Chemical genetic modulation of peptide function

Abstract

Provided herein are methods, compositions, kits, and systems for modulating peptide function. In particular, provided herein are methods, compositions, kits, and systems comprising a nucleic acid construct and a fusion protein comprising a chemically activated protein domain (CAP) that modulates the activity of a fusion peptide in the presence of a ligand.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 329,736, filed April 11, 2022, the disclosure of which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was supported by Grant Nos. R01 AT011652 and R01 HL156989 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Technical Field

[0004] Provided herein are methods, compositions, kits and systems for regulating peptide function. Specifically, provided herein are methods, compositions, kits and systems comprising nucleic acid constructs and fusion proteins comprising a chemically activated protein domain (CAP) that regulates the activity of the fusion peptide in the presence of a ligand. Background Art

[0005] Natural or engineered peptides act as partners in dimerization pairs 1 , inhibitors of enzyme activity 2,3 , and regulators of protein localization 4,5 ,degradation 6,7 and editing 8 To exert biological functions. Activation of peptide functions by user-defined signals (such as light or chemicals) can manipulate a variety of biological processes through temporal control. One way to achieve such control is to use chemically modified synthetic peptides that contain removable protecting groups. 9 or azobenzene-based chromophores 10 While these approaches are useful in vitro, their use in endogenous biological environments is limited due to challenges with delivery to cells and subsequent degradation of the peptide externally. Another way to control peptide function is to use genetically encoded protein domains. 7,11 . Caging and uncaging of peptides can be achieved by fusing the peptide to a protein that changes the conformation of the peptide in the presence of light or small molecules. This approach allows the genetic introduction of peptides into living organisms and the expression of cell type-specific proteins, and is therefore suitable for biological studies. The second light, oxygen, voltage sensing domain from oat (Avenasativa) phototropin 1 (AsLOV2) has been used as a protein domain to control peptide function 2,6,11-16 AsLOV2 works by controlling the accessibility of the N-terminus of a peptide fused to it. In the dark, the peptide is blocked by AsLOV2; under light, a conformational change occurs in AsLOV2, so the peptide is released and becomes accessible.17,18 Although light provides rapid temporal control, its use in opaque organisms is limited due to poor tissue penetration. 19 In contrast, chemicals can be easily administered into animals, and many can penetrate cell membranes and deep tissues. Chemically activated protein domains for controlling peptide function will complement their photocontrolled counterparts and can be used to manipulate cellular processes. Chemically dependent protein domains have been used to provide protein neighbors with 20,21 and conformation 22,23 However, it is not generally applicable to controlling peptide function. One example is the ligand-induced degradation (LID) system. 7 , in which a five-amino acid peptide that can induce protein degradation is blocked by FK506 binding protein (FKBP) until the small molecule shield-1 (https: / / www.takarabio.com / products / inducible-systems / inducible-protein-stabilization / shield1) displaces the peptide from the ligand binding site. However, LID cannot control other peptides and has limited potential for specific manipulation of cellular processes.

[0006] Therefore, novel small-molecule-based protein switches and gating modules are needed to control the functions of other peptides. Summary of the invention

[0007] Provided herein are methods, compositions, kits and systems for regulating peptide function. Specifically, provided herein are methods, compositions, kits and systems comprising nucleic acid constructs and fusion proteins comprising a chemically activated protein domain (CAP) that regulates the activity of the fusion peptide in the presence of a ligand.

[0008] In some embodiments, the present invention provides a method for regulating the activity of a peptide, comprising generating a nucleic acid construct comprising a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site and a nucleic acid encoding a peptide; applying the nucleic acid construct to one or more cells to produce a fusion protein, wherein the fusion protein comprises at least one CAP connected to the peptide; and applying a ligand to the one or more cells, wherein the ligand binds to the ligand binding site of the CAP of the fusion protein, wherein the administration of the ligand increases the activity of the peptide. In some embodiments, at least one CAP comprises a CapN bound to the N-terminus of the peptide, and / or a CapC bound to the C-terminus of the peptide. In some embodiments, the administration of the nucleic acid construct comprises direct injection of the nucleic acid construct, macromolecular-mediated liposome and / or biopolymer gene delivery, plasmid delivery, and / or viral delivery. In some embodiments, viral delivery is selected from the group consisting of: adeno-associated virus (AAV) delivery, adenovirus delivery, lentivirus delivery, vaccinia virus delivery, and retrovirus delivery. In some embodiments, the nucleic acid construct is stably expressed or transiently expressed. In some embodiments, expression is intracellular expression or extracellular expression.

[0009] In some embodiments, the ligand binding site comprises a FKBP binding domain. In some embodiments, the peptide is SsrA, a nuclear localization signal peptide (NLS), a nuclear export signal peptide, or a TEV protease cleavage site. In some embodiments, the peptide is an enzyme activation peptide, an enzyme inhibitory peptide, an enzyme regulatory peptide, a binding peptide, a positioning peptide, or a degradation peptide. In some embodiments, the ligand is shield-1 and / or aquashield-1.

[0010] In some embodiments, administration is parenteral administration and / or non-parenteral administration. In some embodiments, one or more cells are in vitro cells or in vivo cells. In some embodiments, in vivo cells are neuronal cells or hepatocytes. In some embodiments, the nucleic acid construct comprises at least one nucleic acid encoding the group selected from the following: Aga2p, reporter gene, TetR DBD, SspB, Vp16, P2A, joint, FLAG, UAS-mCherry and internal ribosome entry site (IRES). In some embodiments, the nucleic acid construct also comprises a promoter nucleic acid selected from the group consisting of the following: CMV, CAG and synaptic protein.

[0011] In some embodiments, the method of the present invention further comprises a protein of interest (POI). In some embodiments, POI is a transcription factor, a kinase, a gene editing enzyme or a marker. In some embodiments, POI is an evolved green fluorescent protein (EGFP). In some embodiments, the method of the present invention further comprises measuring the activity of the peptide, wherein the measurement comprises measuring the location, structure and / or function of the peptide. In some embodiments, the present invention provides a general mechanism and / or module for the time, user-controlled regulation of the structure, function and location of small peptides, large peptides or proteins in cells.

[0012] In some embodiments, the present invention provides a kit comprising: a nucleic acid construct comprising: a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site; and a nucleic acid encoding a peptide; a gene transfer agent and / or vector; and a ligand that binds to the ligand binding site.

[0013] In some embodiments, the present invention provides a composition comprising: a nucleic acid construct comprising: a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site, wherein the ligand is shield-1 and / or aquashield-1; a nucleic acid encoding a peptide; and a gene transfer agent and / or a vector.

[0014] In some embodiments, the present invention provides a fusion protein comprising a protein expressed by a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site, wherein the ligand is shield-1, rapamycin and / or aquashield-1; and a peptide expressed by the nucleic acid, wherein the peptide is regulated by shield-1, rapamycin and / or aquashield-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Representative CapN systems are shown. a, Design of CAP. CapN and CapC block the N-terminus and C-terminus of the peptide, respectively. Addition of shield-1 releases the binding sequence from the ligand binding site, thereby unblocking the peptide. b, Labeling and library selection of CapN-caged TEV protease cleavage site. CapN-tobacco etch virus protease cleavage site (CapN-TEVcs) is displayed on the yeast surface by fusion with yeast Aga2p protein. The accessibility of TEVcs is confirmed by protease cleavage. FLAG and HA signals indicate protein expression level and TEVcs cleavage, respectively. For library selection, the retained population is shown as a rectangle on the FACS plot. TEV protease cleavage site (ENLYFQ / G, cuts between Q and G). FLAG and HA are epitope tags. c, Labeling scheme of CapN-caged SsrA. CapN-SsrA is displayed on the yeast surface by fusion with yeast Aga2p protein. The accessibility of SsrA is assessed by binding to its binding partner SspB. APEX2 tags proteins in close proximity to biotin-phenol. FLAG and biotin signals represent protein expression levels and SsrA-SspB association, respectively. APEX2 is an engineered ascorbate peroxidase. FLAG is an epitope tag. d, CapN sequences before, during, and after directed evolution. Before directed evolution, the original linker and binding sequence (TRGVEEVAEGVVLL) (SEQID NO: 1) from LID was fused to the C-terminus of FKBP together with TEVcs or SsrA. For directed evolution (libraries 1-4), the last six amino acids of the binding sequence were mutated to 6-9 random amino acids. The evolved sequence is the final CapN used for the rest of this study. Aga2p is a yeast protein used to display CapN on the yeast surface. "X" represents any of the twenty amino acids. Amino acids that differ from the original LID sequence are highlighted in red. e, FACS selection of CapN libraries for improving shield-1 dependence. Four libraries (each with >10 6 CapN variants) were displayed on the yeast surface by fusion with yeast Aga2p protein. After the first round of selection, the libraries were combined. The value is the percentage of cells in Q2 to (Q2+Q4). The sequencing results after the fourth round of libraries are shown in Figure 2. Figure 2 f, FACS analysis of TEVcs caged by CapN before and after directed evolution. Values ​​are median HA intensities of FLAG-positive cells (Q2+Q4). After evolution, CapN showed improved closure and dynamic range. This experiment was performed three times with similar results. Additional FACS plots are shown in Figure 2g, FACS analysis of SsrA caged by CapN before and after directed evolution using the labeling scheme as in c. Values ​​are median biotin intensities of FLAG-positive cells (Q2+Q4). After evolution, CapN showed tighter cages and greater responses to shield-1. This experiment was performed three times with similar results.

[0016] Figure 2 Shows the Figure 1 The ligand binding site of related FKBPs and the directed evolution results of CapN. a, Crystal structure of FKBP12 (PDB: 1FAP). The hydrophobic residues around the ligand binding site are shown in yellow and sticks. b, Figure 1 e, sequencing of forty clones from the 4th round of CapN library. Twenty-three different sequences have been identified. Clone #1 is the final CapN used for the rest of this study. c, FACS analysis of the eight most enriched clones, corresponding to clones #1-#8 shown in b. Values ​​are median HA intensities of FLAG positive cells (Q2+Q4). All eight clones showed similar results. Clone #1 is the final CapN used for the rest of this study. This experiment was performed once.

[0017] Figure 3 Characterization of shield-1 dose response using CapN is shown. a, FACS analysis of CapN-caged SsrA on the surface of yeast treated with different concentrations of shield-1. Three technical replicates were performed for each condition. Values ​​are median biotin intensities of FLAG-positive cells (Q2+Q4). This experiment was performed twice with similar results. b, Dose-response curves obtained using data from a. The median biotin signal is plotted against shield-1 concentration. A half-maximal response was observed at 53 nM. Error, sem

[0018] Figure 4 Figure 2 shows the reversibility characterization of shield-1 using CapN. a, Schematic diagram of the reversibility characterization of shield-1. Yeast cells were incubated with shield-1 for 10 minutes and then washed to remove excess shield-1. Yeast cells were then incubated at room temperature for 0-12 hours and then the accessibility of SsrA was assessed using SspB-APEX2 and biotin-phenol labeling, as shown in Figure 2. Figure 1 c. b, FACS analysis of yeast cells from a. Values ​​are median biotin intensities of FLAG-positive cells (Q2+Q4). No reduction in biotin signal was observed even after incubation of cells for 12 h in the absence of shield-1. This experiment was performed once.

[0019] Figure 5 Engineering of the CapC system is shown. a, CapC sequences before, during, and after directed evolution. Before directed evolution, a flexible linker (SGAGSGGSGTGS GSGGS) (SEQ ID NO:2) and the last six amino acids from the evolved CapN binding sequence (RYSPNL) (SEQ ID NO:3) were fused to the N-terminus of FKBP along with SsrA. For directed evolution (libraries 1-3), the amino acids highlighted in red were randomly mutated to any of the twenty amino acids. The evolved sequence is the final CapC used for the rest of this study. Aga2p is the yeast protein used to display CapC on the yeast surface. "X" represents any of the twenty amino acids. Amino acids that differ from the evolved CapN sequence are highlighted in red. b, Labeling and library selection scheme for CapC-caged SsrA. SsrA-CapC is displayed on the yeast surface, and the accessibility of SsrA is controlled using the same method as in Figure 1 c, FACS selection of the CapC library for improved shield-1 dependency. The pre- and post-evolution CapCs are also shown for comparison. 6 CapN variants) were displayed on the yeast surface by fusion with yeast Aga2p protein. The libraries were combined for selection. The values ​​shown in the figure represent the percentage of cells in Q2 (Q2 Q4). The sequencing results after the second round of libraries and the additional FACS graphs are shown in Figure 6 The experiment was conducted once.

