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 that include a nucleic acid construct and a fusion protein that includes a chemically activated protein domain (CAP) that modulates the activity of the fusion peptide in the presence of a ligand.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 329,736, filed April 11, 2022, the disclosure of which is incorporated by reference herein in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was supported by grant numbers R01 AT011652 and R01 HL156989 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] Provided herein are methods, compositions, kits, and systems for modulating peptide function. In particular, provided herein are methods, compositions, kits, and systems that include a nucleic acid construct and a fusion protein that includes a chemically activated protein domain (CAP) that modulates the activity of the fusion peptide in the presence of a ligand. [Background technology]

[0004] Natural or artificial peptides can be dimerization pair partners. 1 , an inhibitor of enzyme activity 2、3 , and protein localization 4、5 , decomposition 6、7 , and splicing 8 They perform biological functions by acting as regulators of the signal transduction pathway. Activation of peptide function with user-defined signals, such as light or chemicals, allows for the temporally controlled manipulation of diverse biological processes. One way to achieve such control is through the use of removable protecting groups. 9 or azobenzene-based chromophores 10One approach to control peptide function is to use chemically modified synthetic peptides incorporating exogenous peptides. Although these methods are useful in vitro, challenges with intracellular delivery and subsequent degradation of the exogenous peptide limit their use in endogenous biological environments. Another approach to control peptide function is to use genetically encoded protein domains. 7、11 Caging and uncaging of peptides can be achieved by fusing them to proteins that change their conformation in the presence of light or small molecules. This approach is suitable for biological studies, as it allows the genetic transfer of peptides into living organisms and cell-type specific protein expression. A second light-, oxygen-, and voltage-sensing domain from oat phototropin 1 (AsLOV2) has been used as the protein domain that controls the function of the peptide. 2、6、11~16 AsLOV2 functions by controlling the accessibility of the N-terminus of peptides fused to it: in the dark, the peptide is blocked by AsLOV2, and in the light, a conformational change in AsLOV2 occurs, resulting in the peptide being released and accessible. 17、18 Although light offers rapid temporal control, its poor tissue penetration limits its use in opaque organisms. 19 In contrast, chemicals can be easily administered to animals and many can penetrate cell membranes and deep tissues. Chemically activated protein domains to control peptide function complement their light-controlled counterparts and have useful applications in the manipulation of cellular processes. Chemical-dependent protein domains can be activated by a variety of mechanisms, including protein proximity. 20、21 and three-dimensional structure 22、23 Although many systems have been used to chemically control peptides, none have general applicability for controlling peptide function. One example is the ligand-induced degradation (LID) system. 7In , a five-amino acid peptide that induces 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, limiting the possibilities for specifically manipulating cellular processes.

[0005] Therefore, new small-molecule-based protein switches and gating modules are needed to control the functions of other peptides. Summary of the Invention

[0006] Provided herein are methods, compositions, kits, and systems for modulating peptide function. In particular, provided herein are methods, compositions, kits, and systems that include a nucleic acid construct and a fusion protein that includes a chemically activated protein domain (CAP) that modulates the activity of the fusion peptide in the presence of a ligand.

[0007] In some embodiments, the present invention provides a method for modulating 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; administering the nucleic acid construct to one or more cells to generate a fusion protein comprising at least one CAP linked to a fusion peptide; and increasing the activity of the peptide by administering to the one or more cells a ligand that binds to the ligand binding site of the CAP of the fusion protein. In some embodiments, 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. In some embodiments, administering the nucleic acid construct comprises direct injection of the nucleic acid construct, polymer-mediated liposome and / or biopolymer gene delivery, plasmid delivery, and / or viral delivery. In some embodiments, 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. In some embodiments, the nucleic acid construct is stably expressed or transiently expressed. In some embodiments, the expression is intracellular or extracellular.

[0008] In some embodiments, the ligand binding site comprises an FKBP binding domain. In some embodiments, the peptide is SsrA, a nuclear localization signal peptide (NLS), a nuclear transport signal peptide, or a TEV protease cleavage site. In some embodiments, the peptide is an enzyme activating peptide, an enzyme inhibitory peptide, an enzyme regulatory peptide, a binding peptide, a localization peptide, or a degradation peptide. In some embodiments, the ligand is Shield-1 and / or Aquashield-1.

[0009] In some embodiments, the administration is parenteral and / or non-parenteral. In some embodiments, the one or more cells are in vitro cells or in vivo cells. In some embodiments, the in vivo cells are neuronal cells or hepatic cells. In some embodiments, the nucleic acid construct comprises at least one nucleic acid selected from the group encoding Aga2p, a reporter gene, a TetR DBD, SspB, Vp16, P2A, a linker, FLAG, UAS-mCherry, and an internal ribosome entry site (IRES). In some embodiments, the nucleic acid construct further comprises a promoter nucleic acid selected from the group consisting of CMV, CAG, and synapsin.

[0010] In some embodiments, the method of the invention further comprises a protein of interest (POI). In some embodiments, the POI is a transcription factor, a kinase, a gene editing enzyme, or a marker. In some embodiments, the POI is evolved green fluorescent protein (EGFP). In some embodiments, the method of the invention further comprises measuring an activity of the peptide, wherein measuring comprises measuring the localization, structure, and / or function of the peptide. In some embodiments, the invention provides a universal mechanism and / or module for temporal, user-controlled regulation of the structure, function, and localization of small intracellular peptides, large peptides, or proteins.

[0011] 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, a nucleic acid encoding a peptide, a gene transfer reagent and / or vector, and a ligand that binds to the ligand binding site.

[0012] 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 reagent and / or vector.

[0013] In some embodiments, the 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, where the ligand is shield-1, rapamycin and / or aquashield-1, and a peptide expressed by the nucleic acid, where the peptide is regulated by shield-1, rapamycin and / or aquashield-1. [Brief description of the drawings]

