CN108753818B - RNA signal connector, target mRNA translation regulation method, logic gate and application - Google Patents

Abstract

The present application provides an RNA signaling connector. The RNA signaling linker is an oligonucleotide sequence comprising an aptamer domain complementary to a target mRNA sequence and an antisense domain that binds to a ligand as an input signal, optionally comprising a nucleic acid switch. The RNA signaling connector can be used to regulate translation of a target mRNA in a cell. The RNA signal connector can also be used for constructing a biological logic gate, establishing a relation between input and output signals, and having potential application in reconnecting a cell signal path and creating a feedback loop, silencing the expression of survival genes and inhibiting the growth of cancer cells, converting oncogenic signals into cancer inhibitory signals and the like.

Description

RNA signal connector, target mRNA translation regulation method, logic gate and application

Technical Field

The invention relates to the fields of genetic engineering and molecular biology, in particular to an RNA signal connector, a target mRNA translation regulation method, a logic gate and application thereof.

Background

The regulation of gene expression after the induction of extracellular signals is the basic function of cells. Cells fulfill complex physiological functions such as cell survival, behavior and recognition through a natural signaling network. Synthetic biology has rapidly progressed in recent years, and one of the important goals is to remodel cell signaling networks at the level of therapeutic and biotechnological applications. Synthetic elements are widely used to construct various novel gene regulatory circuits, such as genetic switches, digital logic circuits, artificial signal pathways, and feedback loops. Recently, the present inventors have constructed a new class of gene regulation devices, the "CRISPR signal transducers" (see chinese patent application CN 106318973a of the present inventors, entitled "a gene regulation device and gene regulation method based on CRISPR-Cas 9"), which can sense and respond to specified cell signals and regulate transcription of specific endogenous genes through CRISPR interference or activation. The device functions to integrate information about any given biomolecule and to remodel different phenotypes of the cell.

However, as with the previously reported genetic devices, this system still requires the transfer of exogenous genes encoding large proteins (e.g., dCas9), which further increases the complexity of the system. From an application point of view, the use of compact RNA devices may be more desirable to the transgene delivery technology than devices containing encoded large proteins.

Disclosure of Invention

The object of the present invention is to provide a genetic device of simple and efficient construction and its use.

Thus, in a first aspect, the invention provides an RNA signaling linker which is an oligonucleotide sequence comprising an antisense domain linked to a first copy of an aptamer domain and two copies of the aptamer domain, the first copy of the aptamer domain being linked to a second copy of the aptamer domain by a linker sequence, the antisense domain being complementary to a target mRNA sequence, the aptamer domain binding to a ligand which serves as an input signal.

Specifically, the antisense domain is complementary to the 5' -UTR or coding sequence of the target mRNA.

In some embodiments, the ligand is theophylline, tetracycline, β -catenin, VEGF, OPN, or NF-kB, and the aptamer domain is a theophylline, tetracycline, β -catenin, VEGF, OPN, or NF-kB aptamer domain, respectively.

Further, the present invention provides an RNA signal connector, wherein the RNA signal connector is an oligonucleotide sequence, and comprises an antisense domain and two copies of an aptamer domain, and further comprises a nucleic acid switch, the antisense domain is connected to the nucleic acid switch, the nucleic acid switch is connected to the first copy of the aptamer domain through a first linker sequence, the first copy of the aptamer domain is connected to the second copy of the aptamer domain through a second linker sequence, the antisense domain is complementary to a target mRNA sequence, and the aptamer domain binds to a ligand as an input signal.

In some embodiments, the ligand is theophylline, tetracycline, β -catenin, VEGF, OPN, or NF-kB, and the nucleic acid switch is a theophylline, tetracycline, β -catenin, VEGF, OPN, or NF-kB nucleic acid switch, respectively, and the aptamer domain is an eIF4G aptamer domain.

In a specific embodiment, the cDNA of the aforementioned RNA signal connector is represented by any one of the sequences shown in tables 1-17 below and SEQ ID NO 1-40 of the sequence Listing. The sequence numbers in the table below correspond to the sequence numbers of the sequence listing, for example sequence number R1 corresponds to sequence number SEQ ID NO:1, and so on.

TABLE 1 Theine-induced cDNA sequence of a signal linker targeting and inhibiting translation of Renilla luciferase mRNA. Each sequence consists of a complementary sequence (shown in bold), two copies of the theophylline aptamer, and a linker sequence (shown underlined).

TABLE 2 tetracycline-induced cDNA sequence of the signal linker targeting and inhibiting translation of Renilla luciferase mRNA. Each sequence consists of one complementary sequence (shown in bold), two copies of the tetracycline aptamer, and one linker sequence (shown underlined).

TABLE 3 tetracycline-induced cDNA sequence of the signal linker targeting and inhibiting VEGF mRNA translation. Each sequence consists of one complementary sequence (shown in bold), two copies of the tetracycline aptamer, and one linker sequence (shown underlined).