[0020] Figure 6 All-atom molecular dynamics simulations are shown. Results of 2 microsecond molecular dynamics simulations of FKBP and capped ArgTyrSerProAsnLeu (SEQ ID NO: 4) peptide in 150 mM buffer. a, The central configurations of the top 5 clusters (ranked 1-5) obtained from RMSD clustering indicate a direct interaction between Leu6 of the peptide (represented by "licorice"; cap residues are shown in green, other atoms are shown in CPK color, and carbon is gray) and the F36V binding site of FKBP (shown as van der Waals spheres). The secondary structure of the FKBP protein is shown in sketches - helices are red, - folds are yellow. b, The RMSD time trace relative to the structure shown in a indicates the lifetime of the corresponding conformation in the simulation. An RMSD of 0 indicates a simulation time point corresponding to the structure in a. The horizontal dashed line represents the time point used for clustering. c, The time trace of the center-of-mass distance between each individual side chain of the peptide and the side chain of the F36V binding site shows that for most of the simulation trajectories (distance ), Leu6 remains in constant proximity to the binding site. The portion of the simulation trajectory where Leu6 is in close proximity to the F36V binding site includes all configurations associated with the first five clusters shown in a.

[0021] Figure 7 The directed evolution results of CapC are shown. Figure 5 a, Sequencing of twenty clones from the second round of CapC library, as shown in Figure 5 c. Eighteen different sequences have been identified and characterized. One sequence with an early stop codon is not shown. Clone #18 is the final CapC used for the remainder of this study. b, FACS analysis of the eighteen clones shown in a. Values ​​are median HA intensities for FLAG positive (Q2+Q4) cells. Clone #18 is the final CapC used for the remainder of this study. This experiment was performed once.

[0022] Figure 8 A comparison of single CapN, single CapC, and tandem CAPs in caged TEVcs is shown. a, Schematic diagram of the three constructs tested. CapN-TEVcs-CapC is the combined use of two evolved CAPs. Aga2p is a yeast protein used to display the constructs on the yeast surface. TEVcs, TEV protease cleavage site (ENLYFQ / G, cleavage between Q and G). FLAG and HA are epitope tags. b, FACS plots of the three constructs shown in a. Values ​​are median biotin intensities of cells in Q2 and Q4. Figure 1 e and 1f more stringent TEV protease conditions. See Methods section for details.

[0023] Fig. 9The application of CAP for shield-1-induced protein translocation is shown. a, Schematic diagram of shield-1-induced protein translocation to the plasma membrane. When CAP uncaging occurs to release SsrA, the target protein (e.g., EGFP) fused to SspB is translocated to the plasma membrane. mCherry is used as a membrane protein marker. The transmembrane domain is CAAX. POI, target protein. b, Representative fluorescence micrographs of HEK293T cells expressing the construct shown in a. Addition of shield-1 induces EGFP translocation to the plasma membrane within seconds. The right panel shows the intensity spectrum of mCherry and EGFP along the red line in the image. Scale bar, 20 μm. The experiment was performed once. c, Schematic diagram of shield-1-induced protein translocation from the plasma membrane. Protease cleavage of TEVcs allows POI (e.g., EGFP) to be removed from the plasma membrane. mCherry is used as a protease expression marker. The transmembrane domain is CAAX. TEVcs, TEV protease cleavage site (ENLYFQ / M, (SEQ ID NO:5) cleaves between Q and M). POI, protein of interest. d, Representative fluorescence micrographs of HEK 293T cells expressing the constructs shown in c. Addition of shield-1 eliminated EGFP from the plasma membrane. Scale bar, 20 μm. Additional images are shown in Figure 8 As shown. This experiment was performed twice with similar results. e, Schematic diagram of the CAP controlling the nuclear localization signal peptide. Shield-1-dependent uncaging of the NLS brings the POI (e.g., EGFP) from the cytosol into the nucleus. mCherry was used to represent the cytosol. NLS, nuclear localization signal peptide (PKKKRKV) (SEQ ID NO:6). POI, protein of interest. NES, nuclear export signal peptide (LQLPPLERLTLD) (SEQ ID NO:7). PKIt NES, truncated cAMP-dependent protein kinase inhibitor α (PKIt) NES (LALKLAGLDI) (SEQ ID NO:8). f, Left: Representative fluorescence micrographs of HEK 293T cells expressing the construct shown in e. Scale bar, 20 μm. Additional images are shown in Fig.10 As shown. This experiment was performed three times with similar results. Right: Quantification of the total EGFP intensity distribution. The ratio was calculated as the ratio of the total EGFP intensity in the cytosol to the total EGFP intensity in the whole cell. The cytosol and the whole cell were determined by mCherry. The center line indicates the mean of the ratios. P values ​​were determined by unpaired two-tailed t-test. ****P<0.0001.

[0024] Fig.10 Shows Fig. 9Additional images. Left: Four additional views for each condition. Right: Negative control for CAP caging using TEVcs instead of SsrA; addition of shield-1 does not increase the cytoplasmic pattern of EGFP. This further supports the fact that translocation from the membrane is due to TEV protease cleavage. Scale bar, 20 μm.

[0025] Fig.11 The expression of + / -shield-1 was shown. Fig. 9 e Additional fluorescence microscopy of HEK 293T cells with the indicated constructs. Scale bar, 20 μm.

[0026] Fig.12 The application of CAP for shield-1-induced transgene transcription is shown. a, Schematic diagram of shield-1-induced gene transcription. Uncaging of SsrA restores the segmented transcription factor and enables expression of a reporter gene (e.g., mCherry). EGFP was used as an expression marker for SspB and the transcriptional activation domain. For all subsequent experiments, the transcriptional activation domain was VP16. The DNA binding domain (DBD) was specified for each experiment. b, Summary of the main constructs tested. The amino acid sequence of SsrA is highlighted. DBD, DNA binding domain. P2A, self-cleaving peptide. IRES, internal ribosome entry site. c, Quantification of mCherry expression levels of the constructs shown in b. The numbers on the graph are the ratio of the average mCherry intensity of the +shield-1 condition to the average mCherry intensity of the -shield-1 condition for each construct. The center line represents the average of the mCherry intensity. Images as Fig.11 , 13 and 17. For this experiment, Gal4 was used as the DBD and UAS-mCherry was used as the reporter gene. n=12 for all conditions. This experiment was performed three times with similar results. d, Representative fluorescence micrographs of HEK 293T cells expressing the best performing non-single component construct u3. Same DBD and reporter gene as in c. Scale bar, 20 μm. FLAG is an epitope tag. e, Representative fluorescence micrographs of rat cortical neurons expressing the single component construct u4, and quantification of mCherry expression levels. For this experiment, TetR was used as the DBD and TRE-mCherry was used as the reporter gene. The numbers on the graph are the ratio of the average mCherry intensity of the +shield-1 condition to the average mCherry intensity of the -shield-1 condition. The center line indicates the average of the mCherry intensity. n=5 for both conditions. Scale bar, 100 μm. Additional images used for quantification are shown in Fig.18As shown. This experiment was performed twice with similar results. P values ​​were determined by unpaired two-tailed t-test. **P<0.01; ***P<0.001; ****P<0.0001.

[0027] Fig.13 Shows Fig.12 Images of constructs u1 and u2 in b and 12c. Fluorescence micrographs of HEK 293T cells expressing the indicated constructs and the UAS-mCherry reporter gene. These images were used for quantification Fig.12 Constructs u1 and u2 in c. All scale bars, 50 μM.

[0028] Fig.14 Shows Fig.12 Images of construct u3 in b and 12c and two other constructs with different SsrA sequences. Fluorescence micrographs of HEK 293T cells expressing the indicated constructs and the UAS-mCherry reporter gene. These images were used to quantify Fig.12 Construct u3 in c. The two other truncations shown here (one or two N-terminal alanines removed from SsrA) showed no improvement. All scale bars, 50 μM.

[0029] Fig.15 Shown is the effect of shield-1 on the expression of the transcriptional activation domain. No difference was found between +shield-1 and -shield-1 conditions. All scale bars, 50 μM.

[0030] Fig.16 Shows Fig.12 Gene expression of construct u3 in a at different shield-1 concentrations, quantification of mCherry expression levels at different shield-1 concentrations. 50 μM or above induced strong gene expression. P values ​​were determined by unpaired two-tailed t-test. ***P<0.001; NS, not significant. n=12 for all conditions. b, Fluorescence micrographs of HEK293T cells used for quantification in a, showing only mCherry (reporter gene expression). Gal4 was used as DBD and UAS-mCherry was used as reporter gene. Scale bar, 50 μm. c, Fluorescence micrographs of HEK 293T cells in the same experiment in b, showing only EGFP (transcription activation domain expression). It is worth noting that differences in EGFP signal do not indicate actual differences in gene expression. Scale bar, 50 μM.

[0031] Fig.17 Shows the use Fig.12Gene expression of construct u3 in b after incubation with shield-1 for different times. a, Schematic diagram of the experiment. HEK 293T cells expressing construct u3 were incubated with 100nM or 1000nM shield-1 for different times. The cells were then washed and incubated in the absence of shield-1 and imaged 24 hours after the initial shield-1 addition, b, Quantification of mCherry expression levels at different shield-1 incubation times at 100nM or 1000nM shield-1 concentrations. P values ​​were determined by unpaired two-tailed t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS, not significant. n=12 for all conditions. c, Representative fluorescence micrographs of HEK293T cells used for quantification in b. For this experiment, Gal4 was used as DBD and USA-mCherry was used as a reporter gene. Scale bar, 50 μm.

[0032] Fig.18 Shows Fig.12 Images of constructs u4 and u5 in b and 4c. Fluorescence micrographs of HEK 293T cells expressing the indicated constructs and the UAS-mCherry reporter gene. These images were used for quantification Fig.12 Constructs U4 and U5 in C. All scale bars, 50 μm.

[0033] Fig.19 Shows Fig.12 Additional images of E. Four additional views are shown for each shield-1 condition. Scale bar, 100 μm.

[0034] Fig. 20 Shows aqua-shield-1( https: / / cheminpharma.com / product / as1-50-mm- 2 / ) induced transgene expression in mouse tissues. a, Timeline of aquashield-1-induced transgene expression in mouse brain. Aquashield-1 was topically administered to mice (1 μL, 1 mM). b, Representative fluorescence micrographs of brain sections of the lateral hypothalamic region and quantification of the total number of cells expressing mCherry. The numbers on the graph are the ratio of the average cell count of the +aquashield-1 condition to the average cell count of the -aquashield-1 condition. The center line represents the mean of the cell count. n=3 for both conditions. Scale bar, 200 μm. c, Timeline of aquashield-1-induced transgene expression in mouse liver. Aquashield-1 was administered to mice by two intraperitoneal (IP) injections (40 mg / kg) 24 hours apart. d, Representative fluorescence micrographs of liver sections at the injection site and quantification of the total number of cells expressing mCherry. The numbers on the graph are the ratio of the average cell count of the +aquashield-1 condition to the average cell count of the -aquashield-1 condition. Center line indicates mean of cell counts. n = 3 for both conditions. Scale bar, 200 μm. P values ​​were determined by unpaired two-tailed t-test. *P < 0.1; ***P < 0.001.

[0035] Fig.21 Representative gating strategies for single-cell analysis are shown.

[0036] Fig. 22 Schematic diagram of the split NanoLuc assay for detecting methionine-enkephalin activation of CapC cages in a 2-chain design (left panel) and a 1-chain design (right panel) is shown.

[0037] Fig.23 Shows Fig. 22 Quantification of the split NanoLuc assay for the 1-strand design is provided in the right panel. The X-axis provides the different experimental conditions. The Y-axis provides the luminescent signal in relative luminescence units (RLU).

[0038] Fig.24 The activity of CapC caged Met-enkephalin detected by GloSensor cAMP assay is shown. RLU, relative luminescence unit.

[0039] definition

[0040] For ease of understanding of the present disclosure, multiple terms and phrases are defined below. Although the present invention will be described in conjunction with certain representative embodiments, it should be understood that the present invention is not limited to these illustrative examples. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is by no means limited to the methods and materials described.

[0041] Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods, devices and materials similar or equivalent to those described herein may be used in the practice of the present invention, certain methods, devices and materials are described herein. It should also be understood that all base sizes or amino acid sizes and all molecular weight values ​​or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for descriptive purposes.

[0042] As used in this disclosure, including the appended claims, the singular forms "a, an," and "the" include plural referents and are used interchangeably with "at least one" and "one or more," unless the content clearly dictates otherwise.

[0043] As used herein, the term "about" represents an insignificant modification or variation in a value that leaves the basic function of the item to which the value is related unchanged.

[0044] As used herein, "protein" is used synonymously with "peptide," "polypeptide" or "peptide fragment." A "purified" polypeptide, protein, peptide or peptide fragment is substantially free of cellular material or other contaminating proteins from the cell, tissue or cell-free source from which the amino acid sequence was obtained, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0045] As used herein, "regulating" means changing the activity of a gene or protein by increasing or decreasing. As used herein, the term "inhibiting" means preventing or reducing the activity of a gene or protein.

[0046] As used herein, the term "biological activity" refers to an effect on one or more cellular or extracellular processes (eg, by binding, signaling, etc.) that can affect a physiological or pathophysiological process.