[0014] [Figure 1]A representative CapN system is shown. a, Design of CAP. CapN and CapC block the N- and C-termini of the peptide, respectively. Addition of Shield-1 releases the binding sequence from the ligand-binding site and unblocks the peptide. b, Labeling of CapN-retained TEV protease cleavage site and library selection. CapN-tobacco etch virus protease cleavage site (CapN-TEVcs) is displayed on the yeast surface by fusing to the yeast Aga2p protein. Accessibility of TEVcs is confirmed by protease cleavage. FLAG and HA signals indicate protein expression levels and TEVcs cleavage, respectively. For library selection, the retained population is indicated by a rectangle on the FACS plot. (TEV protease cleavage site (ENLYFQ / G, cleaved between Q and G). FLAG and HA are epitope tags. c, Labeling scheme of CapN-retained SsrA. CapN-SsrA is displayed on the yeast surface by fusing it to the yeast Aga2p protein. SsrA accessibility is assessed by binding to its binding partner SspB. APEX2 labels proteins in close proximity with biotin phenol. FLAG and biotin signals indicate the association of SsrA-SspB with protein expression levels, respectively. APEX2 is an artificial ascorbate peroxidase. FLAG is an epitope tag. d, CapN sequences before, during, and after directed evolution. Before directed evolution, the original LID-derived CapN was The linker and binding sequence (TRGVEEVAEGVVLL) (SEQ ID NO: 1) of is fused to TEVcs or SsrA at the C-terminus of FKBP. For directed evolution (libraries 1-4), the last 6 amino acids of the binding sequence are mutated to 6-9 random amino acids. The evolved sequence is the final CapN used for the remainder of this study. Aga2p is the yeast protein that displays CapN on the yeast surface. "X" denotes any of the 20 amino acids. Amino acids that differ from the original LID sequence are highlighted in red. e, FACS selection of CapN libraries to improve shield-1 dependency. Four libraries containing >106 CapN variants were displayed on the yeast surface via fusion with the yeast Aga2p protein.Libraries were combined after the first round of selection. Values ​​are the percentage of cells in Q2 relative to (Q2+Q4). Sequencing results for libraries from the fourth round onwards are shown in Fig. 2. f, FACS analysis of CapN-harboring TEVcs before and after directed evolution. Values ​​are the median HA intensity of FLAG-positive cells (Q2+Q4). Evolved CapN shows improved caging and dynamic range. This experiment was performed three times with similar results. FACS plots of addition are shown in Fig. 2. g, FACS analysis of CapN-harboring SsrA before and after directed evolution using the labelling scheme shown in c. Values ​​are the median biotin intensity of FLAG-positive cells (Q2+Q4). Evolved CapN shows both tighter caging and a greater response to Shield-1. This experiment was performed three times with similar results. [Diagram 2] The ligand-binding site of FKBP and the results of the directed evolution of CapN are shown in Figure 1. a, Crystal structure of FKBP12 (PDB: 1FAP). Hydrophobic residues around the ligand-binding site are shown in yellow and in bars. b, Sequences of 40 clones obtained from the CapN library after the fourth round shown in Figure 1e. 23 different sequences were identified. Clone #1 is the last CapN used for the remainder of this study. c, FACS analysis of the eight most enriched clones, corresponding to clones #1-#8 shown in b. Values ​​are the median HA intensity of FLAG-positive cells (Q2+Q4). All eight clones showed similar results. Clone #1 is the last CapN used for the remainder of this study. This experiment was performed once. [Diagram 3] Dose-response characterization of Shield-1 by CapN. a, FACS analysis of CapN-retained SsrA on yeast surfaces 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 curve using data from a. Median biotin signal is plotted against Shield-1 concentration. Half-maximal response was observed at 53 nM. Error, standard error. [Figure 4]Characterization of the reversibility of shield-1 with CapN is shown. a, Schematic of the reversibility characterization of shield-1. Yeast cells were incubated with shield-1 for 10 min and then washed to remove excess shield-1. Yeast cells were then incubated at room temperature for 0–12 h, after which the accessibility of SsrA was assessed using SspB-APEX2 and biotin-phenol labeling as shown in Fig. 1c. b, FACS analysis of yeast cells in a. Values ​​are the median biotin intensity of FLAG-positive cells (Q2+Q4). No decrease in biotin signal was observed when cells were incubated for 12 h without shield-1. This experiment was performed once. [Diagram 5] Engineering of the CapC system is shown. a, CapC sequence before, during, and after directed evolution. Before directed evolution, a flexible linker (SGAGSGGSGTGSGSGGS) (SEQ ID NO: 2) and the last 6 amino acids of the evolved CapN binding sequence (RYSPNL) (SEQ ID NO: 3) are fused to SsrA at the N-terminus of FKBP. In directed evolution (libraries 1-3), the amino acids highlighted in red are randomly mutated to one of 20 amino acids. The evolved sequence is the final CapC used in the remainder of this study. Aga2p is a yeast protein for displaying CapC on the yeast surface. "X" indicates one of the 20 amino acids. Amino acids that differ from the evolved CapN sequence are highlighted in red. b, Labeling of CapC-retained SsrA and library selection scheme. SsrA-CapC was displayed on the yeast surface and the accessibility of SsrA was evaluated with the same scheme as in Fig. 1c. For library selection, the retained population is shown by a rectangle on the FACS plot. c, FACS selection of CapC libraries to improve shield-1 dependency. For comparison, pre-evolved and post-evolved CapC are also shown. Three libraries, each with >106 CapN variants, are displayed on the yeast surface by fusion with the yeast Aga2p protein. Libraries are combined for selection. Values ​​shown on the plot represent the percentage of cells in Q2 relative to (Q2 Q4). Figure 6 shows the sequencing results of the libraries after the second round and the FACS plot of the spike-in. This experiment was performed once. [Figure 6] All-atom molecular dynamics simulations are shown. Results of 2 microsecond molecular dynamics simulations of FKBP and the capped ArgTyrSerProAsnLeu (SEQ ID NO: 4) peptide in 150 mM buffer. a, The central configuration of the top five clusters (ranks 1-5) obtained from RMSD clustering shows a direct interaction between Leu6 of the peptide (displayed in "licorice" representation, with the cap residue in green and the other atoms in CPK color with grey carbons) and the F36V binding site of FKBP (displayed as van der Waals sphere). The secondary structure of the FKBP protein is shown in a cartoon representation with red helices and yellow sheets. b, RMSD time traces for the structures shown in a show the lifetime of each structure in the simulation. RMSD of 0 indicates the simulation time point corresponding to the structure in a. The horizontal dashed line indicates the 1.5 Å cutoff used for clustering. c, Time traces of the center of mass distance between each side chain of the peptide and the side chain of the F36V binding site show that Leu6 is in persistent proximity to the binding site for the majority of the simulation trajectories (distances 5–6 Å). The fraction of simulation trajectories in which Leu6 is in close proximity to the F36V binding site includes all configurations associated with the top five clusters shown in a. [Figure 7] Results of directed evolution of CapC. Related to Fig. 5. a, Sequence of 20 clones from the CapC library after the second round shown in Fig. 5c. 18 different sequences were identified and characterized. One sequence with a premature stop codon is not shown. Clone #18 is the final CapC used for the remainder of the study. b, FACS analysis of the 18 clones shown in a. Values ​​are the median HA intensity of FLAG positive (Q2+Q4) cells. Clone #18 is the final CapC used for the remainder of the study. This experiment was performed once. [Figure 8]Comparison of single CapN, single CapC, and tandem CAPs in caged TEVcs. a, Schematic of the three constructs tested. CapN-TEVcs-CapC combines both evolved CAPs. Aga2p is a yeast protein for displaying the construct on the yeast surface. TEVcs, TEV protease cleavage site (ENLYFQ / G, cleaved between Q and G). FLAG and HA are epitope tags. b, FACS plot of the three constructs shown in a. Values ​​are median biotin intensities of Q2 and Q4 cells. Stronger TEV protease conditions were used in this experiment than in Figs. 1e and 1f. For details, see the Methods section. [Figure 9]Application of CAP to translocate Shield-1-induced proteins is shown. a, Schematic of protein translocation to the plasma membrane by Shield-1. A protein of interest (EGFP as an example) fused to SspB translocates to the plasma membrane when SsrA is released from the CAP. mCherry is used as a membrane protein marker. The transmembrane domain is CAAX. POI, protein of interest. b, Representative fluorescence microscopy images of HEK293T cells expressing the constructs shown in a. Upon addition of Shield-1, EGFP translocates to the plasma membrane within seconds. The right panel shows the intensity profiles of mCherry and EGFP along the red line in the image. Scale bar is 20 μm. This experiment was performed once. c, Schematic of protein translocation from the plasma membrane by Shield-1. Protease cleavage of TEVcs removes the POI (EGFP as an example) 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), cleaved between Q and M). POI, protein of interest. d, Representative fluorescence microscopy images of HEK293T cells expressing the constructs shown in c. Upon addition of shield-1, EGFP is depleted from the plasma membrane. Scale bar is 20 μm. Additional images are shown in FIG. 8. This experiment was performed twice with similar results. e, Schematic of CAP controlling nuclear localization signal peptide. Shield-1-dependent uncaging of the NLS transports the POI (EGFP as an example) from the cytoplasm to the nucleus. mCherry is used to indicate the cytoplasm. 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 alpha (PKIt) NES (LALKLAGLDI) (SEQ ID NO: 8). f, Left: Representative fluorescence microscopy images of HEK293T cells expressing the constructs shown in e. Scale bar is 20 μm. Additional images are shown in FIG. 10. This experiment was performed three times with similar results. Right: Quantification of EGFP total intensity distribution.The ratio is calculated by the total EGFP intensity in the cytoplasm and the total EGFP intensity in the whole cell, which is determined by mCherry. The center line indicates the average ratio. P values ​​are determined by unpaired two-tailed t-test. ****P<0.0001. [Figure 10] Additional images from Figure 9 are shown. Left: 4 additional views for each condition. Right: Negative control where CAP caged SsrA instead of TEVcs. Addition of Shield-1 did not increase the cytoplasmic pattern of EGFP, further supporting that translocation from the membrane is due to TEV protease cleavage. Scale bar 20 μm. [Figure 11] Additional fluorescence microscopy images of HEK293T cells expressing the constructs shown in Figure 9e under + / -Shield-1 conditions are shown. Scale bar is 20 μm. [Figure 12]Application of CAP to shield-1-induced gene transcription is shown. a, Schematic of shield-1-induced gene transcription. Uncaging of SsrA reconstitutes the split transcription factor and expresses a reporter gene (mCherry as an example). EGFP is used as an expression marker for SspB and the transcription activation domain. In all subsequent experiments, the transcription activation domain is VP16. The DNA binding domain (DBD) is specified for each experiment. b, Overview 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 for constructs shown in b. Numbers on the plots are the ratio of the mean mCherry intensity in +shield-1 conditions to -shield-1 conditions for each construct. The central line indicates the mean mCherry intensity. Images are shown in Figs. 11, 13, and 17. In 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 microscopy image of HEK293T cells expressing the best non-single component construct, u3. Same DBD and reporter gene as in c. Scale bar is 20 μm. FLAG is the epitope tag. e, Representative fluorescence microscopy image of rat cortical neurons expressing the single component construct, u4, and quantification of mCherry expression levels. In this experiment, TetR was used as the DBD and TRE-mCherry was used as the reporter gene. Numbers on the plots are the ratio of mCherry average intensity in +shield-1 condition to -shield-1 condition. The center line indicates the average value of mCherry intensity. n=5 for both conditions. Scale bar is 100 μm. Additional images used for quantification are shown in FIG. 18. This experiment was performed twice with the same results. P values ​​are determined by unpaired two-tailed t-test. **P<0.01, ***P<0.001, ****P<0.0001. [Figure 13]Images of constructs u1 and u2 in Figures 12b and 12c are shown. Fluorescence microscopy images of HEK293T cells expressing the indicated constructs and the UAS-mCherry reporter gene. These images are used for quantification of constructs u1 and u2 in Figure 12c. All scale bars are 50 μM. [Figure 14] Images of construct u3 in Figures 12b and 12c are shown, as well as two other constructs with different SsrA sequences. Fluorescence microscopy images of HEK293T cells expressing the indicated constructs and the UAS-mCherry reporter gene. These images are used to quantify construct u3 in Figure 12c. The other two truncations shown here (truncating one or both of the N-terminal alanines from SsrA) did not result in any improvement. All scale bars are 50 μM. [Figure 15] Figure 1 shows the effect of Shield-1 on the expression of transcription activation domains. No differences were observed between +Shield-1 and -Shield-1 conditions. All scale bars are 50 μM. [Figure 16a] Figure 12a shows gene expression at different Shield-1 concentrations using construct u3. Quantification of mCherry expression levels under different Shield-1 concentrations. Strong gene expression was induced above 50 μM. P values ​​are determined by unpaired two-tailed t-test. ***P<0.001, NS, not significant. n=12 for all conditions. b, Fluorescence microscopy image of EK293T 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 is 50 μm. c, Fluorescence microscopy image of HEK293T cells in the same experiment as b, showing only EGFP (transcription activation domain expression). Note that the difference in EGFP signal does not indicate an actual difference in gene expression. Scale bar is 50 μm. [Figure 16b]Figure 12a shows gene expression at different Shield-1 concentrations using construct u3. Quantification of mCherry expression levels under different Shield-1 concentrations. Strong gene expression was induced above 50 μM. P values ​​are determined by unpaired two-tailed t-test. ***P<0.001, NS, not significant. n=12 for all conditions. b, Fluorescence microscopy image of EK293T 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 is 50 μm. c, Fluorescence microscopy image of HEK293T cells in the same experiment as b, showing only EGFP (transcription activation domain expression). Note that the difference in EGFP signal does not indicate an actual difference in gene expression. Scale bar is 50 μm. [Figure 16c] Figure 12a shows gene expression at different Shield-1 concentrations using construct u3. Quantification of mCherry expression levels under different Shield-1 concentrations. Strong gene expression was induced above 50 μM. P values ​​are determined by unpaired two-tailed t-test. ***P<0.001, NS, not significant. n=12 for all conditions. b, Fluorescence microscopy image of EK293T 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 is 50 μm. c, Fluorescence microscopy image of HEK293T cells in the same experiment as b, showing only EGFP (transcription activation domain expression). Note that the difference in EGFP signal does not indicate an actual difference in gene expression. Scale bar is 50 μm. [Figure 17]Figure 12b shows gene expression after different incubation times with Shield-1 using construct u3. a, Schematic of the experiment. HEK293T cells expressing construct u3 were incubated with 100 nM or 1000 nM Shield-1 for different times. Cells are then washed, incubated without Shield-1, and imaged 24 h after the first Shield-1 addition. b, Quantification of mCherry expression levels at different Shield-1 incubation times with Shield-1 concentrations of 100 nM or 1000 nM. P values ​​are 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 microscopy images of HEK293T cells used for quantification in b. Gal4 was used as DBD and UAS-mCherry was used as reporter gene in this experiment. The scale bar is 50 μm. [Figure 18] Images of constructs u4 and u5 in Fig. 12b and Fig. 4c are shown. Fluorescence microscopy images of HEK293T cells expressing the indicated constructs and the UAS-mCherry reporter gene. These images are used for quantification of constructs u4 and u5 in Fig. 12c. All scale bars are 50 μm. [Figure 19] Additional images from Figure 12e are shown. Four additional views are shown for each Shield-1 condition. Scale bar is 100 μm. [Figure 20]Aquashield-1 (http: / / 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 is administered locally to mice (1 μL, 1 mM). b, Representative fluorescent microscopy images of brain sections in the lateral hypothalamic region and quantification of the total number of cells expressing mCherry. Numbers on the plots are the ratio of the average cell number in the +Aquashield-1 condition to the average cell number in the -Aquashield-1 condition. The central line indicates the average cell number. n=3 for both conditions. Scale bar is 200 μm. c, Timeline of Aquashield-1-induced transgene expression in mouse liver. Aquashield-1 is administered to mice via two intraperitoneal (IP) injections (40 mg / kg) at 24-h intervals. d, Representative fluorescence microscopy images of liver sections from the injection site and quantification of the total number of cells expressing mCherry. Numbers on the plots are the ratio of the average cell number in the +Aquashield-1 condition to the average cell number in the -Aquashield-1 condition. The center line indicates the average cell number. n = 3 for both conditions. Scale bar is 200 μm. P values ​​are determined by unpaired two-tailed t-test. *P < 0.1, ***P < 0.001. [Figure 21] A representative gating strategy for single cell analysis is shown. [Figure 22] FIG. 1 shows a schematic of the split NanoLuc assay for detecting CapC-caged metenkephalin activation in a two-chain (left panel) and one-chain (right panel) design. [Diagram 23] Quantification of the split NanoLuc assay for the one-chain design shown in the right panel of Figure 22 is shown. The X-axis provides the various experimental conditions. The Y-axis provides the luminescence signal in relative light units (RLU). [Figure 24] Activity of CapC-caged metenkephalin detected by GloSensor cAMP assay is shown. RLU, relative light units. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] definition To facilitate understanding of this disclosure, certain terms and phrases are defined below. Although the present invention will be described in conjunction with certain representative embodiments, it will 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 to practice the present invention. The present invention is not limited to the methods and materials described.

[0016] Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used to practice the invention, specific methods, devices, and materials are described herein. Furthermore, it should be understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes.

[0017] As used in this disclosure, including the appended claims, the singular forms "a," "an," and "the" include plural references and are used interchangeably with "at least one" and "one or more," unless the content clearly dictates otherwise.

[0018] As used herein, the term "about" denotes an insignificant modification or variation of a numerical value such that the basic function of the item to which the numerical value is associated is not altered.

