence, and the second site comprising a second nucleotide sequence which is the reverse complementary strand of at least a portion of the first nucleotide sequence. Such constructs may exhibit reduced translation misses compared to other constructs described herein.

[0020] In another general context, this disclosure provides methods for using recombinant nucleic acid molecules (e.g., recombinant IRES elements) described herein in a variety of applications. For example, a method for activating and / or regulating protein expression may include (a) providing a eukaryotic cell engineered to express one of the recombinant nucleic acid molecules described herein, and (b) introducing a trigger RNA molecule into the eukaryotic cell that contains a third nucleotide sequence which is the reverse complementary strand of a first nucleotide sequence of the recombinant nucleic acid molecule, the first nucleotide sequence hybridizing to the third nucleotide sequence under in vivo conditions to fold the group 1 disistroviridae IRES into an activated state. In some contexts, a eukaryotic cell engineered to express a recombinant nucleic acid molecule is provided by introducing one of the recombinant nucleic acid molecules of the claims into a eukaryotic cell. The eukaryotic cell used in any of the methods described herein may be, for example, an animal cell (e.g., a human cell or a primate cell).

[0021] As described herein, the recombinant IRES elements described herein can be used as sensors for detecting external stimuli. Accordingly, the present disclosure provides a method for detecting viral infection in eukaryotic cells, comprising the steps of (a) providing eukaryotic cells engineered to express any of the recombinant nucleic acid molecules of the preceding claims, wherein a first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of the mRNA sequence specific to the virus, and (b) determining whether the eukaryotic cells are infected with a virus by detecting and / or measuring the presence of a protein encoded by a second segment of the recombinant nucleic acid molecule. The virus may be, for example, dengue virus or Zika virus.

[0022] In a further aspect, the present disclosure provides a method for controlling the differentiation of eukaryotic cells, comprising the steps of (a) providing eukaryotic cells engineered to express any of the recombinant nucleic acid molecules described herein, and (b) culturing the eukaryotic cells, wherein a first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of an mRNA sequence specific to a selected cell type, and the protein encoded by a second segment of the recombinant nucleic acid molecule comprises a toxin or protein that induces apoptosis of the selected cell type.

[0023] Various exemplary aspects of the inventions of this disclosure are described in the following appendix. Similar or equivalent methods and materials may be used in carrying out or testing the inventions, but exemplary methods and materials are described herein. Other features, purposes and advantages of the inventions will become apparent from the description and the claims. In this specification and the appendix claims, the singular form includes the plural form unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the inventions pertain. [Brief explanation of the drawing]

[0024] [Figure 1] This figure summarizes conventional IRES-mediated eukaryotic gene expression using unmodified IRESs. [Figure 2] This is a schematic diagram of a Group 1 CrPV IRES, highlighting the three main loops (or domains) of this IRES. The structure of Group 1 IRES elements is conserved among members of the Disistroviridae family (e.g., CrPV, KBV, and ABPV). [Figure 3] This is a schematic diagram of Group 1 CrPV IRES, highlighting eight sites (i.e., "Site 1", "Site 2", ... "Site 8") that can be used as insertion regions for exogenous nucleic acid sequences as described herein. [Figure 4]This figure summarizes eukaryotic gene expression using exemplary recombinant IRESs described herein. [Figure 5] This shows an mRNA construct encoding one of the recombinant IRES elements described herein, and a schematic diagram of the second upstream gene. [Figure 6] This graph shows the activity levels of various IRES modules. From left to right in Figure 6, the modules are, in order: "+T7 pol+GFP (trigger)", "+T7pol-GFP (trigger)", "-T7pol+GFP (trigger)", and "-T7pol-GFP (trigger)". [Figure 7-1] Figure 7 is a graph showing the screening results of recombinant IRES riboswitch constructs having pairs of exogenous nucleotide sequences introduced at various sites. From left to right in Figure 7, the constructs are "+T7 pol+GFP (trigger)", "+T7pol-GFP (trigger)", "-T7pol+GFP (trigger)", and "-T7pol-GFP (trigger)". [Figure 7-2] Refer to the explanation in Figure 7-1. [Figure 8] This graph shows the effect of selecting exogenous nucleotide sequences with corresponding base pairs that disrupt specific fold and pseudoknot regions. From left to right in Figure 8, the sequences are "+T7 pol+GFP (trigger)", "+T7pol-GFP (trigger)", "-T7pol+GFP (trigger)", and "-T7pol-GFP (trigger)". [Figure 9] This graph shows the effects of promoter switching and the addition of an upstream activation sequence for RNA polymerase I. [Figure 10] This graph shows that the exemplary recombinant IRES riboswitches described herein are highly specific to their respective trigger RNAs (trRNAs). From left to right in Figure 10 are the "GFP trigger," the "Azurite trigger," and the "ySUMO trigger." [Figure 11]This graph shows the effect of mutations on the functionality of exemplary recombinant IRES riboswitches described herein. [Figure 12] This graph shows that recombinant IRES riboswitches can be based on the sequences of IRES modules produced by several members of the Disistroviridae family (e.g., KBV and ABPV). [Figure 13] This figure shows the use of recombinant IRESs according to this disclosure in eukaryotic cells as sensors for detecting viral infection. [Figure 14A]Figures 14A–14G illustrate eToehold design and screening. Figure 14A: The eToehold module is in a locked state, with inhibited IRES activity, preventing ribosome recruitment and translation. Trigger RNA activates IRES activity via strand entry and release of the IRES to the activated state, enabling ribosome binding and protein production. Figure 14B: Basic screening method for eToehold. Plasmids encoding upstream IRES or eToehold candidates with a reporter protein, polymerase (e.g., T7), and trigger RNA sequence (e.g., GFP) were co-transfected into HEK293T cells. See Example 3 and Figure 17 for further details. Figure 14C: Activity of various IRES modules in HEK293T with and without co-transfection with T7 polymerase and GFP trigger sequence. Figure 14D: Disistroviridae IRES structure containing three loops important for translational ability and insertion site. Figure 14E: Screening of the eToehold module by inserting two complementary sequences of unequal length into the insertion site shown in Figure 14D. The numbers first indicate the insertion site of the toehold RNA sequence (approximately 40 base pairs complementary to the trigger RNA), and secondly, a relatively smaller fragment (approximately 10-18 base pairs) complementary to the first. Figure 14F: CrPV IRES structure. The region where the insertion overlaps with the stem-loop or pseudoknot (base pair disruption, i.e., BB site) is noteworthy. For clarity, an example of the sequence of the BB site required for CrPV IRES is included. Figure 14G: Effects of choosing to insert the corresponding base pairs (3 base pairs) into the region shown in Figure 14D. See Table 1 for details of the construct. All bars represent mean values. Dots represent individual data points. Error bars represent the standard deviation of three experimental replicates exposed to the same conditions. Each experiment was repeated at least twice. [Figure 14B] Refer to the explanation in Figure 14A. [Figure 14C] Refer to the explanation in Figure 14A. [Figure 14D] Refer to the explanation in Figure 14A. [Figure 14E-1]Refer to the explanation in Figure 14A. [Figure 14E-2] Refer to the explanation in Figure 14A. [Figure 14F-1] Refer to the explanation in Figure 14A. [Figure 14F-2] Refer to the explanation in Figure 14A. [Figure 14G] Refer to the explanation in Figure 14A. [Figure 15A] Figures 15A-15E demonstrate the optimization of eToehold expression. Figure 15A: Effects of switching the promoter-polymerase system and adding an RNA polymerase I upstream activation sequence on the expression of the eToehold-gated transgene (mKate). Figure 15B: eToehold activity evaluated by mKate expression in the presence of designed trRNA and mismatched RNA. From left to right in Figure 15B are the "GFP trigger," "Azurite trigger," and "ySUMO trigger." Figure 15C: Schematic diagram of the eToehold-gated RNA driven by RNA polymerase II, including the stop codon and stem-loop. Figure 15D: eToehold or IRES activity of constructs with and without the features shown in Figure 15C, evaluated by mKate expression. See Table 1 for details of the constructs. Figure 15E: All bar graphs show the mean value. Dots represent individual data points. The error bars represent the standard deviation (SD) of three experimental replicates under the same conditions. Each experiment was repeated at least three times. [Figure 15B] Refer to the explanation in Figure 15A. [Figure 15C] Refer to the explanation in Figure 15A. [Figure 15D] Refer to the explanation in Figure 15A. [Figure 15E-1] Refer to the explanation in Figure 15A. [Figure 15E-2] Refer to the explanation in Figure 15A. [Figure 16A]Figures 16A–16F demonstrate that eToehold can respond to infection status, cellular state, and cell type. Figure 16A: Stable cell lines were constructed using the eToehold module designed to sense Zika virus infection. Figure 16B: Luminescence signals from cells engineered to express nanoluciferase at the time of Zika infection, after mock, Zika, or dengue fever infection. Cells engineered with CrPV-gated nanoluciferase were used as a positive control. Figure 16C: Stable cell lines constructed using the eToehold module designed to sense exposure to heat by detecting heat shock protein mRNA. Figure 16D: HeLa cells were transfected with a construct containing a GFP reporter and eToehold-gated Azurite. Figure 16E: B16, D1, or HEK293T (not shown) cells were transfected with a construct designed to translate Azurite protein in the presence of mouse tyrosinase (Tyr). Figure 16F: Expression of eToehold-gated Azurite in B16, D1, or HEK293T cells after transfection. Bars all represent mean values. Dots represent individual data points. Error bars represent the standard deviation (sd) of three (four in Figure 16F) experimental replicates exposed to the same conditions. Each experiment was repeated at least three times. [Figure 16B] Refer to the explanation in Figure 16A. [Figure 16C] Refer to the explanation in Figure 16A. [Figure 16D] Refer to the explanation in Figure 16A. [Figure 16E] Refer to the explanation in Figure 16A. [Figure 16F] Refer to the explanation in Figure 16A. [Figure 17]This is a schematic diagram of the eToehold screening. HEK293T cells were seeded at 60% confluence and transfected with a plasmid combination containing T7 polymerase, a T7 promoter that drives potential eToehold gated mKate, and a trigger construct (GFP). After incubation at 37°C for 60 hours, the cells were detached and analyzed using flow cytometry. [Figure 18-1] Figures 18A–18H show the gating for flow cytometry experiments. Figures 18A–18D show representative plots for negative controls. Figures 18E–18H show representative plots for positive GFP and mKate controls. [Figure 18-2] See the explanation in Figure 18-1. [Figure 18-3] See the explanation in Figure 18-1. [Figure 18-4] See the explanation in Figure 18-1. [Figure 19] Figures 19A and 19B demonstrate that eToehold activity depends on the thermodynamics of the insertion region. Figure 19A: The construct was based on CrPV eToehold 8-6 designed for GFP sensing. Figure 19B: The construct was based on CrPV eToehold 6-7 designed for GFP sensing. All data are shown as mean values. Dots represent individual data points. Error bars represent the standard deviation of three experimental replicates exposed to the same conditions. Each experiment was repeated at least three times. [Figure 20A]Figures 20A and 20B demonstrate intracellular cytokine staining in cells expressing CrPV IRES with or without additional RNA binding. Figure 20A: Effects of transfection or transduction with CrPV IRES constructs on the production of IL6, CCL5, and CCL2 in primary human fibroblasts and musculoskeletal cells. See Table 1 for construct details. Figure 20B: Expression of IL6, CCL5, and CCL2 in cells transduced to express CrPV IRES constructs and "triggers" compared to cells expressing eToehold and orthogonal non-binding sequences. Error bars indicate sd. * indicates p<0.05 in two-way ANOVA. All experiments were repeated at least three times, and dots represent individual data points. [Figure 20B] Refer to the explanation in Figure 20A. [Figure 21A] Figures 21A to 21C are graphs of the average intensity data shown in Figures 14A to 14G. [Figure 21B] Refer to the explanation in Figure 21A. [Figure 21C] Refer to the explanation in Figure 21A. [Figure 22A] Figures 22A–22C demonstrate experiments that reduce basal expression of the eToehold module. Figure 22A: Constructs with various promoters promoting sfGFP were tested based on Figure 17. Figure 22B: An RNA region designed to recruit RNA polymerase I factor and reduce 5' capping was inserted before CrPV eToehold 6–7. Figure 22C: An RNA polymerase I-responsive promoter was tested based on Figure 17. See Table 1 for details of the constructs. All data are shown as mean values. Dots represent individual data points. Error bars represent the standard deviation of three experimental replicates exposed to the same conditions. Representative flow cytometry plots were selected from three experimental replicates showing similar results. Each experiment was repeated at least twice. [Figure 22B] Refer to the explanation in Figure 22A. [Figure 22C] Refer to the explanation in Figure 22A. [Figure 23A]Figures 23A to 23E are graphs of the average intensity data for Figures 15A to 15E and Figures 16A to 16F. From left to right in Figure 23B, the results are "GFP trigger," "Azurite trigger," and "ySUMO trigger." From left to right in Figure 23D, the results are "37°C" and "42°C." From left to right in Figure 23E, the results are "B16 (high expression Tyr)," "D1," and "HEK293T." [Figure 23B] Refer to the explanation in Figure 23A. [Figure 23C] Refer to the explanation in Figure 23A. [Figure 23D] Refer to the explanation in Figure 23A. [Figure 23E] Refer to the explanation in Figure 23A. [Figure 24] This graph demonstrates the decrease in basal expression of the T7 promoter due to stem-loop insertion. The addition of a stop codon and stem-loop before the eToehold module was tested based on Figure 17. See Table 1 for details of the construct. All data are shown as mean values. Dots represent individual data points. Error bars represent the standard deviation of three experimental replicates exposed to the same conditions. Representative flow cytometry plots were selected from three experimental replicates showing similar results. Each experiment was repeated at least twice. [Figure 25] Figures 25A and 25B demonstrate the sensitivity of eToehold to mismatches and eToehold based on various IRESs. Figure 25A: Effect of mismatch mutations within or outside the annealing region of the inserted RNA sequence on eToehold function. All constructs were based on CrPV eToehold 8-6, designed for GFP sensing. Figure 25B: eToehold constructed based on other dicistroviridae IRESs (i.e., KBV and ABPV) retain functionality. See Table 1 for details of the constructs. All data are shown as mean values. Dots represent individual data points. Error bars represent the standard deviation of three experimental replicates exposed to the same conditions. Each experiment was repeated at least three times. [Figure 26] This graph shows that complementation for relatively small fragment insertions does not activate eToehold. EZ-L287, designed for GFP triggers, was tested using the setup in Figure 17, with various triggers, including a trigger with the reverse complementary strand of a relatively small GFP fragment inserted into ySUMO (see Table 1). All data are shown as mean values. Dots represent individual data points. Error bars represent the standard deviation of three experimental replicates exposed to the same conditions. The experiment was repeated twice. [Figure 27] Figures 27A and 27B demonstrate the function of eToehold in yeast. An eToehold module gating iRFP was incorporated into a yeast strain expressing GFP (trigger RNA) when the carbon source was switched to galactose. iRFP signals (Figure 27A) and GFP signals (Figure 27B) were measured 6 hours after logarithmic growth. Biliverdin was added to the culture medium. See Table 1 for details of the construct. All data are shown as mean values. Dots represent individual data points. Error bars represent the standard deviation (sd) of three experimental replicates exposed to the same conditions. Representative flow cytometry plots were selected from three experimental replicates showing similar results. Each experiment was repeated at least twice. [Figure 28A] Figures 28A and 28B demonstrate that eToehold functions in cell-free lysates. Figure 28A: Wheat germ extract with varying amounts of trigger RNA (transcribed EZ-L366) and 50 nM switch-sfGFP RNA (transcribed from EZ-L214 or EZ-L212 as a control). Figure 28B: Rabbit reticulocyte lysates with 250 nM trigger RNA (transcribed EZ-L366), or without it, and 150 nM switch-sfGFP RNA (transcribed from EZ-L214 or EZ-L212 as a control). See Table 1 for construct details. All data are shown as mean values. Error bars represent the standard deviation of three experimental replicates exposed to the same conditions. Representative flow cytometry plots were selected from three experimental replicates showing similar results. Each experiment was repeated at least twice. [Figure 28B] Refer to the explanation in Figure 28A. [Figure 29] This graph shows the detection of Zika virus concentration using eToehold, under the same experimental setup as in Figures 16A and 16B. [Figure 30A] Figures 30A–30D demonstrate further viral infection sensing using eToehold. Figure 30A: A stable cell line designed for sensing Zika virus infection includes Zika RNA-responsive eToehold gated Azurite translation under the T7 promoter. From left to right in Figure 30A are "uninfected," "infected with Zika virus," and "infected with dengue virus." Figure 30B: Wild-type Vero E6 cells shown in (Figure 30A), and stable cell lines expressing Zika-sensing eToehold gated Azurite, were infected with dengue virus or Zika virus. Sample gates are shown elsewhere. Figure 30C: A stable cell line designed for sensing SARS-CoV-2 infection includes SARS-CoV-2 RNA-responsive eToehold gated nanoluciferase translation. Figure 30D: Stable cell lines containing eToehold, which senses various regions of SARS-CoV-2, were transfected with constructs expressing GFP and two regions of SARS-CoV-2. Luminescence measurements were then performed after the addition of flimazine. From left to right in Figure 30D, the results are "GFP transfected," "SARS-CoV-2 spike transfected," and "SARS-CoV-2 3' transfected." See Table 1 for details on the constructs. All bars represent mean values. Dots represent individual data points. Error bars represent the standard deviation of three experimental replicates exposed to the same conditions. Representative flow cytometry plots were selected from three experimental replicates showing similar results. Each experiment was repeated at least twice. [Figure 30B] Refer to the explanation in Figure 30A. [Figure 30C] Refer to the explanation in Figure 30A. [Figure 30D] Refer to the explanation in Figure 30A. [Figure 31A]Figures 31A and 31B show the gates used for virus detection. Figure 31A: Staining results of virus-infected samples. Figure 31B: Infection level dependence of MOI as measured by staining and subsequent flow cytometry. Representative flow cytometry plots were selected from two experimental replicates showing similar results. Each experiment was repeated at least twice. [Figure 31B] Refer to the explanation in Figure 31A. [Figure 32A] Figures 32A and 32B show exemplary gates for Zika virus detection. Stable cell lines expressing Zika-sensing eToehold-gated Azurite (shown in Figure 23A) were subjected to infection with dengue virus and Zika virus. Flow cytometry data based on the gate shown in Figure 24B are shown. Wild-type Vero E6 cells infected with Zika virus did not show increased Azurite fluorescence. [Figure 32B] Refer to the explanation in Figure 32A. [Modes for carrying out the invention]

