Artificial TRAP cages, their uses, and methods for their preparation

Abstract

The present invention provides artificial TRAP cages that comprise a selected number of TRAP rings held in place by crosslinkers that are selected for their specific characteristics, such that the cages are programmable to open or remain closed on demand under specific conditions.

Description

[Technical field]

[0001] The present invention is in the field of biochemistry. The present invention relates to an artificial protein cage, called a "TRAP cage", that contains a selected number of TRAP rings held in place by molecular crosslinkers, which are selected for their specific characteristics, such that the cage is programmable to open or remain closed on demand under specific conditions. [Background technology]

[0002] Protein complexes in nature are important and highly precise biological nanomachines and nanostructures. Large protein complexes in nature are typically constructed from several individual proteins held together by non-covalent interactions (i.e., hydrogen bonds, hydrophobic packing). This is particularly evident in protein cages such as capsids, where multiple copies of identical protein subunits are held together in this way. In synthetic structural biology, it can be useful to be able to design and construct artificial protein assemblies, potentially allowing the introduction of properties and capabilities that do not exist in nature. Towards this goal, new ways of connecting individual proteins together in a defined manner are desirable.

[0003] Recently, we have been exploring such possibilities using TRAP (trp RNA-binding attenuation protein) from Geobacillus stearothermophilus as a nanometric building block. This TRAP adopts an oligomeric ring structure of 11 subunits in the native state and together with several other ring proteins, and has proven to be a useful bio-nano building block.

[0004] Given the disadvantages of the known processes, the inventors have attempted to find other ways to connect protein subunits. Although there are some disclosures on linking two or any other number of proteins via their cysteine ​​SH groups, the inventors have focused on this field, taking into account the use of gold as a "stitching" reagent.

[0005] The use of gold compounds to incorporate gold particles into nanostructures or to provide nanoparticles, protein cages, as nanoclusters for multiple applications, among others, as targeting molecules in delivery systems, has been well described in the literature as well as in patent documents, and such are prior art to the present invention.For example, International Application No. PCT / KR2013 / 004454 describes a method for preparing hyaluronic acid-gold nanoparticle / protein complexes that can be used as liver-targeted drug delivery systems by surface-modifying gold nanoparticles with excellent stability in the body with hyaluronic acid, which has biocompatibility, biodegradability, and liver tissue-specific delivery properties, and binding protein drugs for treating liver diseases to the unmodified surface of the gold nanoparticles.

[0006] US Patent Application No. US10 / 142,838 discloses the introduction of noble metal atoms, such as gold, into cage-like proteins, such as apoferritin, by modifying the inner structure of the cage-like protein, thus forming noble metal-engineered cage-like protein complexes that are applicable to various microstructures.

[0007] International Application No. PCT / US2011 / 034190 discloses antibody-nanoparticle conjugates comprising two or more nanoparticles (such as gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more thereof) directly linked to an antibody or fragment thereof through a metal-thiol bond.

[0008] Another example is US Patent Application No. US14 / 849,379, which discloses a recombinant self-assembling protein comprising a target-directing peptide fused to the self-assembling protein, and a self-assembled gold ion reducing peptide.

[0009] International Application No. PCT / IB2018 / 056150 discloses a method for the conjugation of free thiol groups containing biomolecules leading to biomolecular complex formation, comprising a reaction of connecting biomolecules using a gold-donor agent, in which a -S-Au-S- bond is formed, characterized in that the gold-donor agent is halogen(triarylphosphine)gold(I).Furthermore, the use of halogen(triarylphosphine)gold(I) molecules as gold-donor agents in the method of biomolecular complex formation is disclosed therein.

[0010] A.D. Malay et al.: "An ultra-stable gold-coordinated protein cage displaying reversible assembly"; Nature 569 (2019): 438-442, a publication hereby incorporated by reference, discloses a TRAP cage bound together by a single gold atom, i.e., Au(I) ion, which forms a linear coordinate bond between the thiol groups of a pair of cysteines.

[0011] In the present invention, the TRAP rings forming the artificial TRAP cage are held in place by crosslinkers that are not made of metal atoms, and the crosslinkers are selected for their specific characteristics, so that the cage can be programmed to open or remain closed on demand under specific conditions, not by -S-Au-S-bonds, a new approach is realized. This approach allows for the control of the assembly and disassembly of capsid-like protein complexes, which is innovative in the state of the art. A new approach of metal ions as crosslinkers is also demonstrated, which also allows for the control of the assembly and disassembly of capsid-like protein complexes, which is innovative in the state of the art. Summary of the Invention [Means for solving the problem]

[0012] The subject of a first aspect of the present invention is an artificial TRAP cage comprising a selected number of TRAP rings held in place by molecular crosslinkers, which are molecules selected for their specific characteristics and not a single atom, e.g. not a metal atom, whereby the cage is programmable to open or remain closed on demand under specific conditions.

[0013] Preferably, said specific conditions correspond to a specific cleavage characteristic of the molecular cross-linker.

[0014] The use of molecular crosslinkers rather than single atom crosslinkers offers a greater degree of design choice and flexibility in designing the cages. These allow for enhanced programmability and control over the crosslinker cleavage characteristics. There is a much wider range of molecular crosslinkers available to choose from than (metal) atomic crosslinkers.

[0015] All known TRAP cage syntheses prior to this have utilized atomic gold or mercury crosslinkers, with no work contemplating the use of molecular crosslinkers. Because all previous teachings have focused on gold atoms, the larger size of the molecules and the different chemistries they require to crosslink the TRAP rings means that it is not clear that molecular crosslinkers would be able to perform the bridging function that results in ordered cage formation.

[0016] Preferably, the specific cleavage characteristics of the molecular crosslinker are: (i) reduction-resistant / insensitive molecular crosslinkers, whereby the cage remains closed under reducing conditions; (ii) reduction-responsive / sensitive molecular crosslinkers, whereby the cage opens under reducing conditions; and (iii) a photoactivatable molecular crosslinker, whereby the cage opens upon exposure to light The compound is selected from the group consisting of:

[0017] Preferably, the reduction-resistant / insensitive molecular crosslinker may be selected from the group including bismaleimideohexane (BMH), bisbromobimane and bis-bromoxylene.

[0018] Preferably, the reduction-responsive / sensitive molecular crosslinker may be selected from the group including dithiobismaleimideoethane (DTME).

[0019] Preferably, the photoactivatable molecular crosslinker may be selected from the group comprising bis-halomethylbenzenes and their derivatives including 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0020] Preferably, the molecular cross-linkers are homobifunctional molecular moieties and derivatives thereof.

[0021] Preferably, the homobifunctional molecular crosslinker is bismaleimidohexane (BMH).

[0022] Preferably, the cage is resistant / insensitive to reducing conditions.

[0023] Preferably, the homobifunctional molecular crosslinker is dithiobismaleimideethane (DTME).

[0024] Preferably, the cage is responsive / sensitive to reducing conditions.

[0025] Preferably, the molecular crosslinker is a bis-halomethylbenzene and its derivatives.

[0026] Preferably, the molecular crosslinker is selected from the group comprising 1,2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0027] Preferably, the molecular crosslinker is photolabile upon exposure to UV light.

[0028] Preferably, the number of TRAP rings in the TRAP cage is 6-60, preferably 7-55, preferably 8-50, preferably 9-45, preferably 10-40, preferably 11-35, preferably 12-34, preferably 13-33, preferably 14-32, preferably 15-31, preferably 16-30, preferably 17-29, preferably 18-28, preferably 19-27, preferably 20-26. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is more than 6, preferably more than 10, preferably more than 15, preferably more than 20.

[0029] Preferably, the number of TRAP rings in the TRAP cage is 12 to 24.

[0030] Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

[0031] Preferably, a cage according to the present invention comprises a mixture of different programmable molecular cross-linkers.

[0032] Preferably, cages according to the present invention encapsulate a cargo that can be programmed to deliver said cargo at a specific time and desired location.

[0033] Preferably, the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including K35C, R64S, and K35C / R64S. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation. Preferably, the crosslinker comprises dithiobismaleimide ethane (DTME), and preferably, the artificial TRAP cage protein is modified to include a K35C / R64S mutation.

[0034] Preferably, the cages according to the invention are hollow. Preferably, the cages according to the invention are approximately spherical in shape, preferably hollow sphere shapes. The cages herein are hollow shapes that roughly approximate hollow spheres. These are close to the shapes that would be obtained if TRAP rings were placed at the vertices or corners of a regular convex polyhedron and then linked together. For any shape, the vertices and edges are imaginary, i.e., there is no actual physical polyhedron on which the TRAP rings are placed, rather the shape of the TRAP cage is as if the rings were placed at the vertices or corners of a regular convex polyhedron and then linked together.

[0035] Preferably, the TRAP cage is stable at elevated temperatures, i.e. when the temperature is elevated above normal room temperature or human / animal body temperature, preferably stable between 0 and 100°C, preferably stable between 15 and 100°C, preferably stable between 15 and 75°C, preferably stable up to 75°C, preferably stable below 75°C.

[0036] Preferably, the TRAP cage is stable at non-neutral pH, preferably above pH 7 and below pH 7, preferably stable at pH 3-11, preferably stable at pH 4-10, preferably stable at pH 5-9.

[0037] Preferably, the TRAP cage is stable in chaotropic agents (agents that disrupt hydrogen bonds in solution, that would disrupt or denature protein or macromolecular structures) or detergents that would otherwise be expected to disrupt or denature protein or macromolecular structures. Preferably, the cage exhibits stability in n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. Preferably, the TRAP cage is stable in up to 4M GndHCl. Preferably, the TRAP cage is stable in up to at least 7M urea. Preferably, the TRAP cage is stable in up to 15% SDS. The stability of the cages described herein may be tested using these agents in standard conditions that would be known to those skilled in the art to demonstrate said stability.

[0038] The cages described herein exhibit unexpected stability in these conditions, providing a more stable TRAP cage than previously demonstrated.

[0039] A subject of the present invention is also the use of a cage according to the invention as defined above in the delivery of a cargo in a controlled period of time and to a desired location.