[0019] 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 is obtained, and if chemically synthesized, is substantially free of chemical precursors or other chemicals.

[0020] As used herein, "modulate" means to alter by increasing or decreasing the activity of a gene or protein. The term "inhibit" as used herein means to block or reduce the activity of a gene or protein.

[0021] As used herein, the term "biological activity" refers to an effect on one or more cellular or extracellular processes (e.g., via binding, signaling, etc.) that may affect physiological or pathophysiological processes.

[0022] Ranges provided herein are understood to be shorthand for all values ​​within the range, for example, a range from 1 to 50 is understood to include 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, or any number, combination of numbers, or subranges thereof (including fractions thereof, unless the context clearly dictates otherwise). Any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range, and fractions thereof, where appropriate (such as tenths and hundredths of integers), unless otherwise indicated. Also, any numerical ranges described herein relating to any physical characteristic, such as polymer subunits, size or thickness, should be understood to include any integer within the stated range, unless specifically indicated otherwise.

[0023] As used herein, the term "cell culture" refers to any in vitro culture of cells. This term includes continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, defined cell lines (e.g., non-transformed cells), and any other cell populations maintained in vitro.

[0024] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes, cell lysates, and the like. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0025] Detailed Description of the Disclosure Provided herein are methods, compositions, kits, and systems for modulating peptide function. In particular, provided herein are methods, compositions, kits, and systems that include a nucleic acid construct and a fusion protein that includes a chemically activated protein domain (CAP) that modulates the activity of the fusion peptide in the presence of a ligand.

[0026] Natural or artificial peptides perform important biological functions. A general approach to achieve chemically-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) to control the accessibility of both the N-terminus and C-terminus of a peptide. In experiments performed in the development of embodiments of the present invention, CAPs are identified through directed evolution of FK506 binding protein (FKBP). By fusing a peptide to one or both CAPs, its function is blocked until a small molecule displaces the peptide from the ligand-binding site of FKBP. The present invention provides CAPs applicable to a variety of short and long peptides, including protease cleavage sites, SsrA (10S RNA, TmRNA) peptides that dimerize with SspB protein, and nuclear localization signal (NLS) peptides, with a SHIELD-1-dependent dynamic range of up to 156-fold. In some embodiments, the CAP systems, methods, compositions, and kits are used in mammalian cell cultures and in a variety of tissues in live animals, thereby providing a chemical genetic approach to control peptide activity in vitro and in vivo.

[0027] In experiments conducted in the development of the present invention, CAPs were generated by directed evolution and contain two protein domains, CapN and CapC, to block the N-terminus and C-terminus of the peptide, respectively (Figure 1A). Addition of shield-1 relieves steric blocking and activates peptide function. In some embodiments, the present invention provides CAPs that carry the tobacco etch virus protease cleavage site (TEVcs), SsrA (tmRNA, or 10SA RNA) peptide, and a nuclear localization signal peptide (NLS). In some embodiments, CAPs are used in culture of mammalian cells, such as HEK cells or neuronal cells. In some embodiments, CAPs are used in tissues of living animals, such as brain tissue and liver tissue, thereby including utility for controlling peptide activity both in vitro and in vivo. CAPs offer broad applicability for caging short and long peptides, including TEVcs, SsrA, and NLS. In some embodiments, CAPs are used to translocate proteins to diverse cellular sites and control gene transcription in a chemical-dependent manner in a variety of biological contexts.

[0028] CAP offers many advantages over existing technologies, primarily light-based. 2、6、11~15、27Compared to existing genetic methods of controlling peptide function, CAP offers an alternative approach by using small molecules, Shield-1 or Aquashield-1. Shield-1 is cell permeable and can be administered systemically to living animals. This allows CAP to be used in most tissues of the body, including those that are difficult or too invasive to reach with light, such as the brain and liver. Although other chemical-dependent protein domains have been described, CAP offers general applicability to a variety of peptides, for example, caging TEVcs and SsrA, regulating NLS, and controlling gene transcription. In some embodiments, tandem CAPs offer improved caging efficiency compared to single CAPs. For example, SsrA caged in tandem by CAPs provides up to 156-fold chemical dependence. Caging strategies using two protein domains offer many advantages. For example, undesired leakage is a long-standing challenge to the use of protein cages, but the tandem caging strategy offers a solution to this problem for protein engineers.

[0029] In some embodiments, the caging mechanism of the CAP is AsLOV2, 13、16、32 Similar to the use of , the peptide or binding sequence can be fine-tuned to obtain a high signal-to-noise ratio and can be transplanted to other peptides, e.g., essential residues of a functional peptide are located in close proximity to the CAP domain. CAP is a peptide that cages AsLOV2. 29、33~35 In parallel with its use as a CP-cage, its ability to cage larger polypeptides or proteins with effector sites near the ends of the sequence offers a different approach to temporally control peptide function. The CP engineering strategy was used to engineer the hepatitis C virus protease NS3A. 36、37 and BCL-xL protein 38 This can also be applied to other chemical-dependent protein domains such as . Engineering these domains as general protein cages could enable multiplexed control of cellular processes, expanding the applicability of chemical genetic techniques.

[0030] method In some embodiments, the methods of the invention are used in research where temporal control is desired to regulate peptide and / or protein localization, structure and function, for example, by administration of small molecules. For example, in some embodiments, CapC is used to regulate the display of opioid peptides on the extracellular membrane, thereby allowing characterization of the activity of endogenous GI-linked opioid receptors. In some embodiments, the methods of the invention are used in therapy where temporal control is desired to regulate peptide and / or protein localization, structure and function, for example, by administration of small molecules to activate target pathways.

[0031] The present disclosure contemplates the use of any genetic manipulation to regulate the expression of a peptide of interest. Delivery of the nucleic acid construct to a cell in vitro or in vivo can be performed using any suitable method. A suitable method is one in which the nucleic acid construct is introduced into the cell so that a desired event (e.g., expression of a CAP-peptide construct) occurs. Introduction of molecules carrying genetic information into cells can be accomplished by any of a variety of methods, including, but not limited to, directional injection of naked DNA constructs and polymer-mediated gene transfer using, for example, liposomes, biopolymers, and the like. In some embodiments, the methods of the present invention provide gene delivery vehicles derived from viruses, including, but not limited to, adenoviruses, retroviruses, lentiviruses, vaccinia viruses, and adeno-associated viruses. The vector can be administered to a subject in a variety of ways. For example, in some embodiments of the present disclosure, the vector is administered into the relevant tissue by direct injection. In other embodiments, administration is via the blood circulation or lymphatic circulation.

[0032] The present disclosure further provides, for example, chemical production modulating compounds and pharmaceutical compositions comprising those compounds as described above. The pharmaceutical compositions of the present disclosure can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the site to be treated. Administration can be topical (including the eye and mucous membranes, including intravaginal and rectal), pulmonary (e.g., inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal, and transdermal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, e.g., intrathecal or intraventricular administration. In certain embodiments, the nucleic acid constructs and / or their products are administered by methods that bypass the blood-brain barrier (BBB), including, for example, direct application to the surface of the CNS, the parenchyma of the CNS, the ventricles of the CNS, and the cerebrospinal fluid (CSF) of the CNS. In particular, intrathecal and epidural administration may be achieved by a single shot, a series of single shots, and / or continuous administration into the CSF. In certain embodiments, continuous administration into the CSF is provided by a programmable external pump. In other embodiments, continuous administration is provided by a programmable implantable pump. Compositions and formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions, which may also include other suitable additives, such as, but not limited to, buffers, diluents, and penetration enhancers, carrier compounds, and other pharma- ceutically acceptable carriers or excipients. Agents that enhance uptake of nucleic acid constructs at the cellular level may also be added to pharmaceutical and other compositions of the present disclosure. For example, cationic lipids such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (WO 97 / 30731) also enhance cellular uptake of oligonucleotides.

[0033] kit The present disclosure provides kits that include components of the chemical genetic peptide activation assay described herein. Such kits include, for example, two or more of a nucleic acid encoding at least one chemically activated protein domain (CAP) that includes a ligand binding site, a nucleic acid encoding a peptide, a ligand that binds to the ligand binding site, a gene transfer vector and / or reagents, stabilizers, buffers, instructions, positive and negative control reagents, and a ligand for identifying possible sources of error and contamination. In some embodiments, the positive control peptide achieves statistical significance that the positive control experiment works as specified. In some embodiments, the kits of the present invention include calibration reagents, including, for example, luminescence calibration reagents, and ligands. In some embodiments, the kits include customizable options. In some embodiments, the kits of the present invention include frozen (e.g., prepared in glycerol for storage at -20°C) and / or dried components and reagents.

[0034] Compositions and reaction mixtures The present disclosure provides compositions and reaction mixtures that include the components of the assays described herein. Such compositions and reaction mixtures may include, for example, two or more of a nucleic acid encoding at least one chemically activated protein domain (CAP) that includes a ligand binding site, a nucleic acid encoding a peptide, a ligand that binds to the ligand binding site, a gene transfer vector and / or reagents, stabilizers, buffers, instructions, positive and negative control reagents, and a ligand for identifying possible sources of error and contamination. In some embodiments, the compositions and reaction mixtures of the present invention include a fusion protein product of one or more of the nucleic acid constructs provided herein. EXAMPLES

[0035] 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.

[0036] Example 1 - Design and directed evolution of CapN. Figure 1a shows CAP for caging the N-terminus of a peptide. The design of CapN is based on the mutant FK506 binding protein FKBP12(F36V) (referred to as FKBP), a LID 7 In LID, a five amino acid peptide degradation sequence is blocked by FKBP via fusion to a binding sequence that interacts with FKBP. The degradation sequence is activated when the FKBP ligand, shield-1, is added to displace the binding sequence. The LID ligand, shield-1, has subnanomolar affinity for FKBP, which is greater than that of wild-type FKBP. 24 Shield-1 has a 1000-fold higher affinity for FKBP than FKBP. Therefore, Shield-1 hardly interferes with the physiological function of endogenous FKBP. Shield-1 is membrane-permeable and has not been used in living animals. 25 .

[0037] We tested the ability of LID to cage two short peptides that have previously been shown to be regulated by the AsLOV2 domain: a heptapeptide, TEVcs 26 , and octapeptide, modified SsrA 27 were displayed on the yeast surface and accessibility was assessed using protease cleavage assays (Fig. 1b) and binding assays (Fig. 1c), respectively. The degradation sequence of the LID (RRRGN) was replaced with TEVcs or SsrA, while the remainder of the binding sequence and linker were retained (Fig. 1d). No shield-1 dependency was observed for either TEVcs or SsrA, indicating that the LID is not effective at introducing steric blocking to peptides other than the original degradation sequence.

[0038] To test whether amino acids near the C-terminus of the binding sequence are important for interaction with the hydrophobic ligand-binding site of FKBP (Fig. 2), these residues were adjusted. Yeast surface-based directed evolution was applied to improve the shield-1 dependency of CapN using TEVcs as a caged peptide. The first library was generated by introducing site-saturation mutations in the last six amino acids of the binding sequence immediately before TEVcs (Fig. 1d). Three more libraries were constructed by adding one, two or three amino acids to the randomized binding sequence to ensure sufficient length for binding and caging (Fig. 1d). Four libraries were selected according to the scheme shown in Fig. 1b and 1e. A first round of negative selection is performed to retain tight-caged CapN variants in the absence of shield-1 (high expression and low cleavage). This ensures that all clones entering the second round of selection fully express the DNA construct without premature stop codons. Then, one round of positive selection is performed to enrich for clones that efficiently uncage TEVcs upon addition of shield-1. After a second round of selection, the library of CapN mutants exhibits shield-1 dependency. To further enrich for targeted clones, a second round of selection was performed.