TABLE 4 cDNA sequence of signal linker targeting and enhancing translation of Renilla luciferase mRNA. The sequence consisted of a complementary sequence (shown in bold), two copies of the eIF4G aptamer, and a linker sequence (shown underlined)

TABLE 5 Theine-induced cDNA sequence of the signal linker targeting and enhancing translation of Renilla luciferase mRNA. Each sequence consisted of one complementary sequence (shown in bold), one copy of the theophylline nucleic acid switch (shown in italics), two copies of the eIF4G aptamer, and two linker sequences (shown underlined)

TABLE 6 tetracycline-induced cDNA sequence of signal linker targeting and enhancing translation of Renilla luciferase mRNA. Each sequence consisted of one complementary sequence (shown in bold), one copy of the tetracycline nucleic acid switch (shown in italics), two copies of the eIF4G aptamer, and two linker sequences (shown underlined)

TABLE 7. cDNA sequence of signal linker for beta-catenin induced targeting and inhibition of translation of c-Myc mRNA. The sequence consists of a complementary sequence (shown in bold), two copies of the β -catenin aptamer, and a linker sequence (shown underlined)

Table 8. beta-catenin-induced cDNA sequences of signal linkers targeting and enhancing translation of renilla luciferase mRNA. The sequence consisted of one complementary sequence (shown in bold), one copy of the beta-catenin nucleic acid switch (shown in italics), two copies of the eIF4G aptamer, and two linker sequences (shown underlined)

Table 9. VEGF-induced cDNA sequence of the signal linker targeting and enhancing OPN mRNA translation. The sequence consisted of one complementary sequence (shown in bold), one copy of the VEGF nucleic acid switch (shown in italics), two copies of the eIF4G aptamer, and two linker sequences (shown underlined)

Table 10. OPN-induced cDNA sequences of signal linkers targeting and enhancing VEGF mRNA translation. The sequence consisted of one complementary sequence (shown in bold), one copy of the OPN nucleic acid switch (shown in italics), two copies of the eIF4G aptamer, and two linker sequences (shown underlined)

Table 11. VEGF-induced cDNA sequence of the signal linker targeting and inhibiting OPN mRNA translation. This sequence consists of a complementary sequence (shown in bold), two copies of the VEGF aptamer and a linker sequence (shown underlined)

TABLE 12 cDNA sequence of the theophylline-induced signal linker targeting and inhibiting translation of c-Myc mRNA. The sequence consists of a complementary sequence (shown in bold), two copies of the theophylline aptamer and a linker sequence (shown underlined)

TABLE 13 Theine-induced cDNA sequence of the signal linker targeting and inhibiting translation of BCL2 mRNA. The sequence consists of a complementary sequence (shown in bold), two copies of the theophylline aptamer and a linker sequence (shown underlined)

Table 14.NF-kB induced cDNA sequence of signal linker targeting and enhancing Bax mRNA translation. The sequence consisted of one complementary sequence (shown in bold), one copy of the NF-kB nucleic acid switch (shown in italics), two copies of the eIF4G aptamer, and two linker sequences (shown underlined)

Table 15. cDNA sequence of NF-kB induced signaling linker targeting and enhancing translation of p21 mRNA. The sequence consisted of one complementary sequence (shown in bold), one copy of the NF-kB nucleic acid switch (shown in italics), two copies of the eIF4G aptamer, and two linker sequences (shown underlined)

Table 16.NF-kB induced cDNA sequence of the signal linker targeting and inhibiting BCL2mRNA translation. The sequence consisted of a complementary sequence (shown in bold), two copies of the NF-kB aptamer, and a linker sequence (shown underlined)

TABLE 17 NF-kB-induced cDNA sequences of signal linkers targeting and inhibiting translation of c-Myc mRNA. The sequence consisted of a complementary sequence (shown in bold), two copies of the NF-kB aptamer, and a linker sequence (shown underlined)

In a second aspect, the invention provides a method of modulating translation of a target mRNA in a cell. The method comprises introducing into the cell the RNA signaling connector of the first aspect and allowing the RNA signaling connector to inhibit or activate translation of the target mRNA in the presence of the ligand.

Specifically, the ligand causes the RNA signaling linker to inhibit or activate translation of the target mRNA in a concentration-dependent manner.

In a third aspect, the invention provides a logic gate. The logic gate consists of 2 RNA signal connectors of the first aspect.

Specifically, the logic gate is an inverter gate, an AND gate, a NAND gate, an OR gate, a NOR gate, an XOR gate and an XOR gate.

In a fourth aspect, the invention provides the use of the RNA signalling connector of the first aspect or the logic gate of the third aspect to reconnect the cellular signalling pathway and create a feedback loop.

In a fifth aspect, the invention provides the use of an RNA signaling connector of the first aspect or a logic gate of the third aspect to silence the expression of a survival gene and inhibit the growth of a cancer cell.

In a sixth aspect, the invention provides the use of the RNA signaling connector of the first aspect or the logic gate of the third aspect to convert an oncogenic signal to a cancer suppressor signal.

The invention has the beneficial effects that: the RNA signaling linker of the invention is a genetic device, is an oligonucleotide sequence comprising an antisense domain and an aptamer domain, optionally comprising a nucleic acid switch, and is free of exogenous genes encoding large proteins (e.g., dCas9), and is simple in structure. The RNA signaling connector can regulate translation of a target mRNA in a cell by providing a ligand exogenous or endogenous to the cell as an input signal. The RNA signal connector can also be used for constructing a biological logic gate, establishing a relation between input and output signals, and having potential application in reconnecting a cell signal path and creating a feedback loop, silencing the expression of survival genes and inhibiting the growth of cancer cells, converting oncogenic signals into cancer inhibitory signals and the like.