[0047] The scope provided herein should be understood as abbreviation of all values ​​in the range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers or sub-ranges (and their fractions, unless the context clearly indicates otherwise) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. Any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer in the range, and also include their fractions (such as one tenth and one hundredth of an integer) when appropriate, unless otherwise indicated. Additionally, unless otherwise indicated, any numerical range described herein relating to any physical characteristic (such as polymer subunits, size or thickness) should be understood to include any integer within the range.

[0048] As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortalized phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell populations maintained in vitro.

[0049] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur within an artificial environment. An in vitro environment can consist of, but is not limited to, a test tube and a cell lysate. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and processes or reactions that occur in a natural environment. DETAILED DESCRIPTION

[0050] Provided herein are methods, compositions, kits and systems for regulating peptide function. Specifically, provided herein are methods, compositions, kits and systems comprising nucleic acid constructs and fusion proteins comprising a chemically activated protein domain (CAP) that regulates the activity of the fusion peptide in the presence of a ligand.

[0051] Natural or engineered peptides have important biological functions. A general method for achieving chemical-dependent activation of short and long peptides provides spatial and temporal control of cellular processes. In some embodiments, the present invention provides a pair of chemically activated protein domains (CAPs) for controlling the accessibility of the N-terminus and C-terminus of a peptide. In experiments conducted in the development of embodiments of the present invention, CAPs were identified by directed evolution of FK506 binding protein (FKBP). By fusion of a peptide with one or two CAPs, the function of the peptide is blocked until a small molecule removes the peptide from the FKBP ligand binding site. The present invention provides CAPs suitable for a series of short and long peptides, including protease cleavage sites, SsrA (10S RNA, TmRNA) peptides dimerized with SspB proteins, and nuclear localization signal (NLS) peptides, with a shield-1-dependent dynamic range of up to 156 times. In some embodiments, CAP systems, methods, compositions, and kits can be used in mammalian cell culture and various tissues of living animals, thereby providing a chemical genetics method for controlling the in vitro and in vivo activity of peptides.

[0052] In the experiments conducted for the development of the present invention, CAP was generated by directed evolution and comprises two protein domains, CapN and CapC, which are used to block the N-terminus and C-terminus of the peptide, respectively ( Figure 1 a). The addition of shield-1 relieves steric blocking and activates peptide function. In some embodiments, the present invention provides CAPs that enclose tobacco etch virus protease cleavage site (TEVcs), SsrA (tmRNA or 10Sa RNA) peptides, and nuclear localization signal peptides (NLS). In some embodiments, CAPs can be used for mammalian cell (e.g., HEK cells and neuronal cells) culture. In some embodiments, CAPs can be used for tissues of living animals, such as brain and liver tissues, thereby including both in vitro and in vivo utilities for controlling peptide activity. CAPs have wide applicability in enclosing short and long peptides (including TEVcs, SsrA, and NLS). In some embodiments, CAPs are used to translocate proteins to different cellular locations and control gene transcription in a chemically dependent manner in a variety of biological backgrounds.

[0053] CAP has several advantages over existing technologies. 2,6,11-15,27Compared to existing genetic methods, CAP provides an alternative approach by using the small molecule shield-1 or aquashield-1. Shield-1 is cell permeable and can be administered systemically in living animals. This makes CAP useful in most tissues of living organisms, including those that are difficult or too invasive for light to reach, such as the brain and liver. Although other chemically dependent protein domains have been described, CAP provides universal applicability for different peptides, for example, caging TEVcs and SsrA, regulating NLS, and controlling gene transcription. In some embodiments, tandem CAPs provide higher caging efficiency compared to individual CAPs. For example, SsrA caged by tandem CAPs provides up to 156 times more chemical dependence. The caging strategy using two protein domains provides multiple advantages. For example, unexpected leakage is a long-standing challenge in the use of protein cages; the tandem caging strategy provides protein engineers with a solution to this problem.

[0054] In some embodiments, the caging mechanism of CAP can be transferred to other peptides, and high signal-to-noise ratios can be obtained by fine-tuning the peptide or binding sequence, similar to AsLOV2. 13,16,32 For example, the use of essential residues of a functional peptide positioned next to the CAP domain. CAP provides a different approach to temporally control peptide function, enabling the cage of larger peptides or proteins where the effector site is positioned near the end of the sequence, similar to the use of AsLOV2 as a cage 29,33-35 The CP engineering strategy is also applicable to other chemically dependent protein domains, such as the hepatitis C virus protease NS3a. 36,37 and BCL-xL protein 38 Engineering these domains into universal protein cages could provide multiplexed control over cellular processes and expand the availability of chemical genetic techniques.

[0055] method

[0056] In some embodiments, when it is necessary to control the localization, structure and function of peptides and / or proteins by time control, such as by the administration of small molecules, the methods of the present invention can be used for research. For example, in some embodiments, CapC is used to control the display of opioid peptides on the extracellular membrane, so that endogenous Gi-coupled opioid receptor activity can be characterized. In some embodiments, when it is necessary to control the localization, structure and function of peptides and / or proteins by time control, such as by the administration of small molecules (to activate the targeted pathway), the methods of the present invention can be used for treatment.

[0057] The present disclosure contemplates the use of any genetic manipulation to regulate the expression of the target peptide. Any suitable method can be used to deliver the nucleic acid construct to the cell in vitro or in vivo. A suitable method is to introduce the nucleic acid construct into the cell so that the desired event (e.g., expression of the CAP peptide construct) occurs. The introduction of the molecule carrying genetic information into the cell can be achieved by any of a variety of methods, including but not limited to the direct injection of naked DNA constructs, and the use of macromolecular-mediated gene transfer such as liposomes, biopolymers, etc. In some embodiments, the method of the present invention provides a gene delivery vehicle derived from a virus, including but not limited to adenovirus, retrovirus, slow virus, vaccinia virus and adeno-associated virus. The vector can be applied to the subject in a variety of ways. For example, in some embodiments of the present disclosure, the vector is applied to the relevant tissue by direct injection. In other embodiments, administration is carried out through blood or lymphatic circulation.

[0058] The present disclosure also provides pharmaceutical compositions, for example, comprising chemical genetic regulation compounds as described above. The pharmaceutical compositions of the present disclosure can be applied in a variety of ways, depending on whether local treatment or systemic treatment is needed and the area to be treated. Application can be epidermal (including eyes and mucous membranes, including vaginal and rectal delivery), pulmonary (for example, by inhalation or blowing into powder or aerosol, including by a nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. In certain embodiments, nucleic acid constructs and / or their products are applied by a method bypassing the blood-brain barrier (BBB), including, for example, direct application to the surface of CNS, the substance of CNS, the ventricles of CNS and the cerebrospinal fluid (CSF) of CNS. In particular, intrathecal and epidural administration can be achieved by a single injection, a series of single injections and / or by continuous administration to CSF. In certain embodiments, continuous administration to CSF ​​is provided by a programmable external pump. In other embodiments, continuous administration is provided by a programmable implantable pump. Compositions and preparations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions, which may also contain buffers, diluents and other suitable additives, such as but not limited to penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. The medicaments that enhance nucleic acid construct uptake at the cellular level may also be added to medicines of the present disclosure and other compositions. For example, cationic lipids such as lipofectin (U.S. Patent number 5,705,188), cationic glycerol derivatives and polycationic molecules such as polylysine (WO 97 / 30731) also enhance the cellular uptake of oligonucleotides.

[0059] Reagent test kit

[0060] The present disclosure provides a kit comprising components of the chemical genetic peptide activation assay described herein. Such kits may include, for example, nucleic acids encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site, nucleic acids encoding peptides, ligands bound to the ligand binding site, gene transfer vectors and / or reagents, stabilizers, buffers, instructions, positive control reagents and negative control reagents, and two or more of the ligands for identifying possible errors and contamination sources. In some embodiments, the positive control peptide achieves statistical significance, which indicates that the positive control experiment meets the specifications. In some embodiments, the kit of the present invention includes calibration reagents (including, for example, luminescent calibration reagents) and ligands. In some embodiments, the kit includes customizable options. In some embodiments, the kit of the present invention includes frozen (e.g., prepared with glycerol for storage at -20°C) components and reagents, and / or dried reagents and components.

[0061] Compositions and reaction mixtures

[0062] The present disclosure provides compositions and reaction mixtures comprising the components of the assays described herein. Such compositions and reaction mixtures may include, for example, nucleic acids encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site, nucleic acids encoding peptides, ligands bound to the ligand binding site, gene transfer vectors and / or reagents, stabilizers, buffers, instructions, positive control reagents and negative control reagents, and two or more of the ligands for identifying possible errors and contamination sources. In some embodiments, the compositions and reaction mixtures of the present invention include one or more fusion protein products in the nucleic acid constructs provided herein.

[0063] Experimental Examples

[0064] The following examples are provided to demonstrate and further illustrate certain embodiments and aspects of the present disclosure and should not be construed as limiting the scope thereof.

[0065] Example 1 - Design and directed evolution of CapN.

[0066] Figure 1 a shows the CAP used to caging the N-terminus of the peptide. The design of CapN is based on the improvement of the FK506 binding protein mutant FKBP12(F36V) (referred to as FKBP) for LID 7 In LID, a degradation sequence of a 5-amino acid peptide is blocked by FKBP by being fused to a binding sequence that interacts with FKBP. When the FKBP ligand shield-1 is added to replace the binding sequence, the degradation sequence is activated. The LID ligand shield-1 has a subnanomolar affinity for FKBP, which is more than 1000 times higher than the affinity for wild-type FKBP. 24Therefore, shield-1 has minimal interference with the physiological function of endogenous FKBP. shield-1 is membrane permeable and has been used previously in living animals 25 .

[0067] The LID was tested for its ability to caging two short peptides, which had previously been shown to be controlled by the AsLOV2 domain. 26 ) and octapeptide (modified SsrA 27 ) were displayed on the yeast surface and the protease cleavage assay was used ( Figure 1 b) and binding assays ( Figure 1 c) to evaluate accessibility. The degradation sequence (RRRGN) in the LID was replaced with TEVcs or SsrA, and the rest of the binding sequence and linker were retained ( Figure 1 d). No shield-1 dependence was observed for TEVcs or SsrA, indicating that LID is ineffective for introducing steric blockade in peptides other than the original degradation sequence.

[0068] To test whether the amino acids near the C-terminus of the binding sequence are critical for interaction with the hydrophobic ligand binding site of FKBP ( Figure 2 ), these residues were adjusted. Yeast surface-based directed evolution was used to improve the shield-1 dependency of CapN, using TEVcs as the caging peptide. The first library was generated by introducing site-saturation mutagenesis into the last 6 amino acids of the binding sequence before TEVcs ( Figure 1 d). To ensure sufficient length for binding and caging, three additional libraries were constructed by adding 1, 2, or 3 additional amino acids to the random binding sequence ( Figure 1 d) According to Figure 1 Four libraries were selected using the schemes shown in Figures 1a and 1b. In the absence of shield-1 (high expression and low cleavage), a round of negative selection was performed to retain CapN variants with tightly closed cages. This also ensured that all clones entering the second round of selection expressed the entire DNA construct without early stop codons. A round of positive selection was then performed to enrich for clones that could effectively uncage TEVcs after the addition of shield-1. After 2 rounds of selection, the CapN variant library showed shield-1 dependence. Two additional rounds of selection were performed to further enrich for target clones.

[0069] After 4 rounds of directed evolution, sequencing of 40 individual clones revealed 23 different sequences rich in hydrophobic amino acids ( Figure 2 ). Eight clones were characterized as appearing more than once and having similar levels of improvement. The most enriched clone was the evolved CapN ( Figure 1d). The evolved protein shows tighter closure and greater response to shield-1 compared to the pre-evolution CapN ( Figure 1 f). Enhanced closure and greater dynamic range were also observed when the same evolved CapN was applied to SsrA ( Figure 1 g). These results demonstrate the general applicability of CapN for controlling short peptides.

[0070] We then characterized the concentration of shield-1 required to activate CapN ( Figure 3 ). Using the same yeast surface binding assay, the dose response curve showed a half-maximal response at 53 nM, indicating that shield-1 has a high affinity for CapN. It was then assessed whether the binding between shield-1 and CapN is reversible. Even after washing away shield-1 for 12 hours, CapN remained open ( Figure 4 ), indicating that shield-1 stably binds to CapN, which provides an irreversible "on" switch for CapN.

[0071] Example 2 - Design and directed evolution of CapC

[0072] After the development of CapN, a similar CAP was designed that can caging the C-terminus of peptides (CapC). Caging of peptides is generally most effective when key residues on the peptide are tightly bound to the CAP protein. Peptides with critical C-terminal sequences can be better caging by CapC than by CapN. In addition, CapN and CapC can be used in tandem to further improve the efficiency of caging and reduce "leakage" in the absence of shield-1. These features are critical for application in living animals because proteins can be expressed for weeks to months before controlled protein activation is needed.