[0039] After four rounds of directed evolution, sequencing of 40 clones revealed 23 distinct sequences enriched in hydrophobic amino acids (Fig. 2). Eight clones were characterized that appeared multiple times and showed similar levels of improvement. The most enriched clone was the evolved CapN (Fig. 1d). Compared to the pre-evolved CapN, the evolved protein showed both tighter caging and a greater response to shield-1 (Fig. 1f). When the same evolved CapN was applied to SsrA, enhanced caging and a wider dynamic range were also observed (Fig. 1g). These results demonstrate the general applicability of CapN in controlling short peptides.

[0040] Next, the concentration of Shield-1 required to activate CapN was characterized (Figure 3). Using the same yeast surface binding assay, a dose-response curve showed a half-maximal response at 53 nM, indicating a high affinity of Shield-1 for CapN. Next, we assessed whether the binding between Shield-1 and CapN was reversible. Even 12 h after washing away Shield-1, CapN remained open (Figure 4), indicating that Shield-1 is stably bound to CapN and provides an irreversible "on" switch for CapN.

[0041] Example 2 - Design and directed evolution of CapC Following the development of CapN, we designed a similar CAP (CapC) that cages the C-terminus of peptides. Peptide caging is often most effective when critical residues on the peptide are tightly bound to the CAP protein. Peptides with critical C-terminal sequences may be easier to cage with CapC than with CapN. Also, using CapN and CapC together can further increase caging efficiency and reduce "leakage" in the absence of shield-1. These features are important for live animal applications where proteins may be expressed for weeks to months before controlled protein activation is desired.

[0042] To test whether the shield-1 binding sequence functions independently of FKBP binding, we fused it to the N-terminus of FKBP using a flexible linker (Fig. 5a) and examined its effectiveness in caging the SsrA peptide using the same binding assay to test CapN (Fig. 5b). SsrA was chosen because the dynamic range of CapN in caging SsrA is larger than that in caging TEVcs (the difference between “+shield-1” and “−shield-1” conditions), making it easier to select during directed evolution. To design the linker, we estimated the distance from the N-terminus of FKBP to its ligand-binding site and used a flexible glycine- and serine-rich linker to cover that distance. The initial CapC construct showed shield-1 dependence (Fig. 5c, first column) with a smaller dynamic range than CapN.

[0043] Directed evolution was used to enhance the dynamic range of CapC. To generate the libraries, site-saturation mutagenesis was applied simultaneously to two key regions (Figure 5A): (1) the first two or three amino acids of the binding sequence, the region adjacent to the peptide target, which are most important in drawing it deeper into the FKBP ligand-binding site, and (2) the first few amino acids on the linker immediately following the binding sequence, which may be important in positioning the binding sequence in the FKBP ligand-binding site. The last three amino acids of the binding sequence (Pro-Asn-Leu) were retained because they provide a hydrophobic surface important for binding. The importance of the hydrophobic leucine residue was supported by all-atom molecular dynamics simulations, which showed that leucine has a high propensity to interact with the FKBP hydrophobic ligand-binding site (Figure 6). The three libraries were combined and selected according to the schemes shown in Figures 5b and 5c. One round of negative selection to retain CapC mutants was performed using tight-caging in the absence of shield-1 (high expression and low biotin signal). Next, one round of positive selection was performed to retain mutants that uncage peptides in the presence of shield-1 (low expression and high biotin signal). After two rounds of sorting, the CapC library became more shield-1-dependent. Twenty clones were characterized, and a clone with tight caging and a wide dynamic range was identified as evolved CapC (Figures 5A, 5c, and 7).

[0044] Since unwanted leakage is a common issue in protein cages, we tested the use of CapN and CapC in combination for more robust caging and reduced background. Using TEVcs as the caged peptide and both CAPs in tandem was found to significantly reduce leakage compared to using either CapN or CapC alone, but still be uncaged by Shield-1 (Figure 8). This indicates that using both CAPs simultaneously improves caging efficiency and reduces unwanted leakage. Therefore, the tandem CAPs are used for the following experiments in cell culture and animals.

[0045] Example 3 - CAP regulates protein translocation to the plasma membrane in HEK293T cells Protein trafficking to different subcellular compartments is closely linked to their function, and regulated localization to the plasma membrane is essential for phagocytosis. 28 and calcium influx 29 It has been used to manipulate cellular processes such as cellular metabolism and cellular metabolism. To test the use of CAPs for protein translocation, we designed a shield-1-induced membrane localization system (Figure 9). In this design, CAPs-retained SsrA peptide is localized on the plasma membrane of HEK293T cells by fusing it to a transmembrane domain. The SspB protein, an interaction partner of SsrA, is fused to a protein of interest (POI). As an example, evolved green fluorescent protein (EGFP) was used as the POI. In the absence of shield-1, binding of SsrA and SspB does not occur, and EGFP is found throughout the cell. Addition of shield-1 releases SsrA from CAPs, allowing it to bind SspB-EGFP, thereby translocating EGFP to the membrane. As shown in Figure 9b, under conditions without shield-1, EGFP localized to the cytoplasm and nucleus, with no obvious membrane pattern as predicted. Upon addition of shield-1, EGFP was observed to translocate to the plasma membrane within a few seconds.

[0046] These results indicate that shield-1 can easily permeabilize the cell membrane and open CAPs within seconds, indicating that the CAPs-shield-1 system is useful for experiments requiring rapid time control. These results further demonstrate that the CAP system retains its functionality when directly transferred from the yeast surface to mammalian cell cultures without the need for reoptimization. By directing CAPs-SsrA to different subcellular compartments with appropriate localization signals, POIs could be recruited to various regions of interest in a shield-1-dependent manner.

[0047] Example 4 - CAP delocalizes proteins from the plasma membrane to the cytoplasm in HEK293T cells Yet another design shows the reverse process of membrane localization, i.e., removing the POI from the cell membrane of interest, for example in the temporally controlled perturbation of proteins that function localized on the plasma membrane. The CAP-caged TEVcs is anchored to the plasma membrane and subsequently to the POI (Figure 9c). In the absence of Shield-1, the TEVcs is inaccessible to the co-expressed TEV protease, and the POI remains localized on the plasma membrane. With Shield-1, the TEVcs is uncaged and cleaved by the TEV protease, thereby releasing the POI from the cell membrane. Figures 9d and 10 show that before the addition of Shield-1, the example POI, EGFP, was almost completely bound to the plasma membrane. Upon the addition of Shield-1, EGFP is depleted from the plasma membrane and its presence in the cytoplasm is significantly increased.

[0048] Thus, this system delocalizes the POI from its membrane location to a cytoplasmic location in a shield-1-dependent manner. Similar to SsrA in the CAP cage, CAP can efficiently cage TEVcs when transferred directly from the yeast surface to mammalian cell culture. The necessary modification is to change the P1' position (last amino acid) of TEVcs from the canonical glycine to a less reactive methionine to reduce unwanted cleavage, since the TEV protease is present in this assay for a much longer time (2-3 days) than in the yeast surface assay (3 hours).

[0049] Example 5 - CAP regulation of nuclear-cytoplasmic distribution of proteins in HEK293T cells The nucleocytoplasmic distribution of many eukaryotic proteins is a general determinant of their function 30 So far, NLS has been 13、14To test whether the NLS is regulated by CAP in a shield-1-dependent manner, a single-chain construct was designed in which the POI is expressed as a fusion protein with the CAP-caged NLS (Figure 9e). In the absence of shield-1, the NLS is sterically blocked and the POI should be found throughout the cell. Upon addition of shield-1, the CAP opens, exposing the NLS to endogenous importins and delivering the POI to the nucleus. Due to the strength of the NLS, the weak nuclear export signal PKIt 13 is added to the construct to reduce nuclear localization of the protein in the basal state. Figures 9f and 11 show that in the absence of shield-1, the example POI, EGFP, is found in both the cytoplasm and the nucleus. Upon addition of shield-1, EGFP is depleted from the cytoplasm and preferentially localizes to the nucleus, with the cytoplasm to whole cell ratio statistically different from the basal state.

[0050] These results demonstrate the general applicability of CAP. CAP can not only cage the initially optimized TEVcs and SsrA, but also transfer to other peptides, such as NLS. The small amount of nuclear localization in the absence of shield-1 (Fig. 9f and Fig. 10) suggests that the NLS is not completely blocked by CAP. A weaker NLS sequence could be used to further eliminate the nuclear pattern in the absence of shield-1. 13 .

[0051] Example 6 - Temporally gated gene transcription in HEK293T cell cultures and neuronal cultures using CAP Gene transcription is a universal process in all living organisms. Temporally controlled gene transcription provides a useful tool to study the functional role of specific proteins in living cells and animals. To test the utility of CAP in controlling gene transcription, we used the two-hybrid system. 31We designed a split transcription factor based on Shield-1 (Figure S12A). In this design, SsrA of the CAP cage is fused to a DNA-binding domain (DBD) and SspB is fused to a transcription activation domain (VP16). In the presence of Shield-1, SspB-VP16 is recruited to DBD-SsrA and initiates transcription of a reporter gene.

[0052] This system was tested in HEK293T cells using the Gal4 DBD and UAS-mCherry reporter gene. For the 8-amino acid SsrA peptide (Fig. S12b), we observed a 4-fold and 25-fold increase in mCherry intensity upon shield-1 induction with CapN and CAP-caged SsrA (Fig. S12c and Fig. S13). The use of CAP in tandem demonstrated lower background and a broader dynamic range than CapN alone, consistent with previous observations of CAP-caged TEVcs on the yeast surface (Fig. S8). The amino acid sequence of SsrA is tunable 1、16 Because transcription systems are very sensitive and minimizing unwanted transcription is often practically useful, we tested more efficient caging by adjusting the SsrA sequence from both the N-terminus and C-terminus (Fig. 12b and Fig. 14) to further reduce background. The seven amino acid SsrA sequence (AANDENY) (Fig. 12d and Fig. 14) showed a 156-fold change in SHIELD-1-dependent reporter gene expression, with a two-fold lower background and a six-fold higher signal-to-noise ratio compared to the original sequence tested (Fig. 12c). The expression level of SspB-VP16, as indicated by the EGFP signal, positively correlates with the amount of SHIELD-1 added and the expression of the reporter gene. Conditions with higher concentrations of SHIELD-1 and higher levels of reporter gene expression resulted in higher EGFP signals. Because addition of Shield-1 alone to EGFP-SspB-VP16 did not increase the level of EGFP (Fig. S4), these results indicate that the SsrA-SspB interaction stabilizes the SspB protein and causes degradation of non-interacting SspB.

[0053] The required concentration of Shield-1 and incubation time of Shield-1 were tested to induce strong gene expression. When incubated overnight, Shield-1 concentrations below 10 nM did not induce gene expression, whereas Shield-1 concentrations above 50 nM induced gene expression more than 30-fold (Figure 16). Consistent with the results above, the same amount of virus was introduced under all conditions, but the EGFP signal was also higher under conditions with higher concentrations of Shield-1, consistent with the stabilizing effect of SspB-SsrA heterodimerization. Regarding the incubation time of Shield-1, an incubation time as short as 30 min was sufficient to induce gene expression at a concentration of 1 μM of Shield-1, whereas longer incubation times of several hours were required for stronger gene expression (Figure 17).

[0054] To test the CAP chemical-dependent transcription system for neuroscience applications, we tested it in cultured neurons. To enable uniform expression of DNA in rigorous experiments, we generated a single viral construct expressing both the DNA-binding and transcription activation domains via the self-cleaving peptide P2A or an internal ribosome entry site (IRES) (Fig. 12b). Upon addition of shield-1, the P2A and IRES constructs increased mCherry expression 83- and 123-fold, respectively, comparable to the two-component system (Fig. 12c and Fig. 18). We then used the P2A construct to deliver the TetR DBD and VP16 transcription activation domains, along with another AAV encoding a TRE-mCherry reporter gene, via adeno-associated virus (AAV) into cultured rat cortical neurons. Shield-1 increased the expression of the mCherry reporter gene 44-fold compared to conditions without shield-1 (Fig. 12e and Fig. 19), indicating that the system works robustly in cultured neurons.