[0025] Detailed explanation There is a need in the art for novel constructs that can be used as platforms for regulating the translation of any protein of interest in eukaryotic cells without requiring sequence-specific redesign. Conventional options (e.g., ribozyme, antisense DNA / RNA, and RNAi-based methods) typically require sequence-specific techniques, potentially limiting their usefulness. More recent developments, such as prokaryotic toehold switches, address some of the shortcomings of conventional options. However, their usefulness is generally limited to prokaryotes due to differences in the translation mechanisms used by prokaryotes and eukaryotes. For example, eukaryotic translation initiation relies on endogenous RNA polymerase II recruitment 5' modification capping, poly-adenosine (poly-A) tails for mRNA stabilization, and Kozak sequences for protein translation regulation. Kozak sequences improve ribosome binding but are not ideal RBS substitutions. Previously developed Kozak-based toeholds have only achieved up to 2x trRNA propulsion induction for translation. Thus, the usefulness offered by toehold switches compatible with eukaryotic cells is limited at this point.

[0026] In recent years, more complex RNA-based switches have been developed utilizing Cas9 expression and guide RNA (gRNA) folding. Unfolding of gRNA leads to activation of the Cas9 enzyme and corresponding repression or activation. However, despite the bulky circuits, these mechanisms (in both eukaryotes and prokaryotes) only induce moderate change ratios. Similarly, recent advances in ribozyme research have led to the development of ribozymes that cleave the poly(A) tail of target mRNA during small molecule induction. However, ribozyme-based mechanisms are currently limited to short-length trRNAs, which restrict their ability to be tuned to specific sequences, and are therefore exclusively limited to "ON-to-OFF" sensors, which are not ideal for leak and induction modulation.

[0027] This disclosure addresses these and other shortcomings of known mechanisms regulating eukaryotic gene expression by providing minimal component RNA-based sensors (i.e., recombinant IRES elements described herein) that respond to specific trRNAs with a high ON-to-OFF change ratio. These constructs are therefore advantageous in expression systems used to produce proteins for industrial or therapeutic use, and in other novel applications (e.g., in biosensors capable of detecting environmental stimuli such as the presence of viral mRNA).

[0028] Eukaryotic translation mechanisms and viral translation mechanisms In eukaryotes, protein translation is typically initiated by a tightly regulated mechanism requiring a modified nucleotide "cap" at the 5' end of mRNA, as well as an initiation factor protein (eIF) that recruits and positions the ribosome. To circumvent this system, many pathogenic viruses employ alternative cap-independent mechanisms that rely on the use of specific RNA secondary (or tertiary) structures to recruit and manipulate ribosomes, instead of the 5' cap and eIF used during the canonical pathway. The RNA elements that drive this process are known as IRESs.

[0029] Figure 1 illustrates the process by which an arbitrary protein (in this case, mKate) can be expressed using an unmodified viral IRES. In summary, conventional IRES-mediated eukaryotic gene expression requires a promoter (e.g., a T7 promoter) functionally linked to a viral IRES and a downstream DNA segment encoding the gene of interest. In this example, the T7 promoter recruits T7 RNA polymerase (without 5' capping the mRNA) to transcribe the mRNA containing the IRES and the segment encoding the protein of interest, i.e., mKate. The viral IRES typically recruits ribosomes (and potentially other components necessary for translation) to result in the expression of the mKate protein. In this example, the IRES is an unmodified viral IRES, not a recombinant IRES according to this disclosure, which is inactive in this example because trRNA is absent.

[0030] Viral IRESs are organized into four distinct groups based on the secondary and tertiary structures of their RNA elements, as well as their mode of action for initiating translation. Within this classification system, Group 1 IRESs are generally more compact and complex than Group 2–4 IRESs. Furthermore, Group 1 IRESs are noteworthy because they can initiate translation on non-AUG start codons, do not require any eIFs, and do not use the initiation factor Met-tRNA. As a result, Group 1 IRESs can facilitate efficient translation initiation requiring only small and large ribosomal subunits. Several members of the Disicstroviridae family (e.g., cricket paralysis virus (CrPV), Casimir wasp virus (KBV), and acute honeybee paralysis virus (ABPV)) are known to encode Group 1 IRESs. Among members of the Disicstroviridae family, Group 1 IRESs are highly conserved in terms of sequence, secondary and tertiary structures. CrPV IRESs are the most well-studied IRESs in this group and represent other Group 1 Disistroviridae IRESs (e.g., KBV IRESs and ABPV IRESs).

[0031] Figure 2 shows a schematic diagram of the secondary structure of a CrPV group 1 IRES. As shown in Figure 2, CrPV group 1 IRESs typically fold into a compact structure having three main loops (or domains) labeled here as loops 1-3, where loops 1-3 contain pseudoknot structures (called PKI, PKII, and PKIII, respectively), as well as an inner loop, bulge, and hairpin motif, respectively. This folded structure is essential for IRES activity. For example, the triple-pseudoknot structure is known to functionally substitute the initiation factor met-tRNA during internal initiation, leading to translation initiation in non-AUG triplets. Normally, the presence of a CrPV group 1 IRES on viral mRNA recruits eukaryotic ribosomes to the mRNA, initiating translation of the encoded viral protein.

[0032] Recombinant IRES Riboswitch In some aspects, this disclosure relates to nucleic acid constructs (e.g., mRNA) that have been modified to incorporate at least one recombinant IRES riboswitch. These embodiments of recombinant IRES riboswitches may be referred to herein as “eToehold” or “hToehold”. Recombinant IRES riboswitches may be derived from or contain sequences naturally present in viral IRESs. Recombinant IRESs may be viral IRESs modified to contain exogenous, e.g., non-endogenous sequences. In some aspects of the disclosure, recombinant viral IRESs may include viral IRESs containing two insertions of exogenous, e.g., non-endogenous sequences. In some aspects of the disclosure, insertion into a viral IRES to produce a recombinant viral IRES riboswitch as described herein may include a deletion of the viral sequence at the insertion site. In some aspects of the disclosure, insertion into a viral IRES to produce a recombinant viral IRES riboswitch as described herein may not include a deletion of the viral sequence at the insertion site.

[0033] The IRES riboswitches described herein may be obtained in the genome or sequence of a virus or derived from any IRES sequence naturally present in the genome or sequence of a virus. In some aspects of this specification, the IRES riboswitches described herein may be obtained in the genome or sequence of a pathogenic virus of a mammal (e.g., human) or a symbiotic virus of a mammal (e.g., human) or derived from any IRES sequence naturally present in the genome or sequence of a pathogenic virus of a mammal (e.g., human) or a symbiotic virus of a mammal (e.g., human). Such viruses and their sequences are known in the art. In some aspects of this specification, the IRES sequence may be a Group 1 IRES. In some aspects of this specification, the IRES sequence may be a Group 1 IRES, a Group 2 IRES, a Group 3 IRES, or a Group 4 IRES. In some aspect of the context, the IRES is derived from or modified from an IRES sequence of a Group 1 dicistroviridae IRES, a hepacivirus IRES, or an enterovirus IRES. Exemplary wild-type IRES sequences and recombinant IRES riboswitch sequences are provided herein, and further wild-type IRES sequences for use in the methods and compositions described herein are readily obtainable and / or identified by those skilled in the art. For example, databases of IRES sequences are available on the World Wide Web at iresite.org.

[0034] In some aspects of the situation, hepacivirus IRES includes hepatitis C virus (HCV); hepatitis B virus; hepatitis F virus; hepatitis I virus; hepatitis J virus; hepatitis K virus; hepatitis L virus; hepatitis M virus; hepatitis N virus; Guereza hepacivirus; hepatitis GB virus B virus; non-primate hepacivirus NZP1 virus; Norway rate hepacivirus 1 virus; Norway rate hepacivirus 2 virus; bat hepacivirus; bovine hepacivirus; equine hepacivirus; hepacivirus P virus; rodent hepacivirus; and Wenling shark virus. IRESs derived from or modified from the IRES sequence of hepatitis c virus (HCV); hepatitis B virus; hepatitis F virus; hepatitis I virus; hepatitis J virus; hepatitis K virus; hepatitis L virus; hepatitis M virus; hepatitis N virus; Gereza hepacivirus; hepatitis GB virus B virus; non-primate hepacivirus NZP1 virus; Norwegian hepacivirus 1 virus; Norwegian hepacivirus 2 virus; bat hepacivirus; bovine hepacivirus; equine hepacivirus; hepacivirus P virus; rodent hepacivirus; and Wenling's shark virus. In some aspect of the context, the hepacivirus IRES is derived from or modified from the IRES sequence of hepatitis c virus (HCV). Such virus sequences are publicly known in the art and are available, for example, on the World Wide Web at ncbi.nlm.nih.gov / genomes / GenomesGroup.cgi?taxid=11102.

[0035] In some aspects of the situation, enterovirus IRES includes poliovirus (PV); enterovirus 71 (EV71); enterovirus A viruses (e.g., coxsackievirus A2; enterovirus A; or enterovirus A114); enterovirus B viruses (e.g., coxsackievirus B3 or enterovirus B); enterovirus C; dromedary camel enterovirus 19CC; enterovirus D viruses (e.g., enterovirus D or enterovirus D68); enterovirus E; enterovirus F viruses (e.g., enterovirus F or porgynathus enterovirus (possum) Enterovirus W1); Enterovirus H (e.g., Enterovirus H or sulcata enterovirus SV4); Enterovirus J; Enterovirus SEV-gx; Rhinovirus A (e.g., human rhinovirus A1 or rhinovirus A); Rhinovirus B (e.g., human rhinovirus B2 or rhinovirus B14); Rhinovirus C (e.g., human rhinovirus NAT001 or rhinovirus C); Picornaviridae viruses (e.g., Picornaviridae rodent / Ee / PicoV / NX2015); Porcine enterovirus (e.g., porcine enterovirus 9); Enterovirus AN12; Goat enterovirus / JL14; Sichuan takin enterovirus; or Yak enterovirus IRES sequences derived from or modified from enteroviruses include poliovirus (PV); enterovirus 71 (EV71); enterovirus A (e.g., coxsackievirus A2; enterovirus A; or enterovirus A114); enterovirus B (e.g., coxsackievirus B3 or enterovirus B); enterovirus C; dromedary camel enterovirus 19CC; enterovirus D (e.g., enterovirus D or enterovirus D68); enterovirus E;Enterovirus F virus (e.g., Enterovirus F or Pine Conure Enterovirus W1); Enterovirus H virus (e.g., Enterovirus H or Monkey Enterovirus SV4); Enterovirus J virus; Enterovirus SEV-gx; Rhinovirus A virus (e.g., Human Rhinovirus A1 or Rhinovirus A); Rhinovirus B virus (e.g., Human Rhinovirus B2 or Rhinovirus B14); Rhinovirus C virus (e.g., Human Rhinovirus NAT001 or Rhinovirus C); Picornaviridae virus (e.g., Picornaviridae Rodent / Ee / PicoV / NX2015); Porcine Enterovirus (e.g., Porcine Enterovirus 9); Enterovirus AN12; Enterovirus Goat / JL14; Schuan Turkin Enterovirus; or IRES sequences of yak enterovirus. In some aspect of the situation, the enterovirus IRES is derived from or a modified IRES sequence of poliovirus (PV) or enterovirus 71 (EV71). Sequences of such viruses are publicly known in the art and are available, for example, on the World Wide Web at ncbi.nlm.nih.gov / genomes / GenomesGroup.cgi?taxid=12059.

[0036] In some aspect of the context, the IRES riboswitch described herein is derived from a group I IRES element used by members of the Disicstroviridae family of viruses (e.g., CrPV, KBV, or ABPV). In some aspects of the situation, Group I Disicstroviridae IRESs include: Cricket Paralysis Virus (CrPV) IRES, Kashmir Bee Virus (KBV) IRES, Acute Honeybee Paralysis Virus (ABPV) IRES, Brown-winged Stink Bug (Plauta Stali) Enteric Virus (PSIV) IRES; Aphid Lethal Paralysis Virus (ALPV) IRES; Black Queen Bee Larva Virus (BQCV) IRES; Drosophila C Virus (DCV) IRES; Himetobi P Virus (HiPV) IRES; Homalodisca coagulata Virus-1 (HoCV-1) IRES; and Rhopalosiphum IRESs derived from or modified from the IRES sequences of the padi virus (RhPV) or the assassin bug virus (TrV) are the cricket paralysis virus (CrPV) IRES, the Kashmir wasp virus (KBV) IRES, the acute honeybee paralysis virus (ABPV) IRES, the brown marmorated stink bug enteric virus (PSIV) IRES, the aphid lethal paralysis virus (ALPV) IRES, the black queen bee brood virus (BQCV) IRES, the fruit fly C virus (DCV) IRES, the small kite P virus (HiPV) IRES, the small leafhopper virus-1 (HoCV-1) IRES, the wheat aphid virus (RhPV) IRES, or the assassin bug virus (TrV) IRES sequences.

[0037] As described above, the naturally occurring forms of these IRES elements recruit ribosomes to the associated mRNA, resulting in mRNA translation (i.e., a constitutively active regulatory pathway). The co-inventors of this invention have surprisingly discovered that dicistroviridae IRES elements can be genetically modified to produce recombinant IRES riboswitches that can be switched "ON" or "OFF" based on the concentration of a distinct trigger RNA (trRNA) molecule. This novel functionality is provided by inserting two or more segments containing exogenous nucleotide sequences into the original sequence of the viral IRES element. As will be described in more detail below, these segments are designed to hybridize in the absence of the corresponding trRNA, causing the recombinant IRES to fold into an inactive state. Upon provision of the trRNA, the hybridization between the two segments is disrupted, allowing the recombinant IRES to fold into a conformation similar to that of the naturally occurring viral IRES, which is constitutively active as described above. As a result, recombinant IRESs function as riboswitches that can be switched "ON" or "OFF" based on the concentration of the corresponding trigger RNA, regulating the translation of a functionally ligated downstream mRNA sequence encoding the protein of interest.

[0038] In some aspects, the IRES riboswitches described herein include a nucleotide sequence that shares at least 70% sequence identity with a viral IRES (e.g., a hepacivirus IRES or an enterovirus IRES). In some aspects, the IRES riboswitches described herein exhibit at least 90, at least 95, at least 98, at least 99, or at least 100% sequence identity with a viral IRES (e.g., a hepacivirus IRES or an enterovirus IRES) at all positions except for two segments containing exogenous nucleotide sequences. In some aspects of any aspect, the IRES riboswitches according to this disclosure include a nucleotide sequence that shares at least 70, at least 80, at least 85, at least 90, at least 95, at least 98, at least 99, or at least 100% sequence identity with any one of the nucleotide sequences of SEQ ID NO: 30-36, except for the presence of exogenous nucleotide sequences inserted at two sites in the sequence indicated by X in the sequence. In some aspects of the present disclosure, the IRES riboswitch comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 30-36, except for the presence of exogenous nucleotide sequences inserted at two sites in the sequence, indicated by X in the sequence. In some aspects of the present disclosure, the IRES riboswitch comprises a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 30-36, except for the presence of exogenous nucleotide sequences inserted at two sites in the sequence, indicated by X in the sequence. In some aspects of the present disclosure, the IRES riboswitch comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 30-36, except for the presence of exogenous nucleotide sequences inserted at two sites in the sequence, indicated by X in the sequence. The exogenous nucleotide sequences inserted at the two sites may be the first and second nucleotide sequences described elsewhere in this specification.

