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WO2012024558A2 - Régulation médiée par la lumière de la dimérisation de protéine - Google Patents

Régulation médiée par la lumière de la dimérisation de protéine Download PDF

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Publication number
WO2012024558A2
WO2012024558A2 PCT/US2011/048360 US2011048360W WO2012024558A2 WO 2012024558 A2 WO2012024558 A2 WO 2012024558A2 US 2011048360 W US2011048360 W US 2011048360W WO 2012024558 A2 WO2012024558 A2 WO 2012024558A2
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rapamycin
polypeptide
compound
protein
photoactivatable
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PCT/US2011/048360
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WO2012024558A3 (fr
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Alexander Deiters
Klaus Hahn
Andrei Karginov
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The University Of North Carolina At Chapel Hill
North Carolina State University
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Publication of WO2012024558A2 publication Critical patent/WO2012024558A2/fr
Publication of WO2012024558A3 publication Critical patent/WO2012024558A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to photoactivatable rapamycin compounds.
  • the invention further relates to the use of photoactivatable rapamycin compounds to regulate the dimerization and/or activity of polypeptides in a light dependent manner.
  • Rapamycin also known as sirolimus, is a complex macrolide natural product isolated from the bacterium Streptomyces hygroscopicus, found in a soil sample on Easter Island in 1975 (Huang et al, Cancer Biol. Ther. 2:222 (2003); Abraham et al, Annu. Rev. Immunol. 74:483 (1996); Pollock et al, Curr. Opin. Biotechnol. 73:459 (2002); Bayle et al, Chem. Biol. 75:99 (2006)).
  • Rapamycin mediates heterodimerization of the proteins FKBP12 (FK506 binding protein 12) and FRB (FKBP12 rapamycin binding domain) (Huang et al, Cancer Biol. Ther. 2:222 (2003)). Due to the excellent physiological properties of rapamycin, including good pharmacokinetics, permeability across the blood-brain barrier, and oral bioavailability (Abraham et al, Annu. Rev. Immunol 74:483 (1996)), it has been used as a small molecule dimerizer for a wide range of applications in mammalian cells and organisms (Pollock et al, Curr. Opin. Biotechnol. 73:459 (2002)).
  • FRB-FKBP12 interaction has proven valuable in a broad range of basic research applications, where it has been engineered to control gene function through rapamycin-induced transcription (Bayle et al, Chem. Biol. 73:99 (2006); Pollock et al, Proc. Natl. Acad. Sci, USA 97: 13221 (2000)), protein localization (Bayle et al, Chem. Biol. 73:99 (2006)), protein degradation (Edwards et al, J. Biol. Chem. 252:13395 (2007); Maynard-Smith et al, J. Biol. Chem.
  • Photo-activatable derivatives of small molecules are typically generated through the installation of a light-removable protecting group, a so called "caging group", at a site crucial for biological activity of the small molecule (Mayer et al, Angew Chem. Int. Ed. 45:4900 (2006); Young et al, Org. Biomol. Chem. 5:999 (2007)). This renders the molecule inactive, until the caging group is removed through light-irradiation, typically with UV light of 365-405 nm (Dong et al, J. Photochem. Photobiol B 55: 137 (2007); Schindl et al, J. Photochem. Photobiol B 44:91 (1998); Robert et al, J.
  • the present invention addresses previous shortcomings in the art by providing the first synthesis of a caged rapamycin analog which enables successful photo- control of the FKBP-FPvB interaction.
  • the caged rapamycin is applicable to regulating polypeptide dimerization and activity in live cells.
  • the present invention is based, in part, on the identification of a technique for producing a photoactivatable rapamycin compound and the use of the compound to regulate polypeptide dimerization and activity in a light dependent manner.
  • the identification of photoactivatable rapamycin permits light dependent regulation of polypeptide activity with high specificity and temporal resolution.
  • the present invention provides tremendous opportunities to study protein and cell function for research, diagnostic, and therapeutic purposes.
  • the invention relates to a photoactivatable rapamycin compound, comprising a caging group linked to rapamycin or an analog thereof, e.g., at position 40.
  • the invention relates to a complex comprising the photoactivatable rapamycin compound of the invention bound to a first polypeptide comprising a rapamycin binding domain (RBD).
  • RBD rapamycin binding domain
  • the invention relates to a method of modulating the activity a polypeptide comprising a RBD in a light-dependent manner, comprising contacting the polypeptide with the photoactivatable rapamycin compound of the invention in the presence of light.
  • the invention relates to a method of dimerizing a first polypeptide and a second polypeptide in a light-dependent manner, comprising contacting a first polypeptide comprising a RBD with the photoactivatable rapamycin compound of the invention in the presence of light to form a complex of the first polypeptide and the rapamycin compound, and contacting the complex with a second polypeptide that binds to the first polypeptide only when the first polypeptide is in a complex with a rapamycin compound and the rapamycin compound is activated.
  • the invention in another aspect, relates to a method of modulating the activity of a first polypeptide in a light dependent manner, comprising contacting a first polypeptide comprising a RBD with the photoactivatable rapamycin compound of the invention in the presence of light to form a complex of the first polypeptide and the rapamycin compound, and contacting the complex with a second polypeptide that binds to the first polypeptide only when the first polypeptide is in a complex with a rapamycin compound and the rapamycin compound is activated, wherein binding of the second polypeptide to the first polypeptide modulates the activity of the first polypeptide.
  • the invention relates to a kit comprising the photoactivatable rapamycin compound of the invention.
  • FIGS 1A-1C show photocaging of rapamycin.
  • Rap Structure of rapamycin
  • cRap caged rapamycin
  • NPOC-NHS nitro-piperonyloxycarbonyl N- hydroxysuccinimide carbonate
  • NPOC-NHS nitro-piperonyloxycarbonyl N- hydroxysuccinimide carbonate
  • NPOC-NHS nitro-piperonyloxycarbonyl N- hydroxysuccinimide carbonate
  • FIGS 2A-2B show light-regulated dimerization between FKBP12 and FRB.
  • HEK293T cells co-transfected with myc-FKBP 12-F AK and GFP-FRB constructs were treated with either the indicated amount of cRap or 0.5 ⁇ of Rap.
  • the cells were irradiated with UV light (365 nm) for 1 min (a) or 5 min (b) and incubated for 1 hour. Control cells were not irradiated.
  • Myc-FKBP- FAK was immunoprecipitated from cell lysates and co-immunoprecipitation of GFP-FRB was detected by Western blot.
  • Figure 3 shows a K-LISA mTor activity assay (EMD Biosciences) of Rap and cRap. In the absence of Rap and FKBP12, maximum mTor kinase activity is observed. The mTor activity is reduced to the same level in the presence of either Rap or cRap, indicating that introduction of the caging group at C-40 does not disrupt formation' of the ternary complex between mTor, FKBP12, and cRap. The assay was conducted according to the manufacturer's instructions and absorbance was measured on a SpectraMax 384 plus plate reader (Molecular Dynamics). The error bars represent standard deviations from two independent assays.
  • Figures 4A-4E show light-regulated dimerization of iFKBP and FRB.
  • (b-d) HEK293T cells co-transfected with GFP- FRB and either myc-iFKBP-FAK (b, c) or myc-FAK-iFKBP413 (d) were treated with either rapamycin (0.5 ⁇ ) or the indicated concentrations of cRap.
  • Ten minutes after addition of cRap or Rap cells were irradiated with 365 nm UV light for 1 min (b, d) or 5 min (c), and incubated for 1 hour. Control cells were not irradiated.