[0055] Example 7 - Regulation of gene transcription in live animals using CAP To test chemical-dependent peptide regulation in vivo, the CAPs system was applied to mouse brain and liver. For brain application, AAV encoding a Shield-1-dependent gene regulatory construct was stereotaxically injected into the lateral hypothalamic area (LHA). Seven days after viral delivery, Aquashield-1 (a water-soluble analogue of the Shield-1 molecule) was administered locally into the LHA. Forty-eight hours after treatment with Aquashield-1 or saline, mice were euthanized, perfused, and brain tissue was processed for analysis (Figure 20A). In saline-treated control brains (-Aquashield-1), only a few sparse neurons showing mCherry expression were observed throughout the LHA region (Figure 20b). In contrast, in large clusters of LHA neurons in Aquashield-1-treated brains (+Aquashield-1), over 16-fold more mCherry than controls was observed (Figure 20b), indicating Shield-1-dependent gene expression.

[0056] To test the application of the CAP chemical-dependent gene control system beyond neural tissue, AAV encoding the CAP construct was injected into the liver of mice. On days 7 and 8 after viral delivery, Aquashield-1 or saline was administered twice by intraperitoneal injection (Figure 20c). Two days after the first Aquashield-1 injection, liver tissue was harvested and analyzed. In saline-injected control mice, few cells expressed mCherry throughout the liver, whereas the liver of animals administered Aquashield-1 had a significant increase in mCherry-expressing cells (Figure 20d). This indicates that systemic injection of Aquashield-1 controls CAP in mouse liver. These results demonstrate the advantage of using small molecules compared to light to control peptide activity. Light is limited by its illumination range and therefore difficult to apply globally in living animals. In contrast, injection delivers small molecules throughout the animal's body, allowing global control of peptide activity in diverse organs in the body. Taken together, these results indicate that CAP Aquashield-1-induced gene regulation functions in multiple organs in living organisms, including the brain and liver. Aquashield-1 can then be easily administered by parenteral injection to activate gene transcription in animal tissues of interest.Compared to light-induced gene transcription systems, these features of the CAP provide global control with less disruption to the animal.

[0057] Example 8 - Regulation of the opioid peptide met-enkephalin by CapC To detect activation of the CapC opioid receptor, half of the assay was directed to the opioid receptor and the other half to the G iWe developed a split-NanoLuc assay by fusing Nb44 to a mimetic nanobody. Opioid receptor activation leads to recruitment of Nb44 to the receptor and reconstitution of split-NanoLuc. Activation of the μ-opioid receptor is monitored using a cyclic adenosine monophosphate (cAMP)-dependent luciferase (GloSensor). By expressing the real-time sensor GloSensor in cells, high luminescence is observed at high cAMP concentrations and low luminescence is observed at low cAMP concentrations. The GloSensor assay complements the split-NanoLuc assay because it measures downstream events, not just G protein-receptor binding: activation of a membrane-bound opioid peptide (M-PROBE) not only leads to G protein recruitment but also downstream signaling events.

[0058] To test the chemical-dependent regulation of the opioid peptide Shield-1, CapC-caged met-enkephalin was fused directly to the N-terminus of the opioid receptor, as shown in the right panel of Figure 22. In this design, M-PROBE cell membrane-bound opioid peptide (M-PROBE) and opioid receptor were expressed in close proximity at a fixed ratio. Figure 23 shows how Shield-1 dependence is used to control the activity of opioid receptors via chemically gated membrane-bound opioid peptides in HEK293T cells. The antagonist "naloxone" condition provides a basal condition in the absence of receptor activation. In the "no drug" condition, no external drug molecules were added but the cells had CapC-caged met-enkephalin on the cell surface, and the signal arose from the basal activity (i.e., background or leakage) of M-PROBE. Loperamide is a full agonist of the μ-opioid receptor (MOR). The "loperamide" condition shows the highest signal. Both Shield-1 and loperamide increase signaling by either inducing release of opioid peptides (i.e., met-enkephalin) from the CapC (i.e., Shield-1-induced met-enkephalin release increases receptor activation) or by loperamide acting directly on the MOR.

[0059] To test whether M-PROBE induces downstream signaling as a free opioid and whether the function of met-enkephalin is well preserved in the membrane-bound structure, the same single-chain met-enkephalin-CapC-MOR was expressed on the membrane of HEK293T cells and the cells were co-transfected with cAMP-dependent luciferase. After overnight protein expression, HEK293T cells were stimulated with forskolin to increase intracellular cAMP concentrations. MOR is an inhibitory G protein-coupled receptor (GPCR) whose activation reduces cAMP levels. Higher cAMP concentrations increase the effect of MOR activation. Figure 24 shows that upon stimulation with forskolin, the cells significantly enhance the luminescence signal. Cells were incubated with no drug, Shield-1, or the MOR agonist [D-Ala 2 , N-MePhe 4 Cells were treated under four different conditions with met-enkephalin (DMSO), met-Gly-ol, or the MOR antagonist naloxone. Untreated cells showed no change in luminescence levels. Cells treated with a high concentration (10 micromolar) of DAMGO showed a large decrease in luminescence (about 80%). The luminescence levels of cells treated with Shield-1 were intermediate between these two conditions, with an approximately 50% decrease in luminescence. Cells treated with a high concentration of naloxone (10 micromolar) showed a rapid increase in luminescence signal. This increase was even greater than that of cells treated with 1 micromolar of forklin. These results demonstrate that in both binding assays and downstream cAMP assays, CapC regulates met-enkephalin to activate MOR in a drug-dependent manner, thereby providing methods, compositions, kits, and systems to modulate target endogenous opioid receptors within specific cell populations or neuronal circuits in the central nervous system.

[0060] Experimental Method Cloning Constructs for yeast surface display were cloned into the pCTCON2 vector. Constructs for protein expression in HEK293T cells were cloned into the pAAV viral vector for transfection or the pLX208 lentiviral vector for transduction. Constructs for protein expression in neuronal cultures, mouse brain, and mouse liver were cloned into the pAAV vector.

[0061] The FKBP of CapN was amplified from YFP-LID (Addgene plasmid #31767, Thomas Wandless lab). The codon-optimized FKBP of CapC was synthesized by IDT.

[0062] For cloning, PCR fragments were amplified using Q5 or TAQ DNA polymerase (New England Biolabs (NEB)). Vectors were double digested with restriction enzymes (NEB), gel purified, and ligated to the gel-purified PCR fragments by T4 ligation, Gibson assembly, or with the In-Fusion HD Cloning Plus kit (Takara Bio). The ligated plasmid products were introduced into competent XL1-Blue E. coli cells by heat shock transformation or, in the case of In-Fusion cloning, into Stellar competent E. coli cells from the kit following the corresponding protocol. For In-Fusion cloning, a modified protocol was used in which the amount of each reagent or competent cells was proportionally reduced by half or up to three-quarters of the recommended amount.

[0063] Expression and purification of TEV protease Full-length TEV protease (TEVp, S219V) was expressed as a fusion with polyhistidine-tagged maltose-binding protein (MBP). His-tag-MBP-TEVp(S219V) in the pYFJ16 vector was introduced into competent BL21-CodonPlus(DE3)-RIPL E. coli cells by heat shock transformation. Cells were grown in 5 mL of Miller LB medium (Bio Basic) supplemented with 100 mg / L ampicillin at 37 °C with shaking at 220 rpm for 6 h. This saturated culture was then transferred to 500 mL of LB containing 100 mg / L ampicillin and incubated at 37 °C with shaking at 220 rpm for approximately 2–3 h until OD 600= 0.4-0.8. IPTG (isopropyl β-D-1-thiogalactopyranoside, EMD Millipore) 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. The following procedures are performed at 4 °C unless otherwise stated. Cells are harvested by centrifugation at 5,000 rpm for 5 min. Cell pellets were lysed and resuspended in 15 mL of ice-cold B-PER Bacterial Protein Extraction Reagent (Thermo Fisher Scientific). DTT (Fisher, freshly made) 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 min and centrifuged at 10,000 rpm for 15 min. The supernatant was incubated with 3 mL of Ni-NTA resin (Thermo Fisher Scientific) for 10 min with rotation 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 then 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 in a 15 mL 10,000 DA cutoff centrifugal unit (Millipore), flash frozen in liquid nitrogen, and stored at -80°C. To obtain effective TEVp, each batch of TEVp was concentrated at least 10-fold. Batch-to-batch variations in TEVp yield and activity were tracked.

[0064] Expression and purification of SspB-APEX2 SspB-APEX2 in pYFJ16 vector was expressed with a polyhistidine tag in competent BL21 E. coli cells similar to the expression of TEV protease described above (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.

[0065] Yeast strains and non-library cultures Non-library yeast cultures were generated by chemically transforming the yeast surface display plasmid pCTCON2 into Saccharomyces cerevisiae strain EBY100 competent cells. Preparation of EBY100 competent cells was described. 39 To transform, 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 min to 2 h, then transferred to 5 mL of SDCAA (Synthetic Dextrose + Casein Amino Acid Medium, 2% dextrose, 0.67% Yeast Nitrogen Base without Amino Acids (BD Difco), 0.5% Casamino Acids (BD Difco), 0.54% Disodium Phosphate, 0.856% Monosodium Phosphate) and grown at 30 °C with shaking at 220 rpm. Initial saturation (OD ) was reached in 2–3 days. 600 After reaching a .DELTA.>10), yeast cultures were passaged at least once or stored at 4°C for up to half a year before experimentation. Passages were performed by adding 500 μL of saturated culture to 5 mL of fresh SDCAA medium and growing overnight at 30°C and 220 rpm. To ensure the selectivity of the medium, a negative control without plasmid DNA was used for each batch of transformation.

[0066] Yeast library generation CapN and CapC mutant libraries were generated by first generating a plasmid library using targeted mutagenesis and then transforming into EBY100 yeast competent cells by electroporation. Targeted mutagenesis was performed by conventional PCR (polymerase chain reaction) using mixed-base (IDT) primers. The first two bases of each codon corresponding to the mutated amino acid were designed to be an equal mixture of A, C, G, and T, and the third base was an equal mixture of G and T. PCR fragments were amplified to include an extra 40 bases beyond the two restriction sites used to linearize the vector.