[0039] In some aspects, the IRES riboswitches described herein include a nucleotide sequence that shares at least 70% sequence identity with a Group I dicistroviridae IRES (e.g., CrPV IRES, KBV IRES, or ABPV IRES). In some aspects, the IRES riboswitches described herein exhibit at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity with a Group I dicistroviridae IRES at all positions except for two segments containing exogenous nucleotide sequences. For example, the IRES riboswitches according to this disclosure may include a nucleotide sequence that shares at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity with the nucleotide sequence of SEQ ID NO:1, except for the presence of exogenous nucleotide sequences inserted at two sites in the sequence.

[0040] In some aspects of the situation, recombinant IRESs are derived from IRES sequences of viruses other than coxsackievirus B3 (CVB3), or modified IRESs are IRES sequences of viruses other than coxsackievirus B3 (CVB3). In some aspects of the situation, recombinant IRESs are not derived from IRES sequences of coxsackievirus B3 (CVB3), or modified IRESs are not IRES sequences of coxsackievirus B3 (CVB3). In some aspects of the situation, recombinant IRESs are derived from IRES sequences of enteroviruses other than coxsackievirus B3 (CVB3), or modified IRESs are IRES sequences of enteroviruses other than coxsackievirus B3 (CVB3).

[0041] The following description of the structure and function of this recombinant IRES riboswitch technology refers to figures illustrating Group 1 Disistroviridae IRESs and recombinant IRES riboswitches derived therefrom. These figures are illustrative of exemplary embodiments and do not imply that the technology is limited to these embodiments.

[0042] Figure 3 shows a schematic diagram of a CrPV group 1 IRES annotated with numerical labels identifying eight potential insertion sites (sites 1-8). As described above, this structure is representative of the structures of other group 1 dicistroviridae IRESs (e.g., KBV group 1 IRESs and ABPV group 1 IRESs). These sites will be referenced herein in various aspects of this disclosure. For example, recombinant IRESs according to this disclosure may include IRESs having a secondary structure identical or substantially similar to the secondary structure shown in Figure 3, but containing at least one exogenous RNA segment inserted into one or more of sites 1-8. For example, recombinant IRESs may include sequences derived from CrPV, KBV, or ABPV viruses in which the exogenous segment is inserted into sites 1 and 2, or sites 8 and 6, or any other combination of two or more sites.

[0043] As used herein, the terms “site 1,” “site 2,” ... “site 8” are defined with reference to Figure 3, which shows a schematic diagram of a CrPV group 1 IRES (representative of group 1 IRESs among members of the Disistroviridae family). “Site 1” refers to the region of the IRES 5' to the first stem of loop 1; “site 2” refers to the region between the second stem of loop 1 and the pseudoknot (PK1); “site 3” refers to the inner loop between the first and second stems of loop 1; “site 4” refers to the region 5' to the first stem of loop 2; “site 5” refers to the region between the first hairpin of loop 2 and the stem immediately following it; “site 6” refers to the single-stranded region between the last stem of loop 2 and PK1; “site 7” similarly refers to the single-stranded region between PK1 and the first stem of loop 3; and finally, “site 8” refers to the single-stranded region 3' to the pseudoknot 3 (PK3).

[0044] In some aspects of the plane, the first and second sites include sites 1 and 2, sites 1 and 8, sites 2 and 7, sites 6 and 7, or sites 8 and 6, respectively. In some aspects of the plane, the first and second sites include sites 6 and 7, or sites 8 and 6, respectively. In some aspects of the plane, the first and second sites include sites 6 and 7, respectively. In some aspects of the plane, the first and second sites include sites 8 and 6, respectively. In some aspects, the exogenous nucleotide sequence inserted into one or more of sites 1-8 may include a first nucleotide sequence which is the reverse complementary strand of at least a portion of the nucleotide sequence of a separate trigger RNA molecule. This first nucleotide sequence may be, for example, 25-80 nt in length. Such a construct may further include a second exogenous nucleotide sequence inserted into a different site selected from sites 1 to 8, which includes a second nucleotide sequence that is the reverse complementary strand of at least a portion of the first nucleotide sequence. This second nucleotide sequence may be, for example, 8 to 25 nt in length. In some aspects, this structure causes the recombinant IRES to fold into an inactive state due to the interaction between a first exogenous nucleotide sequence and a second exogenous nucleotide sequence (for example, these sequences, due to the second exogenous nucleotide sequence containing a segment complementary to at least a portion of the first exogenous nucleotide sequence, at least partially hybridize under in vitro or in vivo conditions, resulting in a reduction or complete loss of the IRES's ability to initiate translation of a functionally linked protein sequence encoded downstream of the IRES). In some aspects, these constructs can be activated by the presence of the aforementioned trigger RNA molecule containing a nucleotide sequence that is the reverse complementary strand of the first nucleotide sequence. In some aspects, the trigger RNA molecule may contain an artificial nucleotide sequence (e.g., to activate translation in an industrial environment), while in other aspects, this trigger RNA may contain endogenous mRNA produced by a eukaryote, prokaryote, or virus (e.g., to enable the use of the IRES as a sensor to detect the presence of a given organism).It is understood that any of the aforementioned nucleotide sequences can consist solely of RNA. However, in some aspects, these constructs (e.g., exogenous nucleotide sequences inserted into one or more of sites 1-8, and / or trigger RNA molecules) may contain non-RNA bases or modified RNA bases at one or more positions.

[0045] In some aspects, the second exogenous nucleotide sequence is the reverse complementary strand of at least a portion of the first exogenous nucleotide sequence. In some aspects, the first exogenous nucleotide sequence includes a first nucleotide sequence which is the reverse complementary strand of at least a portion of the nucleotide sequence of a separate trigger RNA molecule. The first nucleotide sequence may be, for example, 25–80 nt or 40–50 nt in length. The second nucleotide sequence may be, for example, 8–25 nt or 6–15 nt in length. In some aspects of any of the aspects, the first nucleotide sequence is 2.5–8 times the length of the second nucleotide sequence. In some aspects, this structure causes the recombinant IRES to fold into an inactive state due to the interaction between the first exogenous nucleotide sequence and the second exogenous nucleotide sequence (for example, these sequences, due to the second exogenous nucleotide sequence containing a segment complementary to at least a portion of the first exogenous nucleotide sequence, at least partially hybridize under in vitro or in vivo conditions, resulting in a reduction or complete loss of the IRES's ability to initiate translation of a functionally linked protein sequence encoded downstream of the IRES). In some aspects, these constructs cause the aforementioned trigger RNA molecule containing a nucleotide sequence that is the reverse complementary strand of the first nucleotide sequence to fold into an inactive state. It can be activated by the presence of [a specific RNA molecule]. In some aspects, the trigger RNA molecule may contain an artificial nucleotide sequence (e.g., to activate translation in an industrial environment), while in other aspects, this trigger RNA may contain endogenous mRNA produced by a eukaryote, prokaryote, or virus (e.g., to enable the use of IRES as a sensor to detect the presence of a given organism). It is understood that any of the aforementioned nucleotide sequences may consist solely of RNA. However, in some aspects, these constructs (e.g., inserted exogenous nucleotide sequences and / or trigger RNA molecules) may contain non-RNA bases or modified RNA bases at one or more positions.

[0046] In some aspects of the context, the second nucleotide sequence further comprises an IRES pseudoknot sequence. In some aspects of the context, the second nucleotide sequence further comprises a natural IRES pseudoknot sequence obtained from a wild-type IRES, for example, a wild-type IRES modified as described herein. In some aspects of the context, the second nucleotide sequence is inserted into the IRES pseudoknot sequence. The IRES pseudoknot structure and the IRES pseudoknot sequence are known in the art.

[0047] Figure 4 illustrates the underlying mechanism of the recombinant IRES construct described herein. In this example, the mRNA containing the recombinant IRES according to this disclosure is functionally ligated to a downstream segment encoding the protein of interest. The recombinant IRES contains exogenous sequences at two different insertion sites (i.e., selected from sites 1-8 defined above) that inactivate the IRES, thus initially suppressing the translation of the protein of interest. As described above, hybridization between the exogenous nucleotide sequences inserted at these sites disrupts the secondary structure of the recombinant IRES (i.e., keeps the expression switch in the "OFF" state). However, as shown in this figure, when trRNA is provided, the recombinant IRES switches to "ON" and activates translation. The trRNA contains a segment that is the reverse complementary strand of the nucleotide sequence inserted into the first of the two modification sites, and as a result hybridizes with that nucleotide sequence. In doing so, the trRNA disrupts the initial hybridization between the two exogenous nucleotide sequences, allowing the recombinant IRES to refold into an activated state.

[0048] In some aspects, the recombinant IRES constructs according to this disclosure are incorporated into mRNA transcripts produced by T7 RNA polymerase (for example, such constructs may be downstream of the T7 promoter sequence and may be functionally ligated to the T7 promoter sequence). T7 polymerase may be produced by or introduced into eukaryotic cells (e.g., expressed from cellular genomic DNA or plasmids). In other aspects described herein, the recombinant IRES constructs may be incorporated into mRNA transcripts produced by alternative polymerases (e.g., eukaryotic RNA polymerase II).

[0049] As described above, the recombinant IRES constructs described herein can be incorporated into mRNA transcripts produced by viral RNA polymerase (e.g., T7 polymerase without a 5' cap) because these constructs can recruit ribosomes and initiate translation. However, in some cases, the use of viral polymerase may be undesirable (e.g., the host cell may not produce T7 and may require simultaneous transfection with a vector to supply this enzyme). Alternatively, it may be preferable to use endogenous RNA polymerase II for transcription in order to design a riboswitch system using a small number of exogenous components.

[0050] Figure 5 is a schematic diagram of mRNA produced by RNA polymerase II incorporating the recombinant IRES according to this disclosure. As shown in this schematic diagram, the mRNA includes a segment encoding a first protein followed by a series of stop codons. The recombinant IRES according to this disclosure is located downstream of this element and functionally ligated to a segment encoding a second protein. Note that the mRNA transcript in this case has a 5' cap and a poly-A tail resulting from transcription by RNA polymerase II. Translation of the second protein is controlled by the recombinant IRES, as in the case of constructs in other aspects described herein. However, this configuration may be preferred in some examples because it relies on endogenous mammalian mRNA promoters and polymerases rather than viral components. Furthermore, as illustrated in the following examples, this configuration appears to exhibit reduced expression leaks compared to exemplary aspects that omit upstream genes of interest.

[0051] Accordingly, in any aspect of the embodiment, a recombinant mRNA molecule is described herein, comprising a first segment encoding a first protein, a second segment downstream of the first segment encoding a recombinant viral internal ribosome entry site (IRES) which is modified to incorporate exogenous nucleotide sequences at the first and second sites, and a third segment downstream of the second segment and functionally linked to the second segment encoding a second protein such that translation of the second protein is suppressed when the IRES is inactive, wherein the transcription of the recombinant mRNA molecule is polymerase-dependent, the first site comprising a first nucleotide sequence, and the second site comprising a second nucleotide sequence which is the reverse complementary strand of at least a portion of the first nucleotide sequence.

[0052] In any various aspect of this section, a protein-encoding nucleic acid sequence located at either the 5' or 3' position of a recombinant viral IRES riboswitch described herein may encode a protein that is, for example, a reporter protein that generates a detectable signal. A reporter protein is a polypeptide that is not naturally present in host cells and has readily assayable enzymatic activity or a detectable signal. Exemplary but non-limiting reporter proteins include lacZ, catalase, xylE, GFP, RFP, YFP, ySUMO, CFP, EYFP, ECFP, mRFP1, mOrange, GFPmut3b, OFP, mBanana, neomycin phosphotransferase, luciferase, mCherry, and their derivatives or variants. In any several aspect of this section, the reporter protein is suitable for use in colorimetric assays, luminescence assays, or fluorescence assays.

[0053] The recombinant IRES riboswitches described herein can be used, for example, as sensor modules for detecting specific trRNAs. The recombinant IRES riboswitches may be designed such that the trRNA is a sequence present in a target eukaryote, target prokaryote, or target virus. In the presence of a target organism / virus, or a target organism / virus in a specific transcriptional state, the recombinant IRES riboswitch becomes activated, and a protein encoded at the 3' of the modified IRES sequence (e.g., a reporter protein) is expressed, indicating the presence of the target. Such a sensor system is demonstrated herein, for example, in Example 3, where infection by several different viruses is detected. In some embodiments, the target prokaryote or target virus is a pathogen, e.g., a mammalian pathogen or a human pathogen. In some embodiments, the target virus is a Zika virus, dengue virus, or coronavirus (e.g., SARS-CoV-2). In some embodiments, the target prokaryote or target eukaryote may be an organism containing and / or expressing a recombinant IRES riboswitch, and the trRNA may be a non-constitutively expressed RNA, such as RNA expressed only at a particular developmental or differentiation stage, or RNA expressed in response to a particular stimulus and / or stress.

[0054] Recombinant cells engineered to incorporate a riboswitch module In some aspects, this disclosure provides non-plant eukaryotic cells that have been engineered to express proteins under the control of recombinant IRESs described herein.

[0055] Eukaryotic cells can be animal cells, fungal cells, or protist cells. In some cases, eukaryotic cells may contain genomic DNA encoding recombinant IRESs. In other cases, recombinant IRESs may be encoded by vectors (e.g., plasmids) present within eukaryotic cells. Recombinant IRESs may be functionally ligated to endogenous or exogenous promoters and / or genes encoding the protein of interest.

[0056] Use of riboswitch modules in cell-free expression systems In some aspects, the recombinant IRES modules described herein may be used in cell-free expression systems. For example, a kit or assay may utilize cell-free lysates produced from eukaryotic cells containing DNA encoding at least one mRNA incorporating the recombinant IRES module. In other aspects, such a kit or assay may include transcribed mRNA incorporating at least one recombinant IRES module. It is understood that the riboswitch mechanisms described herein may be used as sensors to induce the expression of a protein of interest in a variety of in vitro applications (e.g., as a sensor for detecting the presence of viral mRNA).

[0057] Regulation of translation in eukaryotic or cell-free expression systems using recombinant IRES riboswitch modules The recombinant IRES riboswitch modules described herein may be used, for example, to regulate the expression of a protein of interest in eukaryotic cells or cell-free expression systems.

[0058] In some aspects, eukaryotic cells can be transfected using a vector encoding a protein of interest functionally linked to an upstream IRES riboswitch according to this disclosure. The IRES riboswitch may contain a sequence that shares at least 90, at least 95, at least 98, at least 99, or at least 100% sequence identity with the sequence of a viral IRES, except that two sites contain exogenous nucleotide sequences. In some embodiments, the IRES riboswitch may contain a sequence that shares at least 90, at least 95, at least 98, at least 99, or at least 100% sequence identity with the sequence of a group I dicistroviridae IRES (e.g., a CrPV IRES represented by SEQ ID NO: 1), except that two sites contain exogenous nucleotide sequences (e.g., any combination of sites 1-8 as defined above). This pair of exogenous sequences may include a first nucleotide sequence 25–80 nt long and a second nucleotide sequence 8–25 nt long, where the second nucleotide sequence is the reverse complementary strand of a portion of the first nucleotide sequence, hybridizing the pair of exogenous sequences. As a result of this hybridization, the IRES riboswitch adopts an inactive fold, preventing translation of the downstream protein of interest. Translation can be activated by introducing a trRNA containing the nucleotide sequence that is the reverse complementary strand of the first nucleotide sequence, causing the first nucleotide sequence to hybridize with the trRNA instead of the second nucleotide sequence, thereby allowing the IRES riboswitch to adopt an active fold. In some cases, the trRNA can be introduced by transfection or expressed by a vector.

[0059] In some cases, trRNA can be configured to have unique sequences not found in mRNA expressed by the eukaryotic cells used for expression. The selection of unique sequences can reduce or eliminate off-target effects (e.g., unintended hybridization between trRNA produced by eukaryotic cells and other endogenous mRNAs).

[0060] In some aspects, trRNA may include a portion of mRNA expressed by eukaryotic cells or in response to external stimuli (e.g., viral mRNA produced after viral infection of cells, as shown in Figure 13). In some aspects, increasing or decreasing the concentration of trRNA may regulate the expression of the protein of interest.

[0061] mRNA containing an IRES riboswitch can be functionally linked to a promoter suitable for expression in selected eukaryotic cells. In some cases, the T7 promoter may be used (e.g., when selected eukaryotic cells are engineered to produce T7 polymerase). Alternatively, a eukaryotic promoter (e.g., the RNA polymerase II promoter) may be used. The choice of suitable promoter varies depending on the intended use of the IRES riboswitch described herein. For example, inductive promoters may be preferred in some applications, while constitutive promoters may be preferred in others. Some promoters may also allow for more precise control over mRNA expression (e.g., the T7 promoter may be prone to leakage when used in eukaryotic cells due to low levels of RNA polymerase II recruitment).