  • Myc-iFKBP-FAK was immunoprecipitated from cell lysates and co-immunoprecipitation of GFP-FRB was detected by Western Blot
  • Figures 5A-5F show light-mediated activation of a protein kinase,
  • Cells were treated with the indicated amount of rapamycin (Rap), caged rapamycin (cRap), or DMSO (control). The indicated samples were exposed to UV light (365 nm, 1 min). All cells were incubated at 37°C for 1 hour after treatment.
  • Myc-RapR-FAK was immunoprecipitated using an anti-myc antibody and tested in a kinase assay using an N-terminal fragment of paxillin as a substrate.
  • the level of phosphorylation of paxillin on Tyr31 indicates the kinase activity
  • (c, d) HeLa cells co-transfected with GFP-RapR-FAK and Cherry-FRB were treated with cRap (1 ⁇ ) and imaged before and after UV irradiation (365 nm, 1 min). Arrows indicate formation of large dorsal ruffles stimulated by activated RapR-FAK.
  • Figure 6 shows light-triggered mTOR deactivation using rapamycin dimer (dRap).
  • Figure 7 shows light-activated TEV protease activity using dRap.
  • Figure 8 shows an in vivo luciferase assay of rapamycin derivatives.
  • Figure 9 shows light-activated DiCre Recombinase activity using dRap.
  • Figure 10 shows light-mediated regulation of FKBP-FRB Cre dimerization with dRap.
  • Figure 1 1 shows light-mediated regulation of FKBP-FRB Cre dimerization with dRap.
  • Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. ⁇ 1.822 and established usage.
  • activate refers to an increase in at least one biological activity of a protein of interest of the invention, e.g., an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.
  • activate refers to a decrease in at least one biological activity of a protein of interest of the invention, e.g., a decrease of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • nucleic acid As used herein, "nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • polynucleotide or nucleotide sequence refers to a chain of nucleotides without regard to length of the chain.
  • the nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand.
  • the nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • the present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid or nucleotide sequence of this invention.
  • an "isolated polynucleotide” is a nucleotide sequence (e.g., DNA and/or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated polynucleotide includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence.
  • An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the chromosome.
  • isolated also can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
  • an "isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. "Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.
  • an "isolated" cell refers to a cell that is separated from other components with which it is normally associated in its natural state,
  • an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier of this invention.
  • an isolated cell can be delivered to and/or introduced into a subject.
  • an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then returned to the subject.
  • fragment as applied to a nucleic acid, nucleotide sequence, or polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid and comprising, consisting essentially of, and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g. , 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid.
  • Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive nucleotides of a nucleic acid according to the invention. In other embodiments, such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of less than about 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, or less consecutive nucleotides of a nucleic acid according to the invention.
  • fragment as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or almost identical ⁇ e.g. , 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide.
  • a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide according to the invention. In other embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of less than about 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, or less consecutive amino acids of a polypeptide according to the invention.
  • a "vector” is any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell.
  • a vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence.
  • a "replicon” can be any genetic element ⁇ e.g. , plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., capable of replication under its own control.
  • the term “vector” includes both viral and nonviral ⁇ e.g.
  • plasmid nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo.
  • a large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc.
  • the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini.
  • the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini.
  • Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.
  • a "recombinant" vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences ⁇ i.e. , transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences.
  • Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects.
  • Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus, geminivirus, and caulimovirus vectors.
  • Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers.
  • a vector may also comprise one or more regulatory regions, expression control sequences, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (e.g., delivery to specific tissues, duration of expression, etc.).
  • Vectors may be introduced into the desired cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu et al , J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 2(53: 14621 (1988); and Hartmut et al, Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).
  • methods known in the art e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu et al , J.
  • a polynucleotide of this invention can be delivered to a cell in vivo by lipofection.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a nucleotide sequence of this invention (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413 (1987); Mackey, et al, Proc. Natl. Acad. Sci, U.S.A. 55:8027 (1988); and Ulmer et al, Science 259: 1745 (1993)).
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner et al, Science 337:387 (1989)).
  • Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications W095/18863 and W096/17823, and in U.S. Patent No. 5,459,127.
  • the use of lipofection to introduce exogenous nucleotide sequences into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit.
  • transfection is directed to particular cell types in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey, et al, 1988, supra).
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
  • other molecules can be used for facilitating delivery of a nucleic acid in vivo, such as a cationic oligopeptide (e.g. , W095/21931), peptides derived from nucleic acid binding proteins (e.g., WO96/25508), and/or a cationic polymer (e.g., W095/21931).
  • transfection means the uptake of exogenous or heterologous nucleic acid (RNA and/or DNA) by a cell.
  • a cell has been “transfected” or “transduced” with an exogenous or heterologous nucleic acid when such nucleic acid has been introduced or delivered inside the cell.
  • a cell has been "transformed” by exogenous or heterologous nucleic acid when the transfected or transduced nucleic acid imparts a phenotypic change in the cell and/or a change in an activity or function of the cell.
  • the transforming nucleic acid can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell or it can be present as a stable plasmid.
  • a peptide is a chain of amino acids having a length of about 3 to about 50 residues.
  • a "fusion protein” is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides and/or peptides not found fused together in nature are fused together in the correct translational reading frame.
  • fusion polypeptides include fusions of a polypeptide of the invention (or a fragment thereof) to a polypeptide that is useful for identifying and/or purifying the fusion protein, e.g., all or a portion of glutathione-S- transferase, maltose-binding protein, or a reporter protein (e.g., Green Fluorescent Protein, ⁇ -glucuronidase, ⁇ -galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc.
  • a reporter protein e.g., Green Fluorescent Protein, ⁇ -glucuronidase, ⁇ -galactosidase, luciferase, etc.
  • hemagglutinin c-myc
  • FLAG epitope etc.
  • a “functional" polypeptide or “functional fragment” is one that substantially retains at least one biological activity normally associated with that polypeptide (e.g., catalytic activity, ligand binding).
  • the "functional" polypeptide or “functional fragment” substantially retains all of the activities possessed by the unmodified peptide.
  • substantially retains biological activity, it is meant that the polypeptide retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide).
  • non-functional polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%).
  • Biological activities such as protein binding and catalysis can be measured using assays that are well known in the art and as described herein.
  • express or "expression” of a polynucleotide coding sequence, it is meant that the sequence is transcribed, and optionally, translated. Typically, according to the present invention, expression of a coding sequence of the invention will result in production of the polypeptide of the invention. The entire expressed polypeptide or fragment can also function in intact cells without purification.
  • the term "photoactivatable rapamycin compound” refers to a modified rapamycin compound or an analog thereof that does not substantially exhibit one or more biological activity of rapamycin when bound to a protein comprising a RBD in the absence of light, e.g. , does not promote dimerization of a protein comprising a RBD.
  • substantially exhibit refers to a level of activity that is at least about 10% of the activity level promoted by rapamycin, e.g., at least about 20, 30, 40, or 50% of the activity level.
  • photocleavable rapamycin compound refers to a caged rapamycin compound or an analog thereof wherein the caging group is cleaved from the compound when irradiated with light.
  • photoswitchable rapamycin compound refers to a caged rapamycin compound or an analog thereof wherein the light-switchable group changes its configuration or conformation when irradiated with light without being cleaved.
  • the photo switching process is reversible and the activity of such compounds can be switched on and off by light.
  • analog of rapamycin refers to any molecule that can bind to a RBD and has substantially the same activity as rapamycin when bound to the RBD.