[0067] 500 ng of template DNA plasmid (Aga2p-FLAG-FKBP-binder sequence-TEVcs-HA) was mixed with 100 μmol of forward and reverse primers annealing outside the FKBP gene, 1× Q5 High GC enhancer, 1× Q5 reaction buffer, 1 unit of Q5 High-Fidelity polymerase, and 10 nMol of dNTPs (VWR) in a total volume of 50 μL (two reactions for each library). CapN library: Forward primer: Full Library: SEQ ID NO:9 GGCTCTGGTGCTAGCGACTACAAGGATGACGACGATAAgACTAGT Reverse primer: Library 1: SEQ ID NO:10 GGATCCACCCTGGAAGTAGAGATTTTCMNNMNNMNNMNNMNNMNNCGCCACTTCCTCCACTCCACGC Library 2: SEQ ID NO:11 GGATCCACCCTGGAAGTAGAGATTTTCMNNMNNMNNMNNMNNMNNMNNCGCCACTTCCTCCACTCCACGC Library 3: SEQ ID NO: 12 GGATCCACCCTGGAAGTAGAGATTTTCMNNMNNMNNMNNMNNMNNMNNMNNCGCCACTTCCTCCACTCCACGC Library 4: SEQ ID NO:13 GGATCCACCCTGGAAGTAGAGATTTTCMNNMNNMNNMNNMNNMNNMNNMNNMNNCGCCACTTCCTCCACTCCACGC

[0068] PCR was performed for 20 cycles with an annealing temperature of 60° C. Product DNA was gel purified and amplified with the following primers: Forward primer: Full Library: SEQ ID NO: 14 GGCTCTGGTGCTAGCGACTACAAGGATGACGACGATAAGACTAGT Reverse primer: Full Library: SEQ ID NO: 15 CTCGAGCTATTAAGCGTAATCTGGAACGTCATATGGGTAGGATCCACCCTGGAAGTAGAGATTTTC CapC library: Forward primer: Library 1: SEQ ID NO: 16 GCTAGCGCAGCGAATGATGAAAATTACTTCNNKNNKNNKCCTAATTTGNNKNNKNNKGGATCAGGCGGTTCTGGTACTG Library 2: SEQ ID NO: 17 GCTAGCGCAGCGAATGATGAAAATTACTTCNNKNNKCCTAATTTGNNKNNKNNKNNKTCAGGCGGTTCTGGTACTGG Library 3: SEQ ID NO: 18 CTGCAGCAAGGTCTGCAGG Reverse primer: Library 1: SEQ ID NO: 19 TCAGATCTCGAGCTATTACTTATCGTCGTC Library 2: SEQ ID NO: 20 TCAGATCTCGAGCTATTACTTATCGTCGTC Library 3: SEQ ID NO: 21 GAGATGGTTTCCACCTGCACTCCMNNMNNMNNMNNMNNMNNTCCAGTACCAGAACCGCCTG

[0069] PCR was performed for 20 cycles with an annealing temperature of 60° C. Product DNA was gel purified and amplified with the following primers: Forward primer: Library 1: SEQ ID NO: 22 CTGCAGCAAGGTCTGCAGGCTAGTGGTGGAGGAGGCTCTGGTGCTAGCGCAGCGAATGATGAAAATTACTTC Library 2: SEQ ID NO: 23 CTGCAGCAAGGTCTGCAGGCTAGTGGTGGAGGAGGCTCTGGTGCTAGCGCAGCGAATGATGAAAATTACTTC Library 3: SEQ ID NO: 24 GGAGTGCAGGTGGAAACCATCTC Reverse primer: Full Library: SEQ ID NO: 25 TCAGATCTCGAGCTATTACTTATCGTCGTC

[0070] The template DNA was divided equally into eight parts. Each part was mixed with 100 μM forward and reverse primers, 1×TAQ reaction buffer without magnesium chloride, 2 mM magnesium chloride, 2 units of TAQ polymerase, 10 nMol dNTPs (VWR) in a total volume of 50 μL. Eight PCRs for the same library were gel purified and combined.

[0071] The linearized vector was gel purified. 2 μg of linearized vector was mixed with 8 μg of 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).

[0072] In parallel, fresh electrocompetent EBY100 yeast cells were prepared. Prior to this procedure, the cells were passaged at least twice in YPD (Yeast Extract Peptone Dextrose Medium, 20 g dextrose, 20 g peptone, and 10 g yeast extract in 1 L deionized water) to ensure that the cells were healthy. A saturated culture of yeast was cultured in 200 mL of YPD at an initial OD 600 The cells were seeded until the OD reached 0.3–0.4. 600The yeast were grown for approximately 6 h at 30 °C with shaking at 220 rpm until the AOD reached 1.8–2.2, then centrifuged at 3,000 rpm at 4 °C for 3 min. The cell pellet was resuspended, washed with ice-cold water, centrifuged again, and resuspended in 50 mL of ice-cold sterile lithium acetate (100 mM in water). After this step, the yeast were kept on ice until the end of electroporation. DTT was added to a final concentration of 10 mM. The cells were then grown for 20 min at 30 °C, 220 rpm, centrifuged for 3 min at 3,000 rpm at 4 °C, washed with 50 mL of ice-cold water, then centrifuged again and resuspended in 0.8 mL of electroporation buffer (1 M sorbital / 1 mM CaCl2).

[0073] Immediately after preparing the yeast cells, 400 μL of cells were mixed with 20 μL of concentrated DNA prepared as above, and the mixture was transferred to an electroporation cuvette. Electroporation was performed in a 2 mm cuvette using a Bio-Rad Gene pulser XCell with the following settings: 500 V, 15 ms pulse duration, and only one pulse. Cells were immediately rescued with 1 mL of a 1:1 mixture of sorbitol and YPD medium. The cuvette was washed three times with 1 mL of fresh sorbitol and YPD mixture each time. All cells were mixed and incubated at 30°C for 30 min without shaking, followed by incubation for 30 min with shaking at 220 rpm. 10 μL of cells were plated in 100-, 1,000-, and 10,000-fold serial dilutions on three SDCAA plates to determine library size. The remaining cells were centrifuged at 3,000 rpm for 2 min, 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). Cultures were incubated at OD 600 The cells were grown for 12–24 h at 30°C with shaking at 220 rpm until the OD reached 15 (OD 600 ~1 is approximately 1×10 7(equivalent to yeast cells / mL). For each batch of library generation, a negative control containing no plasmid DNA was included.

[0074] The total library sizes of CapN and CapC (four libraries for CapN and three libraries for CapC) were approximately 1 × 10 7 It was decided that:

[0075] yeast labeling The labeling procedure was used for both non-library and library yeast cultures. (Generation of non-library and library yeast cultures is described in "Yeast strains and non-library cultures" and "Yeast library generation", respectively.) Yeast were freshly passaged in SDCAA medium before the experiment. To induce expression of the pCTCON2 plasmid, 500 μL of yeast cultured overnight in SDCAA medium was added to 5 mL of SGCAA (synthetic galactose and casein amino acid medium, 2% galactose, 0.67% yeast nitrogen base without amino acids (BD Difco), 0.5% casamino acids (BD Difco), 0.54% disodium phosphate, 0.856% monosodium phosphate)) medium and grown overnight at 30 °C with shaking at 220 rpm. Prior to labeling, 250 μL (or 1 mL for the first library selection round) of yeast cultured overnight in SGCAA medium was centrifuged at 8000 rpm for 30 s and the supernatant was discarded. The cell pellet was resuspended and washed twice with 1 mL of PBSB (sterile phosphate-buffered saline supplemented with 0.1% bovine serum albumin) each time.

[0076] Yeast expressing the TEV protease cleavage site (CapN construct) was treated with TEVp and then labeled with an antibody-fluorophore conjugate. Samples were incubated with 200 μL of PBSB containing TEVp (expressed and purified as described in "TEV protease expression and purification") and 5 μM Shield-1 for 3 h at 4°C with rotation. In negative controls, either TEVp or Shield-1, or both, were absent from PBSB. To maintain TEVp under reducing conditions, 1 mM DTT and 30 mM reduced and 3 mM oxidized glutathione were added to all samples.

[0077] Yeast expressing SsrA(CapC construct) was labeled with APEX2. 40 After subjecting the samples to RT-PCR, they were labeled with antibody-fluorophore and streptavidin-fluorophore conjugates. Samples were incubated with 100 μL of PBSB containing SspB-APEX2 (expressed and purified as described in "Expression and purification of SspB-APEX2") and 5 μM Shield-1 for 10 min at room temperature with rotation. In the negative control, either SspB-APEX2 or Shield-1, or both, were absent from PBSB. After incubation, samples were washed twice with 1 mL of PBSB each time and resuspended in 950 μL of PBSB containing 1% BSA (bovine serum albumin). 1 μL of biotin phenol (1 mM in dimethyl sulfoxide) was added and mixed well with the sample. Then, 1 μL of hydrogen peroxide (0.5 mM in water, freshly prepared, EMD chemicals) was added and mixed well. After exactly 2 min of incubation, 200 μL of quenching solution 1 (freshly prepared 30 mM Trolox (Thermo Fisher Scientific), 60 mM sodium ascorbate (Millipore Sigma)) was added. The samples were centrifuged at 8,000 rpm for 30 s and the supernatant was discarded. Then, 400 μL of quenching solution 2 (freshly prepared 5 mM Trolox, 10 mM sodium ascorbate) was added to resuspend the cell pellet. After centrifugation again at 8,000 rpm for 30 s, the supernatant was discarded and the samples were washed twice with 1 mL of PBSB each time.

[0078] After TEVp or APEX2 labeling, samples were labeled with antibody fluorophore and / or streptavidin fluorophore conjugates. To label the FLAG and HA epitope tags, primary anti-FLAG or anti-HA antibodies were used, followed by secondary antibodies conjugated with Alexa Fluor 568 or 647. To detect biotinylated proteins from APEX2 labeling, streptavidin conjugated with PE (phycoerythrin) was used. All antibodies were diluted to 1 μg / mL in PBSB, streptavidin-PE (Jackson Immuno Research) was diluted 200-fold, and each yeast sample was incubated with 100 μL of the mixture for 15 min at room temperature with rotation. After each step of labeling, samples were washed twice with PBSB. All samples were resuspended in PBSB and analyzed or sorted by FACS (fluorescence-activated cell sorting, following the procedure described in "FACS analysis and library selection") within 24 hours after labeling.

[0079] FACS analysis and library selection After labeling according to the procedure described above (below, "Yeast labeling"), non-library yeast samples were analyzed on an LSRFortessa cell analysis flow cytometer (BD Biosciences) equipped with a 640 nM laser and a 670 / 14 emission filter (for Alexa Fluor 647), and a 561 nM laser and a 586 / 15 emission filter (for Alexa Fluor 568 and PE). Library samples were sorted on a FACSAria III cell sorter flow cytometer (BD Biosciences) equipped with a 633 nM laser and a 660 / 20 emission filter (for Alexa Fluor 647), and a 561 nM laser and a 582 / 15 emission filter (for Alexa Fluor 568 and PE). Single yeast cells were selected from the successive gates P1 and further analyzed according to FIG. 21.

[0080] 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, and no Shield-1 was added. A total of four rounds of selection were performed. The number of cells recovered in each round was as follows:

[0081] 1st round (negative selection): The cells recovered from library 1 were 0.034% (1.4 × 10 7 cell) The cells recovered from library 2 were 0.041% (2.2 × 10 7 cell) The cells recovered from library 3 were 0.028% (2.1 × 10 7 cell) The cells recovered from library 4 were 0.025% (1.0 × 10 7 cell) The recovered cells are bound for further selection.

[0082] 2nd round (positive selection): 3.4% of cells were recovered (1.2 × 10 6 cell).

[0083] 3rd round (negative selection): 11.3% of cells were recovered (3.5 × 10 6 cell).

[0084] 4th (Positive Selection): 5.8% of cells were recovered (1.5 × 10 6 cell).

[0085] A CapC library was generated and labeled as described above ("Yeast library generation" and "Yeast labeling", respectively). For positive selection, both SspB-APEX2 and Shield-1 were added. For negative selection, Shield-1 was not added and only SspB-APEX2 was added. A total of two rounds of selection were performed. The number of cells recovered in each round was as follows:

[0086] 1st round (negative selection): 0.3% of cells were recovered (1.5 × 10 4 cell).