Drawings

Fig. 1 shows the design and construction of a signal connector for constructing connections between signal nodes. (A) Domain composition of signal connectors. The device uses the antisense domain to recognize the mRNA and ligand domains of the target gene in response to different signals, thereby controlling translation of the target gene. (B) By this modular approach, cellular gene expression can be controlled using specific exogenous signals. (C) Signal connectors can be used to establish a link between cell inputs and outputs.

FIG. 2 shows that the signaling linker effectively inhibits or activates gene expression. (A) The signal connector is designed to prevent translation of the target gene. The device is designed to target the 5' -UTR of a target mRNA. In the absence of ligand, the 40S ribosomal subunit was scanned until translation of the start codon was initiated. In the presence of the ligand, the ligand-aptamer complex perturbs the scanning of the ribosome and blocks the initiation of translation. (B) Upon binding to the protein coding region of the target mRNA, it blocks ribosome-mediated translation elongation. (C) Different signal connectors were designed to target different regions of the renilla luciferase messenger mRNA. (D) The signal connector inhibited relative luciferase activity only in the presence of 1mm theophylline. pGPU6/GFP/Neo empty as negative control. (E) The signal connectors (R1, R2 and R3) were grown in theophylline at 0, 250, 500, 750 or 1000 μm. This addition of theophylline inhibits translation of renilla messenger RNA in a dose-dependent manner. (F) In HEK293 cells, luciferase was inhibited by four different tetracycline-responsive signal linkers. (G) Design of a signaling linker for enhancing translation of a target gene. The translational activation of the signaling linker is due to the formation of an initiation factor complex that activates eIF 4G. In the absence of theophylline/tetracycline, the antisense domain is unable to bind to the messenger RNA of the target gene. In the presence of theophylline/tetracycline, the ligand stem forms and the antisense domain will bind to its target. (H) The activity of the relative luciferase is increased by the signal connector. (I) The addition of theophylline/tetracycline increased translation of renilla luciferase. pGPU6/GFP/Neo, empty. NC, negative control, contains two repetitive elements, and no target in the human genome. Data reported are from the mean ± SD of at least three experiments.

Fig. 3 shows that the logic gates based on signal integration are constructed from signal connectors. (A) In each logic gate, a signal connector integrates theophylline and tetracycline of the input signal and produces a luciferase output. The not gate (B), and gate (C), nand gate (D), or gate (E), nor gate (F), exclusive or gate (G) and exclusive nor gate (H) functions are performed using the signal connectors.

Fig. 4 shows that the signal connectors effectively reconnect and create a signal path and feedback loop. (A) Signal connectors are designed to reconnect the mechanisms of the signal paths. (B) The relative expression level of c-Myc messenger RNA was assessed in HEK293 cells using real-time qPCR. The level of c-Myc mRNA was increased in cells responding to LTD4 stimulation. (C) Histogram of protooncogenes. (D) The signal path is created using a mechanism of signal connection. (E) Design of signal linker responding to beta-catenin. The device releases the antisense region, targeting the mRNA only in the presence of β -catenin. (F) The relative activity of renilla luciferase was assessed in HEK293 cells responding to LTD4 stimulation. (G) Designed models and experimental results illustrate the hypothetical role of the signal connector in constructing the OPN-VEGF positive feedback loop. (H) Designed models and experimental results illustrate the hypothetical role of the signal connector in constructing the OPN-VEGF negative feedback loop. NC, negative control, contains two repetitive elements, and no target in the human genome. Data reported are from the mean ± SD of at least three experiments. The experiment was repeated at least three times.

FIG. 5 shows that the signal connector specifically inhibits the expression of the survival gene, inhibiting cell growth of the target cancer cell. (A) The mechanism of the signal connector is to selectively kill cancer cells, which control the survival of the cells through activated TERTp and ligand. (B) Schematic representation of genetics and phyla. The promoter and ligand of hTERT (1000 μm theophylline) are the two inputs to the line. (C) Two different signaling linkers were designed to target the 5-UTRs of human c-Myc messenger RNA and BCL2 mRNA. (D and E) quantitative analysis of target protein expression in bladder cancer cells (T24 and 5637) and normal fibroblasts. NC, a negative control with two repetitive elements, no target in the human genome. (F) The growth of the cells was measured at intervals determined by CCK-8. Analysis of variance was used to compare cell growth curves. Values reported are the mean ± SD of three independent experiments. (G) Apoptosis of cells was determined by Hoechst staining. Values reported are the mean ± SD of three independent experiments.

Figure 6 shows the pathway by which the signaling linker simultaneously activates and inhibits cellular genes in response to oncogenic signals, and converts the oncogenic signals into anti-tumors. (A) The oncogene signal NF-kB is redirected to activate two tumor suppressor factors Bax and p21 and suppress two oncogenes BCL2 and c-Myc through a signaling connector in the cancer cell. (B) The relative expression levels of BAX, BCL2, c-Myc and p21 were determined in T24 cells within 48 hours after cell inoculation, using T24 cells as a standard. Values reported are the mean ± SD of three independent experiments. (C and D) 20 days after injection, tumors formed in the signal connector group were much smaller compared to the negative control group. (E) Tumor volumes were calculated every 5 days after injection of T24 cells. Bars indicate Standard Deviation (SD). (F) Tumor weight changes. P < 0.01.