[0040] The subject of the present invention is also the use of any one or more of the homobifunctional molecular moieties and groups comprising bis-halomethylbenzenes and their derivatives as programmable cross-linkers in the construction of programmable TRAP cages.

[0041] The subject of a second aspect of the present invention is a method for preparing an artificial TRAP cage, the method comprising the steps of: (i) obtaining a TRAP ring unit by expression of the TRAP ring unit in a suitable expression system and purification of said unit from the expression system; (ii) conjugation of the TRAP ring unit via at least one free thiol linkage with a programmable molecular crosslinker, where the crosslinker is selected for its specific characteristics; (iii) formation of a TRAP cage; and (iv) Purification and isolation of TRAP cages The present invention is also a method including the steps of:

[0042] Preferably, step (ii) first comprises the conjugation of the TRAP ring unit via at least one metal crosslinker, preferably an atomic metal crosslinker. Step (ii) then comprises replacing the metal crosslinker with a molecular crosslinker. The molecular crosslinker can exchange the metal atom without changing the orientation of the ring in the cage. Preferably, the metal is gold. This modified step (ii) is preferably applied when the crosslinker is a photocleavable linker, preferably when the crosslinker is bromoxyl or bisbromobimane.

[0043] Preferably, the programmable crosslinker comprises (i) reduction-resistant / insensitive linkers, whereby the cage remains closed under reducing conditions; (ii) a reduction-responsive / sensitive linker, whereby the cage opens under reducing conditions; and (iii) a photoactivatable linker, whereby the cage opens upon exposure to light The compound is selected from the group consisting of:

[0044] Preferably, the reduction-resistant / insensitive molecular crosslinker may be selected from the group including bismaleimidohexane (BMH) and bis-bromoxylene.

[0045] Preferably, the reduction-responsive / sensitive molecular crosslinker may be selected from the group including dithiobismaleimideethane (DTME).

[0046] Preferably, the photoactivatable molecular crosslinker may be selected from the group including bis-halomethylbenzenes and their derivatives including 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB). Preferably, the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including K35C, R64S, and K35C / R64S. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation.

[0047] Preferably, the cross-linking agent comprises dithiobismaleimideethane (DTME) and preferably the artificial TRAP cage protein is modified to include K35C / R64S mutations.

[0048] Preferably, the number of TRAP rings in the TRAP cage is 6-60, preferably 7-55, preferably 8-50, preferably 9-45, preferably 10-40, preferably 11-35, preferably 12-34, preferably 13-33, preferably 14-32, preferably 15-31, preferably 16-30, preferably 17-29, preferably 18-28, preferably 19-27, preferably 20-26. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is more than 6, preferably more than 10, preferably more than 15, preferably more than 20.

[0049] Preferably, the number of TRAP rings in the TRAP cage is 12 to 24.

[0050] Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

[0051] Preferably, step (ii) involves conjugation with a mixture of different programmable cross-linkers.

[0052] Preferably, the reduction-resistant / insensitive molecular crosslinker may be selected from the group including bismaleimidohexane (BMH) and bis-bromoxylene.

[0053] Preferably, the reduction-responsive / sensitive molecular crosslinker may be selected from the group including dithiobismaleimideethane (DTME).

[0054] Preferably, the photoactivatable molecular crosslinker may be selected from the group comprising bis-halomethylbenzenes and their derivatives including 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0055] If cysteines are not present in the biomolecule, or they are present but not available for reaction, preferably an -SH group as the group of cysteine ​​can be introduced into the biomolecule.

[0056] The introduction of cysteine ​​can be carried out by any method known in the art.For example, the introduction of cysteine ​​can be carried out by methods known in the art, such as, but not limited to, commercial gene synthesis or PCR-based site-directed mutagenesis using modified DNA primers.The above-mentioned methods are known by those skilled in the art, and ready-to-use kits with protocols are commercially available.

[0057] The -SH moiety can also be introduced into a biomolecule by alteration of other amino acids in the biomolecule, ie, by site-directed mutagenesis or by solid phase peptide synthesis.

[0058] The subject of the present invention is also the TRAP cages produced by this method. These cages may have any of the characteristics or properties described in connection with the first aspect of the invention above, or any others described herein.

[0059] The subject of a third aspect of the present invention is an artificial TRAP cage comprising a selected number of TRAP rings held in place by at least one crosslinker comprising a metal. Preferably, the crosslinker comprises only metals. Preferably, the metal is a metal ion, preferably of a single type of metal.

[0060] Preferably, the metal crosslinker is selected for specific characteristics, whereby the cage is programmable to open or remain closed on demand under said specific conditions.

[0061] Preferably, the metal is selected from the group comprising Ag(I), Cd(II), Zn(II) and Co(II). The metal may be a derivative of these metals.

[0062] Preferably, the metal is a d10 metal having a non-linear coordination structure or shell. Preferably, the d10 metal having a non-linear coordination structure or shell is Zn(II) or Co(II).

[0063] Preferably, the metal is a d10 metal with a two-ligand linear coordination structure or shell. Preferably, the d10 metal with a non-linear coordination structure or shell is Ag(I) or Cd(II).

[0064] Preferably, the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including K35C, K35H, R64S, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C, and S33H / K35C.

[0065] Preferably, the artificial TRAP cage protein is modified to contain a K35C / S33H mutation or a K35H / S33H mutation.

[0066] Preferably, the cross-linking agent comprises silver (Ag(I)), and preferably the artificial TRAP cage protein is modified to include K35C / R64S mutations.

[0067] Preferably, the cross-linking agent comprises cadmium (Cd(II)), and preferably the artificial TRAP cage protein is modified to include K35C / R64S mutations.

[0068] Preferably, the cross-linking agent comprises cobalt (Co(II)), and preferably the artificial TRAP cage protein is modified to include S33H / K35C or S33H / K35H mutations.

[0069] Preferably, the crosslinker comprises zinc (Zn(II)) and preferably the artificial TRAP cage protein is modified to include S33H / K35C or S33H / K35H mutations.

[0070] Preferably, the number of TRAP rings in the TRAP cage is 6-60, preferably 7-55, preferably 8-50, preferably 9-45, preferably 10-40, preferably 11-35, preferably 12-34, preferably 13-33, preferably 14-32, preferably 15-31, preferably 16-30, preferably 17-29, preferably 18-28, preferably 19-27, preferably 20-26. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is more than 6, preferably more than 10, preferably more than 15, preferably more than 20.

[0071] Preferably, the number of TRAP rings in the TRAP cage is 12 to 24.

[0072] Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

[0073] Preferably, the cage according to the invention comprises a mixture of different cross-linking agents.

[0074] Preferably, cages according to the present invention encapsulate a cargo that can be programmed to deliver said cargo at a specific time and desired location.

[0075] Preferably, the cages according to the invention are hollow. Preferably, the cages according to the invention are approximately spherical in shape, preferably hollow spheres. The cages herein are hollow shapes roughly approximating hollow spheres. These are close to the shapes that would be obtained if TRAP rings were placed at the vertices or corners of a regular convex polyhedron and then linked together.

[0076] Preferably, the TRAP cage is stable at elevated temperatures, i.e. when the temperature is elevated above normal room temperature or human / animal body temperature, preferably stable between 0 and 100°C, preferably stable between 15 and 100°C, preferably stable between 15 and 75°C, preferably stable up to 75°C, preferably stable below 75°C.

[0077] Preferably, the TRAP cage is stable at non-neutral pH, preferably above pH 7 and below pH 7, preferably stable at pH 3-11, preferably stable at pH 4-10, preferably stable at pH 5-9.

[0078] Preferably, the TRAP cage is stable in chaotropic agents (agents that disrupt hydrogen bonds in solution, that would disrupt or denature protein or macromolecular structures) or detergents that would otherwise be expected to disrupt or denature protein or macromolecular structures. Preferably, the cage exhibits stability in n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. Preferably, the TRAP cage is stable in up to 4M GndHCl. Preferably, the TRAP cage is stable in up to at least 7M urea. Preferably, the TRAP cage is stable in up to 15% SDS. The stability of the cages described herein may be tested using these agents in standard conditions that would be known to those skilled in the art to demonstrate said stability.

[0079] The cages described herein exhibit unexpected stability in these conditions, providing a more stable TRAP cage than previously demonstrated.

[0080] A subject of the present invention is also the use of a cage according to the invention as defined above in the delivery of a cargo in a controlled period of time and to a desired location.

[0081] The subject of the present invention is also the use of the metals Ag(I), Cd(II), Zn(II) and Co(II) and any one or more of their derivatives as cross-linking agents in the construction of the TRAP cage.

[0082] The subject of a fourth aspect of the present invention is a method for preparing an artificial TRAP cage, the method comprising the steps of: (i) obtaining a TRAP ring unit by expression of the TRAP ring unit in a suitable expression system and purification of said unit from the expression system; (ii) conjugation of the TRAP ring unit via at least one free thiol linkage to a metal crosslinker, where the crosslinker is selected for its specific characteristics; (iii) formation of a TRAP cage; and (iv) Purification and isolation of TRAP cages The present invention is also a method including the steps of:

[0083] Preferably, the metal is selected from the group comprising Ag(I), Cd(II), Zn(II) and Co(II). The metal may be a derivative of these metals.

[0084] Preferably, the metal is a d10 metal having a non-linear coordination structure or shell. Preferably, the d10 metal having a non-linear coordination structure or shell is Zn(II) or Co(II).

[0085] Preferably, the metal is a d10 metal with a two-ligand linear coordination structure or shell. Preferably, the d10 metal with a non-linear coordination structure or shell is Ag(I) or Cd(II).

[0086] Preferably, the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including K35C, K35H, R64S, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C, and S33H / K35C.

[0087] Preferably, the artificial TRAP cage protein is modified to contain a K35C / S33H mutation or a K35H / S33H mutation.

[0088] Preferably, the cross-linking agent comprises silver (Ag(I)), and preferably the artificial TRAP cage protein is modified to include K35C / R64S mutations.