  • the term “has substantially the same activity” refers to a molecule that has at least about 50% of one or more biological activity of rapamycin upon binding to a RBD, e.g., at least about 60, 70, 80, 90, of 95% of the activity of rapamycin.
  • the biological activity is the ability to act as a dimerizer. Rapamycin analogs are well known in the art.
  • Categories or rapamycin analogs include, without limitation, rapamycin substituted in position 40 and/or 16 and/or 32, such as 40-O- substituted rapamycin derivatives, 16-O-substituted rapamycin derivatives, 32- hydrogenated rapamycin derivatives, 40-O-alkyl-rapamycin derivatives, rapamycin derivatives which are substituted in the 40 position by heterocyclyl, 32-deoxo-rapamycin derivatives, 32-hydroxy-rapamycin derivatives, rapamycin derivatives which are acylated at the oxygen in position 40, rapamycin 29-enol derivatives, tetrazole-containing rapamycin derivatives, mono-ester derivatives of rapamycin, di-ester derivatives of rapamycin, 27-oxime derivatives of rapamycin, 42-oxo derivatives of rapamycin, bicyclic derivatives of rapamycin, rapamycin dimers, silyl ether derivatives of rapa
  • rapamycin analogs include, without limitation, biolimus A9, deforolimus (also referred to as ridaforolimus), tacrolimus, pimecrolimus, 40-O- dimethylphosphinyl-rapamycin, 40-O-(2-ethoxy)ethyl -rapamycin, TAFA-93, AP23464, AP23675, AP23841 , 40-O-(2-hydroxy)-ethyl-rapamycin (RAD001 or everolimus), 40- epi-(tetrazolyl)-rapamycin (ABT578 or zotarolimux), 32-deoxorapamycin, 16-pent-2- ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32(S or R) -dihydro-rapamycin, 16- pent-2-ynyloxy-32(S or R)-dihydro-40-O-(2-hydroxyeth
  • rapamycin analogs include those disclosed in International Publication Nos. WO2007/13541 1, WO2004/101583, WO03/64383, WO01/14387, WO98/02441, WO 96/41807, W096/13273, WO 95/16691, WO 95/14023, WO 94/09010, WO 94/02485, WO94/02385, WO94/02136, WO 93/11130, and WO92/05179, U.S. Patent Nos.
  • rapamycin binding domain or RBD refers to a polypeptide having an amino acid sequence capable of binding rapamycin or an analog thereof.
  • the RBD can be a naturally occurring RBD (e.g., a fragment of a naturally occurring polypeptide) or a synthetic RBD (e.g., having an amino acid sequence not found in a naturally occurring polypeptide, e.g., having a sequence that is modified from a naturally occurring polypeptide sequence).
  • heterologous rapamycin binding domain refers to an RBD present in a polypeptide that is not naturally found in that polypeptide.
  • the term encompasses a RBD that has been fused to a polypeptide that does not normally contain an RBD, a RBD from one polypeptide that has been fused to a different polypeptide that has a different RBD that has been deleted or rendered nonfunctional, and a RBD that is naturally found in a polypeptide and has been modified to be different from the natural RBD.
  • the term “caging group” refers to a chemical moiety that, when bound to a compound, inhibits the binding and/or activity of that compound and in the presence of the appropriate wavelength of light is released from the compound, either permanently (e.g., cleaved from the compound) or reversibly (e.g., acting as a switch), permitting the binding and/or activity of the compound.
  • activation of a photoactivatable rapamycin compound refers to the irradiation of the compound to remove the caging group.
  • modulating the activity refers to the increasing (activating) or decreasing (inactivating) of one or more biological activities of a protein of interest relative to the level of activity prior to carrying out the present invention.
  • One aspect of the invention relates to photoactivatable rapamycin compounds.
  • the compound can be prepared from rapamycin or any analog of rapamycin that that can bind to a RBD and has substantially the same activity as rapamycin when bound to the RBD.
  • Rapamycin analogs are well known in the art and include, without limitation, the analogs described above.
  • the rapamycin analog has a reduced level of one or more of the biological activities of rapamycin, e.g., immune- suppressing activities.
  • the photoactivatable rapamycin compound comprises a caging group linked to rapamycin or an analog thereof.
  • the caging group can be linked to rapamycin or an analog thereof at a suitable location on the compound, including, without limitation, C8, C9, CIO, CI 6, C26, C27, C28, C32, or C40.
  • the caging group is linked to rapamycin or an analog thereof at position C40.
  • the photoactivatable rapamycin compound comprises more than one caging group, e.g., 2, 3, 4, 5, or more caging groups.
  • the caging groups consist of a chromophore that can be excited through light irradiation, and subsequently induces a cleavage process.
  • Such chromophores are typically comprised of conjugated pi-electron systems, traditionally found in aromatic systems such a phenyl, benzyl, quinoline, benzophenone, coumarin, and others.
  • aromatic systems such as a phenyl, benzyl, quinoline, benzophenone, coumarin, and others.
  • the caging group can be any group that is effective to prevent the rapamycin or an analog thereof from significantly exhibiting one or more biological activities of rapamycin when bound to a RBD in the absence of light, e.g. , ultraviolet light of about 365 nm.
  • the caging group can be one that is activated by one photon of light or two photons of light.
  • Suitable caging groups include, without limitation, nitro- piperonyloxycarbonyl, or//7o-nitrobenzyl, nitrodibenzofuran, ort 70-nitropropyl, bromohydroxycoumarin, dimethylaminocoumarin, bromohydroxyquinoline, para- hydroxyacetophenone, benzophenone, dimethylaminonitrophenyl, nitroindole, methoxynitroindole, ort/70-hydroxycinnamate, propylmethoxynitrobiphenyl, or derivatives thereof.
  • the caging group is a nitro- piperonyloxycarbonyl group, a 8-bromo-7-hydroxyquinolinyl group, or a 3-nitro-2- ethyldibenzofuran group.
  • Other suitable caging groups can have enhanced photochemical properties, e.g., higher quantum yield, higher two-photon cross section (enabling decaging at wavelengths >600 nm using two-photon excitation), faster decaging kinetics, and/or less toxic byproducts. Additional enhanced properties can include better on/off ratio of rapamycin activity before and after light irradiation, better solubility, better cellular uptake, and/or better cellular stability.
  • the caging group can be linked to the rapamycin or an analog thereof by any suitable linkage including, without limitation, a carbonate, ether, ester, carbamate, or urea linkage.
  • the linkage is a carbonate linkage.
  • photoactivatable rapamycin compounds include compound 3 (wYro-piperonyloxycarbonyl-caged rapamycin), compound 6 (bromohydroxyquinoline-caged rapamycin), and compound 8 (nitrodibenzofuran-caged rapamycin).
  • caging groups can carry function groups, e.g., alkyne, azide, carboxy, hydroxyl, and/or thiol, that can be further modified by natural and unnatural polymers to enhance the properties of light-activated rapamycin.
  • Those polymers can include, without limitation, polyethylene glycols, polypeptides, polyamines, oligonucleotides, etc.
  • the caging group is immobilized, e.g. , by attachment to a solid support.
  • the solid support can be, for example, a particle, bead, surface, or macromolecule.
  • Immobilized photoactivatable rapamycin compounds can be used, for example, to bind and/or separate proteins comprising a RBD. Further, the immobilized compounds can be released by irradiating the rapamycin compounds with light to cleave the compounds from the solid support.
  • a polymer- containing photoactivatable rapamycin compound is compound 11.