[0073] To test whether the shield-1 binding sequence functions independently of its association with FKBP, the shield-1 binding sequence was fused to the N-terminus of FKBP using a flexible linker ( Figure 5 a), and the efficacy of this binding sequence was tested in the caging of the SsrA peptide using the same binding assay as used for CapN ( Figure 5b). SsrA was chosen because CapN showed a greater dynamic range when caging SsrA than TEVcs (difference between “+shield-1” and “-shield-1” conditions), thus facilitating selection during directed evolution. To design the linker, the distance from the N-terminus of FKBP to its ligand binding site was estimated, and a flexible glycine- and serine-rich linker was used to span this distance. The initial CapC construct showed shield-1 dependence ( Figure 5 c, first column), whose dynamic range is smaller than CapN.

[0074] Directed evolution was used to enhance the dynamic range of CapC. To generate the library, site-saturation mutagenesis was performed simultaneously in two key regions ( Figure 5 a): (1) The first 2 or 3 amino acids of the binding sequence; the region adjacent to the peptide target is most critical for pulling the binding sequence deep into the FKBP ligand binding site; (2) The first few amino acids immediately following the binding sequence on the linker; this region can be critical for positioning the binding sequence in the FKBP ligand binding site. The last 3 amino acids of the binding sequence (Pro-Asn-Leu) are retained because they provide a hydrophobic surface that is important for binding. The importance of the hydrophobic leucine residue is supported by all-atom molecular dynamics simulations, which showed a high tendency for leucine to interact with the FKBP hydrophobic ligand binding site ( Figure 6 ).according to Figure 5 The three libraries were combined and selected using the schemes shown in 5b and 5c. In the absence of shield-1 (high expression and low biotin signal), a round of negative selection was performed by tight cage to retain CapC variants. Then, in the presence of shield-1 (low expression and high biotin signal), a round of positive selection was performed to retain variants that uncaged the peptide. After 2 rounds of sorting, the shield-1 dependence of the CapC library was enhanced. We characterized 20 individual clones and identified one clone with tight cage and large dynamic range as the evolved CapC ( Figure 5 a, 5c and Figure 7 ).

[0075] Since undesirable leakage is a common problem with protein cages, CapN and CapC used together were tested for tighter closure and reduced background. Using TEVcs as the caging peptide, it was found that using two CAPs in tandem significantly reduced leakage compared to using either CapN or CapC alone, while still enabling uncaging by shield-1 ( Figure 8 ), which indicates that the simultaneous use of two CAPs can improve the cage closure efficiency and reduce unwanted leakage. Therefore, the tandem CAP was used in the following cell culture and animal experiments.

[0076] Example 3 - CAP controls protein translocation to the plasma membrane in HEK 293T cells

[0077] The trafficking of proteins to different subcellular compartments is closely related to their function, and controlled localization to the plasma membrane has been used to manipulate cellular processes such as phagocytosis. 28 and calcium influx 29 To test the application of CAP in protein translocation, a shield-1-induced membrane localization system was designed ( Fig. 9 ). In this design, the CAP-caged SsrA peptide is localized to the plasma membrane of HEK 293T cells by fusion to a transmembrane domain. The SspB protein, an interacting partner of SsrA, is fused to the protein of interest (POI). As an example, the evolved green fluorescent protein (EGFP) is used as the POI. If shield-1 is not present, binding between SsrA and SspB does not occur, and EGFP is present throughout the cell. Addition of shield-1 uncages SsrA to release the CAP and allows SsrA to bind to SspB-EGFP, resulting in translocation of EGFP to the membrane. Fig. 9 As shown in (b), in the absence of shield-1, EGFP localizes to the cytosol and nucleus, with no apparent membrane pattern, as predicted. Upon addition of shield-1, translocation of EGFP to the plasma membrane is observed within seconds.

[0078] These results show that shield-1 can readily permeate the cell membrane and open the CAP within seconds, suggesting that the CAP-shield-1 system is useful for experiments that require rapid temporal control. These results also show that the CAP system retains its functionality when transferred directly from the yeast surface to mammalian cell culture without the need for reoptimization. By directing CAP-SsrA to different subcellular compartments using appropriate localization signals, POIs can be recruited to various destinations in a shield-1-dependent manner.

[0079] Example 4 - CAP delocalizes proteins from the plasma membrane to the cytosol in HEK 293T cells

[0080] Other designs show the reverse process of membrane localization, i.e., removal of the POI from the plasma membrane where it is used, e.g., to perturb the function of the protein in a time-controlled manner when localized to the plasma membrane. Fig. 9c) In the absence of shield-1, TEVcs cannot be reached by the co-expressed TEV protease and the POI remains localized on the plasma membrane. Under the action of shield-1, TEVcs is uncaged and cleaved by the TEV protease, thereby releasing the POI from the cell membrane. Fig. 9 d and Fig.10 It is shown that, prior to shield-1 addition, an exemplary POI (ie, EGFP) is almost exclusively bound to the plasma membrane. Addition of shield-1 removes EGFP from the plasma membrane and significantly increases the presence of EGFP in the cytoplasm.

[0081] Thus, this system delocalizes the POI from its membrane location to a cytoplasmic location in a shield-1-dependent manner. Similar to CAP-caged SsrA, CAP was able to efficiently caging TEVcs when transferred directly from the yeast surface to mammalian cell culture. The required modification was to change the P1' position (last amino acid) on TEVcs from the typical glycine to the less active methionine to reduce undesired cleavage, as the TEV protease is present for much longer periods of time in this assay (2-3 days) than in the yeast surface assay (3 hours).

[0082] Example 5 - CAP Control of Nuclear-Cytoplasmic Distribution of Proteins in HEK 293T Cells

[0083] The nucleocytoplasmic distribution of many eukaryotic proteins is a common determinant of their function 30 Previously, the NLS was controlled by light through custom engineering of the AsLOV2 domain. 13,14 To test whether the NLS is controlled by CAP in a shield-1-dependent manner, a single-chain construct was designed in which the POI was expressed as a fusion protein fused to the CAP-caged NLS ( Fig. 9 e) In the absence of shield-1, the NLS is sterically blocked and the POI should be present throughout the cell. Addition of shield-1 opens the CAP and exposes the NLS to endogenous importins, which bring the POI into the nucleus. Due to the strength of the NLS, the weak nuclear export signal PKIt 13 was added to the construct to reduce the nuclear localization of the protein in the basal state. Fig. 9 f and Fig.11 It is shown that in the absence of shield-1, the exemplary POI (i.e., EGFP) is present in both the cytosol and the nucleus. After the addition of shield-1, EGFP is eliminated from the cytosol and preferentially localized in the nucleus, with a cytosol to whole cell ratio that is statistically different from that in the basal state.

[0084] These results demonstrate the general applicability of CAP: CAP can be transferred not only to the originally optimized TEVcs and SsrA clades, but also to other peptides (e.g., NLS). In the absence of shield-1, a small amount of nuclear localization ( Fig. 9 f and Fig.10 ) indicates that NLS is not completely blocked by CAP. To further eliminate the nuclear pattern in the absence of shield-1, a weaker NLS sequence can be used 13 .

[0085] Example 6 - Time-gated gene transcription in HEK 293T cell culture and neuronal culture using CAP

[0086] Gene transcription is a ubiquitous process in all living organisms. Temporally controlled gene transcription provides a useful means to study the functional role of specific proteins in living cells and animals. To test the utility of CAP in controlling gene transcription, a split transcription factor based two-hybrid system was designed. 31 ( Fig.12 a). In this design, CAP-caged SsrA is fused to the DNA binding domain (DBD) and SspB is fused to the transcriptional activation domain VP16. In the presence of shield-1, SspB-VP16 is recruited to DBD-SsrA and initiates transcription of the reporter gene.

[0087] The system was tested in HEK 293T cells using the Gal4 DBD and UAS-mCherry reporter gene. Fig.12 b), shield-1-induced mCherry intensity increases of approximately 4-fold and approximately 25-fold were observed in CapN and CAP-caged SsrA. Fig.12 c and Fig.13 The tandem use of CAPs showed lower background and higher dynamic range compared to the use of CapN alone, which is consistent with previous observations of CAP-caged TEVcs on the yeast surface ( Figure 8 ). The amino acid sequence of SsrA is adjustable 1,16 Since transcription systems are highly sensitive and are generally only of practical use when undesired transcription is minimized, we modified the SsrA sequence from the N-terminus and C-terminus to further reduce background ( Fig.12 b and Fig.14 ) to test for more effective closure. The 7-amino acid SsrA sequence (AANDENY) ( Fig.12 d and Fig.14) showed a 156-fold change in shield-1-dependent reporter gene expression, 2-fold lower background, and 6-fold higher signal-to-noise ratio compared to the original sequence tested ( Fig.12 c). The expression level of SspB-VP16 indicated by the EGFP signal is positively correlated with the amount of shield-1 added and the expression of the reporter gene. Conditions with higher shield-1 concentrations and higher reporter gene expression levels show higher EGFP signals. Since shield-1 alone added to EGFP-SspB-VP16 does not increase the level of EGFP ( Fig.15 ), therefore these results suggest that the SsrA-SspB interaction stabilizes the SspB protein and that the non-interacting SspB is degraded.

[0088] The shield-1 concentrations and shield-1 incubation times required to induce strong gene expression were tested. After overnight incubation, shield-1 concentrations below 10 nM did not induce gene expression, while 50 nM or above induced >30-fold gene expression ( Fig.16 ). Consistent with the above results, although all conditions were transduced with the same amount of virus, conditions with higher shield-1 concentrations also showed higher EGFP signals, consistent with the stabilizing effect of SspB-SsrA heterodimerization. Regarding shield-1 incubation time, an incubation period as short as 30 minutes was sufficient to induce gene expression at 1 μM shield-1 concentration, but longer incubation times (on the order of hours) were required for more robust gene expression ( Fig.17 ).

[0089] To test the application of the CAP chemical-dependent transcription system in neuroscience, we performed experiments in cultured neurons. To achieve uniform expression of DNA in a rigorous experiment, we generated a single viral construct that expresses the DNA binding and transcription activation domains via either the self-cleaving peptide P2A or an internal ribosome entry site (IRES). Fig.12 b). After addition of shield-1, the P2A and IRES constructs showed an 83-fold and 123-fold increase in mCherry expression, respectively, which is comparable to the two-component system ( Fig.12 c and Fig.18 ). The TetR DBD and VP16 transcriptional activation domain were then introduced into cultured rat cortical neurons using the P2A construct via adeno-associated virus (AAV) along with another AAV encoding the TRE-mCherry reporter gene. Shield-1 induced a 44-fold increase in mCherry reporter gene expression compared to conditions in which shield-1 was absent ( Fig.12 e and Fig.19), indicating that the system functions robustly in cultured neurons.

[0090] Example 7 - Controlled gene transcription in living animals using CAP

[0091] To test chemical-dependent peptide regulation in living organisms, the CAP system was applied to the mouse brain and liver. For application in the brain, AAV encoding a shield-1-dependent gene regulation construct was stereotaxically injected into the lateral hypothalamic area (LHA). Seven days after viral delivery, aquashield-1, a water-soluble analog of the shield-1 molecule, was topically administered to the LHA. Forty-eight hours after treatment with aquashield-1 or saline, mice were euthanized and perfused, and brain tissue was processed for analysis ( Fig. 20 a). In saline-treated control brains (-aquashield-1), only a few sparse neurons with mCherry expression were observed throughout the LHA region ( Fig. 20 b). In contrast, mCherry was observed in a large cluster of LHA neurons in aquashield-1 treated brains (+aquashield-1) at levels >16-fold higher than controls ( Fig. 20 b), indicating that shield-1-dependent gene expression occurs.

[0092] To test the application of the CAP chemical-dependent gene regulation system outside of neural tissue, AAV encoding the CAP construct was injected into the mouse liver. Aquashield-1 or saline ( Fig. 20 c). Two days after the first aquashield-1 injection, liver tissue was harvested for analysis. In saline-injected control mice, very few cells throughout the liver expressed mCherry, whereas in the livers of aquashield-1-treated animals, there was a large increase in cells expressing mCherry ( Fig. 20d), indicating that systemic injection of aquashield-1 controlled CAP in mouse liver. These results show the advantages of using small molecules to control peptide activity compared to light. Light is limited by its illumination range and is difficult to administer systemically in living animals. In contrast, injection brings small molecules into the entire body of the animal, allowing comprehensive control of peptide activity in multiple organs of the body. In summary, these results show that gene regulation induced by CAP aquashield-1 plays a role in multiple organs of living organisms, including the brain and liver. Subsequently, aquashield-1 can be easily administered by non-parenteral injection to activate gene transcription in the tissues of the target animal. Compared to light-induced gene transcription systems, these characteristics of CAP can provide systemic control with less disturbance to the animal.