[0089] Preferably, the cross-linking agent comprises cadmium (Cd(II)), and preferably the artificial TRAP cage protein is modified to include K35C / R64S mutations.

[0090] Preferably, the cross-linking agent comprises cobalt (Co(II)), and preferably the artificial TRAP cage protein is modified to include S33H / K35C or S33H / K35H mutations.

[0091] Preferably, the crosslinker comprises zinc (Zn(II)) and preferably the artificial TRAP cage protein is modified to include S33H / K35C or S33H / K35H mutations.

[0092] Preferably, the number of TRAP rings in the TRAP cage is 6-60, preferably 7-55, preferably 8-50, preferably 9-45, preferably 10-40, preferably 11-35, preferably 12-34, preferably 13-33, preferably 14-32, preferably 15-31, preferably 16-30, preferably 17-29, preferably 18-28, preferably 19-27, preferably 20-26. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is more than 6, preferably more than 10, preferably more than 15, preferably more than 20.

[0093] Preferably, the number of TRAP rings in the TRAP cage is 12 to 24.

[0094] Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

[0095] If cysteines are not present in the biomolecule, or they are present but not available for reaction, preferably an -SH group as the group of cysteine ​​can be introduced into the biomolecule.

[0096] The introduction of cysteine ​​can be carried out by any method known in the art.For example, the introduction of cysteine ​​can be carried out by methods known in the art, such as, but not limited to, commercial gene synthesis or PCR-based site-directed mutagenesis using modified DNA primers.The above-mentioned methods are known by those skilled in the art, and ready-to-use kits with protocols are commercially available.

[0097] The -SH moiety can also be introduced into a biomolecule by alteration of other amino acids in the biomolecule, ie, by site-directed mutagenesis or by solid phase peptide synthesis.

[0098] Preferably, the method is carried out partially or completely in a HEPES buffer. Preferably, the method is carried out at about pH 8.

[0099] The subject of the present invention is also the TRAP cages produced by this method. These cages may have any of the characteristics or properties described in connection with the first aspect of the invention above, or any others described herein.

[0100] A subject of the present invention is also the use of any of the TRAP cages described herein as a medicament.

[0101] A subject of the present invention is also a method of treating a patient, comprising administering to said patient a TRAP cage as described herein.

[0102] A subject of the present invention is also the use of any of the TRAP cages described herein in treating a disease in a patient.

[0103] The subject of the present invention is also the TRAP cages produced by this method. These cages may have any of the characteristics or properties described in connection with the previous aspects of the invention above, or any others as described herein.

[0104] The subject of further aspects of the invention are artificial TRAP cages comprising a selected number of TRAP rings held in place by at least one crosslinker. These cages may have any of the characteristics or properties described in connection with the previous aspects of the invention above, or any others described herein.

[0105] Preferably, the cross-linking agent is a metal. Preferably, the cross-linking agent comprises only metals. Preferably, the metal is a metal ion, preferably of a single type of metal.

[0106] Preferably, the metal crosslinker is selected for specific characteristics, whereby the cage is programmable to open or remain closed on demand under said specific conditions.

[0107] Preferably, the metal is selected from the group comprising Au(I), Ag(I), Cd(II), Zn(II) and Co(II). The metal may be a derivative of these metals.

[0108] Preferably, the metal is a d10 metal having a non-linear coordination structure or shell. Preferably, the d10 metal having a non-linear coordination structure or shell is Zn(II) or Co(II).

[0109] Preferably, the metal is a d10 metal with a two-ligand linear coordination structure or shell. Preferably, the d10 metal with a non-linear coordination structure or shell is Ag(I) or Cd(II).

[0110] Preferably, the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including K35C, K35H, R64S, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C, and S33H / K35C.

[0111] Preferably, the artificial TRAP cage protein is modified to contain a K35C / S33H mutation or a K35H / S33H mutation.

[0112] Preferably, the cross-linking agent comprises silver (Ag(I)), and preferably the artificial TRAP cage protein is modified to include K35C / R64S mutations.

[0113] Preferably, the cross-linking agent comprises cadmium (Cd(II)), and preferably the artificial TRAP cage protein is modified to include K35C / R64S mutations.

[0114] Preferably, the cross-linking agent comprises cobalt (Co(II)), and preferably the artificial TRAP cage protein is modified to include S33H / K35C or S33H / K35H mutations.

[0115] Preferably, the crosslinker comprises zinc (Zn(II)) and preferably the artificial TRAP cage protein is modified to include S33H / K35C or S33H / K35H mutations.

[0116] Preferably, the crosslinker comprises gold (Au(I)) and preferably the artificial TRAP cage protein is modified to comprise S33C / R64S.

[0117] Preferably, the specific cleavage characteristics of the molecular crosslinker are selected from the group including: (i) reduction-resistant / insensitive molecular crosslinkers, whereby the cage remains closed under reducing conditions; (ii) reduction-responsive / sensitive molecular crosslinkers, whereby the cage opens under reducing conditions; and (iii) photoactivatable molecular crosslinkers, whereby the cage opens upon exposure to light.

[0118] Preferably, the reduction-resistant / insensitive molecular crosslinker may be selected from the group including bismaleimidohexane (BMH), bisbromobimane, and bis-bromoxylene.

[0119] Preferably, the reduction-responsive / sensitive molecular crosslinker may be selected from the group including dithiobismaleimideethane (DTME).

[0120] Preferably, the photoactivatable molecular crosslinker may be selected from the group comprising bis-halomethylbenzenes and their derivatives including 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0121] Preferably, the molecular cross-linker is a homobifunctional molecular moiety and its derivatives. Preferably, the homobifunctional molecular cross-linker is bismaleimidohexane (BMH).

[0122] Preferably, the cage is resistant / insensitive to reducing conditions.Preferably, the homobifunctional molecular crosslinker is dithiobismaleimideethane (DTME).

[0123] Preferably, the cage is responsive / sensitive to reducing conditions.Preferably, the molecular cross-linker is a bis-halomethylbenzene and its derivatives.

[0124] Preferably, the molecular crosslinker is selected from the group comprising 1,2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0125] Preferably, the molecular crosslinker is photolabile upon exposure to UV light.

[0126] Preferably, the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including K35C, R64S, and K35C / R64S. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation.

[0127] Preferably, the cross-linking agent comprises dithiobismaleimideethane (DTME) and preferably the artificial TRAP cage protein is modified to include K35C / R64S mutations.

[0128] Preferably, the number of TRAP rings in the TRAP cage is 6-60, preferably 7-55, preferably 8-50, preferably 9-45, preferably 10-40, preferably 11-35, preferably 12-34, preferably 13-33, preferably 14-32, preferably 15-31, preferably 16-30, preferably 17-29, preferably 18-28, preferably 19-27, preferably 20-26. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is more than 6, preferably more than 10, preferably more than 15, preferably more than 20.

[0129] Preferably, the number of TRAP rings in the TRAP cage is 12 to 24.

[0130] Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

[0131] Preferably, the cage according to the invention comprises a mixture of different cross-linking agents.

[0132] Preferably, the cage according to the present invention encapsulates a cargo, which may be a cargo that is delivered at a specific time and desired location.

[0133] Preferably, the cages according to the invention are hollow. Preferably, the cages according to the invention are approximately spherical in shape, preferably hollow spheres. The cages herein are hollow shapes roughly approximating hollow spheres. These are close to the shapes that would be obtained if TRAP rings were placed at the vertices or corners of a regular convex polyhedron and then linked together.

[0134] Preferably, the TRAP cage is stable at elevated temperatures, i.e. when the temperature is elevated above normal room temperature or human / animal body temperature, preferably stable between 0 and 100°C, preferably stable between 15 and 100°C, preferably stable between 15 and 75°C, preferably stable up to 75°C, preferably stable below 75°C.

[0135] Preferably, the TRAP cage is stable at non-neutral pH, preferably above pH 7 and below pH 7, preferably stable at pH 3-11, preferably stable at pH 4-10, preferably stable at pH 5-9.

[0136] Preferably, the TRAP cage is stable in chaotropic agents (agents that disrupt hydrogen bonds in solution, that would disrupt or denature protein or macromolecular structures) or detergents that would otherwise be expected to disrupt or denature protein or macromolecular structures. Preferably, the cage exhibits stability in n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. Preferably, the TRAP cage is stable in up to 4M GndHCl. Preferably, the TRAP cage is stable in up to at least 7M urea. Preferably, the TRAP cage is stable in up to 15% SDS. The stability of the cages described herein may be tested using these agents in standard conditions that would be known to those skilled in the art to demonstrate said stability.

[0137] The cages described herein exhibit unexpected stability in these conditions, providing a more stable TRAP cage than previously demonstrated.

[0138] A subject of the present invention is also the use of a cage according to the invention as defined above in the delivery of a cargo in a controlled period of time and to a desired location.

[0139] The subject of the present invention is also a TRAP cage comprising a protein modified to contain any one or more of the mutations selected from the group comprising K35C, K35H, R64S, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C, and S33H / K35C.