  • Photoactivatable rapamycin compounds containing photo-switchable groups are also contemplated, enabling the reversible light-regulation of rapamycin activity with light of different wavelengths (e.g. , UV light (365 nm) for activation of rapamycin, and visible light (>420 nm) for deactivation, or vice versa).
  • Suitable photo- switchable groups include, but are not limited to, azobenzene, diazobenzene, spiropyrans, diarylethenes, fulgides, overcrowded alkenes, and derivatives thereof.
  • the light-switchable group can be installed at different positions within the rapamycin structure, including, but not limited to, C8, C9, CI O, CI 6, C26, C27, C28, C32, or C40.
  • the photoactivatable rapamycin compound comprises more than one light-switchable group, e.g., 2, 3, 4, 5, or more light-switchable groups.
  • the invention relates to photoactivatable rapamycin dimers.
  • the dimers can be either photo-cleavable or photo-switchable.
  • the caging group can be any of the caging groups discussed above.
  • the linkers between the two rapamycin molecules can be of any type known in the art that provides a desirable length, stability, or other characteristic.
  • the photo-cleavable dimer is the compound shown below
  • the photo-switchable dimer is a compound of Formula I:
  • X and Y are independently selected from SiMe 2 , (CH 2 ) n , C(O), C(0)(CH2) n , and C(0)NH, and
  • n 1-5.
  • the photoactivatable rapamycin compound is an engineered rapamycin molecule with specificity for different RBD, e.g. , FKBP or FRB variants that have binding-site amino acid mutations.
  • engineered rapamycin molecules and mutated FRB variants are described in Edwards et al. (J. Biol. Chem. 252: 13395 (2007)), incorporated by reference in its entirety. The use of these components provides an additional level of control as the engineered rapamycin molecules do not recognize the native FKBP and/or FRB in a cell.
  • caged engineered rapamycin molecules include compound 15 and compounds of Formulae II and III:
  • One aspect of the invention relates to a complex comprising the photoactivatable rapamycin compound of the invention bound to a first polypeptide comprising a RBD.
  • a RBD Any RBD Icnown in the art can be used.
  • the RBD can be one that is native to the polypeptide or heterologous to the polypeptide.
  • the first polypeptide is a fusion protein comprising a protein of interest and a heterologous RBD.
  • the RBD is FK506 binding protein 12 (FKBP12) or a functional fragment thereof, i.e., a fragment that binds rapamycin.
  • the RBD is iFKBP, which corresponds to Thr22-Glul08 of human FKBP12 (GenBank Accession No. NM_054014.2).
  • the RBD is a mutant of FKBP12 that binds no n- immunosuppressive derivatives of rapamycin, e.g., the F36V mutant of FKBP12 that binds SLF', Shieldl , and Shield2 as described in Grimley et al. (Bioorg. Med. Chem. Lett. 18:759 (2008)), incorporated by reference herein in its entirety.
  • the protein of interest in the fusion proteins of the invention can be any known protein of interest.
  • the term "protein of interest” encompasses full length proteins, modified proteins, fragments of proteins, and functional domains of proteins.
  • the protein of interest is a mammalian protein, e.g., a human protein.
  • the protein of interest or a functional fragment thereof is selected from a family of proteins, e.g., kinases, GTPases (such as Racl and Cdc42), guanine nucleotide exchange factors, transcription factors, integrins, cytoskeletal proteins (e.g.
  • the protein of interest or a functional fragment thereof is a functional domain selected from translocation signals (such as nuclear localization signals, nuclear export signals, and organelle targeting domains), binding domains, the catalytic domain of proteinases, kinases, and other enzymes, the ATP binding pocket of kinases and other enzymes, the regulatory domain of kinases and other enzymes (e.g., the RI or RII domain of protein kinase A), the regulatory light chain and/or the ATPase domains of myosin motor proteins, the regulatory light chain and/or the ATPase domains of microtubule-driven motor proteins, the regulatory and/or catalytic domains of kinases, and SH domains.
  • translocation signals such as nuclear localization signals, nuclear export signals, and organelle targeting domains
  • binding domains such as nuclear localization signals, nuclear export signals, and organelle targeting domains
  • binding domains such as nuclear localization signals, nuclear export signals, and organelle targeting domains
  • binding domains such
  • the protein of interest is a peptide that inhibits the activity of a target protein (e.g. , an inhibitor of protein kinase A, protein kinase C, vinculin, etc.).
  • a target protein e.g. , an inhibitor of protein kinase A, protein kinase C, vinculin, etc.
  • the protein of interest is a first member of a protein binding pair.
  • the heterologous RBD of the fusion protein can be located at any position in the protein of interest such that it is effective in imparting light- dependent regulation of the protein of interest.
  • the RBD can be at the N- terminus, the C-terminus, or inserted internally.
  • the RBD is inserted adjacent to or near a catalytic domain of the protein of interest.
  • the protein of interest is a kinase and the RBD is inserted into the kinase such that the catalytic activity of the kinase becomes dependent on rapamycin binding.
  • the RBD is inserted within a loop on the surface of the kinase.
  • the RBD is inserted within the catalytic domain of the kinase.
  • the RBD is inserted near the catalytic domain of the kinase, e.g., within 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of the N-terminal and/or C-terminal end of the catalytic domain.
  • one or more kinase amino acid residues are removed at the site at which the RBD is inserted, e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or amino acids are removed.
  • the fusion protein comprises one or more linkers between different domains of the fusion protein, e.g. , between the protein of interest and the RBD.
  • the linker can be an amino acid sequence of a length suitable to provide sufficient flexibility between the domains of the fusion protein to allow light-dependent activation of the protein of interest.
  • the linker can comprise, consist essentially of, or consist of a peptide of about 3 to about 20 amino acids or more, e.g. , about 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acids.
  • the linker comprises repeats of the amino acid sequence Gly-Ser, e.g., 2, 3, 4, 5, or 6 repeats or more.
  • the linker comprises repeats of a thermostable helix from ribosomal protein L9, e.g., 2, 3, 4, 5, or 6 repeats or more.
  • the kinase of the fusion protein can be any kinase known in the art.
  • Examples of known kinases include, without limitation, Abl, Aktl, Akt2, ⁇ ⁇ , ⁇ 1 ⁇ 1 ⁇ 2, ⁇ 2 ⁇ 1 ⁇ 1 , Aurora-A, Aurora-B, Aurora-C, B-RAF, Btk, CaMK2a, CDKl/CycA2, CDKl/CycB, CDK2/CycA, CDK2/CycE, CDK2/Cyclin A2,
  • the kinase is FAK. In another embodiment, the kinase is Src. In a further embodiment, the kinase is a p38 kinase. In an additional embodiment, the kinase is a human kinase. In one embodiment, the kinase is one that is not naturally ligand-dependent. In another embodiment, the kinase is one that is naturally ligand-dependent and the native RBD is optionally removed.
  • the kinase is FAK, e.g. , a mammalian FAK such as human FAK or mouse FAK, and the RBD is inserted within the Met442-Ala448 loop, e.g. , before or after Glu445.
  • the numbering of the amino acids is according to the sequence disclosed in GenBank Accession No. NM_153831 for human FAK.
  • the corresponding sequence in mouse FAK is Leu442-Ala448.
  • the RBD replaces the Met442-Ala448 loop.
  • the RBD replacing the loop can comprise linkers at one or both termini.
  • the kinase is Src, e.g. , a mammalian Src such as human Src or mouse Src, and the RBD is inserted before or after Gly288.
  • the numbering of the amino acids is according to the sequence disclosed in GenBank Accession No. NM_001025395 for mouse Src.