[0087] 2nd round (positive selection): 0.1% of cells were recovered (3 × 10 3 cell)

[0088] All yeast cells were harvested in 5 mL of SDCAA medium containing 100 units / mL penicillin, 100 μg / mL streptomycin, and 30 μg / mL kanamycin. Immediately after sorting, cells were grown at 30 °C with shaking at 220 rpm for 2–5 days until saturation. For the next round of sorting, cells were passaged at least once in SDCAA medium and labeled according to the procedure in "Yeast labeling" above. After the last round of sorting, plasmids were extracted with a Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research), following a modification of the manufacturer's protocol. The overnight yeast culture (500 μL) was transferred to 10 mL of fresh SDCAA medium and incubated at 37 °C for 2 h at 37 °C for 1 h. 600The yeast cells were grown until the NA was 1–2. The yeast cells were spun down, washed once with phosphate-buffered saline (PBS), and then resuspended in 200 μL of solution I and 6 μL of Zymolase solution provided in the kit. Vigorous vortexing was performed for >1 min. The yeast cells were placed on a shaker at 37 °C overnight to break down the cell wall. After overnight incubation, the yeast cells were vortexed vigorously for >5 min. Then, 200 μL of solution II was added to the yeast cells, followed by brief vortexing and incubation at room temperature for 5 min. 400 μL of neutralizing solution was added and the cells were vortexed briefly. The cell lysate was spun down at 20,000 × g for 10 min, 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 E. coli by the heat shock method. Individual clones were sequenced, transformed into EBY100 yeast cells (as described above in "Yeast strains and non-library cultures"), labeled (as described above in "Yeast labeling"), and analyzed by FACS (as previously described in this section).

[0089] All-atom molecular dynamics simulation Atomistic molecular dynamics simulations of FKBP and the RYSPNL hexapeptide with capped N- and C-termini (Ac-RYSPNL-NHMe) were performed to analyze the binding pose of the peptide to its binding site. The simulations were performed using the AMBER99SB-ILDN protein force field. 42 and TIP3P Water 43 GROMACS 2018.1 software package using 41 The simulation was performed using DeepView / Swiss-Pdb Viewer based on the structure of FKBP12 protein (PDBID: 1NSG) with the F36V mutation. 44 The peptide was inserted outside the putative FKBP binding site using the Scwrl4 program. 45The peptide was placed into an α-helical conformation with the side chains generated by. The initial distance of the center of mass of the peptide to the center of mass of V36 of FKBP was 16.7 Å. The minimum distance between any atom of the peptide and the FKBP protein was 5.75 Å, which allows for separation by at least two hydration layers.

[0090] The system was placed in a cubic simulation box of 75 Å × 75 Å × 75 Å and solvated with 13444 water molecules in addition to the 67 water molecules resolved in the crystal structure. Thirty-eight sodium ions and 40 chloride ions were added to approximate a physiological salt concentration of 150 mM and neutralize the protein (+1e) and peptide (+1e) charges at pH 7. The protonation states of the protein side chains were estimated by the pdb2gmx tool in GROMACS. The simulations were run using a particle mesh Ewald lattice with a grid constant of 1.2 Å and fourth-order interpolation to handle long-range electrostatic interactions. 46 The algorithm was used with periodic boundary conditions. A cutoff of 10 Å was used for short-range Lennard-Jones and electrostatic interactions with pressure and total energy corrections. The LINCS algorithm 47 is used to constrain the bond lengths of the protein during dynamics simulations, and the SETTLE algorithm 48 was used to constrain the shape of the water molecules.

[0091] After 1000 steps of steepest descent energy minimization, the system is fitted with a simulation time step of 1 femtosecond and a Berendsen 49 The isothermal-isobaric ensemble molecular dynamics simulations were equilibrated at 300 K and 1 bar for 100 ps using a thermostat and a barostat. During this equilibration, non-hydrogen atoms of the protein and peptide were restrained to their initial positions using isotropic position restraints with a force constant of 10 k / (mol Å2). This was followed by a Nose-Hoover thermostat with a time constant of 1 ps. 50、51 and the Parrinello-Rahman barostat with a time constant of 2 picoseconds.52 A 2-microsecond production simulation was performed using a 2-microsecond time step without positional constraints in the isothermal-isobaric ensemble at 300 K and 1 bar using a simple clustering algorithm using non-hydrogen atoms and a cutoff of 1.5 Å. 53 In addition, the distance between the center of mass of each side chain of the peptide and V36 of FKBP was monitored.

[0092] Fluorescence microscopy of cultured cells Confocal imaging was performed on a Nikon inverted confocal microscope equipped with 10x air, 20x air, and 60x oil immersion objectives, a Yokogawa CSU-X1 5000RPM spinning disk confocal head, and a Ti2-ND-P perfect focus system4, a compact 4-line laser source: 405nM (100mW), 488nM (100mW), 561nM (100mW), and 640nM (75mW) lasers. The following laser excitation and emission filter combinations for the various fluorophores were used: DAPI (excitation at 405 nM; emission at 455 / 50), EGFP / Alexa Fluor 488 (excitation at 488 nM; emission at 525 / 36), mCherry / Alexa Fluor 568 (excitation at 568 nM; emission at 605 / 52), Alexa Fluor 647 (excitation at 647 nM; emission at 705 / 72), and differential interference contrast (DIC). An ORCA-Flash 4.0 LT+sCMOS camera was used. Acquisition times ranged from 100 to 1000 ms. All images were collected and processed using the Nikon NIS-Elements hardware control and analysis module.

[0093] Culture and transfection of HEK 293T cells Low-passage HEK293T cells (<20 passages) were cultured in complete growth medium (1:1 DMEM (Dulbecco's modified Eagle's medium, Gibco):MEM (Eagle's minimum essential medium)) in T25 or T75 flasks at 37 °C and 5% CO2. The medium was 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 imaging experiments, 48-well plates were pretreated with 200 μL of 20 μg / mL human fibronectin (Millipore Sigma) for 10 min at 37 °C. HEK293T cells were then seeded into 48-well plates at 60%–90% confluence. The DNA mixture was incubated with 1 μL of 1 mg / mL PEI max solution in 10 μL of serum-free DMEM medium for 15 min at room temperature. Then, complete DMEM growth medium (100 μL) was mixed with the DNA-PEI max solution and added to the fully attached HEK293T cells at the bottom of the wells and incubated for 18 h before further processing.

[0094] Production of lentiviral supernatant for HEK cell transduction Fresh cell culture flasks were incubated with 20 μg / mL human fibronectin (HFN, Millipore Sigma) for at least 10 min at 37 °C to help cells attach to the surface and increase transfection efficiency. After incubation, HFN was aspirated and HEK293T cells were seeded at 70–90% confluence. For T25 flasks, cells were grown for 1–3 h at 37 °C in 5 mL of complete DMEM growth medium, 1:1 DMEM (Dulbecco's Modified Eagle 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). After incubation, 2.5 μg of viral DNA, 0.25 μg of pVSVG, and 2.25 μg of Delta8.9 lentiviral helper plasmid were combined with 250 μL of DMEM and mixed thoroughly. Then, 25 μL of 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 of complete medium, and transferred to a T25 flask. Cells were incubated at 37°C for 48 hours, and the virus-containing supernatant was harvested, flash frozen in liquid nitrogen, and stored at -80°C for up to 1 year.

[0095] Cultivation and infection of HEK 293T cells Low-passage HEK293T cells (less than passage 20) were cultured in complete growth medium in T25 or T75 flasks at 37°C under 5% CO2. For imaging experiments, 24-well glass-bottom plates (Cellvis) were pretreated with 350 μL of 20 μg / mL human fibronectin (Millipore Sigma) for 10 min at 37°C. HEK293T cells were then seeded in 24-well plates at 40%–60% confluence. When infecting a single well of a 24-well plate, 100–200 μL of each supernatant virus was gently added to the top of the medium and cultured for 48 h before further processing.

[0096] HEK293T cell stimulation, imaging, and data analysis of shield-1-induced protein translocation to the plasma membrane HEK293T cells were seeded at 80% confluence in 24-well plates as described above and then transfected with 200 ng of mCherry-CapN-SsrA-CapC-CAAX and 200 ng of SspB-EGFP plasmid. SEQ ID NO: 18 contains a representative DNA sequence including the CapN, SsrA (bold underlined), and CapC (underlined) sequences. (SEQ ID NO:26) [ka]

[0097] SEQ ID NOs: 27-45 are representative sequences containing the met-enkephalin amino acid sequence of the CapC cage expressed as a separate construct (Figure 22, left panel) with a binding sequence for FKBP.

[0098] Protein sequence Surface transport signal peptide SEQ ID NO:27-MKTIIALSYIFCLVFA Met-enkephalin SEQ ID NO:28-YGGFM Binding sequence, linker, and FKBP binding site SEQ ID NO: 29-PNLRPFGSGGSGTGSGSGGSGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE AgeI restriction site SEQ ID NO:30-TG Linker SEQ ID NO:31-GGSGSGSGGSGGSGG Truncated human cluster of differentiation 4 (CD4) transmembrane domain SEQ ID NO:32- LPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRR Linker SEQ ID NO:33-KGSGSTSGSGSGGSRGSGGSSGG CIBN (truncated cryptochrome interacting basic helix-loop helix protein) SEQ ID NO:34- to enhance surface transport MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKAAKFDTETKDCNEAAKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSKVTKELEKTDYIH Hemagglutinin (HA) epitope tag SEQ ID NO: 35-YPYDVPDYA

[0099] DNA sequence: Surface transport signal peptide SEQ ID NO: 36-ATGAAGACCATCATCGCCCTGAGCTACATCTTCTGCCTGGTGTTCGCC Meto-enkephalin SEQ ID NO: 37-TACGGCGGATTTATG Binding sequence, linker, and FKBP binding site SEQ ID NO: 38- CCTAATTTGCGGCCTTTTGGTTCAGGCGGGTTCTGGTACTGGATCTGGTTCTGGAGGTTCTggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgcactacaccgggatgcttgaagatggaaagaaagttgattcctcccgggacagaaaca agccctttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaa AgeI restriction site SEQ ID NO:39-ACCGGT Linker SEQ ID NO: 40-GGTGGAAGTGGATCAGGCAGCGGTGGATCTGGAGGTAGCGGCGGA Truncated human cluster of differentiation 4 (CD4) transmembrane domain SEQ ID NO: 41- CTGCCCACATGGTCCACCCCGGTGCAGCCAATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTCAGGTGCCGGCACCGAAGGCGC Linker SEQ ID NO: 42- AAGGGCTCGGGCTCGACCTCGGGCTCGGGCagcggtggcTCTAGAGGTTCTGGTGGCAGCTCTGGAGGT CIBN (truncated cryptochrome interacting basic helix-loop helix protein) SEQ ID NO: 43- to enhance surface transport Atgaatggagctatataggaggtgaccttttgctcaattttcctgacatgtcggtcctagagcgccaaagggctcacctcaagtacctcaatcccacctttgattctcctctcgccggcttctttgccga ttcttcaatgattaccggcggcgagatggacagctatctttcgactgccggtttgaatcttccgatgatgtacggtgagacgacggtggaaggtgattcaagactctcaatttcgccggaaacgacgc ttgggactggaaatttcaagGCaGCgaagtttgatacagagactaaggattgtaatgagGCgGCgaagaagatgacgatgaacagagatgacctagtagaagaaggagaagaagagaagtcgaaaata acagagcaaaacaatgggagcacaaaaagcatcaagaagatgaaacacaaagccaagaaagaagagaacaatttctctaatgattcatctaaagtgacgaaggaattggagaaaacggattatattcat Hemagglutinin (HA) epitope tag SEQ ID NO: 44-TACCCATACGATGTGCCAGATTACGCC Stop codon SEQ ID NO:45-tag

[0100] Cells were cultured at 37°C for 24 hours before further processing. HEK293T cells were imaged on a Nikon inverted confocal microscope using a 60x oil immersion objective. Shield-1 dissolved in complete growth medium was gently added to 10 μM on top of the medium during imaging. Intensity profiles were acquired with Nikon NIS-Elements analysis module and plotted with GraphPad Prism 7.

[0101] HEK293T cell stimulation, imaging, and data analysis of Shield-1-induced delocalization from the plasma membrane to the cytoplasm HEK293T cells were seeded at 40% confluence in 24-well plates, then transduced with 200 μL of transmembrane domain lentiviral supernatant and 200 μL of mCherry-TEV protease lentiviral supernatant and cultured at 37°C for 48 hours before further processing. Two additional uninfected wells are also seeded for background subtraction. Shield-1 dissolved in complete growth medium was gently added to the top of the medium at 10 μM. Two uninfected wells are treated with and without Shield-1, respectively. HEK293T cells were cultured at 37°C for 18 hours before imaged.