[0093] Example 8 - CapC Regulation of the Opioid Peptide Met-Enkephalin

[0094] To detect activation of the CapC opioid receptor, a split NanoLuc assay was developed, with one half of the assay fused to the opioid receptor and the other half fused to the G i -Mimetic nanobody Nb44. Opioid receptor activation results in the recruitment of Nb44 to the receptor and the restoration of the split NanoLuc. Cyclic adenosine monophosphate (cAMP)-dependent luciferase (GloSensor) is used to monitor the activation of the μ-opioid receptor. By expressing the real-time sensing GloSensor in cells, the greater the luminescence observed when the cAMP concentration is higher, and the smaller the luminescence observed when the cAMP concentration is lower. The GloSensor assay is complementary to the split NanoLuc assay because it measures downstream events, not just the binding between the G protein and the receptor (i.e., membrane-tethered opioid peptide (M-PROBE)), and activation not only recruits the G protein, but also causes downstream signaling events.

[0095] To test shield-1 chemical-dependent regulation of opioid peptides, CapC-caged methionine-enkephalin was fused directly to the N-terminus of opioid receptors, as Fig. 22 In this design, M-PROBE cell membrane tethered opioid peptides (M-PROBE) and opioid receptors are expressed in close proximity at a fixed ratio. Fig.23The use of shield-1 dependence to control opioid receptor activity by chemically gated membrane-tethered opioid peptides in HEK293T cells is shown. The antagonist "naloxone" condition provides a basal condition in the absence of receptor activation. The "drug-free" condition does not add external drug molecules, but there is indeed a methionine-enkephalin in the CapC cage on the cell surface, and its signal comes from the basal activity of M-PROBE (i.e., background or leakage). Loperamide is a full agonist of μ-opioid receptors (MOR). The "loperamide" condition shows the highest signal. Both shield-1 and loperamide increase the signal by the uncaging of opioid peptides (i.e., methionine-enkephalin) from CapC (i.e., shield-1-induced methionine-enkephalin release increases receptor activation), or by the direct action of loperamide on MOR.

[0096] To test whether M-PROBE acts as a free opioid to induce downstream signaling and whether the function of Met-Enkephalin is well preserved in a membrane-tethered geometry, the same single-chain Met-Enkephalin-CapC-MOR was expressed on the membrane of HEK 293T cells and co-transfected with cAMP-dependent luciferase. After overnight protein expression, HEK 293T cells were stimulated with forskolin to increase intracellular cAMP concentrations. MOR is an inhibitory G protein-coupled receptor (GPCR) whose activation reduces cAMP levels. High cAMP concentrations amplify the effects of MOR activation. Fig.24 The cells were treated under four different conditions: no drug, shield-1, MOR agonist [D-Ala 2 、N-MePhe 4 , Gly-ol]-enkephalin (DAMGO) or the MOR antagonist naloxone. Untreated cells showed no change in luminescence levels. Cells treated with high concentrations (10 micromoles / liter) of DAMGO showed a large reduction in luminescence (about 80%). The luminescence levels of shield-1 treated cells were between these two conditions, with a reduction in luminescence levels of about 50%. Cells treated with high concentrations (10 micromoles / liter) of naloxone showed a sharp increase in luminescence signals. This increase was even greater than that of cells treated with 1 micromoles / liter forskolin. These results indicate that in binding assays and downstream cAMP assays, CapC controls methionine-enkephalin to activate MOR in a drug-dependent manner, thereby providing methods, compositions, kits and systems for regulating target endogenous opioid receptors in specific cell populations or neuronal circuits in the central nervous system.

[0097] Experimental methods

[0098] clone

[0099] Constructs for yeast surface display were cloned into pCTCON2 vector. Constructs for protein expression in HEK 293T cells were cloned into pAAV viral vector for transfection, or pLX208 lentiviral vector for transduction. Constructs for protein expression in neuronal culture, mouse brain, and mouse liver were cloned into pAAV vector.

[0100] The FKBP of CapN was amplified from YFP-LID (Addgene plasmid #31767, Thomas Wardless laboratory). The codon-optimized FK BP of CapC was also synthesized by IDT.

[0101] For cloning, PCR fragments were amplified using Q5 or Taq DNA polymerase (New England Biolabs (NEB)). The vectors were double digested with restriction enzymes (NEB), gel purified, and ligated to the gel purified PCR fragments by T4 ligation, Gibson assembly, or In-Fusion HD Cloning Pluss kit (Takara Bio). The ligated plasmid products were introduced into competent XL1-Blue E. coli cells by heat shock transformation or, for In-Fusion cloning, into Stellar competent E. coli cells from the kit according to the corresponding protocols. For In-Fusion cloning, a modified protocol was used that proportionally reduced the amount of each reagent or competent cell by half or up to three quarters of the recommended amount.

[0102] Expression and purification of TEV protease

[0103] The full-length TEV protease (TEVp, S219V) was expressed as a fusion of maltose binding protein (MBP) with a polyhistidine tag. The His tag-MBP-TEVp (S219V) in the pYFJ16 vector was introduced into competent BL21-CodonPlus (DE3)-RIPL E. coli cells by heat shock transformation. The cells were cultured in 5 mL of Miller's LB medium (Bio Basic) supplemented with 100 mg / L ampicillin at 37°C with shaking at 220 rpm for 6 hours. The saturated culture was then transferred to 500 mL LB containing 100 mg / L ampicillin and grown at 37°C with shaking at 220 rpm for approximately 2-3 hours until the OD 600=0.4-0.8. IPTG (isopropyl β-D-1-thiogalactopyranoside, EMDMillipore) was added to the culture to a final concentration of 1 mM, and the culture was grown overnight at 16°C with shaking at 220 rpm. Unless otherwise specified, the following procedures were performed at 4°C. The cells were harvested by centrifugation at 5,000 rpm for 5 minutes. The cell pellet was lysed and resuspended with 15 mL of ice-cold B-PER bacterial protein extraction reagent (Thermo Fisher Scientific). DTT (Fisher, freshly prepared) was supplemented to a final concentration of 1 mM. Benzonase nuclease (Millipore-Sigma) was added to a final concentration of approximately 100 units / ml. The mixture was incubated on ice for 5 minutes and centrifuged at 10,000 rpm for 15 minutes. The supernatant was incubated with 3 mL of Ni-NTA resin (Thermo Fisher Scientific) for 10 minutes and then transferred to a gravity column. The resin was washed with 5 mL of wash buffer (30 mM imidazole, 50 mM Tris, 300 mM NaCl, 1 mM DTT, pH = 7.8), and the protein was eluted with 3 mL of elution buffer (200 mM imidazole, 50 mM Tris, 300 mM NaCl, 1 mM DTT, pH = 7.8). The eluate was concentrated with a 15 mL 10,000 Da cut-off centrifuge (Millipore), snap frozen in liquid nitrogen, and stored at -80°C. In order to obtain effective TEVp, each batch of TEVp was concentrated at least 10 times. The changes in TEVp yield and activity between batches were tracked.

[0104] Expression and purification of SspB-APEX2

[0105] SspB-APEX2 in the pYFJ16 vector was expressed with a polyhistidine tag in competent BL21 E. coli cells, the expression being identical to that described above for the expression of TEV protease (under "Expression and purification of TEV protease"), except that no DTT was added to the cell lysate, wash buffer, or elution buffer. No protein concentration was performed.

[0106] Yeast strains and non-library cultures

[0107] Non-library yeast cultures were generated by chemically transforming the yeast surface display plasmid pCTCON2 into Saccharomyces cerevisiae strain EBY100 competent cells. The preparation of EBY100 competent cells has been described. 39. For transformation, 1 μg of plasmid DNA was mixed with 5 μL of competent cells. 200 μL of Frozen-EZ Yeast Solution 3 (Zymo Research) was added and mixed thoroughly. The mixture was incubated at 30°C for 30 minutes to 2 hours and then transferred to 5 mL of SDCAA (Synthetic Dextrose Plus Casein Amino Acids Medium, 2% Glucose, 0.67% Yeast Nitrogen Base without Amino Acids (BD Difco), 0.5% Casein Amino Acids (BD Difco), 0.54% Disodium Hydrogen Phosphate, 0.856% Sodium Hydrogen Phosphate) without tryptophan and grown at 30°C with shaking at 220 rpm. At initial saturation (OD 600 >10), within 2-3 days, the yeast culture was passaged at least once before the experiment, or stored at 4°C for up to half a year. Passaging was performed by adding 500 μL of saturated culture to 5 mL of fresh SDCAA medium and growing overnight at 30°C and 220 rpm. Each batch of transformation was accompanied by a negative control without plasmid DNA to ensure the selectivity of the medium.

[0108] Yeast library generation

[0109] CapN and CapC mutant libraries were generated in the following manner: first, targeted mutagenesis was used to generate a plasmid library, which was then transformed into EBY100 yeast competent cells by electroporation. Targeted mutagenesis was performed by conventional PCR (polymerase chain reaction) using primers and mixed bases (IDT). The first two bases in each codon corresponding to the mutant amino acid were designed as an equal mixture of A, C, G, T, and the third base was designed as an equal mixture of G and T. The PCR fragment was amplified so that it had 40 additional bases outside the two restriction sites used to linearize the vector.

[0110] 500 ng of template DNA plasmid (Aga2p-FLAG-FKBP-binding sequence-TEVcs-HA) was mixed with 100 μmol of forward primer and reverse primer annealed outside the FKBP gene, 1×Q5 high GC enhancer, 1×Q5 reaction buffer, 1 unit of Q5 high-fidelity polymerase, and 10 nmol dNTP (VWR) in a total volume of 50 μL (two reactions were performed for each library).

[0111] CapN library:

[0112] Forward Primer:

[0113] All libraries:

[0114] SEQ ID. NO:9

[0115] GGCTCTGGTGCTAGCGACTACAAGGATGACGACGATAAGA CTAGT

[0116] Reverse Primer:

[0117] Library 1:

[0118] SEQ ID NO:10

[0119] GGATCCACCCTGGAAGTAGAGATTTTCMNNMNNMNNMMNNMNNMNNCGCCACTTCCTCCACTCCACGC

[0120] Library 2:

[0121] SEQ ID NO:11

[0122] GGATCCACCCTGGAAGTAGAGATTTTCMNNMNNMNNMNNMNNMNNMNNCGCCACTTCCTCCACTCCACGC

[0123] Library 3:

[0124] SEQ ID NO:12

[0125] GGATCCACCCTGGAAGTAGAGATTTTCMNNMNNMNNNMNN MNNMNNMNNMNNCGCCACTTCCTCCACTCCACGC

[0126] Library 4:

[0127] SEQ ID NO:13

[0128] GGATCCACCCTGGAAGTAGAGATTTTCMNNMNNMNNMMNN MNNMNNMNNMNNMNNCGCCACTTCCTCCACTCCACGC

[0129] PCR was performed for 20 cycles with an annealing temperature of 60°C. The product DNA was gel purified and amplified with the following primers:

[0130] Forward Primer:

[0131] All libraries:

[0132] SEQ ID NO:14GGCTCTGGTGCTAGCGACTACAAGGATGA CGACGATAAGACTAGT

[0133] Reverse Primer:

[0134] All libraries:

[0135] SEQ ID NO:15

[0136] CTCGAGCTATTAAGCGTAATCTGGAACGTCATATGGGTAGG ATCCACCCTGGAAGTAGAGATTTTC

[0137] CapC library:

[0138] Forward Primer:

[0139] Library 1:

[0140] SEQ ID NO:16

[0141] GCTAGCGCAGCGAATGATGAAAATTACTTCNNKNNKNNKC CTAATTTGNNKNNKNNKGGATCAGGCGGTTCTGGTACTG

[0142] Library 2:

[0143] SEQ ID NO:17

[0144] GCTAGCGCAGCGAATGATGAAAATTACTTCNNKNNKCCTAA TTTGNNKNNKNNKNNKTCAGGCGGTTCTGGTACTGG

[0145] Library 3:

[0146] SEQ ID NO:18CTGCAGCAAGGTCTGCAGG

[0147] Reverse Primer:

[0148] Library 1:

[0149] SEQ ID NO:19TCAGATCTCGAGCTATTACTTATCGTCGTC

[0150] Library 2:

[0151] SEQ ID NO:20TCAGATCTCGAGCTATTACTTATCGTCGTC

[0152] Library 3:

[0153] SEQ ID NO:21

[0154] GAGATGGTTTCCACCTGCACTCCMMNNMNNMNNMNNMNN MNNTCCAGTACCAGAACCGCCTG

[0155] PCR was performed for 20 cycles with an annealing temperature of 60°C. The product DNA was gel purified and amplified with the following primers:

[0156] Forward Primer:

[0157] Library 1:

[0158] SEQ ID NO:22

[0159] CTGCAGCAAGGTCTGCAGGCTAGTGGTGGAGGAGGCTCTG GTGCTAGCGCAGCGAATGATGAAAATTACTTC

[0160] Library 2:

[0161] SEQ ID NO:23

[0162] CTGCAGCAAGGTCTGCAGGCTAGTGGTGGAGGAGGCTCTG GTGCTAGCGCAGCGAATGATGAAAATTACTTC

[0163] Library 3:

[0164] SEQ ID NO:24GGAGGTGCAGGTGGAAACCATCTC

[0165] Reverse Primer:

[0166] All libraries:

[0167] SEQ ID NO:25TCAGATCTCGAGCTATTACTTATCGTCGTC

[0168] The template DNA was evenly divided into 8 parts. Each part was mixed with 100 μM forward primer and reverse primer, 1× Taq reaction buffer without magnesium chloride, 2 mM magnesium chloride, 2 units of Taq polymerase, 10 nmol dNTP (VWR) in a total volume of 50 μL. For the same library, 8 PCRs were gel purified and combined.