  • the complex comprising the photoactivatable rapamycin compound of the invention bound to a first polypeptide comprising a RBD further comprises a second polypeptide which binds to the first polypeptide only when the first polypeptide is in a complex with a rapamycin compound and the rapamycin compound has been activated.
  • the second polypeptide comprises a domain that binds to a RBD, e.g., the FKBP12 rapamycin binding (FRB) domain of mTOR.
  • the FRB domain corresponds to amino acids 2015-21 14 of human mTOR (Swiss-Prot Accession No. P42345.1).
  • the second polypeptide is a fusion protein comprising a protein of interest and FRB.
  • the protein of interest can be any protein of interest as listed above.
  • the second polypeptide is a polypeptide that naturally dimerizes (forming homodimers or heterodimers) with the first polypeptide
  • Polypeptides of the invention can be modified for use in cells in vitro, ex vivo, or in vivo by the addition, e.g., at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in the cell or in vivo.
  • a blocking agent to facilitate survival of the relevant polypeptide in the cell or in vivo.
  • Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the protein to be administered. This can be done either chemically during the synthesis of the polypeptides or by recombinant DNA technology by methods familiar to artisans of average skill.
  • blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety.
  • the polypeptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins or other molecules (e.g., PEG) prior to administration, e.g. , for use in animal models of disease.
  • polynucleotides encoding the polypeptides (e.g., fusion proteins) of the invention.
  • the polynucleotide comprises, consists essentially of, or consists of a nucleotide sequence that encodes the polypeptides of the invention.
  • Polynucleotides of this invention include RNA, DNA (including cDNAs) and chimeras thereof.
  • the polynucleotides can further comprise modified nucleotides or nucleotide analogs. It will be appreciated by those skilled in the art that there can be variability in the polynucleotides that encode the polypeptides of the present invention due to the degeneracy of the genetic code. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same polypeptide, is well known in the literature.
  • the isolated polynucleotides encoding the polypeptides of the invention will typically be associated with appropriate expression control sequences, e.g., promoters, enhancers, transcription/translation control signals and polyadenylation signals.
  • appropriate expression control sequences e.g., promoters, enhancers, transcription/translation control signals and polyadenylation signals.
  • promoter/enhancer elements can be used depending on the level and tissue-specific expression desired.
  • the promoter can be constitutive or inducible, depending on the pattern of expression desired.
  • the promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.
  • the promoter is chosen so that it will function in the target cell(s) of interest.
  • the polynucleotide encoding the polypeptide can be operatively associated with a cytomegalovirus (CMV) major immediate-early promoter, an albumin promoter, an Elongation Factor 1-a (EFl-a) promoter, a ⁇ promoter, a MFG promoter, or a Rous sarcoma virus promoter.
  • CMV cytomegalovirus
  • EFl-a Elongation Factor 1-a
  • ⁇ promoter a promoter
  • MFG promoter a Rous sarcoma virus promoter
  • Inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements, and other promoters regulated by exogenously supplied compounds, including without limitation, the zinc-inducible metallothionein (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system ⁇ see WO 98/10088); the ecdysone insect promoter (No et al , Proc. Natl. Acad. Sci. USA 93:3346 (1996)); the tetracycline- repressible system (Gossen et al, Proc. Natl. Acad. Sci.
  • MT zinc-inducible metallothionein
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system ⁇ see WO 98/10088
  • the ecdysone insect promoter No et al , Proc. Natl. Ac
  • translational control sequences which can include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.
  • the present invention further provides cells comprising the polynucleotides and polypeptides of the invention.
  • the cell may be a cultured cell or a cell ex vivo or in vivo, e.g., for use in therapeutic methods, diagnostic methods, screening methods, methods for studying the biological action of polypeptides, methods of producing polypeptides, or methods of maintaining or amplifying the polynucleotides of the invention, etc.
  • the cell can be e.g., a bacterial, fungal ⁇ e.g. , yeast), plant, insect, avian, mammalian, or human cell.
  • the polynucleotide can be incorporated into an expression vector.
  • Expression vectors compatible with various host cells are well known in the art and contain suitable elements for transcription and translation of nucleic acids.
  • an expression vector contains an "expression cassette,” which includes, in the 5' to 3' direction, a promoter, a coding sequence encoding a polypeptide operatively associated with the promoter, and, optionally, a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase.
  • Non-limiting examples of promoters of this invention include CYC1, HIS3, GAL1, GAL4, GAL 10, ADH1, PGK, PH05, GAPDH, ADC1 , TRP1, URA3, LEU2, ENO, TPI, and alkaline phosphatase promoters (useful for expression in Saccharomyces); AOX1 promoter (useful for expression in Pichid); ⁇ -lactamase, lac, ara, tet, trp, IP L , IPR, T7, tac, and trc promoters (useful for expression in Escherichia coif); light regulated-, seed specific-, pollen specific-, ovaiy specific-, pathogenesis or disease related-promoters, cauliflower mosaic virus 35S, CMV 35S minimal, cassaya vein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphate carboxylase, shoot-specific promoters, root specific promoters
  • SV40 early (SV40e) promoter region the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EF1) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, a-actin, tubulin and the like), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses (Ad),
  • Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor I (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.
  • CMV cytomegalovirus
  • EF1 elongation factor I
  • yeast enhancers yeast enhancers
  • viral gene enhancers and the like.
  • Termination control regions i.e., terminator or polyadenylation sequences
  • the termination control region may comprise or be derived from a synthetic sequence, a synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.
  • BGH bovine growth hormone
  • Expression vectors can be designed for expression of polypeptides in host cells, e.g. , prokaryotic or eukaryotic cells.
  • polypeptides can be expressed in bacterial cells such as E. coli, insect cells (e.g., the baculovirus expression system), yeast cells, plant cells or mammalian cells.
  • E. coli E. coli
  • insect cells e.g., the baculovirus expression system
  • yeast cells e.g., the baculovirus expression system
  • plant cells e.g., the baculovirus expression system
  • Examples of bacterial vectors include pQE70, pQE60, pQE-9 (Qiagen), pBS, pDI O, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia).
  • Examples of vectors for expression in the yeast S. cerevisiae include pYepSeci (Baldari et al, EMBO J.
  • Baculovirus vectors available for expression of nucleic acids to produce proteins in cultured insect cells include the pAc series (Smith et al, Mol Cell. Biol. 3:2156 (1983)) and the pVL series (Lucklow and Summers, Virology 170:31 (1989)).
  • mammalian expression vectors include pWLNEO, pSV2CAT, pOG44, pXTl , pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed, Nature 329:840 (1987)), pMT2PC (Kaufman et al, EMBO J. 6: 187 (1987)), Gateway ® and pcDNATM vectors (Invitrogen), CheckmateTM/Flexi ® , pCAT ® and HaloTagTM vectors (Promega).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.
  • Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects.
  • Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus, geminivirus, and caulimovirus vectors.
  • Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers.
  • a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).
  • the recombinant expression vector can contain additional nucleotide sequences.
  • the recombinant expression vector can encode a selectable marker gene to identify host cells that have incorporated the vector.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques, including, without limitation, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and viral-mediated transfection.
  • transformation or transfection techniques including, without limitation, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and viral-mediated transfection.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook et al , Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989), and
  • a nucleic acid that encodes a selectable marker e.g., resistance to antibiotics
  • Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that comprising the nucleic acid of interest or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. , cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • the polynucleotide can also be introduced into a plant, plant cell or protoplast and, optionally, the isolated nucleic acid encoding the polypeptide is integrated into the nuclear or plastidic genome. Plant transformation is known in the art. See, in general, Meth. Enzymol. Vol. 153 ("Recombinant DNA Part D") 1987, Wu and Grossman Eds., Academic Press and European Patent Application EP 693554.