Detailed Description

The present invention is described in further detail below by way of examples. It should be understood that these descriptions are for the purpose of illustrating the invention only, and are not intended to limit the invention in any way.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples used herein are illustrative only and are not intended to be limiting unless otherwise specified.

I. Materials and methods

1. Plasmid construction

The cDNA sequence for Renilla luciferase/c-Myc/OPN/VEGF/BCL 2/Bax/p21mRNA signal linker was designed and its RNA secondary structure was analyzed with online software. Screening out RNA expressing antisense region and maintaining aptamer secondary structure, and synthesizing corresponding cDNA sequence to insert into GFP/Neo vector of pGPU 6/Bam HI/BBSII restriction site. In a similar manner, cDNA sequences of ribosomal flanking signal linkers targeting c-myc/bcl2mRNA were designed and synthesized and inserted into the hTERT-neo-bam vector at Sal I/BamH I restriction sites, respectively. To construct plasmids pcDNA3-0-VEGF and pcDNA 3-OPN, the OPN/VEGF truncated cDNA sequences expressing the deletion of the N-terminal signal peptide were inserted into pcDNA 3.0 and bamhi/ecori, respectively. To construct eukaryotic expression plasmids shRNA-NC and shRNA-VEGF, the synthetic shRNA sequences were inserted into pgpu6/gfp/neo digested with Bam HI/Bbs, respectively.

2. Cell lines and cell cultures

T24, 5637 and HEK-293 cells were purchased from the American Type Culture Collection (ATCC) by the laboratory and cultured in DMEM medium containing 10% fetal bovine serum (Invitrogen) in the presence of added 5% CO 2. Normal human primary fibroblasts extracted from the epidermis were subjected to primary culture in the same medium. Stable cell lines were generated from these cell lines as described below. All cell lines were verified to be mycoplasma free.

HEK-293 cells stably expressing Renilla luciferase were obtained by transfecting cells with pcDNA3/Rluc/Neo and selecting positive clones with G418. Specifically, stable selection was performed in 6-well plates seeded with approximately 2X 105 HEK-293 cells per well, and 2mg of linearized plasmid was transfected using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer's instructions. 48 hours after transfection, cell monolayers were trypsinized and transferred to T25 flasks or 100mm diameter dishes. Mixed colonies of stable transfectants were selected by growth in complete medium containing 50mg G418/ml. These polyclonal cell lines were amplified and then validated by luciferase reporter gene assay.

HEK-293 cells stably expressing the signal connector or negative control were selected after transfection of pGPU6/GFP/Puro vector. HEK-293/T24 cells co-expressing multiple signal connectors were constructed by stable transfection of a single pGPU6/GFP/Puro vector driven by a single U6 promoter to generate these devices simultaneously. Specifically, cells were seeded in six-well plates and 2mg of linearized plasmid was transfected using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer's instructions. After transfection, cells were grown in medium supplemented with 4mg/ml puromycin for approximately 14 days to select mixed colonies of stable cell lines. These polyclonal cells were verified by GFP expression. Fluorescent cells in the culture plate were directly observed using an inverted fluorescence microscope.

3. Luciferase reporter assay

HEK-293 cells stably expressing renilla luciferase were seeded in 6-well plates (5 × 105/well). 48 hours after transfection, the medium was discarded and the cells were lysed in 500ml lysis buffer (Analytical Luminescence Laboratories). Renilla luciferase activity was determined using the Renilla luciferase assay System (Promega) according to the manufacturer's instructions. Renilla luciferase activity was corrected for changes in protein concentration (Bio-Rad) in cell extracts. The assays were performed in duplicate and the experiments were repeated three times.

4. Analysis of nuclear and cytoplasmic RNA fractions

Nuclear and Cytoplasmic RNA was isolated using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen, Belmont, CA) according to the manufacturer's instructions.

5. In vitro translation reaction

The purified ligand (theophylline or eIF4G) was incubated with 1mg signal connector and 1mg Renilla luciferase mRNA, and the mixture was then incubated with the components of the Thermo Scientific 1-Step Human Coupled IVT Kit (Waltham, MA, USA) according to the manufacturer's instructions. The activity of in vitro translated renilla luciferase was calculated as described above.

Determination of VEGF/OPN concentration by ELISA method

HEK293 cells were stably transfected with signal connector or control. The concentration of VEGF/OPN protein was then measured by ELISA assay. Briefly, 106 cells were harvested per sample and resuspended in 200ml of lysis buffer. The supernatant of the lysate was collected by centrifugation, the OD value was measured by a microplate reader (Bio-Rad, Hercules, Calif.), and converted to protein concentration using a standard calibration curve.

7. Western blot analysis

Cells were washed with PBS and lysed in RIPA buffer (50mM Tris-HCl pH 7.2,150mM NaCl, 1% NP40, 0.1% SDS, 0.5% DOC,1mM PMSF,25mM MgCl2, supplemented with phosphatase inhibitor cocktail). Protein concentration was determined using BCA protein assay. Equal amounts of whole protein extracts were electrophoresed onto SDS-polyacrylamide gels and then transferred to PVDF membranes (Millipore, Billerica, MA). The samples were blocked with 5% dry milk and then incubated overnight with primary antibody (Abcam, Cambridge, MA). The samples were then incubated with horseradish peroxidase conjugated secondary antibody (Amersham, Piscataway, NJ) and immunoblots developed with Super Signal chemiluminescence reagent (Pierce Chemical Co.). Protein bands were quantified using Image J analysis software (national institute of health, usa) and histograms were generated by normalizing the amount of each protein to the GAPDH levels detected in the same extraction sample. Each experiment was repeated three times.