[0140] The subject of the present invention is also a TRAP protein modified to contain any one or more of the mutations selected from the group comprising K35C, K35H, R64S, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C and S33H / K35C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0141] References herein to a "TRAP protein" refer to the bacterial protein tryptophan RNA-binding attenuating protein, which may be isolated, for example, from wild-type Geobacillus stearothermophilus or other such bacteria. Although the protein may be isolated from a variety of bacteria, TRAP proteins which will function as described herein include those from the following bacteria: Alkalihalobacillus ligniniphilus, Anaerobacillus isosaccharinicus, Anoxybacillus caldiproteolyticus, Anoxybacillus calidus, Anoxybacillus pushchinoensis, Anoxybacillus tepidamans, Anoxybacillus vitaminiphilus, Bacillaceae bacteria, Bacillus alveayuensis, Bacillus sinesaloumensis, Bacillus species FJAT-14578, Bacillus species HD4P25, Bacillus species HMF5848, Bacillus species PS06, Bacillus species REN16, Bacillus species SA1-12, Bacillus species V3-13, Bacillus timonensis, Bacillus timonensis, Bacillus weihaiensis, Bacillus yapensis, Calidifontibacillus erzurumensis, Calidifontibacillus oryziterrae, Cytobacillus luteolus, Fredinandcohnia aciditolerans, Fredinandcohnia humi, Fredinandcohnia onubensis, Fredinandcohnia onubensis, Geobacillus genomosp. 3, Geobacillus sp. 46C-IIa, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillusstearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus thermodenitrificans NG80-2, Halobacillus dabanensis, Halobacillus halophilus, Halobacillus halophilus, Jeotgalibacillus proteolyticus, Litchfieldia alkalitelluris, Litchfieldia salsa, Mesobacillus harenae, Metabacillus, Metabacillus litoralis, Metabacillus sediminilitoris, Oceanobacillus limi, Oceanobacillus species Castelsardo, Ornithinibacillus, Ornithinibacillus bavariensis, Ornithinibacillus contaminans, Ornithinibacillus halophilus, Ornithinibacillus scapharcae, Parageobacillus caldoxylosilyticus, Parageobacillus species, Parageobacillus thermantarcticus, Parageobacillus thermantarcticus, Parageobacillus thermoglucosidasius, Parageobacillus thermoglucosidasius, Paucisalibacillus globulus, Paucisalibacillus species EB02, Priestia abyssalis, Priestia endophytica, Priestia filamentosa, Priestia koreensis, Priestia megaterium, Psychrobacillus glaciei, SalinibacillusIt can be isolated from bacteria such as, for example, xinjiangensis, Sutcliffiella cohnii, and Thermolongibacillus altinsuensis.

[0142] The Trp RNA binding attenuator protein is a bacterial circular homo 11-mer (see A. A. Antson, J. Otridge, A. M. Brzozowski, E. J. Dodson, G. G. Dodson, K. S. Hilson, T. Smith, M. Yang, T. Kurecki, P. Gollnick, hereby incorporated by reference). The structure of the trp RNA binding attenuator protein can be found in the literature (Nature 374, 693-700 (1995), hereby incorporated by reference).

[0143] Suitably, the protein used herein is a modified version of the wild-type TRAP isolated from Bacillus stearothermophilus, which can be seen in Table 1:

[0144] [Table 1]

[0145] The wild-type TRAP Bacillus stearothermophilus gene sequence can be seen in Table 2:

[0146] [Table 2]

[0147] Preferably, the preparation of the protein is carried out by biomolecule expression in a suitable expression system and purification of the expression product, preferably using a modified version of the wild-type TRAP Bacillus stearothermophilus gene sequence described above.

[0148] TRAP proteins form rings, herein "TRAP rings," which are the native state of TRAP proteins. Typically, as in the case of the Geobacillus Stearothermophilus protein demonstrated herein, TRAP monomeric proteins spontaneously assemble into circular bodies or rings made from monomers.

[0149] TRAP cages are only formed under certain conditions, for example as demonstrated herein, with the presence of cysteines that can be bridged, resulting in a ring that associates with the cage. For example, as demonstrated herein, they will form with the presence of a cysteine ​​at position 35 (as a result of the K35C mutation).

[0150] References herein to a "TRAP ring" are synonymous with a TRAP building block, a subunit of a TRAP cage complex, or a TRAP monomer assembly. References herein to an "analog" of a particular protein or nucleotide sequence refer to a protein or nucleotide sequence that has sufficient identity or homology to the protein or nucleotide sequence to be capable of performing a specified function, e.g., TRAP cage formation under conditions described herein, or to encode a protein that is capable of performing a specified function, e.g., TRAP cage formation under conditions described herein.

[0151] To determine the identity / homology percentage of two sequences, the sequence in question and the reference are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). A sequence can be determined to be an analog of a particular one if it has preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acids or nucleotides of the reference sequence of the relevant length. When comparing amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions, if a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap.

[0152] Suitably, the TRAP protein comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% identity or homology to the amino acid sequence of SEQ ID NO: 1. Preferably, the TRAP protein comprises an amino acid sequence having at least 85% identity or homology to the amino acid sequence of SEQ ID NO: 1.

[0153] Reference herein to a "TRAP cage" refers to an associated protein complex formed from a plurality of biomolecules, herein a plurality of TRAP protein rings forming a complex. The TRAP protein rings may be linked together by a crosslinker, herein a molecular crosslinker. "Complex", "association", "aggregate" are used alternatively in this description to refer to a superstructure built by a reaction between biomolecules. The amount of units involved in the complex depends on the nature of the biomolecule. More specifically, it is present in the amount of biomolecule and the amount of -SH groups present in the biomolecule. "TRAP cage" and "artificial TRAP cage" are used interchangeably herein.

[0154] The TRAP protein is a suitable biomolecule model for the methods of the invention, likely due to its high inherent stability, cyclic shape, lack of native cysteine ​​residues (for easier control of the conjugation process), and the availability of residues that can be converted to cysteine, where the resulting cysteine ​​is in the right chemical and spatial environment for proper bond formation.

[0155] References herein to "programmable" are intended to convey that the TRAP cages of the present invention have properties imparted to them or engineered into them that make them prone or predisposed to behave in a specific and selected manner upon exposure to specific environmental conditions or stimuli.

[0156] References herein to "open" are synonymous with a TRAP cage that has fractured, leaked, fragmented, broken, or generally permitted cargo to escape from the interior of the cage.

[0157] References herein to "closed" are synonymous with a TRAP cage remaining intact, unbroken, impermeable, or generally remaining as a cage in its entirety.

[0158] References herein to "bifunctional" refer to molecular crosslinkers that have two functional groups, e.g., herein, molecules that have two functional groups, one for each of the cysteine ​​thiol groups that are to be crosslinked to connect the TRAP rings in the TRAP cage. References herein to "homobifunctional" refer to bifunctional linkers in which the two groups are the same.

[0159] Preferably, the homobifunctional linkers include bismaleimidohexane (BMH), dithiobismaleimidoethane (DTME), bis-halomethylbenzenes and their derivatives, 2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0160] "Unit," "subunit," "molecule," "biomolecule," and "monomer" are used alternatively in this description to mean one molecule that connects to another molecule to form a complex.

[0161] A "molecular crosslinker" is a molecule that acts to connect a unit, subunit, molecule, biomolecule, or monomer to other instances thereof through the formation of one or more chemical bonds. A molecular crosslinker is not a single atom linker that is a separate entity.

[0162] References herein to "reduction-resistant / insensitive molecular crosslinkers" are references to crosslinkers that are not cleaved by reduction reactions such as those typically seen when disulfide bonds are cleaved by reducing agents. These crosslinkers are stable under conditions that would result in rupture of reduction-sensitive bonds. These include bismaleimidohexane (BMH) and bis-bromoxylene.

[0163] References herein to "reduction-responsive / sensitive molecular crosslinkers" are references to crosslinkers that are cleaved by a reduction reaction, such as those typically seen when disulfide bonds are cleaved by reducing agents. These crosslinkers are not stable under conditions that would result in rupture of reduction-sensitive bonds. These include dithiobismaleimidoethane (DTME).

[0164] References herein to "photoactivatable molecular crosslinkers" are to crosslinkers that are photoreactive or photosensitive, i.e., that will be cleaved when exposed to light. The light can be UV or other such light of a specific range of wavelengths. These include 2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0165] To achieve programmable rather than easily triggerable disassembly, protein cages have been proposed in which the building blocks are tethered together by simple crosslinkers through facile chemistry allowing easy interchangeability (Fig. 1a). The disassembly of such cages would depend on the cleavage characteristics of the crosslinkers employed. To test this possibility, TRAP(K35C / R64S) has been used, and attempts have been made to link TRAP with either the simple bifunctional molecular crosslinkers dithiobismaleimidoethane (DTME) or bismaleimidohexane (BMH). They demonstrate that, thanks to a thiol-specific reaction at neutral pH, the gold-mediated assembly (TRAP cage) can be easily achieved. Au(I) ) acted as a connector, like Au(I) in DTME. Since DTME contains a cleavable disulfide bond, whereas BMH does not, the two resulting cages (TRAP cage) DTME and TRAP Cage BMH ) have contrasting disassembly characteristics when exposed to reducing agents (Figure 1a).

[0166] To obtain covalently cross-linked cages, TRAP(K35C / R64S) was mixed with either DTME or BMH in aqueous buffer. Size-exclusion chromatography (SEC) of the resulting reaction mixtures revealed that the TRAP cages Au(I) The crosslinked TRAP cages showed substantial peaks at elution volumes similar to those of the crosslinked TRAP cages, suggesting successful cage formation with both crosslinkers (Figure 1). The isolated fractions were further analyzed by SEC (Figure 1c), dynamic light scattering (DLS), and negative stain transmission electron microscopy (TEM) (Figure 1d, Figure 2), yielding the expected results for monodisperse spherical cage structures with diameters of approximately 25 nm. The assembly appeared essentially complete within 60 min, with a typical yield of around 20% for both crosslinked TRAP cages (Figure 1b). No free cysteines were detected following the reaction. Further analysis of the acquired TRAP cages using size-exclusion chromatography coupled with right- and low-angle light scattering (SEC-RALS / LALS) showed an apparent average molecular mass of both particles as 2.2 MDa, suggesting a 24-ring organization.

[0167] TRAP Cage DTME and TRAP Cage BMH Detailed structures for both the TRAP cage and the TRAP-Cage have been determined using cryo-EM single particle reconstruction. Electron density maps at 4.7 Å and 4.9 Å resolution for both types of cage have been obtained. These show that each structure is Au(I) We revealed that the TRAP complex is composed of 24 TRAP rings organized into two chiral configurations similar to those found for TRAP (Fig. 1e). The TRAP ring model was refined against the map, producing a good fit. Closer examination at the ring-ring interface found two substantial electron densities bridging two adjacent subunits, likely corresponding to bismaleimide crosslinkers (Fig. 1e). The crosslinker appears to be curved into a horseshoe shape between the cysteine ​​residues of the opposing subunits.