  • the polynucleotides or vectors can be targeted to specific cells or tissues in vivo.
  • Targeting delivery vehicles including liposomes and viral vector systems are known in the art.
  • a liposome can be directed to a particular target cell or tissue by using a targeting agent, such as an antibody, soluble receptor or ligand, incorporated with the liposome, to target a particular cell or tissue to which the targeting molecule can bind.
  • a targeting agent such as an antibody, soluble receptor or ligand
  • Targeting liposomes are described, for example, in Ho et al, Biochemistry 25:5500 (1986); Ho et al, J. Biol. Chem. 262: 13979 (1987); Ho et al., J. Biol. Chem.
  • Enveloped viral vectors can be modified to deliver a nucleic acid molecule to a target cell by modifying or substituting an envelope protein such that the virus infects a specific cell type.
  • the gene encoding the attachment fibers can be modified to encode a protein domain that binds to a cell-specific receptor.
  • Herpesvirus vectors naturally target the cells of the central and peripheral nervous system. Alternatively, the route of administration can be used to target a specific cell or tissue.
  • intracoronary administration of an adenoviral vector has been shown to be effective for the delivery of a gene to cardiac myocytes (Maurice et al, J. Clin. Invest. 104:21 (1999)).
  • Intravenous delivery of cholesterol-containing cationic liposomes has been shown to preferentially target pulmonary tissues (Liu et al, Nature Biotechnol. 15: 161 (1997)), and effectively mediate transfer and expression of genes in vivo.
  • Other examples of successful targeted in vivo delivery of nucleic acid molecules are known in the art.
  • a recombinant nucleic acid molecule can be selectively (i.e., preferentially, substantially exclusively) expressed in a target cell by selecting a transcription control sequence, and preferably, a promoter, which is selectively induced in the target cell and remains substantially inactive in non-target cells.
  • the invention relates to methods of producing the polypeptides of the invention, comprising expressing the polypeptides encoded by the polynucleotides and/or vectors described above.
  • the polypeptides can be expressed in vitro, e.g., by in vitro transcription and/or translation.
  • the polypeptides can be expressed in a cell, e.g. , an isolated cell, such as a cell line or a primary cell or a cell in an isolated tissue.
  • the cell can be a bacterial, fungal (e.g., yeast), insect, plant, or animal (e.g. , mammalian) cell.
  • the cell can be present in an animal or plant, e.g., for in vivo production of the polypeptides or for therapeutic or diagnostic purposes.
  • One aspect of the invention relates to a method of modulating the activity of a polypeptide comprising a rapamycin binding domain in a light-dependent manner, comprising contacting the polypeptide with the photoactivatable rapamycin compound of the invention in the presence of light.
  • the invention relates to a method of dimerizing a first polypeptide and a second polypeptide in a light-dependent manner, comprising contacting a first polypeptide comprising a rapamycin binding domain with the photoactivatable rapamycin compound of the invention in the presence of light to form a complex of the first polypeptide and the rapamycin compound, and contacting the complex with a second polypeptide that binds to the first polypeptide only when the first polypeptide is in a complex with a rapamycin compound and the rapamycin compound is activated.
  • the invention relates to a method of modulating the activity of a first polypeptide in a light dependent manner, comprising contacting a first polypeptide comprising a rapamycin binding domain with the photoactivatable rapamycin compound of the invention in the presence of light to form a complex of the first polypeptide and the rapamycin compound, and contacting the complex with a second polypeptide that binds to the first polypeptide only when the first polypeptide is in a complex with a rapamycin compound and the rapamycin compound is activated, wherein binding of the second polypeptide to the first polypeptide modulates the activity of the first polypeptide.
  • the activity is selected from the group consisting of protein binding, signaling, translocation, and enzymatic activity.
  • the modulation of activity can be an increase or decrease in activity.
  • the first polypeptide can be a fusion protein comprising a protein of interest and a rapamycin binding domain, e.g. , FKBP12 or a functional fragment thereof, e.g. , iFKBP.
  • the protein of interest is a kinase.
  • the second polypeptide can comprise the FRB domain of mTOR. In other embodiments, the second polypeptide can be a fusion protein comprising a protein of interest and FRB.
  • the photoactivatable rapamycin compound can be irradiated at an appropriate wavelength or wavelength range and for a sufficient period of time to remove the caging group, e.g., a sufficient period of time to modulate the activity of the polypeptide comprising the RBD.
  • the rapamycin is a photoswitchable compound
  • the compound can be activated repeatedly by supplying and removing irradiation or by using two different wavelengths, one for activation and one for deactivation.
  • the level of activation can be regulated by controlling the level and/or time of irradiation.
  • irradiation can lead to deactivation of the first polypeptide (e.g. , using photoactivatable compounds comprising photoswitchable groups).
  • irradiation at one wavelength can lead to activation of the polypeptide while irradiation at a second wavelength leads to deactivation of the polypeptide.
  • Irradiation can be provided by any means known in the art, e.g., by using a broad spectrum ultraviolet light (e.g., in the range of about 10 nm to about 400 nm) or a light providing a narrower wavelength range, such as UV-A light (about 315 nm to about 400 nm), or a single wavelength (e.g., about 365 nm), e.g., using a UVP UVGL-25 transilluminator.
  • a broad spectrum ultraviolet light e.g., in the range of about 10 nm to about 400 nm
  • a light providing a narrower wavelength range such as UV-A light (about 315 nm to about 400 nm)
  • a single wavelength e.g., about 365 nm
  • the polypeptide and photoactivatable rapamycin compound can be irradiated, for example, for about 1 , 2, 3, 4, or 5 minutes or longer (e.g. , about 10, 15, 20, 25, or 30 minutes) depending on the circumstances.
  • two photon excitation can be used with certain caging groups as discussed above, e.g., using a suitable laser setup.
  • the steps can occur in any order.
  • the first polypeptide, second polypeptide, and photoactivatable rapamycin compound can be combined in any order.
  • the first polypeptide can be irradiated before, during, and/or after contact with the photoactivatable rapamycin compound and before, during, and/or after contact with the second polypeptide.
  • the polypeptides may be present outside a cell or in a cell.
  • the cell may be an isolated cell, a cell in an isolated tissue, or a cell in an animal or other organism.
  • the cell can be on a substrate, in a position to be photographed, filmed, viewed, stained, observed, etc., such as before, during, and/or after addition and/or removal of a photoactivatable rapamycin compound and/or light.
  • the polypeptides may be contacted with the photoactivatable rapamycin compound by directly adding the compound to a fluid comprising the polypeptides, exposing cells comprising the polypeptides to the compound, or administering the compound to an animal or other organism.
  • the entire cell is irradiated such that the activity of most or all of the first polypeptide present in the cell is modulated.
  • one or more portions of a cell are irradiated such that the modulation occurs in only a portion of the cell.
  • one or more areas outside the cell are irradiated, e.g.,. to create a gradient of uncaged rapamycin.
  • the polypeptides and/or cell can be contacted with other agents, e.g. , kinase inhibitors, other research tools, etc.
  • two or more different first polypeptides and or two or more different photoactivatable rapamycin compounds are irradiated.
  • different compounds respond to a different wavelength so that the binding of polypeptides can be individually induced.