[0169] The linearized vector was gel purified. We combined 2 μg of the linearized vector with 8 μg of the PCR fragment and concentrated using pellet paint (Millipore) according to the manufacturer's protocol on the day of electroporation. The precipitated DNA was resuspended in 20 μL of ultrapure water (Thermo Fisher Scientific).

[0170] Meanwhile, prepare fresh electrocompetent EBY100 yeast cells. Prior to this procedure, passage the cells at least twice in YPD (yeast extract peptone dextrose medium, 20 g glucose, 20 g peptone, and 10 g yeast extract in 1 L deionized water) to ensure cell health. Inoculate 200 mL of YPD with a saturated yeast culture to an initial OD of 0.3-0.4. 600 The cells were grown at 30 °C with shaking at 220 rpm for approximately 6 hours until the OD 600 Reach 1.8-2.2, then centrifuge at 4 ℃ and 3,000rpm for 3 minutes. The cell pellet is resuspended and washed with ice water, centrifuged again, and resuspended in 50mL ice-cold sterile lithium acetate (100mM, soluble in water). After this step, the yeast is placed on ice until after electroporation. Add DTT to a final concentration of 10mM. The cells are then incubated at 30 ℃ and 220rpm for 20 minutes, centrifuged at 4 ℃ and 3,000rpm for 3 minutes, washed with 50mL ice water, centrifuged again, and resuspended in 0.8mL electroporation buffer (1M sorbitol / 1mM CaCl2).

[0171] Immediately after yeast cell preparation, 400 μL of cells were mixed with 20 μL of concentrated DNA prepared as described above, and the mixture was then transferred to the electroporation sample pool. Electroporation was performed using a Bio-Rad Gene pulser XCell, set as follows: 500V, 15 millisecond pulse duration, only one pulse, 2 mm sample pool. Immediately rescue cells with a mixture of 1 mL 1:1 sorbitol and YPD culture medium. The sample pool was washed 3 times, each time with 1 mL of fresh sorbitol and YPD mixture. All cells were combined and incubated at 30°C for 30 minutes (without shaking), then shaken at 220 rpm for 30 minutes, 10 μL of cells were plated on three SDCAA plates, and serial dilutions of 100×, 1,000× and 10,000× were performed to determine the library size. The remaining cells were centrifuged at 3,000 rpm for 2 minutes, resuspended in 5 mL of SDCAA medium, centrifuged again, and transferred to 200 mL of SDCAA medium supplemented with 1% penicillin-streptomycin (50 units / mL penicillin and 50 μg / mL streptomycin, Gibco) and 30 μg / mL kanamycin (DOT Scientific). The culture was grown at 30°C with shaking at 220 rpm for 12-24 hours until the OD 600 Reach 15 (OD 600 About 1 is equivalent to about 1×10 7 For each batch of library generation, a negative control containing no plasmid DNA was included.

[0172] The combinatorial library sizes of CapN and CapC (four library combinations for CapN and three library combinations for CapC) were determined to be approximately 1 × 10 7 .

[0173] Yeast marker

[0174] The labeling procedure was applied to non-library yeast culture and yeast library culture. (The generation of non-library and library yeast culture was performed as described in "yeast strains and non-library culture" and "yeast library generation" above, respectively.) Yeast was freshly passaged in SDCAA medium before the experiment. In order to induce the expression of pCTCON2 plasmid, 500 μL of overnight yeast in SDCAA medium was added to 5 mL SGCAA (synthetic galactose plus casamino acid medium, 2% galactose, 0.67% yeast nitrogen base (BD Difco) without amino acids, 0.5% casamino acids (BD Difco), 0.54% disodium hydrogen phosphate, 0.856% sodium dihydrogen phosphate) medium, and grown overnight at 30 ° C under 220 rpm shaking. Before labeling, 250 μL (or 1 mL for the first round of library selection) of overnight yeast in SGCAA medium was centrifuged at 8000 rpm for 30 seconds, and the supernatant was discarded. The cell pellet was resuspended and washed twice with 1 mL PBSB (sterile phosphate-buffered saline supplemented with 0.1% bovine serum albumin) each time.

[0175] Yeast expressing the TEV protease cleavage site (CapN construct) was treated with TEVp before labeling with an antibody-fluorophore conjugate. The samples were incubated with 200 μL PBSB containing TEVp (expressed and purified as described above in "Expression and purification of TEV protease") and 5 μM shield-1 at 4°C for 3 hours with rotation. For negative controls, PBSB did not contain TEVp or shield-1, or both. 1 mM DTT and 30 mM reduced glutathione and 3 mM oxidized glutathione were added to all samples to maintain TEVp under reducing conditions.

[0176] APEX2 labeling of yeast expressing SsrA (CapC construct) prior to labeling with antibody-fluorophore and streptavidin-fluorophore conjugates 40. The samples were incubated with 100 μL PBSB containing SspB-APEX2 (expressed and purified as described above in "Expression and Purification of SspB-APEX2") and 5 μM shield-1 at room temperature for 10 minutes with rotation. For negative controls, PBSB did not contain SspB-APEX2 or shield-1, or both. After incubation, the samples were washed twice, each time with 1 mL PBSB, and resuspended in 950 μL PBSB containing 1% BSA (bovine serum albumin). 1 μL biotin-phenol (1 mM, dissolved in dimethyl sulfoxide) was added and mixed thoroughly with the sample. Then, 1 μL hydrogen peroxide (0.5 mM, dissolved in water, freshly prepared, EMD Chemicals) was added and mixed thoroughly. After incubation for exactly 2 minutes, 200 μL quenching solution 1 (30 mM Trolox (Thermo Fisher Scientific), 60 mM sodium ascorbate (Millipore Sigma), freshly prepared) was added. The sample was centrifuged at 8,000 rpm for 30 seconds and the supernatant was discarded. Then 400 μL of quenching solution 2 (5 mM Trolox, 10 mM sodium ascorbate, freshly prepared) was added to resuspend the cell pellet. After centrifugation again at 8,000 rpm for 30 seconds, the supernatant was discarded and the sample was washed twice, each time using 1 mL of PBSB.

[0177] After TEVp or APEX2 labeling, the samples were labeled with antibody-fluorophore and / or streptavidin-fluorophore conjugates. To label FLAG and HA epitope tags, anti-FLAG primary antibody or anti-HA primary antibody was used, followed by a secondary antibody conjugated to Alexa Fluor 568 or Alexa Fluor 647. To detect biotinylated proteins from APEX2 labeling, streptavidin conjugated to PE (phycoerythrin) was used. All antibodies were diluted to 1 μg / mL in PBSB, streptavidin-PE (Jackson Immuno Research) was diluted 200 times, and each yeast sample was rotated and incubated with 100 μL of the mixture at room temperature for 15 minutes. Wash twice with PBSB after each labeling step. All samples were resuspended in PBSB and analyzed or sorted by FACS (fluorescence activated cell sorting, as described in "FACS analysis and library selection") within 24 hours of labeling.

[0178] FACS analysis and library selection

[0179] After labeling according to the procedure described above (under "Yeast Labeling"), non-library yeast samples were analyzed using an LSRFortessa cell analysis flow cytometer (BD Biosciences) equipped with a 640nM laser and a 670 / 14 emission filter (for Alexa Fluor 647) and a 561nm laser and a 586 / 15 emission filter (for Alexa Fluor 568 and PE). Library samples were sorted using a FACSAria III cell sorting flow cytometer (BD Biosciences) equipped with a 633nM laser and a 660 / 20 emission filter (for Alexa Fluor 647) and a 561nm laser and a 582 / 15 emission filter (for Alexa Fluor 568 and PE). According to Fig.21 Individual yeast cells were selected by continuous gating P1 and further analyzed.

[0180] The CapN library was generated and labeled as described above (under "Yeast library generation" and "Yeast labeling", respectively). For positive selection, both TEVp and shield-1 were added. For negative selection, only TEVp was added, but shield-1 was not added. A total of 4 rounds of selection were performed. The number of cells collected in each round was as follows:

[0181] Round 1 (negative selection):

[0182] 0.034% of cells (1.4×10 7 cells)

[0183] From library 2, 0.041% of cells (2.2 × 10 7 cells)

[0184] 0.028% of cells (2.1×10 7 cells)

[0185] 0.025% of cells (1.0×10 7 cells)

[0186] The collected cells were combined for further selection.

[0187] Round 2 (positive selection):

[0188] 3.4% of cells (1.2×10 6 cells)

[0189] Round 3 (negative selection):

[0190] 11.3% of cells (3.5×10 6 cells)

[0191] Round 4 (positive selection):

[0192] 5.8% of cells (1.5×10 6 cells)

[0193] The CapC library was generated and labeled as described above (according to "Yeast library generation" and "Yeast labeling", respectively). For positive selection, both SspB-APEX2 and shield-1 were added. For negative selection, only SspB-APEX2 was added, but shield-1 was not added. A total of 2 rounds of selection were performed. The number of cells collected in each round was as follows:

[0194] Round 1 (negative selection):

[0195] Collect 0.3% of the cells (1.5×10 4 cells)

[0196] Round 2 (positive selection):

[0197] Collect 0.1% of the cells (3×10 3 cells)

[0198] All yeast cells were collected in 5 mL SDCAA medium containing 100 units / mL penicillin, 100 μg / mL streptomycin and 30 μg / mL kanamycin. After sorting, the cells were immediately grown at 30 ° C under 220 rpm shaking for 2-5 days until saturation. For the next round of sorting, the cells were passaged at least once in SDCAA medium and labeled according to the procedure described in "yeast labeling" above. After the last round of sorting, plasmids were extracted according to the manufacturer's protocol modified by Zymoprep yeast plasmid miniprep II kit (Zymo Research). μ overnight yeast culture (500 L) was transferred to 10 mL fresh SDCAA medium and grown to OD 600=1-2. The yeast cells were centrifuged and washed once with phosphate buffered saline (PBS), and then resuspended with 200 μL solution I and 6 μL digestion enzyme solution in the kit. Vortex vigorously for >1 minute. The yeast cells were placed in a 37°C oscillator overnight to degrade the cell wall. After overnight incubation, the yeast cells were vortexed vigorously for >5 minutes. Then, 200 μL solution II was added to the yeast cells, followed by a brief vortex and incubation at room temperature for 5 minutes. 400 μL neutralization solution was added and the cells were vortexed briefly. The cell lysate was centrifuged at 20,000 × g for 10 minutes, and the supernatant was loaded onto a DNA column (Epoch Life Science) to purify the plasmid DNA. The plasmid DNA extracted from the yeast library was transformed into XL1-Blue Escherichia coli by heat shock. Individual clones were sequenced, transformed into EBY100 yeast cells (as described above under "Yeast strains and non-library cultures"), labeled (as described above under "Yeast labeling"), and analyzed by FACS (as described earlier in this section).

[0199] All-atom molecular dynamics simulations

[0200] Atomistic molecular dynamics simulations were performed on FKBP and the N-terminal and C-terminal capped RYSPNL hexapeptide (Ac-RYSPNL-NHMe) to analyze the binding pose of the peptide to its binding site. The GROMACS2018.1 software package was used to simulate the binding of the peptide to the binding site. 41 Using the AMBER99SB-ILDN protein force field 42 and TIP3P water 43 The simulation was performed based on the structure of FKBP12 protein (PDBID: 1NSG), in which the F36V mutation was introduced using DeepView / Swiss-Pdb Viewer. 44 The peptide was placed in an alpha-helical conformation by the Scwrl4 program 45 The generated side chains are outside the putative FKBP binding site. The initial distance of the peptide mass center relative to the mass center of V36 of FKBP is The minimum distance between any atom of the peptide and the FKBP protein is 5. This allows separation of at least 2 hydration layers.

[0201] Place the system in The cubic simulation box was solvated with 13,444 water molecules in addition to the 67 water molecules isolated in the crystal structure. 38 sodium ions and 40 chloride ions were added to approach the physiological salt concentration of 150 mM and to neutralize the charges of the protein (+1e) and peptide (+1e) at pH 7. The protonation states of the protein side chains were predicted by the pdb2gmx tool in GROMACS. The simulations were performed under periodic boundary conditions with the particle-mesh Ewald 46 Algorithm usage Grid constants and fourth-order interpolation are used to handle long-range electrostatic interactions. For short-range Lennard-Jones and electrostatic interactions, use During the dynamics simulation, the LINCS algorithm 47 Used to constrain the bond lengths of proteins and to apply the SETTLE algorithm 48 Used to define the geometry of water molecules.