[0168] TRAP Cage DTME and TRAP Cage BMHBoth of the TRAP cages showed similarly high stability in response to elevated temperature, chaotropic agents, and detergents. Specifically, they did not exhibit significant morphological changes after incubation at 75°C, pH in the range of 2-11, up to 4 M GndHCl, up to at least 7 M urea, and 7% SDS for 10 min. However, the TRAP cage DTME The TRAP cage readily disassembles upon addition of the reducing agents tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) (Figure 2a, c). In contrast, the TRAP cage BMH The high stability at high temperature, large pH range, and in the presence of high concentrations of chaotropic agents and detergents also contributed to the high stability of the TRAP cage. DTME (Figure 2d-g) and the TRAP cage BMH DTT-dependent disassembly was further investigated at the single cage level in real time using high-speed atomic force microscopy (HSAFM, Fig. 2l). BMH However, the TRAP cage was resistant to disassembly in the presence of DTT. DTME The TRAP cage readily disassembled, with individual patches of TRAP subunits appearing to be “peeled off” from the cage surface, eventually leading to the opening of the entire structure approximately 3 min after the initial ring detachment (Fig. 2d). DTME Such a stepwise disassembly process of the TRAP cage shows a more coordinated disassembly on a much shorter time scale. Au(I) In stark contrast to.

[0169] To efficiently track the cage stability against various thiol-containing reagents, we employ spectroscopic methods to develop real-time monitoring of bulk cage disassembly in solution. In this system, two fluorescent proteins, mOrange2 and mCherry, which act as donor and acceptor of Förster resonance energy transfer (FRET), respectively, are encapsulated. These fluorescent proteins were genetically fused to the N-terminus of TRAP, which faces the cage interior in the assembled structure. To avoid steric hindrance during the cage formation process, they were co-produced with unmodified TRAP to form a "patchwork" ring.

[0170] The resulting TRAP rings were then assembled into cage structures using either Au(I) or DTME (Figure 3a). After purification using size-exclusion chromatography, the isolated particles were analyzed by native PAGE (Figure 3b) and TEM imaging (Figure 3c), which confirmed guest encapsulation in the lumen of the monodisperse spherical cages. The presence of both proteins in the constrained volume of the TRAP cage lumen should enable efficient FRET (22, 23). Indeed, TRAP cages copackaging both guests Au(I) and TRAP Cage DTME The normalized fluorescence spectra of showed approximately 1.5-fold higher signal in mCherry emission at 610 nm compared to the corresponding control samples containing cages encapsulating only mOrange2 or mCherry and mixed in solution (Fig. 4a, b). However, the addition of DTT, which induces disassembly for both types of cages, led to FRET cancellation observed in the resulting spectra, closely resembling the control samples (Fig. 4a, b). These results indicated efficient energy transfer between the encapsulated guests and their cancellation upon release, demonstrating that the FRET cage is suitable for monitoring the TRAP cage disassembly process.

[0171] We next investigated the cleavage of the TRAP cage upon the addition of DTT. Au(I)and TRAP Cage DTME The disassembly kinetics for both the TRAP cages were measured. The change in the fluorescence intensity ratio at 568 / 610 nm was utilized as an indicator of the time-dependent disassembly process (Figure 4c). For both cages, the increase in fluorescence reached a plateau approximately 5 min after the addition of DTT, indicating complete cage disassembly and guest release. However, the mechanism of this process appeared to differ depending on the cage type. Au(I) In contrast, the TRAP cage DTME exhibited a sigmoidal disassembly behavior indicating multiple steps involved in the guest release procedure. These results are consistent with the results of the TRAP cage, respectively, observed by HSAFM. Au(I) or TRAP cage DTME This coincided well with the coordinated versus gradual dismantling of

[0172] Protein cages that can carry cargo and disassemble in the presence of reducing agents have the potential for intracellular delivery. Ideal nano-vehicles used for this purpose should remain assembled under extracellular conditions but disassemble to release their cargo upon exposure to intracellular conditions. We assessed this possibility by monitoring the cage disassembly kinetics in the presence of cysteine ​​and glutathione, employed as model thiols. A time-dependent increase in FRET cancellation indicates the reversal of the TRAP cage upon the addition of cysteine. Au(I) 4, reaching a plateau after approximately 15 min, indicating complete cage disassembly (Fig. 4d, black circles). In contrast, the TRAP cage containing the same FRET pair DTME showed little fluorescence change, suggesting that the cage does not disassemble under these conditions (Fig. 4d, red circle). This stability to cysteines is consistent with the TRAP cage. Au(I) This is likely due to the slow kinetics of disulfide cleavage catalyzed by this free thiol, in contrast to the presumably much more rapid ligand exchange on the TRAP cage. DTME It could still be disassembled by using higher concentrations of free thiol compounds: with 50 mM reduced glutathione, the TRAP cage wasAu(I) Although the disassembly rate was significantly slower than that of the TRAP cage. DTME Disassembly of and release of cargo from it was observed (Fig. 4e, red and black circles).

[0173] According to an embodiment, the cages described herein can be used as medicines. This can be in treating a patient, such as administering the cages described herein to a patient, or including the cages described herein for use in treating a disease in a patient. This can be cages designed to carry cargo and disassemble in the presence of a reducing agent, particularly for intracellular delivery. These cages can be administered with or in the presence of a pharma- ceutical acceptable carrier, adjuvant, or excipient. Cages for use as medicines or to treat a patient would be beneficial to said patient. For example, as a drug delivery system (DDS) for active molecules (biological macromolecules such as RNA, DNA, peptides, and proteins, among others), they offer advantages since biological macromolecules are often easily destroyed or digested by conditions such as those found in vivo. Biological macromolecules are too large to diffuse through the holes in TRAP, which is a large protein, and the TRAP cage can continue to undergo significant changes without destroying the overall structure. This means that it can be modified to capture therapeutic cargo and simultaneously modified externally to target therapeutic targets. A programmable linker can be used that cleaves in a desired situation that correlates with arrival at the site of action. For example, light can be shone on the target site to cleave the open photocleavable TRAP cage. When the TRAP cage penetrates a cell, the one connected by the reducible linker will spontaneously open and release the cargo as the cell's cytoplasm becomes highly reduced. The cage can also be used in conjunction with or acting as a vaccine, where an antigen (i.e., a peptide) that is expected to stimulate a T cell response is captured inside the TRAP cage and then targeted to the T cell, followed by triggered opening. [Brief description of the drawings]

[0174] [Figure 1] Diagram of molecular crosslinker-mediated TRAP cage formation. a, Schematic of the crosslinking reaction with dithiobismaleimidoethane (DTME) or bismaleimidohexane (BMH). The TRAP(K35C / R64S) rings shown on the left, with cysteines depicted as circles on the exterior, are covalently connected to each other via a reaction between cysteines and a bismaleimide compound (line above the first arrow, with detailed chemical structure below) to form a cage-like structure. Addition of dithiothreitol (DTT) leads to disassembly of the DTME-mediated cage (TRAP cage DTME, top), but has no effect on cages assembled with BMH (TRAP cage BMH, bottom). b, Native PAGE gels for TRAP cage DTME and TRAP cage BMH formation, with black arrowheads marking the height of the cages formed. c, Size-exclusion chromatography profiles for purified TRAP cage DTME (light gray line) and TRAP cage BMH (gray line). A profile of TRAP cage Au(I) (black line) is provided as a control. mAu, milliabsorbance units. d, Transmission electron microscopy (TEM) images for TRAP cage DTME (right) and TRAP cage BMH (left). Scale bar, 50 nm. e, Cryo-electron microscopy density maps for the left-handed (top) and right-handed (bottom) morphologies of TRAP cage DTME, refined to 4.7 Å and 4.9 Å resolution, respectively. Insets show amplified images at the ring-ring interface with fitted crosslinker models highlighted for DTME (middle) and BMH (right) shown in side-view (top) and top-view (bottom) images. [Diagram 2]Figure 1. Stability of cross-linked TRAP cages. Redox responsiveness. a, b, Native PAGE analysis of TRAP cage DTME (a) and TRAP cage BMH (b) in the presence of DTT and tris(2-carboxyethyl)phosphine (TCEP). The TRAP cage appears as a prominent band running between 1048 and 1236 kDa. C = TRAP cage DTME (a) and TRAP cage BMH (b). M = molecular weight marker. c, TEM images showing TRAP cage DTME after treatment with 0.1 mM (left) and 1 mM (middle) TCEP, and TRAP cage BMH after treatment with 10 mM TCEP (right). Scale bar, 50 nm. d-g, Stability of TRAP cage DTME. d, Native PAGE showing the thermal stability of TRAP cage DTME over the indicated incubation times and temperatures. The image below the gel shows that the TRAP cage retains its structure after incubation at 95 °C for 10 min, scale bar, 100 nm. e, Native PAGE showing the effect of pH on stability: the cage is stable at pH 3-11 using native PAGE. The images below the gel are TEM images of samples after incubation at the indicated pH. Scale bar, 100 nm. f, Structure of TRAP cage DTME assessed using native PAGE showing that it is unaffected in the presence of guanidine hydrochloride (GdnHCl), urea, and g, SDS, over the range tested. TEM images (bottom) were acquired after incubation of the cage with 4 M GndHCl, scale bar, 100 nm. h-k, Stability of TRAP cage BMH, as for TRAP cage DTME results (panels d-g), except that TRAP cage BMH was used. The black arrowhead indicates the position of the intact TRAP cage on the gel. l, Frame from an HSAFM movie showing the effect of adding 4 mM DTT to TRAP-caged DTME at times after addition of DTT as indicated. [Diagram 3]Loading of TRAP cages with FRET pairs. a, Schematic of TRAP cages loaded with fluorescent proteins. Patchwork TRAP rings fused at the N-terminus with either mCherry (black cylinders) or mOrange2 (grey cylinders) were mixed together with either DTME or triphenylphosphine monosulfate (TPPMS)-Au(I)-Cl. b, Native PAGE showing the fluorescent properties of purified TRAP cages coupled with fluorescent cargo. The gel was visualized using InstantBlue protein stain (right) and fluorescence (left) with excitation at 532 nm and emission at 610 nm. Note that the exact position of the prominent band corresponding to the TRAP cage, running approximately from 1028 to 1236 kDa, varies slightly according to the presence / absence of cargo and the nature of the crosslinker used. c, TEM images of empty (left) TRAP cages and those loaded with fluorescent proteins (right) assembled using either Au(I) (top) or DTME (bottom). Scale bar, 50 nm. [Figure 4] Figure 1. Guest emission. a, b, Normalized emission spectra of TRAP cage Au(I) (a) and TRAP cage DTME (b) loaded with both mOrange2 and mCherry upon excitation at 510 nm before and after addition of 10 mM DTT. mOrange2 emission peak at 568 nm, mCherry emission peak at 610 nm. Additional lines show the spectra of cages loaded with only mOrange2 or mCherry proteins mixed together immediately before measurement in the absence or presence of DTT, respectively. c, d, e, Time-dependent disassembly of TRAP cage Au(I) (black circles) and TRAP cage DTME (gray circles) after addition of 10 mM DTT (c), 2.5 mM cysteine ​​(Cys) (d), or 50 mM glutathione (GSH) (e). 100% leakage represents the highest donor intensity, following 10 mM DTT treatment for 10 min after each experiment. [Diagram 5]Diagram of TRAP cages with various metal linkers. a. Native PAGE analysis of TRAP cages assembled with Ag(I) or Cd(II); b. TEM image of TRAP cage Ag(I); c. TEM image of TRAP cage Cd(I); d. Native PAGE analysis of TRAP cages assembled with Co(II) or Zn(II); e. TEM image of TRAP cage Co(II); f. TEM image of TRAP cage Zn(II); For a. and d., samples were run on 3–12% native Bis-Tris acrylamide gels. Protein bands were visualized by Coomassie blue staining. For all TEM images, scale bar, 100 nm. [Figure 6]Figure 1. Native PAGE of template reaction; M-marker; R-TRAP ring; C-TRAP cage control; C+T-TRAP cage+10 mM TCEP, showing cage disassembly due to the presence of Au(I); D=TRAP ring+DBX-no cage association; DC-TRAP cage+DBX; DCT-TRAP cage+DBX+10 mM TCEP-cage is still present, suggesting successful exchange reaction; a. Structure of 1,3-dibromoxylene; c. DLS showing the approximate size of the DBX TRAP cage, 24 nm; d. Purified DBX after treatment with 10 mM DTT. TEM images of TRAP cages, scale bar, 100 nm; e. Cryo-EM structures for the leavo (left) and dextro (right) structures of the DBX cross-linked cage; f. Wireframe models of the Au(I)-induced cage (left) and the DBX cage (right) with zoomed in nature of the ring-to-ring connections; g. Structure of 1,2-bisbromomethyl-3-nitrobenzene (BBN); h. SDS PAGE before and after the "template reaction" with BBN; M-marker, R-TRAP ring, C-Au(I)-induced TRAP cage, BBN-TRAP cage after mixing with BBN - appearance of additional bands indicates the presence of covalently linked TRAP dimers; i. TEM images of purified BBN TRAP cages, scale bar, 100 nm (left), 50 nm (right); j. Dependence of photocleavage on the presence of different quencher (DTT) concentrations after 10 min of UV irradiation, k. 10 mM j. Native PAGE showing various time points after the start of UV irradiation in the presence of DTT; for both gels: M-marker, C-TRAP cage, C+ or CDTT-crosslinked TRAP cage with reducing agent added to indicate its resistance to reducing conditions. [Figure 7] Cryo-EM density maps showing the structure of a TRAP cage made using TRAP S33C / R64S, resulting in a 20-ring cage. From left to right: image centered on the 4x hole; image centered on the bowtie hole; perspective image; scale bar -5 nm. EXAMPLES