  • the present invention finds use in research applications, as well as diagnostic and medical applications. Suitable subjects include all organisms, e.g., bacteria, fungi, plants, insects, avians, fish, and mammals.
  • avian as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants.
  • mammal as used herein includes, but is not limited to, humans, bovines, ovines, caprines, equines, felines, canines, murines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, and adults. In other embodiments, the subject is an animal model of disease.
  • kits for carrying out the methods of the invention can comprise one or more photoactivatable rapamycin compounds.
  • the kits can comprise the polypeptides, polynucleotides, vectors, and/or cells of the invention.
  • the kits can comprise further components useful for carry out the methods of the invention, including without limitation, containers, buffers, ligands, reagents, fluorescent dyes, antibodies, cells, probes, primers, vectors, etc.
  • NPOC-Rap N/Yro-piperonyloxycarbonyl-caged rapamycin
  • rapamycin (Rap) 1 (20.0 mg, 0.0219 mmol) was dissolved in dry DCM (0.6 ml).
  • DMAP 5.4 mg, 0.0438 mmol
  • NPOC-NHS 2 39.0 mg, 0.109 mmol
  • the reaction mixture was stirred at rt for 24 h, the volatiles were evaporated, and the product was purified by column chromatography on Si0 2 (eluted with DCM/ethyl acetate 10: 1 , 5 : 1 , 2: 1 , 1 :1), delivering 9.1 mg (36% yield) of NPOC-Rap 3 as a light yellow solid.
  • BHQ-rap 6 was synthesized in two steps from rapamycin 1 via chemoselective acylation with the TIPS protected-BHQ-NHS 4 in dichloromethane at room temperature in 32% yield, followed by removal of TIPS protecting group by KF in 69% yield.
  • the identity of the BHQ-caged rapamycin 6 was confirmed by 1H NMR and MS.
  • NDBF-rap 8 3-Nitro-2-ethyldibenzofuran (NDBF)-caged rapamycin (NDBF - rap).
  • NDBF-rap 8 was synthesized in one step from rapamycin 1 via chemoselective acylation with the mixed carbonate of 3-nitro-2-ethyldibenzofuran alcohol and N- hydroxysuccinimide 7 in dichloromethane (DCM) at room temperature.
  • DCM dichloromethane
  • DMAP Dimethylaminopyridine
  • the identity of the NDBF-caged rapamycin 8 was confirmed by 1H NMR and MS.
  • PEGylated-caged rapamycin PEGylated-caged rapamycin.
  • PEGylated, caged rapamycin 12 was synthesized by linking rapamycin 1 to a photocleavable PEG group of 500 and 5,000 Da, The carbonate caging group 9 was selectively reacted with the C-40 hydroxy group of rapamycin 1 in the presence of DMAP as a catalyst, providing the caged rapamycin 10 in 41% yield. The alkyne moiety in 10 was then reacted with the 5000 Da PEG azide reagent 11 (or a corresponding 500 Da PEG azide reagent) in a [3+2] cycloaddition reaction, providing the PEGylated, caged rapamycin 12.
  • the PEGylated (500 Da) rapamycin analog was characterized by NMR and MS.
  • iRap 13 was synthesized in one step from rapamycin via acid-mediated carbocation formation at C-16, which was subsequently quenched with 3-methylindole (Bayle et al., Chem. Biol. 13:99 (2006). The iRap 13 was then reacted with the carbonate caging group MeNPOC-NHS 2 in the presence of DMAP as a catalyst, providing caged iRap 14 in 24% yield. The caged iRap analog was characterized by NMR and MS.
  • NDBF-caged iRAp [0122] NDBF-caged iRAp.
  • NDBF-caged iRap 15 was synthesized in one step from iRap 13 via chemoselective acylation with NDBF-NHS 7 in the presence of DMAP as a catalyst in 24%> yield.
  • the caged iRap analog was characterized by NMR and MS.
  • Photocleavable rapamycin dimer (dRap).
  • the photocleavable rapamycin dimer 25 was synthesized in one step from the already prepared caged rapamycin 10 via a double [3+2] cycloaddition reaction with 1,4-diazidobutane (24) in 81% yield.
  • the identity of the rapamycin dimer 25 was confirmed by 'HNMR and MS. Photolysis (365 nm) of 25 releases two equivalents of native rapamycin 1.
  • Anti-phospho-paxillin (Tyr31), anti-phospho-FAK (Tyr397), and anti- GFP antibodies were purchased from Invitrogen.
  • Anti-myc antibodies and IgG-coupled agarose beads were purchased from Millipore.
  • Anti-paxillin antibodies were a gift from Dr. Michael Schaller. Rapamycin was purchased from Sigma.
  • HEK293T cells were co-transfected with myc-tagged FAK constructs and GFP-FRB. Transfected cells were treated with either rapamycin or cRap or equivalent volumes of DMSO (solvent control). Ten minutes after addition of cRap, cells were irradiated with 365 nm light using a UVP LMW-20 transilluminator.
  • Lysis Buffer (20 mM Hepes-KOH, pH 7.8, 50 mM KC1, 100 mM NaCl, 1 mM EGTA, 1% NP40, 1 mM NaF, 0.1 mM Na 3 V0 4 ). Cleared lysates were incubated for 2 hours with IgG-linked agarose beads prebound with anti-myc antibody (4A6, Millipore).
  • the beads were washed 2 times with Wash Buffer (20 mM Hepes-KOH, pH 7.8, 50 mM KC1, 100 mM NaCl, 1 mM EGTA, 1% NP40) and two times with Kinase Reaction Buffer (25 mM HEPES pH 7.5, 5 mM MgCl 2 , 5 mM MnCl 2 , 0.5 mM EGTA, 0.005% BRJJ-35). 20 uL of bead suspension were used in kinase assays using the N-terminal fragment of paxillin as previously described (Cai, X. et al. Mol Cell Biol 28, 201-14 (2008)). For immunoprecipitation only experiments the washing with Kinase Reaction Buffer and the kinase reactions were omitted.
  • the 468 nm line from an omnichrome series 43 Ar/Kr laser and the 598 nm line from Cobolt Mambo continuous-wave diode-pumped solid-state laser were used for TIRF imaging.
  • Epifluorescence images were taken using a high pressure mercury arc light source.
  • Cells expressing GFP-RapR-FAK constructs and mCherry-FRB were selected using epifluorescence imaging. Top 50% GFP expressing cells were selected for quantification of phenotype induced by RapR-FAK activation. Time-lapse movies were taken at 1 min time intervals.
  • GFP-RapR-FAK expression level quantification and other image analysis were performed using Metamorph software.
  • To uncage cRap cells were irradiated on stage with 365 nm light using a UVP UVGL-25 transit luminator.
  • the hydroxyl groups at C-28 and C-40 can be protected with silyl groups (Nelson et al, Tetrahedron Lett. 35:7557 (1994); Ayral- Kaloustian et al, J. Med. Chem. 53:452 (2010)), and a trifluoromethylsulfonyl group (Wagner et al., Bioorg. Med. Chem. Lett. 15:5340 (2005)).
  • the lactone at C-34 can be hydrolyzed and eliminated (Adams et al, Annu. Rev. Physiol. 55:755 (1993); Goard et al, Chem. Biol. 72:685 (2005); Cambridge et al, Nat.
  • C-40 represents the most suitable site for chemical modification with a carbonate-linked caging group that can provide facile installation and quick photolysis.