[0062] In any aspect of the situation, the IRES riboswitch may be functionally linked to one or more of the following: (a) an IRES pseudoknot sequence; (b) an IRES pseudoknot sequence found in the naturally occurring viral wild-type sequence; (c) a promoter and / or upstream activator binding sequence; (d) a stop codon; (e) a stem-loop (e.g., SEQ ID NO:10); (f) a 5' cap; (g) a reporter gene; and (h) a poly-A tail, where one or more of elements a-h are located independently at the 5' or 3' of the IRES riboswitch. In any aspect of the situation, the IRES riboswitch may be functionally linked to one or more of the following: (a) an IRES pseudoknot sequence; (b) an IRES pseudoknot sequence found in the naturally occurring viral wild-type sequence; (c) a promoter and / or upstream activator binding sequence; (d) a stop codon; (e) a stem-loop; (f) a 5' cap; and (g) a reporter gene, with one or more of elements a-g individually located at the 5' of the IRES riboswitch.

[0063] Promoters for use in the methods and compositions described herein may be RNA polymerase II; polymerases other than RNA polymerase II; T7 polymerase; T3 promoter, araBAD promoter, trp promoter, lac promoter, Ptac promoter, pL promoter, and / or SP6 polymerase. The upstream activator-binding sequence may be the upstream activator-binding DNA sequence (UAF2) derived from Saccharomyces cerevisiae (e.g., SEQ ID NO: 11).

[0064] In any aspect of the embodiments, a vector, for example, a plasmid or viral vector containing a recombinant nucleic acid molecule, expression construct, recombinant mRNA molecule, or recombinant IRES riboswitch described herein, is described herein. In any aspect of the embodiments, a eukaryotic cell (e.g., an animal cell, a human cell, or a primate cell) containing DNA encoding a recombinant nucleic acid molecule, expression construct, or recombinant mRNA molecule described herein, wherein the eukaryotic cell is not a plant cell, and the DNA is either integrated into the genomic DNA of the eukaryotic cell or present on a vector (e.g., a plasmid or viral vector) present within the eukaryotic cell is described herein. In any aspect of the embodiments, a prokaryotic cell containing DNA encoding a recombinant nucleic acid molecule, expression construct, or recombinant mRNA molecule described herein, wherein the DNA is either integrated into the genomic DNA of the prokaryotic cell or present on a vector (e.g., a plasmid or viral vector) present within the prokaryotic cell is described herein. Such cells are thought to be engineered by the introduction of recombinant nucleic acid molecules, expression constructs, recombinant mRNA molecules, or recombinant IRES riboswitches, and a method for activating and / or regulating protein expression comprising the steps of providing eukaryotic cells engineered to express a recombinant nucleic acid molecule according to any one of the claims, and introducing a trigger RNA molecule into the eukaryotic cells comprising a third nucleotide sequence which is the reverse complementary strand of a first nucleotide sequence of a recombinant nucleic acid molecule, wherein the first nucleotide sequence hybridizes to the third nucleotide sequence under in vivo conditions, causing the recombinant IRES riboswitch to fold into an activated state, and furthermore, the eukaryotic cells can be used in a manner that is not that of plant cells.

[0065] It is understood that the IRES riboswitches described herein may also be used for cell-free expression. For example, a kit may include mRNA containing an IRES riboswitch functionally linked to a segment encoding the protein of interest, and components necessary for in vitro protein expression (e.g., cell lysates).

[0066] The IRES riboswitches described herein may be used in a variety of therapeutic and industrial applications. For example, a subject may be subjected to gene therapy, in which nucleic acids are introduced into the target cells to express a therapeutic protein under the control of an IRES riboswitch. The level of protein expression may be regulated by administering trRNA to the patient. IRES riboswitches may also be used in the laboratory or medical field as a means of controlling cell differentiation. For example, stem cells may be engineered to incorporate an IRES riboswitch triggered by mRNA produced by a specific cell type, and the riboswitch controls the expression of apoptosis-inducing toxins or proteins. Such mechanisms may be used to maintain the purity of stem cell lines by eliminating undesirable cell types that may be produced unintentionally.

[0067] In another embodiment, the IRES riboswitch described herein may be used in a method for detecting viral infection of cells. For example, a eukaryotic cell is engineered to express a recombinant nucleic acid containing the IRES riboswitch described herein, wherein the first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of the mRNA sequence specific to the virus, and it is determined whether the eukaryotic cell is infected with a virus by detecting and / or measuring the presence of a protein encoded by the second segment of the recombinant nucleic acid molecule, where the eukaryotic cell is not a plant cell. The virus may be, for example, dengue virus, Zika virus, or coronavirus.

[0068] In another embodiment, the IRES riboswitch described herein may be used in a method for controlling or monitoring the differentiation of eukaryotic cells. For example, eukaryotic cells are engineered to express recombinant nucleic acids containing the IRES riboswitch described herein, the cells are cultured, the first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of the mRNA sequence specific to a selected cell type, the protein encoded by the second segment of the recombinant nucleic acid molecule contains a toxin or protein that induces apoptosis of the selected cell type, and furthermore, the eukaryotic cells are not plant cells.

[0069] In some aspects of any aspect, a kit or system comprising one or more plasmids or viral vectors, recombinant nucleic acid molecules, expression constructs, recombinant mRNA molecules, recombinant IRES riboswitches and / or trRNAs described herein is described herein. A kit is a collection of materials or components comprising at least one of the aforementioned elements described herein. The exact nature of the components comprising a kit depends on its intended purpose. In some aspects of any aspect, a kit includes instructions for use. The "instructions for use" typically include specific expressions describing the techniques used when using the components of the kit, for example, to detect organisms or RNA. Furthermore, according to the present invention, the "instructions for use" may include specific expressions describing the preparation of recombinant IRES riboswitches, cells or expression systems described herein, for example, instructions for reconstitution, dilution, mixing or incubation typically for the intended purpose. Optionally, a kit also includes other useful components, such as measuring tools, diluents, buffers, syringes, pharmaceutically acceptable carriers, or other useful equipment readily recognizable to those skilled in the art.

[0070] Materials or components assembled into a kit may be provided to a practitioner, stored in any convenient and suitable manner that maintains operability and practicality. For example, components may be in a dissolved, dehydrated, or freeze-dried form. They may be provided at room temperature, refrigerated, or frozen temperature. Components are typically housed in suitable packaging. As used herein, the phrase “packaging” refers to one or more physical structures used to house the contents of the kit, such as the compositions of the present invention. The packaging is preferably constructed by known methods to provide a sterile and contaminant-free environment. The packaging may also preferably provide an environment that protects from light, humidity, and oxygen. As used herein, the term “packaging body” refers to a suitable solid matrix or material capable of holding individual kit components, such as glass, plastic, paper, foil, polyester (such as polyethylene terephthalate or Mylar), etc. Thus, for example, a packaging body may be a glass vial used to house a suitable amount of the compositions described herein. The packaging generally has an external label indicating the contents and / or purpose of the kit and / or its components.

[0071] For convenience, the meanings of some terms and phrases used in this specification, the examples, and the appended claims are provided below. Unless otherwise specified or implied by the context, the following terms and phrases have the meanings provided below. The definitions are provided to aid in the description of particular embodiments and are not intended to limit the scope of the claims, as the scope of the invention is limited solely by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. In the event of any apparent inconsistency between the use of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0072] For convenience, specific terms used in this specification, examples, and appended claims are set forth herein.

[0073] The terms “decrease,” “reduced,” “reduction,” and “inhibit” are all used herein to mean a statistically significant reduction. In some embodiments, “reduce,” “reduction,” “decrease,” or “inhibit” typically mean a reduction of at least 10% compared to a reference level (e.g., the absence of a given treatment or agent), and may include reductions of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition compared to a reference level.

[0074] The terms “increased,” “increase,” “enhance,” and “activate” are all used herein to mean an increase of a statistically significant amount. In some embodiments, the terms “increased,” “increase,” “enhance,” or “activate” may mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100%, or an increase of 10 to 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase of 2 to 10 times or more compared to a reference level.

[0075] As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a set of amino acid residues linked to one another by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms “protein” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used in reference to relatively large polypeptides, the term “peptide” is often used in reference to small polypeptides; however, the use of these terms in the art is often redundant. The terms “protein” and “polypeptide” are used interchangeably as used herein when referring to gene products and their fragments. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologues, paralogs, fragments, and other equivalents, variants, fragments, and analogs of those described above.

[0076] As used herein, the terms “nucleic acid” or “nucleic acid sequence” refer to polymer molecules incorporating units of any molecule, preferably ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid may be one nucleic acid strand of denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid may be a single-stranded nucleic acid that does not originate from any double-stranded DNA. In one aspect, a nucleic acid may be DNA. In another aspect, a nucleic acid may be RNA. Suitable DNA may include, for example, genomic DNA or cDNA. Suitable RNA may include, for example, mRNA.

[0077] The term “expression” refers to the production of RNA and proteins, and, where appropriate, the secretion of proteins, and, where applicable, without limitation, cellular processes involved in transcription, transcript processing, translation, and protein folding, modification, and processing. Expression may refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from one or more nucleic acid fragments of the present invention, and / or the translation of mRNA into polypeptides.

[0078] In some embodiments, the expression of nucleic acid sequences and / or proteins described herein is tissue-specific. In some embodiments, the expression of nucleic acid sequences and / or proteins described herein is global. In some embodiments, the expression of nucleic acid sequences and / or proteins described herein is systemic.

[0079] "Expression products" include RNA transcribed from genes and polypeptides obtained by translation of mRNA transcribed from genes. The term "gene" means a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when functionally ligated to appropriate regulatory sequences. A gene may or may not include the regions before and after the coding region, e.g., the 5' untranslated (5'UTR) or "leader" sequence and the 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0080] "Functionally linked" refers to an arrangement of elements configured so that the components described in this way perform their normal functions. Therefore, control elements functionally linked to a coding sequence can express the coding sequence. Control elements do not need to be contiguous with the coding sequence, as long as they function to guide its expression. For example, an intervening untranslated but transcribed sequence can exist between a promoter sequence and a coding sequence, and the promoter sequence can still be considered "functionally linked" to the coding sequence.

[0081] In some aspects, the methods described herein relate to measuring, detecting, or determining the level of at least one target. As used herein, the terms “detection” or “measurement” refer to indicating the presence of an analyte in a sample by, for example, observing a signal from a probe, label, or target molecule. Any method known in the art for detecting a particular labeled portion may be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. In some aspects of the aspect, the measurement may be a quantitative observation.

[0082] In some aspect of any aspect, polypeptides, nucleic acids, or cells described herein can be manipulated. As used herein, “manipulated” refers to an aspect that has been manipulated by human hands. For example, a polypeptide is considered “manipulated” if at least one aspect of it, e.g., its sequence, has been manipulated by human hands to be different from the original aspect. As is common practice and as understood by those skilled in the art, the offspring of a manipulated cell are typically still referred to as “manipulated,” even if the actual manipulation was performed on the previous entity.

[0083] The term “exogenous” refers to a substance present in a cell or nucleic acid sequence other than its natural source. As used herein, “exogenous” may refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that is not normally found and is desired to be introduced into such a nucleic acid molecule, cell, or organism, such as a cell or organism, by a process involving human intervention. In contrast, the term “endogenous” refers to a substance intrinsic to a nucleic acid molecule, organism, or cell.

[0084] In some embodiments, the nucleic acids described herein are included in a vector. In some aspects described herein, the nucleic acid sequences, or any modules thereof, are functionally linked to a vector. As used herein, the term “vector” refers to a nucleic acid construct designed for delivery to a host cell or for introduction between different host cells. As used herein, a vector may be viral or nonviral. The term “vector” encompasses any genetic element that can be replicated, in conjunction with appropriate regulatory elements, and whose gene sequence can be introduced into a cell. Vectors may include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, and the like.

[0085] In some aspects of the aspect, the vector or nucleic acid is recombinant and includes, for example, a sequence derived from at least two different sources. In some aspects of the aspect, the vector or nucleic acid includes a sequence derived from at least two different species. In some aspects of the aspect, the vector nucleic acid includes a sequence derived from at least two different genes. The sequence can be modified to be recombinant to provide a recombinant sequence by methods well known in the art, for example by the use of restriction enzymes and ligases, or the sequence can be incorporated into another sequence.

[0086] In some aspects of this section, the vectors or nucleic acids described herein are codon-optimized, for example, the natural or wild-type sequence of the nucleic acid sequence is modified or manipulated to include alternative codons so that the modified or manipulated nucleic acid encodes the same polypeptide expression product as the natural / wild-type sequence, but is transcribed and / or translated with improved efficiency in the desired expression system. In some aspects of this section, the expression system is an organism other than the source of the natural / wild-type sequence (or cells obtained from such an organism). In some aspects of this section, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in mammals or mammalian cells, e.g., mice, mouse cells, or human cells. In some aspects of this section, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in human cells. In some aspects of this section, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in yeast or yeast cells. In some aspects of this section, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in bacterial cells. In some aspects of the context, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression within Escherichia coli (E. coli) cells.

[0087] As used herein, the term “expression vector” refers to a vector that leads to the expression of RNA or polypeptides from a sequence ligated to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often heterogeneous to the cell, though not necessarily. An expression vector may include additional elements; for example, an expression vector may have two replication systems, thus allowing the expression vector to be maintained in two organisms, for example, in a human cell for expression and in a prokaryotic host for cloning and amplification.

[0088] As used herein, the term “viral vector” refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. Viral vectors may contain nucleic acids encoding polypeptides described herein, instead of non-essential viral genes. Vectors and / or particles may be used to introduce any nucleic acid into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0089] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapeutic agents. In some embodiments, the vector is an episome. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in high copy number extrachromosomal DNA, thereby eliminating the potential impact of chromosome integration.

[0090] As used herein, “contact” refers to any suitable means for delivering or exposing an active substance to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture media, perfusion, injection, or other delivery methods well known to those skilled in the art. In some embodiments, contact includes physical human activity, such as injection; acts of dispensing, mixing, and / or decantation; and / or operation of a delivery device or machine.

[0091] The term "statistically significant" or "significantly significant" refers to statistical significance, generally meaning a difference of 2 standard deviations (2SD) or more.

[0092] Except as shown in the operational examples, or unless otherwise indicated, any figures representing quantities of components or reaction conditions used herein should be understood to be modified in any case by the term “approximately.” When used in relation to proportions, the term “approximately” may mean ±1%.

[0093] As used herein, the term “comprising” means that other elements may exist in addition to the defined elements presented. The use of “comprising” indicates inclusion, not limitation.

[0094] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not described in the description of the embodiments.

[0095] As used herein, the term "consisting essentially of" refers to an element necessary for a given embodiment. The term allows for the presence of additional elements that do not substantially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0096] As used herein, the term “corresponding” means an amino acid or nucleotide at an enumerated position in the first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in the second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by aligning candidate sequences using homology programs known in the art, such as the degree of BLAST.

[0097] The singular terms “a,” “an,” and “the” refer to multiple objects unless otherwise explicitly indicated by the context. Similarly, the phrase “or” is intended to include “and” unless otherwise explicitly indicated by the context. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the disclosure, but preferred methods and materials are described below. The abbreviation “eg” is derived from the Latin “exempli gratia” and is used herein to indicate non-limiting examples. Thus, the abbreviation “eg” is a synonym for the term “for example.”

[0098] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each group component may be referenced and claimed individually or in any combination with other components of the group or other elements found herein. For convenience and / or patentability reasons, one or more components of a group may be included in or removed from a group. In any such case, this specification shall be deemed to include the modified groups and therefore satisfy the described description of all Markush groups used in the appended claims.

[0099] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings generally understood by those skilled in the art to which this disclosure pertains. It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and that these are subject to change. Terms used herein are for the sole purpose of describing specific aspects and are not intended to limit the scope of the present invention as defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in: The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier,2006;Janeway's Immunobiology,Kenneth Murphy,Allan Mowat,Casey Weaver(eds.),WWNorton&Company,2016(ISBN 0815345054,978-0815345053);Lewin's Genes XI,published by Jones&Bartlett Publishers,2014(ISBN-1449659055);Michael Richard Green and Joseph Sambrook,Molecular Cloning:A Laboratory Manual,4th ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,USA(2012)(ISBN 1936113414);Davis et al.,Basic Methods in Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(2012)(ISBN 044460149X);Laboratory Methods in Enzymology:DNA,Jon Lorsch(ed.)Elsevier,2013(ISBN This information can be found in Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), all of which are incorporated herein by reference in their entirety.

[0100] In any aspect of the context, the disclosures described herein do not relate to processes for cloning humans, processes for altering the genetic identity of human germlines, the use of human embryos for industrial or commercial purposes, or animals that would not provide any substantial medical benefit to humans or animals and would likely cause them disease, or processes for altering the genetic identity of animals resulting from such processes.

[0101] Other terms are defined herein in connection with descriptions of various aspects of the present invention.

[0102] All patents and other publications, including references, granted patents, published patent applications, and concurrently pending patent applications cited in this application, are expressly incorporated herein by reference for the purpose of illustrating and disclosing methods described in such publications, which may be used, for example, in connection with the art described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an acknowledgment by the inventors that they have no prior rights to such disclosures, either on the grounds of prior invention or for any other reason. Any statements regarding the dates or descriptions regarding the contents of these documents are based on information available to the applicant and do not constitute an acknowledgment that the dates or contents of these documents are accurate.