[0175] Techniques Employed in Realizing the Invention Transmission Electron Microscopy (TEM) Samples were typically diluted to a final protein concentration of 0.025 mg / ml, centrifuged briefly in a tabletop centrifuge, and the supernatants were applied to hydrophilized carbon-coated copper grids (STEM Co.), negatively stained with 4% phosphotungstic acid, pH 8, and visualized using a JEOL JEM-1230 80 kV instrument.

[0176] Native PAGE Samples were run on 3-12% native Bis-Tris gels according to the manufacturer's recommendations (Life Technologies). Samples were mixed with 4x native PAGE sample buffer (200 mM BisTris, pH 7.2, 40% w / v glycerol, 0.015% w / v bromophenol blue). NativeMark unstained protein standards (Life Technologies) were used as a qualitative guide to the molecular weight of migrated bands. When blue native PAGE was performed, protein bands were visualized according to the manufacturer's protocol (Life Technologies), otherwise InstantBlue™ protein stain (Expedeon) was used.

[0177] Protein expression and purification In a typical purification, E. coli BL21(DE3) cells (Novagen) transformed with pET21b plasmid carrying the TRAP(K35C / R64S) gene were cultured at OD 2000 in 3 L of LB medium containing 100 μg / ml ampicillin. 600=0.6, induced with 0.5 mM IPTG, then further shaken for 4 h. Cells were harvested by centrifugation and pellets were kept at -80°C until use. Cells were lysed by sonication at 4°C in 50 ml of 50 mM Tris-HCl, pH 7.9, 50 mM NaCl in the presence of proteinase inhibitors (Thermo Scientific) and with or without 2 mM DTT, and the lysate was centrifuged at 66,063g for 0.5 h at 4°C. The supernatant fraction was heated at 70°C for 10 min, cooled to 4°C, and centrifuged again at 66,063g for 0.5 h at 4°C. The supernatant fraction was purified by ion exchange chromatography on an AKTA purifier (GE Healthcare Life Sciences) using a 4 × 5 ml HiTrap QFF column bound in 50 mM Tris-HCl, pH 7.9, 0.05 M NaCl, ± 2 mM DTT buffer and eluted with a 0.05-1 M NaCl gradient. Fractions containing TRAP protein were pooled and concentrated using an Amicon Ultra 10 kDa MWCO centrifugal filter unit (Millipore), and the sample was subjected to size exclusion chromatography on a HiLoad 16 / 60 Superdex 200 column in 50 mM Tris-HCl, pH 7.9, 0.15 M NaCl at room temperature. Protein concentration was calculated using the BCA protein assay kit (Pierce Biotechnology).

[0178] Cage Stability The stability of the TRAP cage against chemicals and heat was tested using methods similar to those previously described. 1 All agents used in the assay (DTT, TCEP, SDS, Gdn-HCl, and urea) were reconstituted in PBS pH 7.4 and mixed with the TRAP cage samples overnight at room temperature. Thermostability checks were performed by heating the samples at varying temperatures for 10 min. Samples were then subjected to native PAGE. These experiments were repeated twice, each giving uniform results.

[0179] Cryo-EM Vitreous ice preparation was performed using 4 μL of protein sample at approximately 1 mg / mL in PBS. After blotting the samples onto EM grids (Quantifoil 1.2 / 1.3, Cu, 300 mesh), they were frozen by immersion in liquid ethane using an FEI Vitrobot with the following parameters: blot force=0, blot time=4 sec, wait time=0 sec, drain time=0 sec. Photomicrographs were collected using an FEI TitanKrios cryo-microscope operating at 300 kV and a Falcon III camera at 75k magnification. 4942 and 10169 photomicrographs were captured, respectively, in the TRAP cage. DTME and TRAP Cage BMH All micrographs were motion corrected using MotionCorr2. 6 ,CTF estimation was performed using CTFFIND4. 7 The particles were first analyzed in manual mode (approximately 4000 particles) and then in automatic mode using cryoSPARC v2.12.4. 8 The first 2D classes served as templates. The extracted particles were re-classified in 2D to select the best particles for the subsequent reconstruction step. The 3D reconstructions were performed using EMD-4443 and EMD-4444 (TRAP cage) as search models. Au(I) ) using the Heterogenous Refinement protocol.

[0180] Example 1. TRAP Cage DTME and TRAP Cage BMH preparation Molecular cloning For all cloning steps, the E. coli NEB 5 alpha strain was used. Plasmid sequences were confirmed by Sanger sequencing performed by Eurofins. Tetracycline-inducible protein expression vectors were constructed by subcloning the gene segment encoding TRAP(K35C) into pACTet_H-mCherry or pACTet_H-mOrange. The gene for TRAP(K35C) was amplified by PCR using pET21b_TRAP-K35C as template and oligonucleotides FW_XhoI_TRAP and RV_MluI_TRAP as primers (see Table 3). The amplified PCR product was used directly as template for a second PCR, which introduced a linker gene segment using FW_BsrGI_tev and RV_MluI_TRAP oligonucleotides as primers. The PCR products were cloned into pACTet_H-mCherry or pACTet_H-mOrange via the BsrGI and MluI sites to give pACTet_H-mCherry-TRAP-K35C and pACTet_H-mOrange-tev-TRAP-K35C. The previously used R64S was the only important site to avoid gold nanoparticle binding. 1 , here, we introduced only the single point mutation K35C, which is essential for subunit linkage, into the TRAP sequence.