  • NPOC-Rap The caged rapamycin (NPOC-Rap) was synthesized in one step from rapamycin (Rap) via chemoselective acylation with the mixed carbonate of 6-nitro- piperonyl alcohol and N-hydroxysuccinimide (NPOC-NHS, Fig. la). The identity of purified NPOC-Rap was confirmed by ⁇ NMR and HRMS analysis.
  • NPOC-Rap could mediate heterodimerization of iFKBP and FRB in a light dependent manner. Indeed, NPOC-Rap (at concentrations of up to 20 ⁇ ) failed to mediate interaction between iFKBP-FAK and GFP-FRB), while irradiation of NPOC-Rap-treated cells with 365 nm UV light successfully removed the caging group and induced iFKBP-FRB dimerization (Fig. 4b-4d). Uncaging kinetics were dependent on both light dosage and NPOC-Rap concentration.
  • NPOC-Rap can effectively mediate light- dependent protein heterodimerization when used with iFKBP rather than FKBP 12.
  • RapR-FAK rapamycin-regulated FAK
  • FIG. 5a Myc-tagged RapR-FAK was co-expressed with GFP-FRB in HEK293T cells.
  • RapR-FAK the ability of rapamycin to induce RapR-FAK phosphorylation of the N-terminal fragment of paxillin was examined (Deakin et al, J. Cell Sci. 121:2435 (2008); Schaller, J. Cell Biol. 166: 157 (2004)), a signal transduction adaptor protein and natural FAK substrate.
  • NPOC-Rap was completely inactive at concentrations of up to 20 ⁇ , producing no detectable paxillin Tyr31 phosphorylation.
  • UV irradiation alone did not activate RapR-FAK in the absence of NPOC-Rap, but irradiation in the presence of NPOC-Rap (1 -20 ⁇ ) induced activation of RapR-FAK through protein complex formation with FRB (Fig. 5b), leading to robust phosphorylation of paxillin.
  • Light-mediated interaction between RapR-FAK and GFP-FRB was further confirmed by co-immunoprecipitation of the two proteins (Fig. 5b) and by translocation of FRB to focal adhesions (Fig. 4f) upon NPOC-Rap irradiation.
  • the iFKBP protein Due to its higher structural plasticity, the iFKBP protein is able to deform and create additional contacts with NPOC-Rap, which is unachievable by the more rigid and stable FKBP12. Based on these simulations, it is suggested that strong binding between iFKBP and NPOC-Rap distorts the FRB-binding interface in FKBP12, and thus prevents further binding to FRB.
  • a new photocaged analog of rapamycin has been developed which can be used together with iFKBP, an engineered version of FKBP12, for the light-mediated regulation of protein dimerization.
  • Successful use of this new dimerization system was demonstrated by modulating protein interactions for two different FAK-iFKBP fusions in living cells.
  • light-mediated activation of an engineered protein kinase, FAK was achieved and light-induced changes in the cell behavior characteristic of this kinase was demonstrated.
  • Rapamycin-mediated protein dimerization and regulation of kinases have lacked the precise spatial and temporal control of light-mediated processes.
  • this new caged rapamycin approach will significantly enhance the many existing methodologies that use rapamycin for the regulation of protein interaction and protein kinase activity in cells and multicellular organisms.
  • mTOR phosphorylates p70S6K, which is detected by antibodies and Horseradish Peroxidase (mTOR -/+ UV).
  • mTOR -/+ UV Horseradish Peroxidase
  • rapamycin and FKBP-12 When rapamycin and FKBP-12 are added, mTOR, rapamycin and FKBP-12 form a complex that inhibits phosphorylation, leading to lower absorbance (FKBP-12 + WT Rap -/+ UV).
  • NPOC-Rap and dimer rapamycin (dRap) inhibit the formation of the complex, thus allowing more phosphorylation and more luminescence.
  • Fig. 7 shows the concept for light-activated TEV protease using dRap.
  • Fig. 8 shows luminescence results for the assay of rapamycin derivatives using split TEV protease ⁇ Nature Methods 3:985 (2006)) and the luciferase reporter system.
  • HEK293T cells were transfected with FRB-TEVnt, FKBP12-TEVct, and circularly permutated luciferase plasmids (100 ng of each) using Lipofectamine 2000. Wild type rapamycin, NPOC-Rap (denoted pRap in Fig.
  • dimer rapamycin starting material (dRapSM, 10) were added to a concentration of 100 nM, dimer rapamycin (dRap) was added to a concentration of 50 nM 24 hours post-transfection. All were incubated for 1 hour before a 3 -minute irradiation at 365 nm by a transluminator. After irradiation, samples were incubated for another 1.5 hours at 37°C with 5% C0 2 . Cells were lysed using an equal volume of BrightGlo reagent to media volume, which includes the substrate for luciferase. Luminescence was read on a plate reader,
  • the principle of the DiCre system is to split Cre recombinase into two fragments to render its enzymatic activity inactive, which could be restored by dimerization.
  • the first DiCre system was reported by Jean-Paul Hermans' lab in 2003, which split Cre between amino acids 59 and 60, and fused each moiety to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12- rapamycin-associated protein), respectively.
  • FKBP12 FK506-binding protein
  • FRB binding domain of the FKBP12- rapamycin-associated protein

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Abstract

La présente invention concerne des composés de rapamycine photoactivables. L'invention concerne en outre l'utilisation de composés de rapamycine photoactivables pour réguler la dimérisation et/ou l'activité de polypeptides d'une façon dépendante de la lumière.
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CN113980959A (zh) * 2021-10-27 2022-01-28 闽江学院 一种“y型”多功能dna纳米组装体、制备方法及其应用
CN115531535A (zh) * 2021-06-28 2022-12-30 香港大学 用于光化学控制mTOR信号传导和mTOR依赖性自噬的组合物和方法
WO2023067491A1 (fr) 2021-10-18 2023-04-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Modulateur de canal ionique photoactivable

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Cited By (11)

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Publication number Priority date Publication date Assignee Title
JP2017531624A (ja) * 2014-09-11 2017-10-26 ザ・リージエンツ・オブ・ザ・ユニバーシテイ・オブ・カリフオルニア ｍＴＯＲＣ１阻害剤
US10646577B2 (en) 2014-09-11 2020-05-12 The Regents Of The University Of California mTORC1 inhibitors
JP2020125320A (ja) * 2014-09-11 2020-08-20 ザ・リージエンツ・オブ・ザ・ユニバーシテイ・オブ・カリフオルニア ｍＴＯＲＣ１阻害剤
JP7057798B2 (ja) 2014-09-11 2022-04-20 ザ・リージエンツ・オブ・ザ・ユニバーシテイ・オブ・カリフオルニア ｍＴＯＲＣ１阻害剤
US11452780B2 (en) 2014-09-11 2022-09-27 The Regents Of The University Of California Mtorc1 inhibitors
US12097262B2 (en) 2014-09-11 2024-09-24 The Regents Of The University Of California mTORC1 inhibitors
CN115531535A (zh) * 2021-06-28 2022-12-30 香港大学 用于光化学控制mTOR信号传导和mTOR依赖性自噬的组合物和方法
WO2023067491A1 (fr) 2021-10-18 2023-04-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Modulateur de canal ionique photoactivable
WO2023067366A1 (fr) 2021-10-18 2023-04-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Modulateur de canal ionique photoactivable
CN113980959A (zh) * 2021-10-27 2022-01-28 闽江学院 一种“y型”多功能dna纳米组装体、制备方法及其应用
CN113980959B (zh) * 2021-10-27 2023-10-24 闽江学院 一种“y型”多功能dna纳米组装体、制备方法及其应用

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