8. Cell proliferation assay

Cells were treated with 0.25% trypsin (15min,37 ℃), counted and then analyzed at 0, 24, 48 and 72 hours with an electronic cell counter (Beckman Coulter). The experiments were repeated at least three times independently.

9. Apoptosis assay

Signaling connector-induced apoptotic cells were visualized using the Hoechst 33258 staining kit (Life, Eugene, OR). Briefly, treated cells were fixed in 4% paraformaldehyde for 10 minutes and washed twice in PBS. Then, the cells were stained with 0.5ml of Hoechst 33258 staining solution for 5 minutes, and photographed at a wavelength of 350nm with a fluorescence microscope. Each assay was repeated three times.

RNA extraction and real-time quantitative PCR

Total RNA was isolated from cells using trizol (invitrogen) according to the suggested protocol. cDNA strands were synthesized from total RNA in a volume of 2ml using the RevertAIdTM First Strand cDNA Synthesis Kit (Fermentas, Hanover, Md.). Real-time Quantitative PCR was performed on the ABI PRISM 7000Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, Calif.) using All-in-one qPCR Mix (GeneCopoea Inc, Rockville, Md.) in a 20ml reaction volume. The PCR cycle parameters were: 95 ℃ for 15min, then 94 ℃ for 15s, 55 ℃ for 30s, 72 ℃ for 30s, 40 cycles. The relative fold change in expression was determined by the 2- Δ Δ Ct method.

11. Tumor formation experiment in nude mice

All experiments involving animals were approved by the institutional review board. Female BALB/c nude mice at 4 weeks of age were obtained in the animal center of the academy of sciences. 107T 24 cells stably expressing the signal connector or negative control were resuspended in 100ml PBS and injected subcutaneously into the left and right axilla of 3 4-week-old female balb/c nude mice. Tumor growth was examined every 5 days and tumor volume was calculated using the following formula: 0.5 length by width 2. Mice were euthanized 20 days after injection and the subcutaneous weight of each tumor was measured.

12. Data analysis

The sample volume is not predetermined using statistical methods. Investigators assigned blindly during the course of the experiment and the evaluation of the results. Statistical analysis student t-test or analysis of variance was used and P <0.05 was considered statistically significant. All statistical tests were performed using SPSS version 17.0 software (SPSS, Chicago, IL).

Results II

1. Design and construction of RNA-based signaling connectors

Previous studies have demonstrated that the insertion of RNA aptamers into the 5 'untranslated region (5' -UTR) of messenger RNA (mrna) in the presence of ligands can reduce the rate of translation initiation, a process that is generated by blocking ribosome binding. We speculate that: a reverse complementary RNA sequence is designed to bind to the target mRNA and, in the presence of a specific signal, will regulate mRNA translation in trans by means of the RNA aptamer. To test this, we designed an RNA device containing an antisense domain (18-22nt sized antisense RNA, preferably 20nt sized) that recognizes a given mRNA and an aptamer domain to control translation (fig. 1A). We couple a signaling linker to the translation activation domain to enhance translation, or use the aptamer domain alone to inhibit translation. In principle, these modular devices, which we call "signal connectors", can respond to any given signal molecule (FIG. 1B) and control the translation of any given target mRNA, thereby connecting a given endogenous signal (input) to a specific cellular signal (output) (FIG. 1C).

2. Signal connector for effectively inhibiting target gene expression

To test whether this approach inhibited translation initiation (FIG. 2A) and extension (FIG. 2B), we designed 12 signal linkers complementary to different regions of the Renilla luciferase reporter mRNA sequence, which bind to the 5' -UTR or coding sequence (FIG. 2C). These signal linkers each contain two fragments: 20nt of antisense RNA sequence complementary to the target mRNA sequence and two copies of theophylline aptamer (Jenison et al, 1994). Each of the 12 connectors was stably transfected into HEK293 cells expressing renilla luciferase, and 11 resulted in a significant reduction in luciferase expression under the action of 1000mM theophylline (fig. 2D). In the absence of theophylline induction, the relative levels of luciferase activity in cells containing the signal linker did not change significantly.

Furthermore, the inhibitory activity appears to be inversely related to the distance of the binding site from the 5' cap of the mRNA, suggesting that expression of the target gene may be more effectively inhibited early in translation. We then observed that the inhibitory effect of luciferase activity was dose-dependent on theophylline (fig. 2E), and therefore we speculated that the effect of the signal linker was influenced by the number of ligands recruited to each mRNA target. To test this possibility, we introduced one or three theophylline aptamers at the 3' end of the device, and then measured luciferase expression levels and compared their effect to that of 2 signal linkers. The inhibition of luciferase expression by the device with three aptamers was similar to 2 signal linkers, probably due to saturation effects. However, a device containing only one aptamer can only reduce reporter gene expression very weakly.