[0181] [Table 3]

[0182] Protein expression and purification TRAP proteins were produced using essentially the same protocol previously described in A.D. Malay et al., "An ultra-stable gold-coordinated protein cage displaying reversible assembly," Nature 569, 438-442 (2019), which is hereby incorporated by reference, except that 2 mM DTT was kept in the buffer for the first purification step to avoid undesired cysteine ​​oxidation. To produce patchwork TRAP rings, E. coli strain BL21(DE3) cells were co-transformed with either pACTet_H-mOrange-TRAPK35C or pACTet_H-mCherry-TRAP-K35C and pET21_TRAP-K35C (see Table 4 in Materials and Methods). Cells were grown at OD at 37°C in 100 ml LB medium supplemented with ampicillin and chloramphenicol. 600=0.5-0.7. At this point, protein expression was induced by the addition of 0.2 mM IPTG and 10 ng / ml tetracycline in the case of pACTet_H-mCherry-TRAP-K35C or 30 ng / ml tetracycline in the case of pACTet_H-mOrange-TRAP-K35C, followed by 20 h cell culture at 25 °C. The cells were then harvested by centrifugation at 5,000 × g for 10 min. The cell pellets were stored at -80 °C until purification. They were then resuspended in 40 ml lysis buffer (50 mM sodium phosphate buffer, 600 mM NaCl, 10 mM imidazole, pH 7.4) supplemented with a spatula tip of DNase I and lysozyme, one tablet of protease inhibitor cocktail, and 2 mM DTT, and stirred at room temperature for 30 min. The samples were then sonicated and clarified by centrifugation at 10,000×g and 4° C. for 20 min. The supernatant was then incubated with 4 ml Ni-NTA resin pre-equilibrated in lysis buffer in a gravity-flow column for 20 min. The resin was then washed over 10 column volumes in lysis buffer containing 20 and 40 mM imidazole. His-tagged proteins were eluted using 5 ml of 50 mM sodium phosphate buffer (pH 7.4) containing 500 mM imidazole. The protein samples were then buffer exchanged into 2× phosphate buffered saline (PBS) supplemented with 5 mM ethylenediaminetetraacetic acid (EDTA), hereafter referred to as 2× PBS-E, using Amicon Ultra-15 centrifugal filter units (50k molecular weight cut-off (MWCO)) (Merck Millipore). The protein was then subjected to size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (GE Healthcare) at a flow rate of 0.8 ml / min. The major peaks absorbing at 548 nm or 587 nm were pooled and concentrated using an Amicon Ultra-15 (50k MWCO). Protein purity was checked by SDD-PAGE and protein concentration was determined by the extinction coefficient: ε mCherry 587 =72000M -1cm -1 , ε mOrange 548 =58000M -1 cm -12 , ε TRAP 280 =8250M -1 cm -1 The protein was determined by absorbance measured using a UV-1900 UV-Vis spectrophotometer (Shimadzu Corporation) using the ELISA kit (http: / / expasy.org / tools / protparam.html). The protein was stored at 4°C until use.

[0183] [Table 4]

[0184] Free thiol concentration measurement TRAP Cage DTME and TRAP Cage BMH The free thiol concentration of either 2-nitro-5-thiobenzoic acid or 5,5'-dithiobis-(DTNB) was assessed using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent according to the manufacturer's protocol. Both samples were concentrated to 0.3 mM using Amicon Ultra-4 centrifugal filter units (100k MWCO). The absorbance at 412 nm was measured using a Spectramax 190 UV / VIS plate reader (Molecular Devices). The concentration of free thiols in the samples was determined using the molar extinction coefficient of 2-nitro-5-thiobenzoic acid (14150 M -1 cm -1 ) and the TRAP cage DTME and TRAP Cage BMH was undetectable.

[0185] Cage assembly and purification For crosslinker-induced cage association, TRAP(K35C / R64S) (100–500 μM) in 2× PBS-E was mixed with a 5-fold molar excess of either DTME or BMH and stirred at room temperature for 1 h. The final DMSO concentration in the solution was kept no greater than 12.5%. After the reaction, the insoluble fraction, possibly caused by the low solubility of the crosslinker in aqueous solution, was removed by centrifugation at 12,000×g for 5 min. The supernatant was then purified by size-exclusion chromatography using a Superose 6 Increase 10 / 300 GL column (GE Healthcare) at a flow rate of 0.5 ml / min in an AKTA purifier (GE Healthcare). The fractions containing crosslinked TRAP cages were pooled and concentrated using an Amicon Ultra-4 (100k MWCO) centrifugal filter unit. The typical yield of crosslinked TRAP cages obtained was approximately 20%. Gold(I)-induced TRAP cage formation and purification was carried out as previously described. 1 Cage formation with fusion proteins was performed using the same protocol as described for both cross-linked and gold(I)-derivatized cages, with an additional Ni-NTA purification step before size-exclusion chromatography to purify the sample from only partially assembled cages (His-tagged mCherry and mOrange2, which are not protected inside the cage, bind to a Ni-NTA column). Protein concentrations and ratios of encapsulated guests were estimated using the absorbance ratios at 280 / 548 nm or 280 / 587 nm. The extinction coefficient used in the calculation was ε mCherry 587 =72000M -1 cm -1 , ε mOrange2 548 =58000M -1 cm -1 , ε TRAP 280 =8250M -1 cm -1Due to the spectral overlap between mCherry and mOrange2, to properly calculate the concentrations of both encapsulated guests, the mCherry extinction coefficient was also estimated at 548 nm (ε mCherry 548 =42538M -1 cm -1 Similarly, the extinction coefficients of mCherry and mOrange2 at 280 nm are ε mCherry 280 =56744M -1 cm -1 and ε mOrange2 280 =52200M -1 cm -1 It was experimentally determined that.

[0186] Example 2. Confirmation of the TRAP cage structure using cryo-EM TRAP Cage DTME and TRAP Cage BMH The detailed structures of both the TRAP cage and the TRAP-Cage have been determined using cryo-EM single particle reconstruction, which has obtained electron density maps at 4.7 Å and 4.9 Å resolution for both types of cage. These indicate that each structure is similar to the TRAP cage. Au(I) The authors revealed that TRAP is composed of 24 TRAP rings organized into two chiral configurations similar to those found for TRAP. The TRAP ring model was refined against the map, producing a good fit. Closer examination at the ring-ring interface found two substantial electron densities bridging two adjacent subunits, likely corresponding to a bismaleimide crosslinker. The crosslinker appears to be curved into a horseshoe shape between the cysteine ​​residues of the opposing subunits (Figure 1).

[0187] Example 3. Specific cleavage characteristics of molecular crosslinkers used in the TRAP cage (Figures 2-4) TRAP Cage DTME and TRAP Cage BMHBoth of the TRAP cages showed similarly high stability in response to elevated temperature, chaotropic agents, and detergents. Specifically, they did not exhibit significant morphological changes after incubation at 75°C, pH in the range of 2-11, up to 4 M GndHCl, up to at least 7 M urea, and 7% SDS for 10 min. However, the TRAP cage DTME The TRAP cage readily disassembles upon addition of the reducing agents tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT). In contrast, the TRAP cage BMH was unaffected. DTT-dependent disassembly was further investigated at the single cage level in real time using high-speed atomic force microscopy (HSAFM). BMH However, the TRAP cage was resistant to disassembly in the presence of DTT. DTME The TRAP cage readily disassembled, with individual patches of TRAP subunits appearing to "peel off" from the cage surface, eventually leading to the opening of the entire structure approximately 3 min after the initial ring detachment. DTME Such a stepwise disassembly process of the TRAP cage shows a more coordinated disassembly on a much shorter time scale. Au(I) This is in stark contrast to the TRAP cage that contains the FRET protein pari crog. DTME The assembly of the complex (Figure 3) also allows its disassembly in the presence of reducing agents to be kinetically characterized (Figure 4).

[0188] Example 4. Cage association with various metals Protein expression and purification TRAP(K35C / R64S) protein was expressed and purified as previously described. TRAP(S33H / K35C) and TRAP(S33H / K35H) proteins were expressed and purified following the same protocol as TRAP(K35C / R64S), but all buffers had a pH of 8.5.

[0189] Protein concentration was determined by measuring absorbance at 280 nm.

[0190] Cage association using various metals Formation of TRAP cages was performed by mixing purified TRAP variants (final concentration 0.1 mM for monomeric subunits) with salts of relevant metals at TRAP monomer:metal ion ratios of 4:1 to 2:1 in appropriate buffers: AgNO3 in 50 mM Tris, pH 7.9, 0.15 M NaNO3; Cd(NO3)2 in 50 mM Tris, pH 7.9, 0.15 M NaCl; CoCl2 or ZnCl2 in 50 mM HEPES, pH 7.9, 0.15 M NaCl. Reactions were typically incubated at room temperature for 3 days. Formation of TRAP cages was confirmed using native PAGE and TEM. Any precipitated material was removed by centrifugation at 12,045 g for 5 min.

[0191] Gold-propelled TRAP cage [TRAP(K35C / R64S)+Au(I)] The tryptophan RNA-binding attenuator protein double mutant TRAP(K35C / R64S) can assemble into hollow sphere structures by reaction with monovalent gold ions. Cryo-EM single-particle reconstructions revealed that the resulting 22 nm cage is composed of 24 cyclic 11-mer subunits linked together via linear sulfur-Au(I)-sulfur bridges between opposing cysteines.

[0192] Silver and cadmium propelled TRAP cages [TRAP(K35C / R64S)+Ag(I) or Cd(II)] Cage formation can be promoted by other metals besides Au(I), namely Hg(II), Ag(I), and Cd(II), suggesting that metal-driven cage formation requires a water-soluble d10 metal ion with a preferred two-ligand linear configuration.

[0193] The Ag(I)-TRAP cage forms and remains stable only in the absence of chloride ions.

[0194] The TRAP(K35C / R64S) cages created by the addition of Ag(I) or Cd(II) showed bands on native PAGE with similar mobility as the Au(I)-mediated TRAP cages (Figure 5a). Cage formation was further confirmed by negative staining transmission electron microscopy (TEM), which showed spherical hollow structures with diameters of 22-24 nm (Figures 5b and 5c). These results suggested that the silver- and cadmium-propelled cages could form structures with similar morphologies as the Au(I)-TRAP cages.

[0195] Cobalt and zinc-propelled TRAP cages [TRAP(S33H / K35C) or TRAP(S33H / K35H)+Co(II) or Zn(II)] The TRAP metal binding site has been reengineered to target metal ions with tetrahedral coordination preferences. Based on the crystal structure, a histidine or a cysteine ​​and histidine pair was introduced at positions i and i+2 of the β-sheet motif around the periphery of the TRAP ring, yielding TRAP(S33H / K35C) and TRAP(S33H / K35H), whereby each monomer unit provides two ligands to coordinate divalent metals. These variants assembled into a cage structure upon addition of Zn(II) and Co(II).