[0103] The description of the aspects of this disclosure is not intended to be exhaustive, nor to limit the disclosure to the strictly disclosed form. While specific aspects and examples of the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art. For example, while the steps or functions of a method are shown in a given order, alternative aspects may involve functions in a different order, or functions occurring substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods as needed. Further aspects may be provided by combining the various aspects described herein. Aspects of this disclosure may be modified, if necessary, to use the compositions, functions, and concepts of the above-mentioned references and applications to provide further aspects of this disclosure. Furthermore, some modifications to the protein structure may be made without affecting the biological or chemical action in terms of type or quantity, due to considerations of biological function equivalence. These and other modifications may be made in light of the detailed description. All such modifications are intended to fall within the scope of the appended claims.

[0104] In some embodiments, the technology may be defined by any of the following numbered sections: 1. (a) A first segment encoding a recombinant group 1 dicistroviridae internal ribosome entry site (IRES), which has been modified to incorporate exogenous nucleotide sequences at the first and second sites; (b) A second protein-coding segment located downstream of the first segment and functionally linked to the first segment, such that protein translation is suppressed when the IRES is in an inactive state. Recombinant nucleic acid molecules containing, The first region includes a first nucleotide sequence, The second site includes a second nucleotide sequence which is the reverse complementary strand of at least a portion of the first nucleotide sequence. Recombinant nucleic acid molecules. 2. The recombinant nucleic acid molecule according to item 1, wherein the nucleic acid molecule is mRNA. 3. The recombinant nucleic acid molecule of item 1, wherein the second nucleotide sequence is substantially the entire reverse complementary strand of the first nucleotide sequence. 4. The recombinant nucleic acid molecule according to item 1, wherein the group 1 disicstroviridae IRES is cricket paralysis virus (CrPV) IRES, cashimir wasp virus (KBV) IRES, acute honeybee paralysis virus (ABPV) IRES, or brown marmorated stink bug enteric virus (PSIV) IRES. 5. The recombinant nucleic acid molecule according to item 1, wherein the first and second sites are independently selected from site 1, site 2, site 3, site 4, site 5, site 6, site 7, and site 8, respectively. 6. The recombinant nucleic acid molecule of claim 4, wherein the first and second sites each include site 1 and site 2, site 1 and site 4, site 1 and site 5, site 1 and site 6, site 1 and site 7, site 1 and site 8, site 2 and site 6, site 2 and site 7, site 4 and site 6, site 5 and site 6, site 5 and site 7, site 6 and site 7, site 8 and site 2, site 8 and site 6, or site 8 and site 7, respectively. 7. A recombinant nucleic acid molecule according to item 1, wherein the first nucleotide sequence has a length of 25 to 80 nt. 8. A recombinant nucleic acid molecule according to item 1, wherein the second nucleotide sequence is 8 to 25 nt in length. 9. The recombinant nucleic acid molecule of item 1, wherein the first and second nucleotide sequences hybridize when expressed in a eukaryotic cell under in vivo or in vitro conditions, and can fold the group 1 dicistroviridae IRES into an inactive state, and the eukaryotic cell is not a plant cell. 10. The recombinant nucleic acid molecule of item 1, wherein the group 1 dicistroviridae IRES is configured to fold into an activated state in the presence of a trigger RNA molecule containing a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule of item 1. 11. The recombinant nucleic acid molecule of item 10, wherein the first nucleotide sequence, when expressed in a eukaryotic cell under in vivo conditions, hybridizes to the third nucleotide sequence, and can fold the group 1 dicistroviridae IRES into an activated state, and the eukaryotic cell is not a plant cell. 12. A plasmid encoding any one of the recombinant nucleic acid molecules described in the preceding items. 13. A eukaryotic cell containing DNA encoding any one of the recombinant nucleic acid molecules described in the preceding paragraph, wherein the eukaryotic cell is not a plant cell, The aforementioned DNA, (a) incorporated into the genomic DNA of the eukaryotic cell; or (b) Plasmids or viral vectors present in the eukaryotic cells, eukaryotic cells. 14. The eukaryotic cell of item 13, wherein the cell is (a) an animal cell; (b) a human cell; or (c) a primate cell. 16. (a) any of the recombinant nucleic acid molecules described in the preceding paragraph; (b) A trigger RNA molecule comprising a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule A system for controlling gene expression, including [specific components / functions]. 17. (a) Plasmid of item 12; (b) A trigger RNA molecule comprising a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule A kit that includes this. 18. (a) a first segment encoding the first protein; (b) A second segment downstream of the first segment, which encodes a recombinant group 1 dicistroviridae internal ribosome entry site (IRES), modified to incorporate exogenous nucleotide sequences at the first and second sites, (c) A third segment encoding the second protein, located downstream of the second segment and functionally linked to the second segment, such that the translation of the second protein is suppressed when the IRES is in an inactive state. Recombinant mRNA molecules containing, The transcription of the recombinant mRNA molecule is polymerase-dependent. The first region includes a first nucleotide sequence, The second site includes a second nucleotide sequence which is the reverse complementary strand of at least a portion of the first nucleotide sequence. Recombinant mRNA molecule. 19. The transcription of the recombinant mRNA molecule is as follows: (a) RNA polymerase II; (b) Polymerases other than RNA polymerase II; (c) T7 polymerase; and / or (d) SP6 polymerase A recombinant mRNA molecule that depends on item 18. 20. (a) a step of providing eukaryotic cells that have been manipulated to express any of the recombinant nucleic acid molecules described in the preceding items; and (b) The step of introducing a trigger RNA molecule into the eukaryotic cell, the trigger RNA molecule containing a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule. A method for activating and / or regulating protein expression, comprising: The first nucleotide sequence hybridizes to the third nucleotide sequence under in vivo conditions, causing the group 1 dicistroviridae IRES to fold into an activated state. Furthermore, the eukaryotic cells mentioned above are not plant cells. method. twenty one. The method of item 20, wherein the eukaryotic cells manipulated to express the recombinant nucleic acid molecules are provided by introducing any of the recombinant nucleic acid molecules of the preceding item into the eukaryotic cells. twenty three. (a) A step of providing a eukaryotic cell manipulated to express any of the recombinant nucleic acid molecules described in the preceding paragraph, wherein the first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of the mRNA sequence specific to the virus; and (b) A step of determining whether the eukaryotic cell is infected with the virus by detecting and / or measuring the presence of a protein encoded by a second segment of the recombinant nucleic acid molecule, wherein the eukaryotic cell is not a plant cell. A method for detecting viral infection in eukaryotic cells, including [specific example]. twenty four. The method of item 23, wherein the virus is dengue virus or Zika virus. twenty five. (a) a step of providing eukaryotic cells that have been manipulated to express any of the recombinant nucleic acid molecules described in the preceding items; and (b) Steps to culture the eukaryotic cells. A method for controlling the differentiation of eukaryotic cells, including, The first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of the mRNA sequence specific to the selected cell type. The protein encoded by the second segment of the recombinant nucleic acid molecule comprises a toxin or protein that induces apoptosis of the selected cell type. Furthermore, the eukaryotic cells mentioned above are not plant cells. method. 27. (a) DNA encoding mRNA, including recombinant nucleic acid molecules A vector containing, The recombinant nucleic acid molecule (i) A first segment encoding a recombinant group 1 dicistroviridae internal ribosome entry site (IRES), which has been modified to incorporate exogenous nucleotide sequences at the first and second sites; (ii) A second protein-coding segment located downstream of the first segment and functionally linked to the first segment, such that protein translation is suppressed when the IRES is in an inactive state. Includes, The first region includes a first nucleotide sequence, The second region includes a second nucleotide sequence which is the reverse complementary strand of at least a portion of the first nucleotide sequence. The group 1 disicstroviridae IRES is configured to activate the expression of the protein in response to the presence of mRNA containing a segment that is the reverse complementary strand of the first nucleotide sequence. vector. 28. A prokaryotic cell comprising DNA encoding any one of the recombinant nucleic acid molecules described in the preceding paragraph, The aforementioned DNA, (a) incorporated into the genomic DNA of the prokaryotic cell; or (b) Plasmids or viral vectors present in the prokaryotic cells, Prokaryotic cells.

[0105] In some embodiments, the technology may be defined by any of the following numbered sections: 1. (a) A first segment encoding a recombinant group 1 dicistroviridae internal ribosome entry site (IRES), which has been modified to incorporate exogenous nucleotide sequences at the first and second sites; (b) A second protein-coding segment located downstream of the first segment and functionally linked to the first segment, such that protein translation is suppressed when the IRES is in an inactive state. Recombinant nucleic acid molecules containing, The first region includes a first nucleotide sequence, The second site includes a second nucleotide sequence which is the reverse complementary strand of at least a portion of the first nucleotide sequence. Recombinant nucleic acid molecules. 2. (a) A first segment encoding a recombinant viral internal ribosome entry site (IRES), which is modified to incorporate a first exogenous nucleotide sequence at the first site and a second exogenous nucleotide sequence at the second site; (b) A second protein-coding segment located downstream of the first segment and functionally linked to the first segment, such that protein translation is suppressed when the IRES is in an inactive state. A recombinant nucleic acid molecule containing from 5' to 3', The second nucleotide sequence is the reverse complementary strand of at least a portion of the first nucleotide sequence. Recombinant nucleic acid molecules. 3. The recombinant nucleic acid molecule according to item 2, wherein the modified IRES is a group 1 disistroviridae IRES, a hepacivirus IRES, or an enterovirus IRES. 4. A recombinant nucleic acid molecule according to any of items 1 to 3, wherein the modified IRES is an IRES derived from a mammalian pathogenic virus or a mammalian symbiotic virus. 5. The recombinant nucleic acid molecule according to item 4, wherein the modified IRES is an IRES derived from a human pathogenic virus or a human symbiotic virus. 6. A recombinant nucleic acid molecule, any of items 1 to 5, wherein the nucleic acid molecule is mRNA. 7. A recombinant nucleic acid molecule according to any of items 1 to 6, wherein the second nucleotide sequence is substantially the entire reverse complementary strand of the first nucleotide sequence. 8. Recombinant nucleic acid molecules of any of items 1-7, wherein the Group 1 Disistroviridae IRES is cricket paralysis virus (CrPV) IRES, Casimir wasp virus (KBV) IRES, acute honeybee paralysis virus (ABPV) IRES, brown marmorated stink bug enteric virus (PSIV) IRES; aphid lethal paralysis virus (ALPV) IRES; black queen bee brood virus (BQCV) IRES; fruit fly C virus (DCV) IRES; dwarf kite P virus (HiPV) IRES; leafhopper virus-1 (HoCV-1) IRES; wheat aphid virus (RhPV) IRES; and assassin bug virus (TrV) IRES. 9. The aforementioned hepacivirus IRES is a hepatitis c virus (HCV) IRES, and is a recombinant nucleic acid molecule according to any of items 2 to 7. 10. A recombinant nucleic acid molecule according to any of items 2 to 7, wherein the enterovirus IRES is poliovirus (PV) IRES or enterovirus 71 (EV71) IRES. 11. A recombinant nucleic acid molecule according to any of items 1 to 10, wherein the first and second sites are independently selected from site 1, site 2, site 3, site 4, site 5, site 6, site 7, and site 8, respectively. 12. The recombinant nucleic acid molecule according to claim 11, wherein the first and second sites each comprise site 1 and site 2, site 1 and site 4, site 1 and site 5, site 1 and site 6, site 1 and site 7, site 1 and site 8, site 2 and site 6, site 2 and site 7, site 4 and site 6, site 5 and site 6, site 5 and site 7, site 6 and site 7, site 8 and site 2, site 8 and site 6, or site 8 and site 7, respectively. 13. A recombinant nucleic acid molecule according to any of items 11 to 12, wherein the first and second sites each include site 1 and site 2, site 1 and site 8, site 2 and site 7, site 6 and site 7, or site 8 and site 6, respectively. 14. A recombinant nucleic acid molecule according to any of items 11 to 13, wherein the first and second sites each include site 6 and site 7, or site 8 and site 6, respectively. 15. A recombinant nucleic acid molecule according to any of items 1 to 14, wherein the first nucleotide sequence described above is 25 to 80 nt in length. 16. The recombinant nucleic acid molecule according to item 15, wherein the first nucleotide sequence is 40 to 50 nt in length. 17. The recombinant nucleic acid molecule according to item 15, wherein the second nucleotide sequence is 8 to 25 nt in length. 18. A recombinant nucleic acid molecule according to any of items 1 to 17, wherein the second nucleotide sequence is 6 to 15 nt in length. 19. A recombinant nucleic acid molecule according to any of items 1 to 18, wherein the first nucleotide sequence is 2.5 to 8 times longer than the second nucleotide sequence. 20. A recombinant nucleic acid molecule according to any of items 1 to 19, wherein the first nucleotide sequence is the reverse complementary strand of a sequence found in a target eukaryote, target prokaryote, or target virus. twenty one. The recombinant nucleic acid molecule according to item 20, wherein the target prokaryote or target virus is a human pathogen. twenty two. The recombinant nucleic acid molecule according to item 21, wherein the target virus is Zika virus or coronavirus. twenty three. The recombinant nucleic acid molecule according to item 22, wherein the coronavirus is SARS-CoV-2. twenty four. The protein is a recombinant nucleic acid molecule of any of items 1 to 23, which generates a detectable signal. twenty five. A recombinant nucleic acid molecule according to any of items 1 to 24, wherein the first and second nucleotide sequences hybridize when expressed in a eukaryotic cell under in vivo or in vitro conditions, causing the IRES to fold into an inactive state, and the eukaryotic cell is not a plant cell. 26. A recombinant nucleic acid molecule according to any of items 1 to 25, wherein the IRES is configured to fold into an activated state in the presence of a trigger RNA molecule containing a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule according to item 1. 27. The recombinant nucleic acid molecule according to item 26, wherein the first nucleotide sequence, when expressed in a eukaryotic cell under in vivo conditions, hybridizes to the third nucleotide sequence, causing the IRES to fold into an activated state, and the eukaryotic cell is not a plant cell. 28. A sequence comprising a recombinant nucleic acid molecule or a sequence containing one of items 1 to 27, The 5' and / or 3' of the sequence encoding or containing any recombinant nucleic acid molecule from items 1 to 27 is as follows: (a) IRES pseudoknot arrangement; (b) The IRES pseudoknot sequence found in naturally occurring viral wild-type sequences; (c) Promoter and / or upstream activator binding sequence; (d) Terminal codon; (e) Stem loop; (f) 5' cap; (g) reporter gene; and (h) Poly A Tail An expression construct further comprising one or more of the following. 29. The following is added to the 5' of the sequence encoding or containing any recombinant nucleic acid molecule from items 1 to 27: (a) IRES pseudoknot arrangement; (b) The IRES pseudoknot sequence found in naturally occurring viral wild-type sequences; (c) Promoter and / or upstream activator binding sequence; (d) Terminal codon; (e) Stem loop; (f) 5' cap; and (g) Reporter gene An expression construct according to item 28, comprising one or more of the following. 30. (a) The promoter is selected from the SP6 promoter, T3 promoter, araBAD promoter, trp promoter, lac promoter, Ptac promoter and pL promoter; and / or (b) The upstream activator binding sequence is an upstream activator binding DNA sequence (UAF2) derived from Saccharomyces cerevisiae. An expression construct from any of items 28-29. 31. The transcription of the recombinant nucleic acid molecule is as follows: (a) RNA polymerase II; (b) Polymerases other than RNA polymerase II; (c) T7 polymerase; and / or (d) SP6 polymerase An expression construct or recombinant nucleic acid sequence that depends on any of items 1-30. 32. (a) a first segment encoding the first protein; (b) A second segment downstream of the first segment, which encodes a recombinant group 1 dicistroviridae internal ribosome entry site (IRES), modified to incorporate exogenous nucleotide sequences at the first and second sites; (c) A third segment encoding the second protein, located downstream of the second segment and functionally linked to the second segment, such that the translation of the second protein is suppressed when the IRES is in an inactive state. Recombinant mRNA molecules containing, The transcription of the recombinant mRNA molecule is polymerase-dependent. The first region includes a first nucleotide sequence, The second site includes a second nucleotide sequence which is the reverse complementary strand of at least a portion of the first nucleotide sequence. Recombinant mRNA molecule. 33. The transcription of the recombinant mRNA molecule is as follows: (a) RNA polymerase II; (b) Polymerases other than RNA polymerase II; (c) T7 polymerase; and / or (d) SP6 polymerase A recombinant mRNA molecule as described in item 32, which depends on the recombinant mRNA molecule. 34. A plasmid encoding any of the recombinant nucleic acid molecules, expression constructs, or recombinant mRNA molecules described in the preceding paragraphs. 35. A eukaryotic cell comprising DNA encoding any of the recombinant nucleic acid molecules, expression constructs, or recombinant mRNA molecules described in the preceding paragraph, The aforementioned eukaryotic cells are not plant cells, The aforementioned DNA, (a) incorporated into the genomic DNA of the eukaryotic cell, or (b) Plasmids or viral vectors present in the eukaryotic cells, eukaryotic cells. 36. The eukaryotic cell according to item 35, wherein the cell is (a) an animal cell; (b) a human cell; or (c) a primate cell. 37. (a) any of the recombinant nucleic acid molecules described in the preceding paragraph; (b) A trigger RNA molecule comprising a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule A system for controlling gene expression, including [specific components / functions]. 38. (a) Plasmid described in item 34; (b) A trigger RNA molecule comprising a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule A kit that includes this. 39. (a) a step of providing eukaryotic cells that have been manipulated to express any of the recombinant nucleic acid molecules described in the preceding items; and (b) The step of introducing a trigger RNA molecule into the eukaryotic cell, the trigger RNA molecule containing a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule. A method for activating and / or regulating protein expression, comprising: The first nucleotide sequence hybridizes to the third nucleotide sequence under in vivo conditions, causing the IRES to fold into an activated state. Furthermore, the eukaryotic cells mentioned above are not plant cells. method. 40. The method according to item 39, wherein the eukaryotic cells manipulated to express the recombinant nucleic acid molecules are provided by introducing any of the recombinant nucleic acid molecules of the preceding item into the eukaryotic cells. 41. (a) A step of providing a eukaryotic cell manipulated to express any of the recombinant nucleic acid molecules described in the preceding paragraph, wherein the first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of the mRNA sequence specific to the virus; and (b) A step of determining whether the eukaryotic cell is infected with the virus by detecting and / or measuring the presence of a protein encoded by a second segment of the recombinant nucleic acid molecule, wherein the eukaryotic cell is not a plant cell. A method for detecting viral infection in eukaryotic cells, including [specific example]. 42. The method according to item 41, wherein the virus is dengue virus or Zika virus. 43. (a) a step of providing eukaryotic cells that have been manipulated to express any of the recombinant nucleic acid molecules described in the preceding items; and (b) Steps to culture the eukaryotic cells. A method for controlling the differentiation of eukaryotic cells, including, The first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of the mRNA sequence specific to the selected cell type. The protein encoded by the second segment of the recombinant nucleic acid molecule comprises a toxin or protein that induces apoptosis of the selected cell type. Furthermore, the eukaryotic cells mentioned above are not plant cells. method. 44. Sequences encoding any of the recombinant nucleic acid molecules described above A vector containing, The modified IRES is configured to activate the expression of the protein in response to the presence of mRNA containing a segment that is the reverse complementary strand of the first nucleotide sequence. vector. 45. A prokaryotic cell comprising DNA encoding any of the recombinant nucleic acid molecules described in the preceding paragraph, The aforementioned DNA, (a) incorporated into the genomic DNA of the prokaryotic cell; or (b) Plasmids or viral vectors present in the prokaryotic cells, Prokaryotic cells.