We also performed in vitro translation reactions using macromolecular components (ribosomes, trnas, aminoacyl-tRNA synthetases, initiation, elongation and termination factors), purified ligand (theophylline), in vitro transcribed renilla luciferase mRNA, and in vitro transcribed RNA signaling linker (R1 used in fig. 2C) or negative controls. In vitro data suggest that the observed silencing effect of the signaling linker is indeed caused by the ligand-aptamer complex, suggesting a roadblock mechanism.

We then used a simple mathematical model to better understand the relationship between various input parameters and outputs, this equation describing the dose or concentration effect relationship and the maximum effect, which are key features of many biological phenomena. Based on this equation and the experimental results we observed, we will use 2 signal connectors and in the presence of sufficient amount of ligand to perform the subsequent gene knockdown experiments.

To demonstrate the modular design nature of this approach, we constructed several signaling connectors targeting the human Vascular Endothelial Growth Factor (VEGF) gene and replaced the theophylline aptamer with a tetracycline ligand (Muller et al, 2006). Relevant experimental data support the modularity of the signaling linker for the different aptamer domains (fig. 2F). As predicted earlier, these devices (R13-R15 and R17-R20) only exerted potent silencing effects under 100. mu.M tetracycline conditions. The level of VEGF mRNA expression in the cells containing the signal connector did not change significantly in the absence or presence of tetracycline, indicating that the signal connector is primarily translationally repressive and does not affect mRNA levels (FIG. 2).

These results indicate that the signaling linker can be used as a gene switch to down-regulate the expression of a target gene.

3. Signal connector for efficient activation of endogenous gene expression

We then used the RNA aptamer corresponding to eukaryotic translation initiation factor 4G (eIF4G) (Miyakawa et al, 2006) to determine if the signal linker could also enhance translation of the target gene by promoting initiation factor complex formation (fig. 2G). eIF4G is able to recruit ribosomal 40S subunits and activate mRNA translation (Moore, 2005). We selected the renilla luciferase gene as the target gene and the results of the luciferase reporter gene assay showed that the specific signal linker with the two aptamers induced a 15-fold increase in luciferase protein activity relative to the control group (fig. 2H). After elimination of one aptamer copy from the device, the efficiency of induction activation decreased significantly, while addition of another copy of aptamer resulted in little increase in expression (fig. 2).

We also constructed a simple mathematical model to elucidate the relationship between the various input parameters and the output, and the equations revealed that the relationship was non-linear and saturable (fig. 2). Based on the predictions and observed results of the new equation for gene activation, we performed gene expression activation experiments with 2 signal connectors designed in the presence of sufficient amounts of ligand. To achieve dynamic regulation of translation initiation, we used a combination of one aptamer that recognizes theophylline and two aptamers that recognize eIF4G to regulate gene expression, where the antisense domain was designed to bind to the theophylline aptamer (fig. 2G). Theophylline binding stabilizes the aptamer and results in allowing the antisense domain to bind to the mRNA of the target gene. The results of the luciferase reporter gene assay showed that the addition of theophylline increased luciferase activity in a dose-dependent manner (fig. 2I).

These results indicate that the signaling linker can be used as a gene switch to upregulate expression of endogenous target genes

4. Constructing logic gates of all basic types using signal connectors

In the construction of electronic circuits, logical operations and digital systems may be implemented using logic gates, including not gates, nand gates, or gates, exclusive or gates, and exclusive nor gates. Many aspects of biological cell information processing are similar to signal integration of electronic circuits. We have then presented a question of whether signal connectors can be used to construct complex programmable logic gates. Due to the powerful gene-regulatory capabilities of the signal connectors, we attempted to construct various logic gates that generated output signals in response to multiple input signals by stably transfecting the devices (fig. 3A). We constructed all basic types of two-input Boolean logic gates in HEK293 cells stably expressing 5' -capped and uncapped Renilla luciferase mRNA, recognizing exogenous theophylline or tetracycline signals through the use of ligands. In the construction of expressing 5' uncapped luciferase mRNA, an Open Reading Frame (ORF) encoding Renilla luciferase was placed downstream of the primary ORF. The major ORF contains a stop codon at the end and is long enough to prevent translation restart.

First, we build two not gates, each producing a high output at low input. As shown in fig. 2D and F, the location of the antisense RNA target sequence of the mRNA is important for the inhibitory efficiency of the signaling linker. We used two signal linkers (R1 and R13) to maximize the inhibition of translation of the 5' capped Renilla luciferase mRNA in the presence of 1000 μm theophylline or 100 μm tetracycline. As shown in fig. 3B, each gate exhibits a high fluorescein output in the absence of an input signal.

We then build an and gate that produces a high output only if the input signals are all high. As shown in figure 2I, since activation efficiency is inversely proportional to the distance of the target from the 5 'end of the mRNA, we used two signal linkers (R25 and R29), each of which produced only minor translational activation of the 5' uncapped renilla luciferase mRNA in the presence of the ligand. As shown in FIG. 3C, although each individual signal (1000 μm theophylline or 100 μm tetracycline) did not significantly stimulate the expression of the target luciferase gene, the presence of both signal inputs resulted in a strong activation through synergy between the two devices.

We also construct a nand gate that will show a higher output value if any of the inputs are low. We used two signal linkers (R12 and R16) to minimally inhibit the 5' capped renilla luciferase messenger mRNA in the presence of ligand. As shown in FIG. 3, although the introduction of a single signal (1000 μm theophylline or 100 μm tetracycline) did not significantly inhibit the expression of the target luciferase gene, the synergistic effect induced a strong inhibition in the presence of both input signals.