[0202] After 1000 steps of steepest descent energy minimization, the system was equilibrated for 100 ps in a molecular dynamics simulation at 300 K and 1 bar in an isothermal-isobaric ensemble with a simulation time step of 1 femtosecond. 49 The time constant of the thermostat and barostat is 1 picosecond. In this equilibrium, the non-hydrogen atoms of the protein and peptide are constrained to their initial positions using a force constant of Then, a 2-microsecond production simulation was performed in an isothermal-isobaric ensemble at 300 K and 1 bar without position restraints using a 2-fs time step, a Nosé-Hoover thermostat, and a 2-microsecond time step. 50,51 The time constant of the Parrinello-Rahman barostat is 1 picosecond. 52 The time constant of is 2 picoseconds. The trajectories are clustered using a simple clustering algorithm 53 Using non-hydrogen atoms and In addition, the distance between the center of mass of each side chain of the peptide and V36 of FKBP was also monitored.

[0203] Fluorescence microscopy of cultured cells

[0204] Confocal imaging was performed on a Nikon inverted confocal microscope with 10× air, 20× air, and 60× oil immersion objectives, equipped with a Yokogawa CSU-X15000RPM spinning disk confocal head and Ti2-ND-P Perfect Focus System 4, a compact 4-line laser source: 405nm (100mW), 488nm (100mW), 561nm (100mW), and 640nm (75mW) lasers. The following laser excitation and emission filter combinations were used for various fluorophores: DAPI (405nm excitation; 455 / 50 emission), EGFP / Alexa Fluor 488 (488nm excitation; 525 / 36 emission), mCherry / Alexa Fluor 568 (568nm excitation; 605 / 52 emission), Alexa Fluor 647 (647nm excitation; 705 / 72 emission), and differential interference contrast (DIC) filters. ORCA-Flash 4.0LT+ sCMOS camera. Acquisition time ranges from 100 to 1000 ms. All images were collected and processed using Nikon NIS-Elements hardware control and analysis module.

[0205] HEK 293T cell culture and transfection

[0206] Low-passage HEK 293T cells (less than 20 generations) were cultured in T25 or T75 flasks at 37°C, 5% CO2 in complete growth medium, i.e., 1:1 DMEM (Double's Modified Eagle's Medium, Gibco):MEM (Eagle's Minimum Essential Medium) supplemented with 10% FBS (fetal bovine serum, Sigma), 50mM HEPES (Gibco) and 1% penicillin-streptomycin (50 units / mL penicillin and 50μg / mL streptomycin, Gibco). For imaging experiments, 48-well plates were pretreated with 200μL 20μg / mL human fibronectin (Millipore Sigma) at 37°C for 10 minutes. HEK 293T cells were then plated in 48-well plates at 60%-90% confluence. The mixture of DNA was incubated in 10μL serum-free DMEM medium at room temperature for 15 minutes with 1μL 1mg / mL PEI max solution. Complete DMEM growth medium (100 μL) was then mixed with the DNA-PEI max solution and added to the HEK 293T cells that were fully adhered to the bottom of the wells and incubated for 18 hours before further treatment.

[0207] Generation of lentiviral supernatant for HEK cell transduction

[0208] New cell culture flasks were incubated with 20 μg / mL human fibronectin (HFN, Millipore Sigma) at 37°C for at least 10 minutes to promote cell adhesion to the surface and improve transfection efficiency. After incubation, HFN was aspirated and HEK293T cells were plated at 70%-90% confluence. For T25 flasks, cells were grown at 37°C in 5 mL complete DMEM growth medium, i.e., 1:1 DMEM (Dub's Modified Eagle's Medium, Gibco):MEM (Eagle's Minimum Essential Medium) supplemented with 10% FBS (fetal bovine serum, Sigma), 50 mM HEPES (Gibco) and 1% penicillin-streptomycin (50 units / mL penicillin and 50 μg / mL streptomycin, Gibco) for 1-3 hours. After incubation, 2.5 μg viral DNA, 0.25 μg pVSVG and 2.25 μg delta8.9 lentiviral helper plasmid were combined with 250 μL DMEM and mixed thoroughly. Then, 25 μL PEI max solution (polyethyleneimine HCl Max, pH 7.3, 1 mg / mL, Polysciences) was added. The mixture was incubated at room temperature for at least 10 minutes, mixed with 1 mL complete medium, and transferred to a T25 flask. The cells were incubated at 37 ° C for 48 hours, and the supernatant containing the virus was collected, quickly frozen in liquid nitrogen, and stored at -80 ° C for up to one year.

[0209] HEK 293T cell culture and infection

[0210] Low-passage HEK 293T cells (less than 20 passages) were cultured at 37°C and 5% CO 2 , in T25 or T75 flasks, in complete growth medium. For imaging experiments, 24-well glass bottom plates (Cellvis) were pretreated with 350 μL 20 μg / mL human fibronectin (Millipore Sigma) at 37°C for 10 minutes. HEK 293T cells were then plated in 24-well plates at 40%-60% confluence. For infection of a single well in a 24-well plate, 100-200 μL of each supernatant virus was slowly added to the top of the culture medium and incubated for 48 hours before further processing.

[0211] HEK 293T cell stimulation, imaging, and data analysis of shield-1-induced protein translocation to the plasma membrane

[0212] HEK 293T cells were plated at 80% confluence in 24-well plates and then transfected with 200 ng mCherry-CapN-SsrA-CapC-CAAX and 200 ng SspB-EGFP plasmids as described above. SEQ ID NO: 18 comprises a representative DNA sequence comprising a CapN sequence, a SsrA sequence (bold underline), and a CapC sequence (underlined).

[0213] SEQ ID NO:26:

[0214] GGCGTCCAAGTCGAGACAATATCTCCCGGCGACGGACGAACGTTTCCGAAGCGGGGACAGACTTGCGTCGTTCACTACACAGGAATGCTGGAGGATGGCAAAAAGGTCGATTCCAGCCGGGACAGGAATAAGCCGTTCAAATTCATGCTGGGAAAACAGGAGGTAATACGAGGTTGGGAGG AGGGTGTGGCGCAAATGTCTGTCGGTCAGCGGGCGAAACTGACCATTTCTCCTGATTATGCTTACGGAGCGACAGGCCATCCGGGGATCATTCCGCCCCACGCTACCCTGGTATTCGATGTAGAACTCTTGAAGCTCGAAACCAGAGGGGTTGAAGAAGTAGCCAGATATTCCCCCAACCTG GCAGCGAATGATGA AAATTACGGTACTCCTAATTTGCGGCCTTTTGGTTCAGGCGGTTCTGGTACTGGATTCTGGTTCTGGAGGTTCTGGAG TGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACC GGGATGCTTGAAGATGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCA GGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAG ATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTA AAACTGGAA

[0215] SEQ ID NOs: 27-45 comprise representative sequences comprising a CapC caged methionine-enkephalin amino acid sequence expressed as a separate construct with a binding sequence for FKBP ( Fig. 22 , left panel).

[0216] Protein sequence:

[0217] Surface trafficking signal peptide SEQ ID NO: 27-MKTIIALSYIFCLVFA

[0218] Methionine-enkephalin SEQ ID NO: 28-YGGFM

[0219] Binding sequence, linker and FKBP binding site SEQ ID NO: 29-

[0220] PNLRPFGSGGSGTGSGSGGSGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0221] AgeI restriction site SEQ ID NO: 30-TG

[0222] Linker SEQ ID NO:31-GGSGGSGGSGGSGG

[0223] Truncated human cluster of differentiation 4 (CD4) transmembrane domain SEQ ID NO: 32-

[0224] LPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRR

[0225] Linker SEQ ID NO:33-KGSGSTSGSGSGGSRGSGGSSGG

[0226] CIBN (truncated cryptochrome interacting basic helix-loop-helix protein) for enhanced surface transport SEQ ID NO: 34-

[0227] MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKAAKFDTETKDCNEAAKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIH

[0228] Hemagglutinin (HA) epitope tag SEQ ID NO: 35-YPYDVPDYA

[0229] DNA sequence:

[0230] Surface transport signal peptide SEQ ID NO: 36-

[0231] ATGAAGACCATCATCGCCCTGAGCTACATCTTCTGCCTGGT GTTCGCC

[0232] Methionine-enkephalin SEQ ID NO:37-TACGGCGGATTTATG

[0233] Combined sequence, linker, and FKBP binding site SEQ ID NO:38 -

[0234] CCTAATTTGCGGCCTTTTGGTTCAGGCGGTTCTGGTACTGGATCTGGTTCTGGAGGTTCTggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgcactacaccgggatgcttgaagatggaaagaaagttgattcctcccgggacagaaacaagccctttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaa

[0235] AgeI restriction site SEQ ID NO:39 - ACCGGT

[0236] Linker SEQ ID NO:40 -

[0237] GGTGGAAGTGGATCAGGCAGCGGTGGATCTGGAGGTAGCG GCGGA

[0238] Truncated human cluster of differentiation 4 (CD4) transmembrane domain SEQ ID NO:41 -

[0239] CTGCCCACATGGTCCACCCCGGTGCAGCCAATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTCAGGTGCCGGCACCGAAGGCGC

[0240] Linker SEQ ID NO:42 -

[0241] AAGGGCTCGGGCTCGACCTCGGGCTCGGGCagcggtggcTCTAGAGGTTCTGGTGGCAGCTCTGGAGGT

[0242] CIBN (truncated cryptochrome interacting basic helix-loop-helix protein) for enhanced surface transport SEQ ID NO: 43-

[0243] Atgaatggagctataggaggtgaccttttgctcaattttcctgacatgtcggtcctagagcgccaaagggctcacctcaagtacctcaatcccacctttgattctcctctcgccggcttctttgccga ttcttcaatgattaccggcggcgagatggacagctatctttcgactgccggtttgaatcttccgatgatgtacggtgagacgacggtggaaggtgattcaagactctcaatttcgccggaaacgacgc ttgggactggaaatttcaagGCaGCgaagtttgatacagagactaaggattgtaatgagGCgGCgaagaagatgacgatgaacagagatgacctagtagaagaaggagaagaagagaagtcgaaaata acagagcaaaacaatgggagcacaaaaagcatcaagaagatgaaacacaaagccaagaaagaagagaacaatttctctaatgattcatctaaagtgacgaaggaattggagaaaacggattatattcat

[0244] Hemagglutinin (HA) epitope tag SEQ ID NO:44-TACCCATACGATGTG CCAGATTACGCC

[0245] Stop codon SEQ ID NO:45-tag

[0246] Cells were incubated at 37°C for 24 hours before further processing. HEK293T cells were imaged using a 60× oil immersion objective on a Nikon inverted confocal microscope. During imaging, shield-1 dissolved in complete growth medium was slowly added to the top of the medium to 10 μM. Intensity spectra were acquired using the Nikon NIS-Elements analysis module and plotted by GraphPad Prism 7.

[0247] HEK 293T cell stimulation, imaging, and data analysis of shield-1-induced delocalization from the plasma membrane to the cytosol

[0248] HEK 293T cells were plated at 40% confluence in 24-well plates and then transduced with 200 μL of transmembrane domain lentiviral supernatant and 200 μL of mCherry-TEV protease lentiviral supernatant and incubated at 37°C for 48 hours before further treatment. Two additional wells without any infection were plated for background subtraction. Shield-1 dissolved in complete growth medium was slowly added to the top of the medium to reach 10 μM. Two non-infected wells were treated with and without shield-1, respectively. HEK 293T cells were incubated at 37°C for 18 hours before imaging.

[0249] HEK 293T cell stimulation, imaging, and data analysis of shield-1-controlled nuclear-cytoplasmic protein distribution

[0250] HEK 293T cells were plated at 40% confluence in 24-well plates and then transduced with 50 μL of PKIt NES-EGFP-CapN-NLS-CapC lentiviral supernatant and 150 μL of NES-mCherry lentiviral supernatant. Cells were incubated at 37°C for 24 hours and replated in 24-well plates. Shield-1 dissolved in complete growth medium was slowly added to the top of the medium to reach 10 μM. HEK 293T cells were incubated at 37°C for 18 hours before imaging. HEK293T cells were imaged using a 60× oil immersion objective on a Nikon inverted confocal microscope. Individual cells and nuclei were determined by mCherry signal. Average intensity and area were acquired using the Nikon NIS-Elements analysis module. The background average intensity was subtracted from the average intensity, and the resulting number was then multiplied by the area to obtain the total intensity. The EGFP distribution ratio was calculated by the ratio of the total EGFP intensity in the cytosol to the total EGFP intensity in the whole cell. P values ​​were determined by unpaired two-tailed t-test.

[0251] HEK 293T cell stimulation, imaging, and data analysis of shield-1-dependent gene transcriptional activation

[0252] HEK 293T cells were plated at 40% confluence in 24-well plates and then transduced with 100 μL UAS-mCherry lentiviral supernatant, 100 μL Gal4 DBD lentiviral supernatant, and 50 μL VVP16 lentiviral supernatant and incubated at 37°C for 48 hours before further treatment. Two additional wells were plated without any infection for background subtraction. Shield-1 dissolved in complete growth medium was slowly added to the top of the medium to reach 10 μM. Two non-infected wells were treated with and without shield-1, respectively. HEK 293T cells were incubated at 37°C for 18 hours before imaging. HEK 293T cells were imaged using a 20× air objective on a Nikon inverted confocal microscope. Twelve fields of view were acquired for each condition. Average intensity was acquired from each image using the Nikon NIS-Elements analysis module. The average intensity of the twelve background images was subtracted from the average intensity and plotted by Prism 7. Several intensities were negative and not included in the Fig.12 c, but they were included in the mean for each condition. P values ​​were determined by unpaired two-tailed t-test.