[0106] Certain elements of any of the embodiments described above may be combined with elements of other embodiments or replaced by elements of other embodiments. Furthermore, while advantages related to certain embodiments of the Disclosure have been described in the context of those embodiments, other embodiments may also demonstrate such advantages, and not all embodiments are necessarily required to demonstrate such advantages in order to fall within the scope of the Disclosure.

[0107] The technologies described herein are further illustrated by the following examples, which should not be construed as being further limiting. [Examples]

[0108] The following non-limiting examples are provided to further illustrate this disclosure.

[0109] Example 1: Screening of recombinant IRES riboswitches The process of designing recombinant IRES riboswitches began with the selection of IRES modules from a viral database and their testing in transfection assays based on human fetal kidney 293 (HEK293) cells. Co-transfecting these constructs resulted in a dramatic enhancement of mKate expression by the IRES module, as shown in Figure 6, rather than T7 polymerase reaching the 5' cap mRNA. Hepatitis C virus (HCVd20) IRES was used as a control in this assay but was not pursued due to its extensive structural differences compared to the rest of the selected IRES modules and the reported reliability of its activity against small RNAs. Based on this initial screening, and focusing on CrPV IRES due to the abundance of existing structural information, the cricket paralysis virus (CrPV) IRES module, the Kashmiri wasp virus (KBV) IRES module, and the acute honeybee paralysis virus (ABPV) IRES module were selected for further testing.

[0110] To test whether disrupting the structure of these IRES modules could reduce their translation initiation ability, we manipulated mutants of CrPV, KBV, and ABVP IRES modules incorporating exogenous nucleotide sequences. We hypothesized that insertion of two reverse complementary DNA segments into two sites of an IRES module could be used to distort the functional configuration / structure of the IRES module (e.g., due to hybridization of these two segments). We further designed two reverse complementary DNA segments so that one of them is the reverse complementary of the other segment and is released by a trigger RNA sequence (trRNA) containing a duplicate portion longer than the duplicate between the first and second segments. Specifically, the longer segment (40-50 base pairs) of the inserted DNA was the reverse complementary of a portion of the target trigger, and the shorter segment (10-15 base pairs) was the reverse complementary of a portion of the first segment. We selected eight sites (i.e., sites 1-8 shown in Figure 3) where insertion would not individually disrupt IRES module activity, thus avoiding loop 3, whose complete functionality is critical for all levels of IRES module activity.

[0111] Using the same assay for IRES activity determination, a series of 15 fold combinations were tested (Figure 7). Each fold combination was named using the format (long segment site number - short segment site number). In this initial testing, GFP mRNA was used as the trigger RNA. Folds 1-2, 1-8, and 2-7 yielded reproducible multiplier changes in this initial screening, but further testing showed a lack of translocation of the same folds to other target triggers. We decided to continue working with folds 6-7 and 8-6, both of which showed a 1.7-fold increase in the percentage of mKate-positive cells when simultaneously transfected with trigger RNAs.

[0112] Example 2: Optimization of Recombinant IRES Riboswitches The practical applications of currently disclosed recombinant IRES riboswitches often require a large multiplier of change in downstream protein translation after trigger induction. Therefore, reducing the leakage of two hits from the initial screening (i.e., folds 6-7 and 8-6) was required. Previous studies have suggested that the IRES pseudoknot is important for ribosome recruitment. Thus, we conducted tests to determine whether the strain on the IRES module associated with pseudoknot disruption at the same locations (after insertion site 7 in folds 6-7 and after insertion site 6 in folds 8-6) reduces IRES module leakage. We designed a novel recombinant IRES riboswitch such that the base pair at insertion 1 following the annealing site of insertion 2 is the reverse complementary strand of the base pair corresponding to the pseudoknot. In the absence of a trigger, this annealing prevents correct pseudoknot folding, i.e., the formation of a pseudoknot disruption site (PB site). When a recombinant IRES riboswitch with a PB portion was designed, as shown in Figure 8, in fold 8-6, the "OFF" (untriggered) state was reduced to the level observed when the IRES module is absent, and the change ratio from "ON" (triggered) to "OFF" (untriggered) increased from 1.7 to 2.5 times. Further optimization of the melting temperature of the annealing portion demonstrated the dependence of the IRES riboswitch behavior on this parameter, but did not show an improvement in the change ratio from "ON" to "OFF".

[0113] Based on these findings, we hypothesized that the remaining leakage is caused by nonspecific binding of the T7 promoter sequence by endogenous RNA polymerase II, resulting in 5' capping and bypass of IRES-mediated translational initiation. To find suitable polymerase-promoter substitution pairs, sfGFP was placed under different promoters with high RNA expression levels and transfected into HEK293T cells (without corresponding polymerases). SP6 So, P T7It was found to exhibit significantly less leakage than its analogues. For RNA polymerase I, which has been shown to reduce RNA polymerase II binding, upstream activating sequences were tested. Our findings demonstrate that an upstream activator-binding DNA sequence from Saccharomyces cerevisiae reduces leakage (named UAF2). By combining these two methods / techniques / techniques, leakage was substantially minimized, achieving a 15.9-fold "ON" to "OFF" trigger-mRNA-based induction, as shown in Figure 9. Similar induction folds could be achieved by adding a stop codon and stem-loop before the recombinant IRES riboswitch. Further increases in the "ON" to "OFF" change fold could be achieved using a recombinant IRES riboswitch that obscures the Kozak sequence downstream of the riboswitch; however, this method requires extensive screening (only one out of nine Kozak toeholds improved the change fold).

[0114] To further characterize the recombinant IRES riboswitches of this disclosure, their ability to sense trigger mRNAs from several sources was tested. In particular, the inventors designed folds for detecting GFP, Azurite, and yeast SUMO mRNA. By exposing each fold to each of the three mRNAs, it is demonstrated that the recombinant IRES riboswitches are orthogonal and can be designed for most target mRNAs (Figure 10). The sensitivity of the inventors' folds was further tested by mutating the insertion inside insertions 1 and 2 (Mut Anneal) or inside insertion 1 instead of insertion 2 (Mut Outside). The recombinant IRES riboswitches were found to be highly sensitive to mutations inside insertion 2, indicated by a threefold decrease in activation level due to a single mutation, but not sensitive to mutations outside insertion 2 (Figure 11). Thus, this test confirmed that recombinant IRES riboswitches can be designed for various mRNAs while maintaining specificity.

[0115] Subsequently, the inventors aimed to expand the range of possible PB sites for easier recombinant IRES riboswitch design. They attempted to achieve this by fabricating folds using various IRES modules, taking into account the differences in arrangement and structural similarities between IRES modules. Similar magnifications of trigger-inducible translation were observed when fabricating recombinant IRES riboswitches using both KBV and ABPV IRES modules compared to CrPV IRES modules (Figure 12). These alternative IRES eToeholds effectively expand the range of possible PB sites.

[0116] Example 3: Modular RNA-responsive element for eukaryotic translational regulation In biotechnology, robust and easily programmable RNA-responsive modules are highly desirable for a variety of applications. In eukaryotes, however, simple RNA-responsive elements with translational control over transgenes remain unrealized. 1~3 This specification describes a eukaryotic toehold switch (eToehold) as a modular riboregulator based on an internal ribosome entry site (IRES) sequence that provides translational control of an introduced gene in response to a specific RNA sequence. Designed with optimized RNA annealing, eToehold is demonstrated herein to sense and respond to the presence of trigger RNA in a range of eukaryotic cells, resulting in up to 16-fold induction of the introduced gene. eToehold is also demonstrated to be able to distinguish infection status, cellular state, and cell type in mammalian cells based on the presence of exogenous or endogenous RNA transcripts.

[0117] Synthetic biology techniques for sensing and responding to specific intracellular RNAs are desirable for therapeutic and diagnostic applications because they provide a means for identifying and targeting specific cells, tissues, and organisms, and can serve as fundamental elements for advanced gene circuits. To detect specific RNA transcripts, RNA-based prokaryotic modules called toehold switches have been developed.1、4 A toehold switch selectively represses in-cis translation of a reporter gene by capturing the ribosome-binding site (RBS) upstream of the reporter gene within a stem-loop structure in the absence of trigger RNA (trRNA). The RBS is released when trRNA binds to the toehold switch, opening the stem-loop structure and initiating translation of the reporter gene. However, eukaryotic translation is far more complex and is typically regulated by several factors, including RNA polymerase II, poly-adenosine (poly-A) tails for mRNA stabilization, and 5' modification capping mobilized by the Kozak consensus sequence for protein translation regulation. While the Kozak sequence improves ribosome binding, it is not as crucial to translation as the prokaryotic RBS. Previously developed Kozak-based toehold switches have only achieved up to twice the trRNA propulsion induction of eukaryotic translation. 5 The 5' cap governs translational regulatory mechanisms and poses a significant challenge for eukaryotic RNA-sensing riboswitches that function at the translational level.

[0118] By utilizing Cas9 expression and the manipulated folding of guide RNA (gRNA), more complex RNA-based switches are being developed to hide sequences essential for function. 2、3 Unfolding of gRNA by trRNA leads to activation of the Cas9 enzyme and corresponding downstream regulation. However, these mechanisms only induce moderate change ratios (in both eukaryotes and prokaryotes). Another technique involves the use of ribozymes that cleave the poly(A) tail during small molecule induction. 6、7 This method has not yet been extended to larger nucleotide sequences and results in RNA degradation. This makes the sequence-induced reaction irreversible, making it impossible to sense temporal changes at the RNA level. Recent studies have used ribozymes to respond to short nucleotide oligomers. 8、9、The sensors based on these ribozymes are not yet compatible with the detection of longer trRNAs, including endogenous transcripts.

[0119] IRES is an endogenous and viral eukaryotic mRNA element that has evolved to initiate protein translation independent of mRNA 5'-capping and polyadenylation. The development of an RNA-based eukaryotic module called eToehold, which enables regulated translation of an in cis reporter gene by the presence of a specific trRNA, is described herein. eToehold incorporates a modified IRES that is designed to be inactive until a sense-antisense interaction with a specific trRNA triggers activation (Figure 14A). Using this system, up to 16-fold trRNA-induced translation of a transgene is achieved. eToehold has been demonstrated to be functional in human and yeast cells, as well as in mammalian cell-free lysates. Further demonstration shows that stable cell lines expressing eToehold can sense natural viral infection (by Zika virus) and viral transcripts (SARS-CoV-2 constructs). It has also been shown that eToehold has the ability to discriminate between various cell states and cell types by selectively activating protein translation based on endogenous RNA levels.

[0120] To engineer RNA-sensing riboswitches that function in eukaryotic cells, the structural induction translation activity 10~14 of a viral IRES module was modified (Figure 14B). The IRES is adapted to sense small molecules 15IRES-based systems have not been previously designed to respond to trRNA. The IRES module was initially selected and tested in a transfection assay based on human embryonic kidney 293 (HEK293) cells (Figure 17). To avoid 5' capping, T7 polymerase was co-transfected into the cells, and these transcripts, which do not undergo 5' capping, were used to generate the IRES sequence. The presence of the IRES module resulted in an approximately 9-fold enhancement of incis mKate expression (Figure 14C, Figures 18A-18H). Due to its well-characterized structure and reported functionality in a wide range of eukaryotic systems, we decided to focus on the CrPV IRES and pursue the cricket paralysis virus (CrPV) IRES module, the Kashmiri wasp virus (KBV) IRES module, and the acute honeybee paralysis virus (ABPV) IRES module as a basis for further development of eToehold. 10~12 .

[0121] We hypothesized that by confirming IRES activity and then inserting a short complementary RNA segment into the IRES sequence, the secondary structure would be disrupted by the formation of a new loop, thereby reducing translation initiation ability, and that IRES functionality would be rescued when these introduced loops were disrupted by the sense-antisense action of trRNA. We tested this hypothesis by inserting reverse complementary DNA fragments (two fragments) into a site within the IRES template and theorized that base pairing between these sequences in the resulting transcript would distort the functional structure of IRES. To allow for the restoration of IRES function in the presence of trRNA, these introduced complementary DNA segments were designed to bind to a selected trRNA sequence. The insertions were designed to be of unequal lengths, as the goal was for base pairing between trRNA and the new insertion to be sufficient to disrupt the IRES disruption loop. The longer fragment (40-50 base pairs) was selected to be the reverse complementary of a portion of the trRNA, and the shorter fragment (6-15 base pairs) was selected to be the reverse complementary of a portion of the first fragment. Eight sites where insertion would not eliminate CrPV IRES activity if there were no alteration to the entire secondary structure. 11、16 I selected (Figure 14D).

[0122] We screened various CrPV IRES sequences with complementary sequences inserted into eight possible sites. GFP mRNA was selected and used as the trRNA, and the IRES was designed so that the GFP mRNA could degrade the newly formed loop. To monitor IRES activity, we used the downstream incis mKate gene of the modified IRES sequence and co-transfected cells with these constructs and the GFP plasmid (Figure 14E). Each site combination is named in the format long fragment site number-short fragment site number (e.g., 1-2). Several site combinations (1-2, 1-8, 2-7, 6-7, and 8-6) behaved as predicted and were found to generate even higher mKate signals when co-expressed with GFP. Based on these results, we decided to focus on site combinations 6-7 and 8-6, which reliably showed a 1.7-fold increase in the percentage of mKate-positive cells in the presence of the trigger RNA, as confirmed via GFP fluorescence.

[0123] Modified IRESs appeared to retain significant translational capacity despite the newly introduced insertions. IRES pseudoknots may be important for ribosome recruitment. 10、17It was hypothesized that simultaneously distorting the IRES module as described above and disrupting the IRES pseudoknot using the same insertion sequence would reduce the basal expression of the module. To test this hypothesis, a novel eToehold was designed by selecting relatively short insertions that included sequences present in the pseudoknot. It was hypothesized that, in the absence of a trigger, annealing between insertions would impair correct pseudoknot folding. The insertion sites where this occurs were named base-pair disruption sites (BB sites; Figure 14F). Designing eToeholds with BB sites reduced the off (no trigger) state to background levels, and for site combination 8-6, the on-to-off change ratio increased from 1.7 to 2.5 (Figure 14G, Figures 21A-21C). To further characterize the thermodynamic requirements of eToehold switching, the length of the short insertions at site 8-6 was changed, and consequently, the annealing temperature was also changed. Some eToehold sequences showed a correlation between annealing temperature and output, while others did not (Figures 19A-19B). Since relatively long eToehold sequences may be long enough to potentially induce an RNAi response, RNA levels of IRES constructs designed to bind to “trigger” RNA sequences were evaluated and compared to unbound orthogonal sequences. No significant difference in IRES construct RNA levels was observed in two of the three cell types evaluated, but primary human fibroblasts showed approximately a twofold decrease in the presence of “trigger” RNA (Table 2). No significant difference in the production of inflammation-related cytokines was observed between cells transfected with “trigger” and orthogonal RNAs, but transfection alone significantly increased cytokine production levels (Figures 20A-20B). 18 .