[0196] Native electrophoresis revealed that TRAP(S33H / K35H) bearing both Zn(II) and Co(II) migrated similarly to the Au(I)-mediated TRAP cage (Figure 5d). The formation of a cage with a diameter of around 22 nm was confirmed by negative staining transmission electron microscopy (TEM), suggesting the similarity of this structure to the Au(I)-TRAP cage (Figures 5e and 5f).

[0197] Example 5. TRAP cage assembled using a photocleavable crosslinker material and method: Gold-derived TRAP cages were prepared as previously described (Malay et al., Nature, 2019, hereby incorporated by reference). 1,3-Dibromoxylene and 1,3-bisbromomethyl-4-nitrobenzene were purchased from commercial suppliers and dissolved in N,N-dimethylformamide (DMF). A 2 molar excess (relative to the TRAP monomer) of either crosslinker was mixed with freshly purified gold-derived TRAP cages in 50 mM sodium phosphate buffer, pH 7.4, containing 5 mM EDTA, with stirring at room temperature for 1 h. 10 mM dithiothreitol (DTT) was then added to the reaction to capture Au(I). Samples were then purified by size-exclusion chromatography using a Superose 6 Increase 10 / 300 GL column (GE Healthcare).

[0198] Light-induced disassembly of the 1,3-bisbromomethyl-4-nitro-benzene TRAP cage was examined by exposing samples to 365-nm wavelength light for varying periods of time in the presence of 10 mM dithiothreitol (DTT) to quench free radical species. Cage morphology and crosslinker cleavage processes were monitored using dynamic light scattering (DLS) on a Zetasizer (Malvern), SDS, native PAGE, and TEM.

[0199] result In a first attempt, we tried a similar method of cage assembly as we did for the BMH / DTME crosslinker (simply mixing the TRAP rings with an excess of crosslinker) and were unsuccessful, likely due to the difficulty of controlling the correct orientation of the rings in the structure.

[0200] We found a novel way to overcome this challenge, using pre-assembled Au(I)-derived TRAP cages instead of just rings, which allows the dibromo crosslinker to exchange gold atoms without changing the orientation of the rings in the cage, which we call "template reaction". We further used 1,3-dibromoxylene (DBX) crosslinker (Figure 6b) as a basis for the optimization of this method. The gold-derived TRAP cages, possessing Au(I) as a linker between the rings, tend to disassemble in the presence of reducing conditions (Figure 6a, lane CT). We used this property to check whether DBX was incorporated into the structure of the TRAP cage as a result of the "template reaction", due to its lack of disassembly property in the same conditions. Indeed, after mixing DBX with the Au(I)-derived TRAP cage and further purification, the TRAP cage became resistant to disassembly in reducing conditions, providing for the presence of a different type of linkage between the rings. We further characterized the structure of the obtained DBX TRAP cage by DLS method (Fig. 6c), which showed that the size of DBX TRAP cage is approximately 24 nm with high monodispersity. DBX TRAP cage is 2 nm larger than Au(I)-induced TRAP cage, also suggesting the presence of a crosslinker in the structure that extends their size with the maintenance of the cage-like structure that can be observed by TEM (Fig. 6d). Finally, we solved the structure of DBX TRAP cage proving the presence of DBX crosslinker between the rings. The cryo-EM structure of the resulting cage (Fig. 6e) revealed other interesting properties of the aggregate. Indeed, as in the case of Au(I)-induced cage, we could distinguish two chiral forms of the cage (leavo and dextro), which was not surprising due to the chiral properties of the gold-induced cage. The noticeable difference between the template cage (gold-induced) and the resulting one (DBX-containing) is the number of connections between the TRAP rings. For the Au(I)-derived cage, 120 connections were identified (-S-Au-S-bridges), whereas for the DBX cage, the number of connections drops to half that number. 60 linker molecules and the same overall configuration (based on a distorted cube) imposes a slightly different TRAP ring orientation.Wireframe models of the Au(I)-induced and DBX cages show the difference between the TRAP ring orientations and the transition from edge-to-edge (Au(I)) to apex-to-apex (DBX) (Figure 6f).

[0201] Obtaining good results with the basic dibromo bridge of the TRAP cage, we decided to replace DBX with the photolabile crosslinker 1,2-dibromo-3-nitro-benzene, which is very similar in structure but can be cleaved after UV (365 nm) light irradiation due to the presence of a nitro group (Fig. 6g). We optimized the conditions of the "template reaction" with the BBN crosslinker, which turned out to be identical to the DBX used previously. SDS PAGE showed the clear appearance of TRAP dimers after the "template reaction", providing the presence of a covalent bond (Fig. 6h), suggesting that the BBN crosslinker is incorporated into the TRAP cage structure in a similar manner to the DBX used previously. TEM confirmed the presence of monodisperse TRAP cages with a diameter of approximately 24 nm (Fig. 6i).

[0202] To further explore the potential of the acquired photolabile TRAP cages, we tested their ability for disassembly under UV light. Such a reaction depends on the presence of a quencher that does not make the reaction reversible. We tested various concentrations of the quencher (DTT) and showed such a dependency in the case of the BBN TRAP cage as well. The BBN TRAP cage was successfully disassembled under UV light and in the presence of 10 mM DTT (Figure 6j). We also tested how long it took to completely disassemble the BBN TRAP cage. Native PAGE showed a progressive disappearance of the cage band in time, and the complete disassembly was estimated to occur within approximately 2 min from the start of UV irradiation (Figure 6k).

[0203] Throughout the description and claims of this specification, the words "comprise" and "containing" and variations thereof mean "including, but not limited to," and they are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural, unless the context otherwise requires. In particular, where the indefinite article is used, the specification should be understood to contemplate the plural as well as the singular, unless the context otherwise requires.

[0204] It should be understood that any feature, integer, characteristic, compound, chemical moiety, or group described in conjunction with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All of the features disclosed herein (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel one or any novel combination of the features disclosed herein (including any accompanying claims, abstract, and drawings), or to any novel one or any novel combination of the steps of any method or process so disclosed.

[0205] Example 6. TRAP cage made of 20 rings material and method The TRAP protein was expressed as described above, except that the expression plasmid encoded the TRAP protein with the mutation S33C instead of K35C (also with the mutation R64S). Incubation with a source of Au(I) was additionally as described above. Subsequent purification of the resulting TRAP cage formed was as described above. The structure of the resulting TRAP cage was determined using cryo-EM as described above.

[0206] result Structural analysis of the assembled cage (Figure 7) revealed that it is composed of 20 TRAPS33C / R64S rings connected with a cross-link density reminiscent of that seen in the cage using the TRAPK35C mutant, giving confidence that, despite the lower resolution in this case, the connections between adjacent rings are the same gold staples seen in previous TRAP cages with Au(I) ions acting as cross-links between two opposing Cys residues.

Claims

1. An artificial TRAP cage comprising a selected number of TRAP rings held in place by homobifunctional molecular crosslinkers, the crosslinkers being selected for their specific characteristics such that the cage is programmable to open or remain closed on demand under specific conditions.

2. 2. The cage of claim 1, wherein the specific cleavage characteristics of the molecular crosslinker are selected from the group comprising: (i) to (iii) above: (i) a reduction-resistant / insensitive molecular crosslinker such that the cage remains closed under reducing conditions; optionally, the crosslinker is bismaleimidohexane (BMH) or bis-bromoxylene; (ii) a reduction-responsive / sensitive molecular crosslinker such that the cage opens under reducing conditions, optionally the crosslinker is dithiobismaleimidoethane (DTME); and (iii) a photoactivatable molecular crosslinker, whereby the cage opens upon exposure to light; optionally, the crosslinker is a bis-halomethylbenzene and derivatives thereof, including 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB); and further optionally, the crosslinker is photolabile upon exposure to UV light.

3. 3. The cage according to claim 1 or 2, wherein the number of TRAP rings in the TRAP cage is between 6 and 60, optionally the number of TRAP rings in the TRAP cage is 12, 20 or 24, preferably 24.

4. 4. The cage of claim 1 , comprising a mixture of different programmable cross-linking agents.

5. 5. The cage of claim 1 , encapsulating a cargo that can be programmed to deliver the cargo at a specific time and desired location.

6. 6. The cage of any one of claims 1 to 5, wherein the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group comprising K35C, R64S and K35C / R64S.

7. 7. The cage of any one of claims 1 to 6, which is stable at elevated temperatures, stable at non-neutral pH, and / or stable in chaotropic agents, and / or is approximately spherical in shape.

8. 8. A cage according to any one of claims 1 to 7 for use as a medicine or for treating a disease in a patient.

9. The use of any one or more of the groups comprising homobifunctional molecular moieties, bis-halomethylbenzenes, and derivatives thereof, as programmable cross-linkers in the construction of programmable TRAP cages.

10. (a) obtaining a TRAP ring unit by expression of the TRAP ring unit in a suitable expression system and purification of said unit from the expression system; (b) conjugation of the TRAP ring unit via at least one free thiol linkage with a programmable homobifunctional molecular crosslinker, where the crosslinker is selected for its specific characteristics; (c) formation of a TRAP cage; and (d) Purification and isolation of TRAP cages A method for preparing an artificial TRAP cage, comprising:

11. The method of claim 10, wherein the programmable crosslinker is selected from the group comprising: (i) a reduction-resistant / insensitive linker, whereby the cage remains closed under reducing conditions; (ii) a reduction-responsive / sensitive linker, whereby the cage opens under reducing conditions; and (iii) a photoactivatable linker, whereby the cage opens upon exposure to light.

12. 12. The method of claim 10 or 11, wherein step (b) first comprises conjugating the TRAP ring units via at least one metal crosslinker, optionally an atomic metal crosslinker, and then replacing the metal crosslinker with a programmable homobifunctional molecular crosslinker.

13. 13. The method of any one of claims 10 to 12, wherein step (b) comprises conjugation with a mixture of different programmable molecular crosslinkers.

14. A TRAP cage produced by the method of any one of claims 10 to 13.

15. An artificial TRAP cage comprising a protein modified to include any one or more of the mutations selected from the group including K35C, K35H, R64S, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C, and S33H / K35C.

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