[0102] HEK293T cell stimulation, imaging, and data analysis of nucleocytoplasmic protein distribution with Shield-1 control HEK293T cells were seeded 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 cultured at 37°C for 24 hours and replated in 24-well plates. Shield-1 dissolved in complete growth medium was gently added to the top of the medium at 10 μM. HEK293T cells were cultured at 37°C for 18 hours before imaging. HEK293T cells were imaged using a 60x oil immersion objective on a Nikon inverted confocal microscope. Individual cells and nuclei are determined by mCherry signal. Mean intensity and area are acquired using Nikon NIS-Elements analysis module. The background mean intensity was subtracted from the mean intensity and the resulting number was multiplied by the area to obtain the total intensity. The EGFP distribution ratio was calculated from the total intensity of EGFP in the cytoplasm to the total intensity of EGFP in the whole cell. P values ​​were determined by unpaired two-tailed t-test.

[0103] HEK293T cell stimulation, imaging, and data analysis of shield-1-dependent gene transcription activation HEK293T cells were seeded at 40% confluence in 24-well plates, then transduced with 100 μL UAS-mCherry lentiviral supernatant, 100 μL Gal4 DBD lentiviral supernatant, and 50 μL VP16 lentiviral supernatant, and cultured at 37°C for 48 h before further processing. Two uninfected wells were also seeded for background subtraction. Shield-1 dissolved in complete growth medium was gently added to the top of the medium at 10 μM. Two uninfected wells were treated with and without Shield-1, respectively. HEK293T cells were imaged after 18 h of culture at 37°C. HEK293T cells were imaged using a 20x air objective on a Nikon inverted confocal microscope. Twelve fields were acquired for each condition. Mean intensity was acquired from each image using the Nikon NIS-Elements analysis module. The mean intensity was subtracted from the average of the intensities of the 12 background images and plotted with Prism 7. Some intensities were negative and are not shown in Figure 12c, but were counted in the average for each condition. P values ​​were determined by unpaired two-tailed t-tests.

[0104] Generation of AAV supernatant AAV supernatant was used for neuronal culture experiments. 6-well plates were pretreated with human fibronectin for 10 min at 37 °C. Then, HEK293T cells were seeded in 6-well plates at 60–90% and transfected 2–3 h 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 helper 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), incubated at room temperature for 15 min, and then 2 mL of complete growth medium was added and mixed. The DNA mix was gently added to the top of the cells. After culturing HEK293T cells at 37 °C for 40 h, the supernatant (containing the secreted AAV) was collected. Viral supernatants were stored in sterile Eppendorf tubes (0.5 mL / tube), flash frozen in liquid nitrogen, and stored at −80°C.

[0105] Concentrated AAV manufacturing AAV was prepared for in vivo use as previously described. HEK293T cells less than passage 10 were transfected into three T150 flasks at 80% confluence. For each T150 flask, 5.2 μg of construct plasmid, 4.35 μg of AAV1 serotype plasmid and 4.35 μg of AAV2 serotype plasmid, 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 for 10 min at room temperature. The DNA mixture was further suspended in 10 mL of complete medium and added to the cells. HEK293T cells were cultured at 37 °C and 5% CO2 for 40-48 h. Cells were harvested with a cell scraper and resuspended in 10 mL of DPBS before being collected by centrifugation at 1200 rpm for 5 min at room temperature. The supernatant was discarded and the pellet was resuspended in 20 mL of 100 mM NaCl, 20 mM Tris (pH = 8.0). One mL of freshly prepared 10% aqueous sodium deoxycholate (Sigma-Aldrich) 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 h and then centrifuged at 8000 rpm for 10 min. The heparin column was first equilibrated with 10 mL of 100 mM NaCl, 20 mM Tris (pH = 8.0) using a peristaltic pump and then loaded with the viral supernatant. The column was washed with 25 mL of 100 mM NaCl, 20 mM Tris (pH = 8.0) using a peristaltic pump, followed by 1 mL of 200 mM NaCl, 20 mM Tris (pH = 8.0) and 1 mL of 300 mM NaCl, 20 mM Tris (pH = 8.0) using a 5 mL syringe. To elute the virus, 1.5 mL of 400 mM NaCl, 20 mM Tris (pH = 8.0), 3.0 mL of 450 mM NaCl, 20 mM Tris (pH = 8.0), and 1.5 mL of 500 mM NaCl, 20 mM Tris (pH = 8.0) were sequentially injected into the column with a 5 mL syringe.The eluted virus was concentrated using an Amicon Ultra 15mL centrifugation unit with a 100,000 molecular weight cutoff at 8000 rpm for 6 min to a final volume of 500 μL. For buffer exchange, 1mL of sterile 20mM Tris, 150mM NaCl, 0.05% PF68 solution was added to the filter unit and the column was centrifuged again until the virus volume was approximately 500 μL. The buffer exchange step was repeated two more times, with a final volume of approximately 100 μL. The concentrated AAV was aliquoted in 5μL aliquots into 0.6mL low-retention microcentrifuge tubes (Thermo Fisher Scientific) and stored at -80°C.

[0106] Neuronal cell culture experiments Rat cortical neurons (Thermo Fisher Scientific, Cat. No. A1084001) were seeded according to the user protocol. 96-well half-area glass plates (Corning, CLS4580-10EA) were coated with 50 μl 0.1 mg / ml poly-D-lysine (Gibco) for 1 h and then washed twice with ultrapure water. Frozen rat cortical neurons were quickly thawed in a 37°C water bath until small ice chips formed. The cells were transferred to a 50 ml conical tube. 1 ml of a pre-warmed 3:1 ratio mixture of complete neurobasal medium (nM) and glial enrichment medium (GEM) was added dropwise very slowly to the cells with gentle mixing. nM consisted of Neurobasal medium (Thermo Fisher Scientific) supplemented with 2% B27 (Thermo Fisher Scientific), 50 mM HEPES (Thermo Fisher Scientific), 1% penicillin-streptomycin (50 units / mL penicillin and 50 μg / mL streptomycin, Thermo Fisher Scientific), and 1% GlutaMAX (Thermo Fisher Scientific). GEM consisted of DMEM (Gibco) supplemented with 10% FBS (fetal bovine serum, Sigma), 2% B27 (Thermo Fisher Scientific), 50 mM HEPES (Thermo Fisher Scientific), 1% penicillin-streptomycin (50 units / mL penicillin and 50 μg / mL streptomycin, Thermo Fisher Scientific), and 1% GlutaMAX (Thermo Fisher Scientific). Additionally, 2 ml of complete Neurobasal medium was added to the cells. Viable cell density was determined by adding 10 μL of cell suspension to 10 μl of 0.4% trypan blue followed by cell counting using a hemocytometer. 5 Viable cells were seeded and the cells were grown at 37 °C and 5% CO2. Half of the medium was replaced with fresh complete neurobasal medium within 4–24 h after seeding. To maintain the cells, half of the medium was replaced every 3 days.

[0107] For neuronal infection, either concentrated AAV or supernatant AAV was added to neurons at DIV5–DIV10 (days in vitro). Five days post-infection, neurons were treated with 2 μM Shield-1 for 24 h and imaged live. The mean intensity was subtracted from the mean intensity of five background images and plotted by Prism 7. P values ​​were determined by unpaired two-tailed t-test.

[0108] animal All procedures were approved by the University of Michigan Committee on Animal Use and Care. C57BL / 6 mice were housed on a 12-h light / dark cycle and had food and water available ad libitum. Adult mice of both sexes were used.

[0109] Stereotaxic injection of AAV into the mouse brain The stereotactic injection procedure was performed as previously described. 54 Adult mice were anesthetized with isoflurane (5% for induction, 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 from the midline, −1.40 mm posterior, and −5.25 mm ventral from the corona) at a rate of 50 nL / min. The pipette was left in place in the brain for 10 min after injection to allow pressure to equalize and to avoid a vacuum effect upon removal of the pipette. Mice were injected subcutaneously with 1 mL saline and allowed to recover from surgery. An additional subcutaneous dose of carprofen 5 mg / kg was administered the day after surgery.

[0110] AAV virus injection into mouse liver Adult mice with shaved abdomens were placed in a stereotaxic apparatus and maintained under anesthesia with 1.5% isoflurane. Prior to surgery, the abdomen was disinfected with povidone followed by alcohol. A ~2 cm midline incision was made in the abdomen to expose the liver. One μL of AAV encoding a shield-1-dependent gene regulatory construct under the CMV promoter was delivered at a rate of 500 nL / min via a micropipette inserted directly into the liver.

[0111] Shield-1 Administration and Histology For mouse brains, 7 days after viral vector injection, 1 μL of 1 mM Aquashield-1 or saline control was administered locally into the LHA by stereotaxic injection. For mouse livers, 7 days after viral vector expression, animals were injected intraperitoneally with 40 mg / kg Aquashield-1 or saline twice at 24-h intervals. 48 h 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, followed by cryoprotection in 30% sucrose for 48 h at 4°C. Fixed tissues were then embedded in optimal cutting temperature compound and sectioned at 30 μm. Sections were rinsed in 0.1% PBS Tween-20 and stained with DAPI (1:10,000, Invitrogen, D1306) for 10 min at room temperature. Sections were then washed again and mounted with Prolong Gold mounting medium (Invitrogen, P36930). Confocal images were acquired with a Nikon A1 confocal microscope.

[0112] Equivalent The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0113] Incorporation by Reference All publications, published patent documents, and patent applications cited in this specification are herein 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 herein.

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Claims

1. 1. A method of modulating the activity of a peptide, comprising: a) a nucleic acid construct, 1) a nucleic acid encoding at least one chemically activated protein domain (CAP) that comprises a ligand binding site; and 2) generating said nucleic acid construct comprising a nucleic acid encoding a peptide; b) administering said nucleic acid construct to one or more cells to produce a fusion protein, said fusion protein comprising said at least one CAP linked to said peptide; and c) administering to the one or more cells a ligand, the ligand binding to the ligand binding site of the CAP of the fusion protein, wherein administering the ligand increases the activity of the peptide.

2. The method of claim 1 , wherein the at least one CAP comprises CapN, which binds to the N-terminus of the peptide, and / or CapC, which binds to the C-terminus of the peptide.

3. 10. The method of claim 1, wherein said administering said nucleic acid construct comprises direct injection of said nucleic acid construct, polymer-mediated liposomal and / or biopolymer gene delivery, plasmid delivery, and / or viral delivery.

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

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

6. The method of claim 5 , wherein the expression is intracellular or extracellular expression.

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

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

9. The method of claim 1 , wherein the peptide is an enzyme activating peptide, an enzyme inhibitory peptide, an enzyme regulatory peptide, a binding peptide, a localization 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 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. 2. The method of claim 1, wherein the nucleic acid construct comprises at least one nucleic acid selected from the group encoding Aga2p, a reporter gene, a 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. 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 an activity of the peptide, said measuring comprising measuring the localization, structure and / or function of the peptide.

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

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

22. 1. A composition comprising: a) a nucleic acid construct, 1) a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site, said ligand being shield-1 and / or aquashield-1; 2) the nucleic acid construct, comprising a nucleic acid encoding a peptide; C) a gene transfer reagent and / or a vector.

23. A fusion protein comprising: a) a protein expressed by a nucleic acid encoding at least one chemically activated protein domain (CAP) comprising a ligand binding site, said ligand being Shield-1 and / or Aquashield-1; b) a peptide expressed by a nucleic acid, said peptide being regulated by said Shield-1 and / or said Aquashield-1.

Images