[0124] T7 promoter (P T7 ) is generally considered to be highly specific to T7 polymerase, but mammalian RNA polymerase II is P T7 It has been shown to bind to and initiate a significant level of transcription. 19It was hypothesized that some of the unexpected translation from eToehold in the absence of trRNA could be due to the recruitment of endogenous RNA polymerase II and the subsequent generation of transcripts with a 5' cap that independently induce translation and negate the need for functional IRES. Furthermore, P T7 The recruitment of mammalian RNA polymerase II by P T7 This may be due to the accidental similarity of the primary sequence between the IRES regulatory reporter and the natural mammalian transcription start site. Therefore, to test whether off-state translation of the IRES regulatory reporter is reduced, P T7 For transcription systems orthogonal to this, we screened for exogenous promoter sequences. The SP6 promoter (P SP6 ) is P T7 It was found to result in significantly lower basal expression than that of its analogues (Figure 22A). Next, it has been shown to reduce RNA polymerase II binding. 20~22 We then tested upstream activator-binding DNA sequences from RNA polymerase I to see if they could further reduce basal expression. We identified an upstream activator-binding DNA sequence from Saccharomyces cerevisiae (named UAF2, Figure 22B) that successfully reduced basal expression. By combining these components, we achieved a 15.9-fold on-to-off trigger-mRNA-based induction with substantially minimized basal expression (Figures 15A and 23A-23E). While maintaining the use of T7 polymerase and promoter, we added a stop codon and stem-loop before the IRES module. 23 By adding this, we were able to achieve a similar rate of change during induction (Figure 24).

[0125] We developed a method to achieve a robust multiplier of change in transgene expression upon trRNA induction, and then tested the specificity of this system for desired trRNA sequences. eToehold was designed to detect GFP, Azurite, and yeast SUMO mRNA, and these designs were found to specifically sense their respective trRNA sequences (Figure 15B). The sensitivity of eToehold to these congeneral trRNAs was further tested by introducing mismatches into two insertion sequences. eToehold was found to be sensitive to mismatches within the annealing region (Figure 25A). Furthermore, the generalizability of eToehold designs for other IRESs was tested. eToehold was synthesized using both the KBV IRES module and the ABPV IRES module, and similar multipliers of change in trigger-induced translation were observed compared to CrPV IRES module-based eToehold (Figure 25B). Additionally, RNA sequences that can bind to short eToehold insertions but not to longer insertions were found to be insufficient to activate eToehold (Figure 26). In summary, these findings demonstrate that eToehold can be easily designed for a variety of mRNAs with high levels of specificity, and that sense-antisense activation is broadly generalizable to IRES-mediated translation.

[0126] Next, we attempted to adapt the eToehold system to expression using endogenous eukaryotic polymerase. This eliminated the dependence on exogenous polymerase for producing uncapped transcripts, thereby reducing the size of the construct and increasing the compatibility of our eToehold system with current cell engineering and gene therapy techniques. RNA polymerase I-responsive promoter 24、25Although it has been shown to produce uncapped transcripts, it was found that the on / off ratio was significantly reduced and basal expression increased in a RNA polymerase I-dependent manner (Figure 22C). Therefore, we decided to explore ways to reduce translational activity in the presence of canonical 5' capping. Stop codons and stem loops follow genes regulated by constitutive promoters. 23 By adding this component, it was possible to reduce the basal expression of downstream mRNA despite the reliance on RNA polymerase II for transcription and the presence of 5' capping (Figure 15C, Figure 15D). Furthermore, by inserting an IRES or eToehold module between the stem-loop and the coding sequence of the desired gene and constructing a bicistronic construct, it was possible to maintain trRNA-mediated control over translation. To test the applicability of this system to IRES modules that have evolved to utilize the mammalian translation system, specifically the human translation system, hepatitis C virus (HCV) 26 , poliovirus (PV) 27 We also designed an eToehold module adapted from the IRES sequence of enterovirus 71 (EV71). 28、29 These novel eToeholds also demonstrated the ability to sense specific trRNAs. Compared to the CrPV construct, these novel eToeholds, called human Toeholds (hToeholds), produced orders of magnitude more output protein. However, monocistronic 5' cap mRNA yielded higher protein output than these novel eToeholds, consistent with the finding that monocistronic constructs yield higher levels of mRNA and protein output than disicistronic constructs. 30 .

[0127] Next, we attempted to test the functionality of the eToehold switch in various eukaryotic systems. To test eToehold in single-cell eukaryotes, we created a yeast strain (Saccharomyces cerevisiae) that expresses GFP upon galactose induction, and an eToehold designed to produce iRFP670 in the presence of GFP mRNA. Although IRES-mediated expression was low, eToehold was induced by galactose-regulated GFP expression, whereas the control constructs (unmodified KBV and CrPV, Figures 27A-27B) were not. Next, we tested eToehold in eukaryotic cell extracts (wheat germ and rabbit reticulocyte lysates) to evaluate their functionality in cell-free systems. We transcribed various target RNAs and their corresponding eToeholds and confirmed that eToehold-mediated translation depends on the presence of specific triggers (Figures 28A-28B). Furthermore, this induction was observed to be dose-dependent, suggesting that eToehold may provide relative intracellular RNA level readout.

[0128] Since eToehold demonstrated the ability to detect exogenously introduced transcripts in mammalian cells, it was hypothesized that they could function as live-cell biosensors for viral infection. Lentiviral constructs containing eToehold specific to the Zika and SARS-CoV-2 sequences, respectively, were generated, producing either nanoluciferase or Azurite fluorescent protein as readout. These constructs were transduced into Vero E6 cell lines, a host cell line commonly used for several viral diseases. Infection resulted in up to a 9.2-fold increase in luminescence signaling in cells engineered to express Zika-specific eToehold (Figures 16A, 16B), and furthermore, they were found to exhibit a dose-dependent responsiveness to Zika virus infection with higher sensitivity than existing live-cell biosensor methods. 31 (Figure 29). To test the specificity of these eToeholds, transduced cells were also tested for related flaviviruses, including dengue virus. 32When infected with Zika, the eToehold response was found to be specific to Zika infection. Similar results were observed using eToehold encoding the Azurite fluorescent protein (Figures 30A, 30B, 31A–31B, and 32A–32B). To test the ability of eToehold to function as a living cell sensor for SARS-CoV-2, stable cell lines were engineered with eToehold designed to sense SARS-CoV-2 transcripts. When transfected with constructs expressing SARS-CoV-2 fragments, cells engineered with these eToeholds were found to be able to distinguish between SARS-CoV-2 trRNA and other non-target RNAs (Figures 30C and 30D).

[0129] Finally, we explored the potential of the eToehold system for sensing endogenous transcripts, enabling applications that identify and target specific cell states and cell types. To evaluate eToehold's ability to determine cell state, we designed eToehold to produce an Azurite protein reporter in response to transcripts of the heat shock proteins hsp70 and hsp40, which are upregulated upon exposure to high temperatures in HeLa cells. The eToehold construct was found to increase Azurite production by up to 4.8 times after growth at 42°C for 24 hours compared to routine 37°C culture (Figure 16C, Figure 16D). Next, to evaluate eToehold's ability to distinguish between various cell types, we designed eToehold to sense mouse tyrosinase (Tyr) mRNA, which is abundant in melanin-producing cells. When Azurite was again used as a reporter, a 3.6-fold increase in signaling was observed using Tyr-sensing eToehold transfected into B16-F10 mouse melanoma cells compared to two control cell lines (HEK293T cells and D1 bone marrow stromal cells, Figure 16E, Figure 16F). These results demonstrate that eToehold can regulate gene and RNA expression based on the level of endogenous transcripts within cells, demonstrating their applicability for targeted therapies against specific cell types.

[0130] This specification describes eToehold, a novel RNA-based eukaryotic sense and response module based on a modified IRES element that enables the translation of a desired protein in the presence of a specific RNA trigger sequence. This approach, which alters the secondary structure of the IRES module for translational control using sense-antisense interactions, surpasses existing RNA-based sensor systems by expanding the trigger sequence length and thus specificity, and further enables dose-responsiveness to varying trigger amounts. In biotechnology, the proven functionality of eToehold in multiple domains of eukaryotes, including fungal and mammalian systems, suggests potential for broad utility.

[0131] Although eToehold is RNA-based, DNA transfection and transduction techniques relied on producing them intracellularly, given the widespread use of these strategies in the biotechnology environment. Doing so revealed that incorporating elements that reduce basal mRNA translation was crucial for designing the RNA-sensing riboswitch. Endogenous 5' capping has a strong impact on translation, and it was found that transcribing eToehold in vivo using exogenous polymerase systems (T7 or SP6) avoided 5' capping, allowing for a substantial difference between the on and off states. Since 5' capping is beneficial for improving mRNA stability and nuclear export, means of reducing basal translation levels in the presence of 5' capping were also investigated. Incorporating stem-loops and stop codons between the 5' cap and the eToehold module preserved 5' capping, but still limited basal translation levels. An RNA-only strategy, in which pre-transcribed eToehold constructs are directly transfected into cells, allows for greater control over the chemistry and composition of the eToehold molecule and eliminates design considerations associated with the endogenous transcription process.

[0132] eToehold can detect a variety of intracellular RNAs, including those exogenously introduced by transfection or infection, and endogenous transcripts such as those indicating cellular state or cell type. As demonstrated herein by eToehold, the ability to initiate translation of a desired protein in response to the presence of cell type-specific or cell state-specific RNA transcripts holds great potential for improving biotechnology therapies. While many molecules retain therapeutic potential, their clinical utility is often hindered by severe off-target toxicity. The ability of the eToehold module to translate proteins or protein-based precursors in response to mRNA signatures can be an extremely useful tool for addressing this challenge by limiting the activation of the desired therapy to specific target cells. The eToehold design implementing logic gates allows for the calculation of cellular state from multiple trRNAs, further enhancing the specificity and utility of this system, as intended herein.

[0133] material and method Construction of DNA constructs. The promoter-gene-polyA tail sequence was cloned into pCAG-T7pol (Addgene#59926) or pXR1. The pCAG-T7pol-based plasmid was cleaved at the 5' and 3' ends, respectively, with EcoRI and NotI to insert the gene for T7 polymerase substitution. pXR1 was cleaved with NotI and NcoI to replace the IRES module. pXR1 was cleaved with PacI and BglII to replace the promoter sequence. pXR1 was cleaved with NcoI and XhoI to replace the reporter gene. Lentiviral vectors were cloned from pLenti CMV Puro DEST (Addgene#17452). pLenti CMV Puro DEST was cleaved with BspDI and PshAI to insert the promoter (especially P SFFVThe reporter gene was altered by cleaving pLenti CMV Puro DEST with PshAI and XmaI. The eToehold regulatory gene construct was added by cleaving pLenti CMV Puro DEST with XmaI and SalI. The entire eToehold construct was constructed so that the downstream gene is in-frame with the non-canonical start codon within the IRES element. 33 The yeast plasmid was constructed as described above. 34 .

[0134] Plasmids or fragments were extracted and purified using Qiagen Miniprep or Qiagen Gel Extraction purification kits. The entire insert was ordered as a gBlock gene fragment from Integrated DNA Technologies, or amplified from experimental plasmids or human genomic DNA by PCR to include homology arms for Gibson isothermal assembly (using 2× reaction mixture purchased from New England Biosciences). The entire vector was sequenced using Sanger sequencing from GENEWIZ prior to the experiment. Tandem repeats were avoided to prevent recombination events.

[0135] HEK293T cells were transfected, and constructs were tested. Plasmid concentrations were determined using a NanoDrop OneC® Microvolume UV-Vis Spectrophotometer. For eToehold construct testing, 70% confluent HEK293T cells were transfected in 96-well plates using Lipofectamine 3000® Transfection Reagent and a standard protocol. Each well was transfected with 30 ng of T7 / SP6 polymerase expression construct, 70 ng of eToehold-mKate construct, and 50 ng of trigger RNA expression construct. For samples not requiring T7 / SP6 polymerase, 75 ng of eToehold construct and 75 ng of trigger RNA expression construct were added. After incubation at 37°C for 60 hours, cells were detached using TripLE Express® and resuspended in 2% FBS in PBS for flow cytometry using a Cytoflex LX® flow cytometer.

[0136] Yeast transformation and experiments. Yeast transformation was performed as previously described. 34EZ-L1 was cleaved with PmeI and transformed into CEN.PK2-1C to generate strain EZy1. Subsequently, EZy1 was transformed using EZ-L183, EZ-L184, EZ-L185, EZ-L186, and EZ-L187 to generate strains EZy13, EZy14, EZy15, EZy16, and EZy17, respectively. Liquid yeast cultures were grown in 24-well plates at 30°C and shaken at 200 rpm in SC dropout medium supplemented with 2% glucose and 50 ng / mL biliverdin (VWR International, LLC for iRFP imaging). Fluorescence and OD600 measurements were performed using a Biotek Synergy H1® Plate Reader. For GFP fluorescence measurements, excitation and emission wavelengths of 485 nm and 535 nm were used, respectively. For iRFP fluorescence measurements, excitation and emission wavelengths of 640 nm and 675 nm were used, respectively. Growth and fluorescence normalization were performed as previously described. 34 .

[0137] Cell-free lysate analysis. RNA was synthesized using constructs containing a T7 promoter preceding the desired RNA (EZ-L212, EZ-L214, EZ-L366) and the HiScribe® T7 High Yield RNA Synthesis Kit (including DNase I treatment) from New England Biolabs, according to the manufacturer's instructions. After purification using the Zymo Research RNA Clean and Concentrator Kit, the RNA was added to wheat germ extract from Promega or reticulocyte lysate IVT Kit from ThermoFisher Scientific, according to the manufacturer's instructions. Fluorescence changes were measured over 6 hours using a Biotek Synergy H1® Plate Reader.

[0138] Lentivirus generation and transduction. As previously described. 35Lentiviruses were generated by cloning a transvestment based on pLenti CMV Puro DEST(w118-1) using psPAX2 and pMD2.G as helper plasmids. psPAX2 was donated by Didier Trono (available on the World Wide Web as Addgene plasmid #12260; n2t.net / addgene:12260; RRID:Addgene_12260). pMD2.G is Addgene plasmid #12259; available on the World Wide Web as n2t.net / addgene:12259; RRID:Addgene_12259. pLenti CMV Puro DEST(w118-1) is Addgene plasmid #17452; available on the World Wide Web as n2t.net / addgene:17452; RRID:Addgene_17452. EZ-L521, EZ-L534, and EZ-L536 were cloned from pLenti CMV Puro DEST(w118-1) via Gibson assembly. After transduction of Vero E6 cells with lentivirus, transduced cells were sorted for GFP fluorescence using a Sony SH800™ cell sorter.

[0139] Zika virus infection test. Vero E6 cells (maintained in DMEM 10% FBS), dengue virus serotype 2 (DENV2 strain New Guinea C, accession AAA42941), and Zika virus isolate (ZV, Pernambuco isolate 243, accession MF352141) were used. Cell lines were seeded and grown overnight to a 90% confluent monolayer, and then infected with ZV (in DMEM 2% FBS; MOI=0.02 for stable cell lines prepared using EZ-L536 or EZ-L521, MOI=2 for stable cell lines prepared using EZ-L534) or DENV2 (MOI=2, in DMEM 2% FBS). 32, fixed at 48 hpi using Cytofix™ from BD Sciences, followed by two washes in 2% FBS in DPBS. Cell replicas were fixed and stained with anti-NS1 antibodies 7724.323 (diluted 1:1000, anti-dengue NS1 mAb 323); 7944.644 (diluted 1:2000; anti-chikungunya NS1 mAb 644). 36、37 . Other cell replicas were either run on a CytoFlex LX™ flow cytometer or utilized in a Nano-Glo™ luciferase assay using Promega according to the manufacturer's instructions and measured using a Biotek Synergy H1™ Plate Reader.

[0140] Heat shock sensing eToehold test. HeLa cells were transfected with EZ-L512 (ABPV positive control), EZ-L548 (eToehold sensing hsp70) or EZ-L554 (eToehold sensing hsp40). 36 hours after transfection, a portion of the sample was brought to 42°C over 24 hours. Cells were detached using TripLE™ Express and resuspended in 2% FBS in PBS for flow cytometry on a Cytoflex LX™ flow cytometer.