[0253] AAV supernatant production

[0254] AAV supernatant was used for neuronal culture experiments. Six-well plates were pretreated with human fibronectin for 10 minutes at 37°C. HEK 293T cells were then plated in 6-well plates at 60%-90% and transfected 2-3 hours later. For each well, 0.35 μg of viral DNA, 0.29 μg of AAV1 serotype, 0.29 μg of AAV2 serotype plasmid, and 0.7 μg of auxiliary plasmid pDF6 were mixed with 80 μL of serum-free DMEM and 10 μL of PEI max (PEI Max, pH 7.3 1 mg / mL, Polysciences) and incubated at room temperature for 15 minutes, then 2 mL of complete growth medium was added and mixed. The DNA mixture was slowly added to the top of the cells. HEK293T cells were incubated at 37°C for 40 hours, and the supernatant (containing secreted AAV) was then collected. The viral supernatant was stored in sterile Eppendorf tubes (0.5 mL / tube), quickly frozen by liquid nitrogen, and stored at -80°C.

[0255] Production of concentrated AAV

[0256] AAV was prepared as described above for in vivo use. HEK 293T cells (passaged less than 10 generations) in three T150 flasks were transfected at 80% confluence. For each T150 flask, 5.2 μg of construct plasmid, 4.35 μg of AAV1 and 4.35 μg of AAV2 serotype plasmids, 10.4 μg of pDF6 adenovirus helper plasmid and 130 μL of PEI (PEI Max, pH 7.3 1 mg / mL, Polysciences) were mixed in 500 μL of serum-free DMEM at room temperature for 10 minutes. The DNA mixture was further suspended in 10 mL of complete medium and added to the cells. HEK293T cells were incubated at 37°C, 5% CO 2The cells were incubated at 37 °C for 40 to 48 hours. The cells were collected using a cell scraper, resuspended in 10 mL DPBS, and then collected at room temperature by centrifugation at 1200 rpm for 5 minutes. The supernatant was discarded and the precipitate was resuspended in 20 mL 100 mM NaCl, 20 mM Tris (pH = 8.0). 1 mL of freshly prepared 10% sodium deoxycholate (Sigma-Aldrich) dissolved in water was added to the resuspended cells. Benzonase nuclease (Millipore-Sigma) was added to a final concentration of 50 units / ml. The solution was incubated in a 37 °C water bath for 1 hour, and then centrifuged at 8000 rpm for 10 minutes. A peristaltic pump was first used to balance the heparin column with 10 mL 100 mM NaCl, 20 mM Tris (pH = 8.0), and then the viral supernatant was loaded. Use peristaltic pump, wash column with 25mL 100mM NaCl, 20mM Tris (pH=8.0), then use 5mL syringe to wash column with 1mL 200mM NaCl, 20mM Tris (pH=8.0) and 1mL 300mM NaCl, 20mM Tris (pH=8.0).For virus elution, 1.5mL 400mM NaCl, 20mM Tris (pH=8.0); 3.0mL 450mM NaCl, 20mM Tris (pH=8.0) and 1.5mL 500mM NaCl, 20mMTris (pH=8.0) are added to column successively by 5mL syringe.Use Amicon Ultra 15mL centrifugal unit to concentrate the eluted virus, the molecular weight cutoff of this centrifugal unit is 100,000, centrifuge at 8000rpm for 6 minutes, to reach the final volume of 500μL. For buffer exchange, 1 mL of sterile 20 mM Tris, 150 mM NaCl, 0.05% PF68 solution was added to the filter unit and the column was centrifuged again until the virus volume was about 500 μL. The buffer exchange step was repeated 2 more times with a final volume of about 100 μL. The concentrated AAV was aliquoted in 5 μL portions into 0.6 mL low retention microcentrifuge tubes (Thermo Fisher Scientific) and stored at -80°C.

[0257] Neuron culture experiments

[0258] Rat cortical neurons (Thermo Fisher Scientific, Cat#A1084001) were plated according to the protocol. Half-area 96-well glass plates (Corning, CLS4580-10EA) were coated with 50 μl 0.1 mg / ml poly-D-lysine (Gibco) for 1 hour and then washed twice with ultrapure water. Frozen rat cortical neurons were rapidly thawed in a 37°C water bath until small ice cubes appeared. The cells were transferred to a 50 ml conical tube. 1 ml of a preheated 3:1 ratio of complete neurobasal medium (NM) and glial enriched medium (GEM) mixture was slowly dripped into the cells and gently vortexed. NM is composed of the neurobasal medium (Thermo Fisher Scientific) supplemented with 2% B27 (Thermo Fisher Scientific), 50mM HEPES (Thermo Fisher Scientific), 1% penicillin-streptomycin (50 units / mL penicillin and 50μg / mL streptomycin, Thermo Fisher Scientific) and 1% GlutaMAX (Thermo Fisher Scientific). GEM is composed of the DMEM (Gibco) supplemented with 10% FBS (fetal bovine serum, Sigma), 2% B27 (Thermo Fisher Scientific), 50mM HEPES (Thermo Fisher Scientific), 1% penicillin-streptomycin (50 units / mL penicillin and 50μg / mL streptomycin, Thermo Fisher Scientific) and 1% GlutaMAX (Thermo Fisher Scientific). In addition, 2ml of complete neurobasal medium was added to the cells. The viable cell density was determined by adding 10μL cell suspension to 10μL 0.4% trypan blue, and then using a hemocytometer to count cells. 0.25×10 5 Live cells were plated in each well and the cells were incubated at 37°C with 5% CO 2 Within 4-24 hours after plating, replace half of the medium with fresh complete neurobasal medium. For maintenance of cells, replace half of the medium every three days.

[0259] For neuronal infection, neurons were added to concentrated AAV or supernatant AAV at DIV5-DIV10 (days in vitro). Five days after infection, neurons were treated with 2 μM shield-1 for 24 hours and then subjected to live imaging. The intensity average of 5 background images was subtracted from the average intensity and plotted by Prism 7. P values ​​were determined by unpaired two-tailed t-test.

[0260] animal

[0261] All procedures were approved by the University of Michigan Committee on the Use and Care of Animals. C57BL / 6 mice were maintained under a 12-h light / dark cycle and provided with free access to food and water. Adult mice of both sexes were used.

[0262] Stereotaxic injection of AAV into the mouse brain

[0263] The stereotactic injection procedure was performed as described above. 54 . Adult mice were anesthetized with isoflurane (5% for induction and 1.5% for maintenance), injected with 5 mg / kg carprofen, and placed in a stereotaxic apparatus. Body temperature was maintained at 35-37°C. 400 nL of concentrated AAV encoding a shield-1-dependent gene regulatory construct (under the hSyn promoter) was stereotaxically injected into the lateral hypothalamic region (±0.95 mm lateral to the midline, -1.40 mm posteriorly, and -5.25 mm ventral to the bregma) at a rate of 50 nL / min. After injection, the pipette was left stationary in the brain for 10 minutes to balance the pressure and prevent a vacuum effect when the pipette was removed. Mice were injected subcutaneously with 1 mL of saline and allowed to recover from surgery. 5 mg / kg carprofen was additionally administered subcutaneously on the second day after surgery.

[0264] Injection of AAV virus into mouse liver

[0265] Adult mice with shaved abdominal hair were placed on a stereotaxic device and maintained under 1.5% isoflurane anesthesia. The abdomen was disinfected with povidone before surgery and then disinfected with alcohol. A midline incision of about 2 cm was made in the abdomen to expose the liver. 1 μL of AAV encoding a shield-1-dependent gene regulatory construct (under the CMV promoter) was delivered directly to the liver by a micropipette at a speed of 500 nL / min.

[0266] Shield-1 administration and histology

[0267] For mouse brain, 1 μL 1mM Aquashield-1 or saline control was topically applied to LHA ​​by stereotaxic injection 7 days after viral vector injection. For mouse liver, 7 days after viral vector expression, animals were intraperitoneally injected with 2 doses of 40mg / kg aquashield-1 or saline, 24 hours apart. 48 hours after the first injection of aquashield-1 or saline, animals were euthanized and perfused with PBS and 4% paraformaldehyde (PFA). Brain and liver tissues were harvested and fixed overnight in 4% PFA, then cryoprotected in 30% sucrose at 4°C for 48 hours. The fixed tissues were then embedded in the optimal cutting temperature compound and sliced ​​at 30 μm. The slices were rinsed in 0.1% PBS Tween-20 and stained with DAPI (1:10,000, Invitrogen, D1306) for 10 minutes at room temperature. The sections were then rinsed again and mounted with Prolong Gold mounting medium (Invitrogen, P36930). Confocal images were taken on a NikonA1 confocal microscope.

[0268] Equivalent solutions

[0269] The present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the foregoing embodiments should be considered illustrative in all respects, rather than limiting the present invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the above description, and all changes within the meaning and scope of the equivalents of the claims are intended to be included therein.

[0270] Incorporated by Reference

[0271] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

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Claims

1. A method for regulating the activity of a peptide, comprising: a) producing a nucleic acid construct comprising: 1) a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site; as well as 2) a nucleic acid encoding a peptide; b) administering the nucleic acid construct to one or more cells to produce a fusion protein, wherein the fusion protein comprises the at least one CAP linked to the peptide; as well as c) administering a ligand to said one or more cells, wherein said ligand binds to said ligand binding site of said CAP of said fusion protein, wherein said administration of said ligand increases said activity of said peptide. 2 . The method of claim 1 , wherein the at least one CAP comprises CapN bound to the N-terminus of the peptide, and / or CapC bound to the C-terminus of the peptide.

3. The method of claim 1, wherein the administering of the nucleic acid construct comprises direct injection of the nucleic acid construct, macromolecule-mediated liposome and / or biopolymer gene delivery, plasmid delivery, and / or viral delivery.

4. The method of claim 3, wherein the viral delivery is selected from the group consisting of adeno-associated virus (AAV) delivery, adenovirus delivery, lentivirus delivery, vaccinia virus delivery, and retrovirus delivery.

5. The method of claim 1, wherein the nucleic acid construct is stably expressed or transiently expressed. The method of claim 5 , wherein the expression is intracellular expression or extracellular expression.

7. The method of claim 1, wherein the ligand binding site comprises a FKBP binding domain.

8. The method of claim 1, wherein the peptide is SsrA, a nuclear localization signal peptide (NLS), a nuclear export signal peptide, a methionine-enkephalin, or a TEV protease cleavage site.

9. The method of claim 1, wherein the peptide is an enzyme activating peptide, an enzyme inhibiting peptide, an enzyme regulating peptide, a binding peptide, a localizing peptide, or a degradation peptide.

10. The method of claim 1, wherein the ligand is shield-1 and / or aquashield-1.

11. The method of claim 1, wherein the administration is parenteral administration and / or non-parenteral administration.

12. The method of claim 1, wherein the one or more cells are in vitro cells or in vivo cells.

13. The method of claim 12, wherein the in vivo cell is a neuronal cell or a hepatic cell.

14. The method of claim 1, wherein the nucleic acid construct comprises at least one nucleic acid encoding selected from the group consisting of Aga2p, a reporter gene, TetR DBD, SspB, Vp16, P2A, a linker, FLAG, UAS-mCherry, and an internal ribosome entry site (IRES).

15. The method of claim 1, wherein the nucleic acid construct further comprises a promoter nucleic acid selected from the group consisting of: CMV, CAG, and synapsin.

16. The method of claim 1, comprising a protein of interest (POI).

17. The method of claim 16, wherein the POI is a transcription factor, a kinase, a gene editing enzyme, or a marker.

18. The method of claim 16, wherein the POI is evolved green fluorescent protein (EGFP).

19. The method of claim 1, further comprising measuring the activity of the peptide, wherein the measuring comprises measuring the localization, structure and / or function of the peptide.

20. A kit comprising: a) a nucleic acid construct comprising: 1) a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site; and 2) a nucleic acid encoding a peptide; c) Gene transfer agents and / or vectors: and d) a ligand that binds to the ligand binding site.

21. The kit of claim 20, comprising one or more control ligands.

22. A composition comprising: a) a nucleic acid construct comprising: 1) a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site, wherein the ligand is shield-1 and / or aquashield-1; 2) a nucleic acid encoding a peptide; and C) Gene transfer agents and / or vectors.

23. A fusion protein comprising a) a protein expressed from a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site, wherein the ligand is shield-1 and / or aquashield-1; and b) a peptide expressed from a nucleic acid, wherein said peptide is regulated by said shield-1 and / or aquashield-1.

Images