Next, we construct an OR gate that produces a high output if one or both of the inputs are high. During this construction we still used two signal linkers (R22 and R26), each of which strongly activated translation of 5' uncapped renilla luciferase mRNA in the presence of ligand. As shown in FIG. 3E, the luciferase signal can be generated from either of these signals (1000 μm theophylline or 100 μm tetracycline).

We also constructed a nor gate. We used two signal connectors (R1 and R13) to maximally inhibit translation of renilla luciferase mRNA. Since the introduction of a single signal significantly suppressed the expression of the target gene, luciferase was produced only when neither signal (1000 μm theophylline or 100 μm tetracycline) was present. (FIG. 3F)

We also build an exclusive or gate. We designed two signal connectors (R22 and R27) targeting two different regions of the renilla luciferase mRNA without a cap at the 5' end, while their RNA sequences are complementary to each other. The results show that each device strongly activates luciferase expression in the corresponding ligand. In contrast, the simultaneous introduction of both devices did not significantly activate the expression of the luciferase gene of interest due to the specific base pairing of the antisense domains of both devices (fig. 3G).

Finally, we construct an exclusive nor gate. Using a similar design strategy, we used two signal linkers (R1 and R14) that strongly inhibited translation of 5' capped renilla luciferase mRNA in the corresponding ligands. The XOR gate can produce an output with high luciferase signal when 1000 μm theophylline or 100 μm tetracycline are present at the same time or not (FIG. 3H).

These results indicate that the signal connector can logically connect the input signal with the desired cell output signal.

5. The signal connector is capable of effectively reconnecting the signal path and creating a feedback loop

In eukaryotic cells, signaling proteins often activate transcription factors to initiate transcription of downstream genes. Because, in theory, signaling linkers could link transcription factors to inhibit downstream gene translation, we engineered the molecular network by rejoining the original cellular signaling pathway (fig. 4A). Beta-catenin is a multifunctional protein that normally aggregates in the nucleus of cancer cells, activating transcription of the oncogene c-Myc gene (He et al, 1998). We synthesized a signal linker that contained a β -catenin ligand (Culler et al, 2010) and located a region within the 5' UTR of c-Myc mRNA (Table S7) by antisense sequence. We investigated the effect of stimulating the β -catenin pathway with leukotriene D4(LTD4) in HEK293 cells. Both cell lines showed expression of C-Myc mRNA (FIG. 4B) after stable expression of the signal connector or negative control, whereas the expression of C-Myc protein was significantly reduced in cells stably expressing the signal connector compared to cells of the negative control group (FIG. 4C). These results indicate that our signal connector can effectively reconnect the signal pathway by establishing a negative connection between the transcription factor and the mRNA of the downstream gene.

We also tested whether the signal connector could cause a specific regulatory factor to activate a downstream target gene (fig. 4), thereby creating a new signaling pathway. A particular signaling linker uses one ligand domain to recognize the β -catenin signal and two other ligand domains to form a translation initiation factor complex. In the absence of β -catenin signaling, the stem of the β -catenin aptamer may "turn off" the antisense domain. In the presence of β -catenin signaling, this signal linker can interact with the target renilla luciferase mRNA (fig. 4E). The effect of leukotriene D4(LTD4) was investigated by stable transfection of signaling linkers or negative controls in HEK 293T cells. Luciferase assay results showed that luciferase activity was significantly increased in cells expressing the signal connector (FIG. 4F), whereas activity was not affected by LTD4 in cells expressing the negative control. These results indicate that β -catenin signaling can efficiently activate the expression of renilla luciferase with the help of a designed signaling linker.

Next, we tested the ability of the signal connector to incorporate a feedback loop into the gene-gene interaction network. A positive feedback loop may amplify the signal received from the sender and transition the system from the initial state. We designed a feedback loop with a signaling connector in which bone protein (OPN) and VEGF are activators of each other (fig. 4G). OPN and VEGF are cytokines that secrete proteins, regulate cellular activity and angiogenesis (Ferrara et al, 2003; Lyle et al, 2014), and their RNA ligands have been proposed in previous literature (Ng et al, 2006; Mi et al, 2009). We inserted two copies of eIF4G ligand into the 3' end of OPN or VEGF nucleic acid switch (riboswitch) and constructed a signal linker to recognize VEGF or OPN. The results show that OPN and VEGF are independent of each other in bladder cancer. We transfected OPN or VEGF over-expression plasmids into T24 cells expressing signal connectors and found that transfection of the corresponding over-expression plasmids effectively increased the expression level of the regulated genes (fig. 4G). We also investigated whether the signal connector could be used to construct a negative feedback loop between OPN and VEGF (fig. 4H), making the system more stable. Since OPN can induce expression of VEGF through the signaling connector, we need only to demonstrate that VEGF can also inhibit OPN expression by a similar method. We inserted two copies of VEGF ligand into the 3' end of the antisense RNA, constructing a signaling linker that regulates OPN. Using T24 cells stably expressing this signaling linker, we demonstrated that transient expression of VEGF can reduce OPN concentrations, which in turn increases OPN levels following VEGF gene knockout (fig. 4H).

These results indicate that signal connectors are an effective tool for constructing regulatory circuits and gene-gene networks.