[0141] Mouse Tyr eToehold test. B16-F10 melanoma cells, D1 bone marrow stromal cells, and HEK293T cells (ATCC) were maintained at subconfluence in DMEM 10% FBS. Following manufacturer's instructions (Mirus Bio), cells were transfected with plasmids encoding eToehold or a control sequence using Mirus TransIT-2020® transfection reagent. 48 hours after transfection, cells were detached, stained for viability (Fisher Scientific), and analyzed using flow cytometry with LSRFortessa® (BD Biosciences) including HTS. FACSDiva® software (BD Biosciences) was used for sample analysis. Only viable cells were included in the analysis.

[0142] Intracellular cytokine staining. HEK293T, human skeletal muscle cells (ATCC), and human dermal fibroblasts (ATCC) were cultured in DMEM 10% FBS, Primary Skeletal Muscle Growth Kit in Mesenchymal Stem Cell Basal Media (ATCC), and Fibroblast Growth Media 2 (Promocell), respectively. Cells were transfected or transduced using EZ-L793 to introduce eToehold, and either EZ-L1061 or EZ-L1062. Cells were fixed 24 hours after transfection, or after antibiotic selection following transduction, permeabilized with Cytofix / Cytoperm® (BD Biosciences), stained with either PE / Dazzle 594 anti-human IL-6 (501121), PE anti-human CCL5 (515503), or PE anti-human CCL2 (505903), and analyzed using LSRFortessa® (BD Biosciences), including HTS. FACSDiva® software (BD Biosciences) was used for sample analysis.

[0143] qPCR. Cells were pelleted, frozen according to the manufacturer's instructions using the RNEasy® Plus Mini Kit (Qiagen), and then RNA was isolated and stored at -80°C or used immediately. RNA was quantified by a NanoDrop® spectrophotometer, and reverse transcription and amplification were performed using the Luna Universal One-Step RT-qPCR Kit (New England BioLabs) on a CFX96® RT-PCR instrument (Bio-Rad). PrimePCR primers (Bio-Rad; human glyceraldehyde-3-phosphate dehydrogenase, qHsaCED0038674 for GAPDH) or custom primers either TIFF2026097945000002.tif13150 were used. Custom primers were validated by calculating primer efficiency from the standard curve of nanoluciferase template RNA. Relative gene expression was calculated by the delta-delta Ct method comparing Ct values to control samples (orthogonal triggers) and reference gene (GAPDH).

[0144] Statistics. Statistical significance was determined using a standard t-test to calculate p-values. Equation: T-score was calculated by TIFF2026097945000003.tif9128. P-values were calculated using a two-tailed t-test calculator with 2 degrees of freedom. For qPCR, delta-Ct values, which are relative expression normalized to the reference gene GAPDH, were statistically processed using two-way ANOVA followed by Sidak multiple comparison test in PRISM.

[0145] References TIFF2026097945000004.tif92165TIFF2026097945000005.tif253166TIFF2026097945000006.tif213166

[0146] (Table 1) Plasmids used in this study TIFF2026097945000007.tif28166TIFF2026097945000008.tif251170TIFF20260979450 00009.tif254164TIFF2026097945000010.tif237166TIFF2026097945000011.tif254170 TIFF2026097945000012.tif242170TIFF2026097945000013.tif232162TIFF20260979450 00014.tif251170TIFF2026097945000015.tif251170TIFF2026097945000016.tif139158

[0147] (Table 2) Relative change in intracellular IRES RNA levels containing a 45 bp sequence complementary to the IRES transcript, compared to orthogonal sequences. TIFF2026097945000017.tif24158

[0148] In SEQ ID NO:12, residues 153-160 and 218-259 are GFP RNA insertions. In SEQ ID NO:13, residues 152-186 and 197-207 are GFP RNA insertions. In SEQ ID NO:14, residues 153-162 and 220-259 are Azurite RNA insertions. In SEQ ID NO:15, residues 153-160 and 218-262 are ySUMO RNA insertions. In SEQ ID NO:16, residues 159-171 and 257-302 are GFP RNA insertions. In SEQ ID NO:17, residues 15-168 and 254-297 are ySUMO RNA insertions. In SEQ ID NO:18, residues 153-158 and 216-254 are Dika RNA insertions. In SEQ ID NO:19, residues 153-162 and 220-266 are SARS-CoV-2 spike RNA insertions. In SEQ ID NO:20, residues 159-168 and 254-301 are SARS-CoV-2 spike RNA insertions. In SEQ ID NO:21, residues 153-164 and 250-289 are hsp70 RNA insertions. In SEQ ID NO:22, residues 153-161 and 219-259 are hsp40 RNA insertions. In SEQ ID NO:23, residues 159-166 and 252-292 are mouse tyrosinase RNA insertions. In SEQ ID NO:24, residues 153-159 and 217-256 are mouse tyrosinase RNA insertions. In SEQ ID NO:25, residues 83-117 and 317-328 are iRFP RNA insertions. In SEQ ID NO:26, residues 117-151 and 204-218 are iRFP RNA insertions. In SEQ ID NO:27, residues 78-108 and 142-156 are mouse metalloproteinase 9 RNA insertions. In SEQ ID NO:28, residues 1-40 and 674

[0149] SEQ ID NO:30~36 shows exemplary modified IRES sequences, where the "X" residue indicates the site for insertion of the first and second nucleotide sequences.

Claims

1. (a) A first segment encoding a recombinant group 1 dicistroviridae internal ribosome entry site (IRES), which has been modified to incorporate exogenous nucleotide sequences at the first and second sites; (b) A second protein-coding segment located downstream of the first segment and functionally linked to the first segment, such that protein translation is suppressed when the IRES is in an inactive state. Recombinant nucleic acid molecules containing, The first region includes a first nucleotide sequence, The second site includes a second nucleotide sequence which is the reverse complementary strand of at least a portion of the first nucleotide sequence. Recombinant nucleic acid molecules.

2. (a) A first segment encoding a recombinant viral internal ribosome entry site (IRES), which is modified to incorporate a first exogenous nucleotide sequence at the first site and a second exogenous nucleotide sequence at the second site; (b) A second protein-coding segment located downstream of the first segment and functionally linked to the first segment, such that protein translation is suppressed when the IRES is in an inactive state. A recombinant nucleic acid molecule containing from 5' to 3', The second nucleotide sequence is the reverse complementary strand of at least a portion of the first nucleotide sequence. Recombinant nucleic acid molecules.

3. The recombinant nucleic acid molecule according to claim 2, wherein the modified IRES is a group 1 dicistroviridae IRES, a hepacivirus IRES, or an enterovirus IRES.

4. The recombinant nucleic acid molecule according to any one of claims 1 to 3, wherein the modified IRES is an IRES derived from a mammalian pathogenic virus or a mammalian symbiotic virus.

5. The recombinant nucleic acid molecule according to claim 4, wherein the modified IRES is an IRES derived from a human pathogenic virus or a human symbiotic virus.

6. The recombinant nucleic acid molecule according to any one of claims 1 to 5, wherein the nucleic acid molecule is mRNA.

7. The recombinant nucleic acid molecule according to any one of claims 1 to 6, wherein the second nucleotide sequence is substantially the reverse complementary strand of the first nucleotide sequence.

8. The recombinant nucleic acid molecule according to any one of claims 1 to 7, wherein the group 1 Disicstroviridae IRES is cricket paralysis virus (CrPV) IRES, Casimir wasp virus (KBV) IRES, acute honeybee paralysis virus (ABPV) IRES, brown marmorated stink bug (Plauta Stali) enteric virus (PSIV) IRES; aphid lethal paralysis virus (ALPV) IRES; black queen bee brood virus (BQCV) IRES; fruit fly (Drosophila) C virus (DCV) IRES; Himetobi P virus (HiPV) IRES; leafhopper (Homalodisca coagulata) virus-1 (HoCV-1) IRES; wheat aphid (Rhopalosiphum padi) virus (RhPV) IRES; and assassin bug (Triatoma) virus (TrV) IRES.

9. The recombinant nucleic acid molecule according to any one of claims 2 to 7, wherein the hepacivirus IRES is a hepatitis c virus (HCV) IRES.

10. The recombinant nucleic acid molecule according to any one of claims 2 to 7, wherein the enterovirus IRES is a poliovirus (PV) IRES or an enterovirus 71 (EV71) IRES.

11. The recombinant nucleic acid molecule according to any one of claims 1 to 10, wherein the first and second sites are independently selected from site 1, site 2, site 3, site 4, site 5, site 6, site 7, and site 8, respectively.

12. The recombinant nucleic acid molecule according to claim 11, wherein the first and second sites each include site 1 and site 2, site 1 and site 4, site 1 and site 5, site 1 and site 6, site 1 and site 7, site 1 and site 8, site 2 and site 6, site 2 and site 7, site 4 and site 6, site 5 and site 6, site 5 and site 7, site 6 and site 7, site 8 and site 2, site 8 and site 6, or site 8 and site 7.

13. The recombinant nucleic acid molecule according to any one of claims 11 to 12, wherein the first and second sites each comprise site 1 and site 2, site 1 and site 8, site 2 and site 7, site 6 and site 7, or site 8 and site 6, respectively.

14. The recombinant nucleic acid molecule according to any one of claims 11 to 13, wherein the first and second sites each comprise site 6 and site 7, or site 8 and site 6, respectively.

15. A recombinant nucleic acid molecule according to any one of claims 1 to 14, wherein the first nucleotide sequence is 25 to 80 nt in length.

16. The recombinant nucleic acid molecule according to claim 15, wherein the first nucleotide sequence is 40 to 50 nt in length.

17. The recombinant nucleic acid molecule according to claim 15, wherein the second nucleotide sequence is 8 to 25 nt in length.

18. The recombinant nucleic acid molecule according to any one of claims 1 to 17, wherein the second nucleotide sequence is 6 to 15 nt in length.

19. A recombinant nucleic acid molecule according to any one of claims 1 to 18, wherein the first nucleotide sequence is 2.5 to 8 times longer than the second nucleotide sequence.

20. The recombinant nucleic acid molecule according to any one of claims 1 to 19, wherein the first nucleotide sequence is the reverse complementary strand of a sequence found in a target eukaryote, target prokaryote, or target virus.

21. The recombinant nucleic acid molecule according to claim 20, wherein the target prokaryote or target virus is a human pathogen.

22. The recombinant nucleic acid molecule according to claim 21, wherein the target virus is Zika virus or coronavirus.

23. The recombinant nucleic acid molecule according to claim 22, wherein the coronavirus is SARS-CoV-2.

24. The recombinant nucleic acid molecule according to any one of claims 1 to 23, wherein the protein generates a detectable signal.

25. The recombinant nucleic acid molecule according to any one of claims 1 to 24, wherein the first and second nucleotide sequences hybridize when expressed in a eukaryotic cell under in vivo or in vitro conditions, causing the IRES to fold into an inactive state, and the eukaryotic cell is not a plant cell.

26. The recombinant nucleic acid molecule according to any one of claims 1 to 25, wherein the IRES is configured to fold into an activated state in the presence of a trigger RNA molecule containing a third nucleotide sequence which is the inverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule according to claim 1.

27. The recombinant nucleic acid molecule according to claim 26, wherein the first nucleotide sequence, when expressed in a eukaryotic cell under in vivo conditions, hybridizes to the third nucleotide sequence, causing the IRES to fold into an activated state, and the eukaryotic cell is not a plant cell.

28. A sequence comprising a recombinant nucleic acid molecule according to any one of claims 1 to 27, The 5' and / or 3' of the sequence encoding or containing a recombinant nucleic acid molecule according to any one of claims 1 to 27 is: (a) IRES pseudoknot arrangement; (b) IRES pseudoknot sequences found in naturally occurring viral wild-type sequences; (c) Promoter and / or upstream activator binding sequence; (d) Terminal codon; (e) Stem loop; (f) 5' cap; (g) reporter gene; and (h) Poly A Tail An expression construct further comprising one or more of the following.

29. The 5' of the sequence encoding or containing a recombinant nucleic acid molecule according to any one of claims 1 to 27 is: (a) IRES pseudoknot arrangement; (b) IRES pseudoknot sequences found in naturally occurring viral wild-type sequences; (c) Promoter and / or upstream activator binding sequence; (d) Terminal codon; (e) Stem loop; (f) 5' cap; and (g) Reporter gene The expression construct according to claim 28, comprising one or more of the following.

30. (a) The promoter is selected from the SP6 promoter, T3 promoter, araBAD promoter, trp promoter, lac promoter, Ptac promoter and pL promoter; and / or (b) The upstream activator binding sequence is an upstream activator binding DNA sequence (UAF2) derived from Saccharomyces cerevisiae. An expression construct according to any one of claims 28 to 29.

31. The transcription of the recombinant nucleic acid molecule is as follows: (a) RNA polymerase II; (b) Polymerases other than RNA polymerase II; (c) T7 polymerase; and / or (d) SP6 polymerase An expression construct or recombinant nucleic acid sequence according to any one of claims 1 to 30, depending on the following.

32. (a) the first segment encoding the first protein; (b) A second segment downstream of the first segment, which encodes a recombinant group 1 dicistroviridae internal ribosome entry site (IRES), modified to incorporate exogenous nucleotide sequences at the first and second sites; (c) A third segment encoding a second protein, located downstream of the second segment and functionally linked to the second segment, such that the translation of the second protein is suppressed when the IRES is in an inactive state. Recombinant mRNA molecules containing, The transcription of the recombinant mRNA molecule is polymerase-dependent. The first region includes a first nucleotide sequence, The second site includes a second nucleotide sequence which is the reverse complementary strand of at least a portion of the first nucleotide sequence. Recombinant mRNA molecule.

33. The transcription of the recombinant mRNA molecule is as follows: (a) RNA polymerase II; (b) Polymerases other than RNA polymerase II; (c) T7 polymerase; and / or (d) SP6 polymerase A recombinant mRNA molecule according to claim 32, which depends on the recombinant mRNA molecule.

34. A plasmid encoding a recombinant nucleic acid molecule, expression construct, or recombinant mRNA molecule according to any one of the above claims.

35. A eukaryotic cell comprising DNA encoding a recombinant nucleic acid molecule, expression construct, or recombinant mRNA molecule according to any one of the above claims, The aforementioned eukaryotic cells are not plant cells, The aforementioned DNA, (a) either incorporated into the genomic DNA of the eukaryotic cell, or (b) Plasmids or viral vectors present in the eukaryotic cells, eukaryotic cells.

36. The eukaryotic cell according to claim 35, wherein the cell is (a) an animal cell; (b) a human cell; or (c) a primate cell.

37. (a) a recombinant nucleic acid molecule according to any one of the claims; (b) A trigger RNA molecule containing a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule A system for controlling gene expression, including [specific components / functions].

38. (a) the plasmid according to claim 34; (b) A trigger RNA molecule containing a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule A kit that includes this.

39. (a) a step of providing eukaryotic cells manipulated to express the recombinant nucleic acid molecule described in any one of the claims; and (b) The step of introducing a trigger RNA molecule into the eukaryotic cell, the trigger RNA molecule containing a third nucleotide sequence which is the reverse complementary strand of the first nucleotide sequence of the recombinant nucleic acid molecule. A method for activating and / or regulating protein expression, comprising: The first nucleotide sequence hybridizes to the third nucleotide sequence under in vivo conditions, causing the IRES to fold into an activated state. Furthermore, the eukaryotic cells mentioned above are not plant cells. method.

40. The method according to claim 39, wherein the eukaryotic cells manipulated to express the recombinant nucleic acid molecule are provided by introducing the recombinant nucleic acid molecule according to any one of the claims into the eukaryotic cells.

41. (a) A step of providing a eukaryotic cell manipulated to express a recombinant nucleic acid molecule according to any one of the claims, wherein the first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of the mRNA sequence specific to the virus; and (b) A step of determining whether the eukaryotic cell is infected with the virus by detecting and / or measuring the presence of a protein encoded by a second segment of the recombinant nucleic acid molecule, wherein the eukaryotic cell is not a plant cell. A method for detecting viral infection in eukaryotic cells, including [specific example].

42. The method according to claim 41, wherein the virus is dengue virus or Zika virus.

43. (a) a step of providing eukaryotic cells manipulated to express the recombinant nucleic acid molecule described in any one of the claims; and (b) The step of culturing the eukaryotic cells. A method for controlling the differentiation of eukaryotic cells, including, The first nucleotide sequence of the recombinant nucleic acid molecule is configured to be the reverse complementary strand of at least a portion of the mRNA sequence specific to the selected cell type. The protein encoded by the second segment of the recombinant nucleic acid molecule comprises a toxin or protein that induces apoptosis of the selected cell type. Furthermore, the eukaryotic cells mentioned above are not plant cells. method.

44. Sequence encoding a recombinant nucleic acid molecule according to any one of the above claims A vector containing, The modified IRES is configured to activate the expression of the protein in response to the presence of mRNA containing a segment that is the reverse complementary strand of the first nucleotide sequence. vector.

45. A prokaryotic cell comprising DNA encoding a recombinant nucleic acid molecule according to any one of the above claims, The aforementioned DNA, (a) incorporated into the genomic DNA of the prokaryotic cell; or (b) Plasmids or viral vectors present within the prokaryotic cells, Prokaryotic cells.

Images