6. Signal connector capable of silencing survival gene expression and inhibiting cancer cell growth

To examine whether signaling linkers could be used to identify cells, reprogram cell status and behavior, we used the human telomerase reverse transcriptase (hTERT) promoter to drive expression of signaling linkers that silence surviving genes (Gao et al, 2014), and selected bladder cancer cells as target cells (fig. 5). hTERT

The promoter (hTERTp) is very active in more than 85% of human cancers, but is inactive in most normal cells (Takakura et al, 1999). Therefore, we constructed a logical and gate in which the activated hTERTp and ligand must combine to inhibit the survival gene (fig. 5B). Signaling connectors were generated as described previously that inhibited the human C-Myc gene (Sardi et al, 1998) and BCL2 gene (Kunze et al, 2012), and stably transfected cysts or normal fibroblasts epithelial cells (fig. 5C). In bladder cancer cell lines, the corresponding devices were able to show a significant decrease in gene expression in the presence of theophylline compared to the absence of ligand (fig. 5D). These devices did not produce inhibition of the fibroblasts, whether or not the ligand was present (fig. 5E). Growth curves of these cell lines also showed that this line effectively inhibited proliferation of the target bladder cancer cells without affecting the fibroblasts (fig. 5F). Furthermore, we investigated whether these devices could induce apoptosis of cancer cells. Bladder cancer cells treated with the signal connector exhibited strong blue fluorescence, showing typical apoptotic characteristics. In contrast, the signal connector had no such effect in normal cells (fig. 5G).

These results indicate that the signal connector-based and gate line can suppress gene expression of the target cell line.

7. Conversion of carcinogenic signals to cancer suppressive signals via signal connectors

The successful use of signal-linked mediator for translational control in human cells opens a way to simultaneously turn on or off a multi-gene translation process in which some genes are activated and others are repressed. We hypothesize that these devices should potentially alter the output of oncogenic pathways and control the fate of cancer cells by simultaneously activating and repressing endogenous genes.

NF-kB is an oncogenic signal that promotes the development of cancer. NF-kB promotes cell proliferation by activating downstream target genes such as cyclin D1, c-fos and c-jun (Li et al, 2015). Therefore, we attempted to reconnect NF-kB signals from the proliferation pathway to the rest/death pathway by using a signal connector. We constructed 4 signal connectors recognizing NF-kB (p65) (Urster et al, 2008) to activate bax (R37) and p21(R38) and to inhibit two oncogenes Bcl2(R39) and c-Myc (R40). High levels of NF-kB signaling were expressed in human bladder cancer T24 cells (FIG. 6A). Two copies of eIF4G ligands were inserted at the 3 'end of the NF-kB nucleic acid switch to construct signaling connectors that activate Bax or p21, while two NF-kB ligands were ligated to the antisense RNA3' end to construct signaling connectors that inhibit Bcl2 and c-Myc. The results showed that the protein expression levels of Bax and p21 could be simultaneously enhanced, reducing Bcl2 and c-Myc in T24 cells stably expressing the signaling linker (fig. 6B). Finally, we determined whether the signal connector could simultaneously inhibit tumor growth in vivo. T24 cells stably expressing the signaling connector or its negative control were injected into male nude mice. At 20 days post-injection, we found that tumors formed in the signaling connector group were much smaller than those of the negative control group (FIGS. 6C-E). In addition, the mean tumor weight was significantly lower for the signal connector group compared to the negative control group at the end of the experiment (fig. 6F).

These results indicate that signaling can inhibit tumor growth in vivo by redirecting oncogenic signaling pathways.

The present invention has been described above using specific examples, which are only for the purpose of facilitating understanding of the present invention, and are not intended to limit the present invention. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

Claims (6)

1. An RNA signal connector, which is characterized in that the RNA signal connector is an oligonucleotide sequence, the cDNA of the RNA signal connector is shown as any sequence in SEQ ID NO 1-40, wherein,

comprising an antisense domain linked to a first copy of an aptamer domain and two copies of the aptamer domain linked to a second copy of the aptamer domain by a linker sequence,

alternatively, the first and second electrodes may be,

comprising an antisense domain and two copies of an aptamer domain, and a nucleic acid switch, said antisense domain being linked to said nucleic acid switch, said nucleic acid switch being further linked to a first copy of the aptamer domain by a first linker sequence, said first copy of the aptamer domain being linked to a second copy of the aptamer domain by a second linker sequence,

wherein the antisense domain is complementary to a target mRNA sequence and the aptamer domain binds to a ligand as an input signal.

2. A method of modulating translation of a target mRNA in a cell for non-therapeutic purposes, the method comprising introducing into the cell the RNA signaling connector of claim 1 and allowing the RNA signaling connector to inhibit or activate translation of the target mRNA in the presence of a ligand.

3. A logic gate, characterized in that it consists of 2 RNA signal connectors according to claim 1.

4. Use of the RNA signal connector of claim 1 or the logic gate of claim 3 to reconnect a cell signal pathway and create a feedback loop for non-therapeutic purposes.

5. Use of the RNA signaling connector of claim 1 or the logic gate of claim 3 for a non-therapeutic purpose of silencing the expression of a survival gene and inhibiting the growth of a cancer cell.

6. Use of the RNA signal connector of claim 1 or the logic gate of claim 3 for a non-therapeutic purpose of converting an oncogenic signal into a cancer suppressor signal.

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