Bioluminescence-induced photocatalytic activation

Abstract

This specification provides systems, methods, and compositions for bioluminescence-induced photocatalytic activation of molecular entities in a proximity-dependent manner, which can be operated within biological systems. In particular, this specification provides activatable molecular entities, systems thereof, including bioluminescent proteins or complexes, their luminescent substrates, photocatalysts, and activatable molecular entities, which incorporate photoresponsive moieties that limit their activity, as well as methods for catalytically activating activatable molecular entities via bioluminescence-induced catalysis.

Description

[Technical Field]

[0001] Cross-reference of related applications This invention claims priority under U.S. Provisional Patent Application No. 63 / 492,061, filed on 24 March 2023, which is incorporated herein by reference in its entirety.

[0002] This specification provides systems, methods, and compositions for bioluminescence-induced photocatalytic activation of molecular entities in a proximity-dependent manner, which can be operated within biological systems. In particular, this specification provides activatable molecular entities, systems thereof, including bioluminescent proteins or complexes, their luminescent substrates, photocatalysts, and activatable molecular entities, which incorporate photoresponsive moieties or conformations that limit their activity, as well as methods for catalytically activating activatable molecular entities via bioluminescence-induced catalysis. [Background technology]

[0003] The need to study dynamic microenvironments, signaling pathways, and molecular processes in physiologically relevant contexts has created a demand for new functional biological tools that enable such analyses non-destructively in living cells and complex models. [Overview of the project]

[0004] This specification provides systems, methods, and compositions for bioluminescence-induced photocatalytic activation of molecular entities in a proximity-dependent manner, which can be operated within biological systems. In particular, this specification provides activatable molecular entities, systems thereof, including bioluminescent proteins or complexes, their luminescent substrates, photocatalysts, and activatable molecular entities, which incorporate photoresponsive moieties that limit their activity, as well as methods for catalytically activating activatable molecular entities via bioluminescence-induced catalysis.

[0005] In some embodiments, photocatalysts are provided herein that utilize bioluminescence as a light source to activate activatable molecules (e.g., molecules incorporating photosensitive moieties that limit their activity, photoswitchable molecules capable of photo-driven conformational changes from inactive to active conformations). Such molecules include molecules having photodissociable protecting groups (photocages), photoswitchable molecules (photoswitches), and the like. In some embodiments, the activated molecules can be used for subsequent interaction with and / or detection of target molecules (e.g., biomacromolecules). However, while the activatable molecules are activated by the systems and methods described herein, the activated molecules do not contain highly reactive and / or short-lived functional groups (e.g., for labeling biomolecules). Alternatively, exposure to light emitted from a luminescent phosphatid / bioluminescent protein or complex may (1) facilitate the removal of blocking portions from the caged entity, thereby allowing the molecular entity to participate in its chemical / biological function, or (2) cause a conformational change in the molecular entity, causing portions within the molecular entity to be re-established in an activated state (e.g., allowing those portions to interact).

[0006] The components of bioluminescent-driven photocatalytic systems described herein include a bioluminescent light source (e.g., a luminescent phore and luciferase or bioluminescent complex), and a pair comprising a photosensitive catalyst (photocatalyst) and an activatable molecular entity (e.g., a caged molecule, a photoswitchable molecule). In some embodiments, upon exposure to a photostimulus from a bioluminescent light source, the excited catalyst engages in the activation of adjacent activatable molecules for subsequent interactions, detection, etc., within the surrounding environment. Activation of the catalyst by absorption of visible light results in temporal control of catalytic reactivity. The use of bioluminescence to induce photocatalysis in a proximity-dependent manner (e.g., requiring localization of the bioluminescent light source and photocatalyst) results in a mild, minimally destructive light source, reduced phototoxicity, and efficient light delivery for inducing catalysis in intact cells, as well as spatial and temporal (+luminescent phore) control over catalytic activation, thereby increasing the overall spatiotemporal resolution of downstream molecular activation. In some embodiments, the bioluminescent light source and photocatalyst are bioconjugated to induce proximity between the light source and the catalyst.

[0007] One aspect of this technology involves the use of bioluminescence, i.e., light produced by the interaction of a bioluminescent protein or peptide(s) and / or polypeptide complex with a luminescent phoset, to activate a photocatalyst. Another aspect of this technology involves the activation of activatable molecules by a bioluminescent-activated photocatalyst, and increases specificity and reduces toxicity by assembling the components of a bioluminescent-driven system / method using a conjugate of one or more components of the system herein that drives intracellular photocatalytic activation using spatiotemporally arranged components (e.g., via protein fusions, scavengers / elements, linkers, etc.).

[0008] In one exemplary embodiment, exposure of a bioluminescent protein to a suitable luminescent photic generates light that induces localized photocatalytic activation of the molecular entity (e.g., decaching via bond cleavage and removal of protecting groups, or conformational changes of photoswitchable molecules). The activated molecule is then available for interaction with and / or detection of biomacromolecules in its surrounding environment. Such activated molecules can be utilized for a wide range of spatiotemporalally controlled phenotypic, proteomic, and genomic analyses, including detection, activation, inactivation, and degradation of proximal proteins and nucleic acids, as well as exploration and modification of biological processes.

[0009] In some embodiments, an appropriate proximity between the bioluminescent protein and the photocatalyst is achieved by linking the photocatalyst to the bioluminescent protein (directly or indirectly). In certain embodiments, the bioluminescent protein is prepared as a fusion with a capture agent (e.g., a capture protein), and the photocatalyst is conjugated to a capture element (e.g., via a linker). The binding of the capture agent to the capture element brings the bioluminescent protein and photocatalyst into close proximity, allowing the light generated by the bioluminescent protein and luminescent phosphatid to activate the photocatalyst.

[0010] In some embodiments, instead of using bioluminescent proteins, multi-component bioluminescent complexes can be used as light sources for the photocatalytic systems or methods herein. The use of bioluminescent complexes that generate light upon complementation of two or more components (e.g., peptides and / or polypeptides) offers several advantages to some systems and methods herein. For example, by directly or indirectly conjugating (e.g., fusion, linking, etc.) one or more components of the bioluminescent complex to other components of the system (e.g., photocatalysts, activatable molecules, targets, etc.), it is ensured that these components are in close proximity to the bioluminescent complex during photogeneration. By linking two components of the system to separate components of the bioluminescent complex, it is ensured that these components are in close proximity during photogeneration by the complex. When a photocatalyst is linked to a first component of a bioluminescent complex (e.g., LgBiT or circularly permuted LgBiT (see, for example, U.S. Patent Application No. 17 / 105,925; the whole is incorporated by reference)), and the first component has a high affinity for a second component of the bioluminescent complex (e.g., HiBiT), and the second component is genetically fused to the target of interest, then proximity between the photocatalyst and the second component of the bioluminescent complex is required for the initiation of photocatalytic activity, thereby providing greater spatiotemporal control over photocatalytic activation and a modality-independent approach to targeting the photocatalytic system to the desired site (i.e., complementation and addition of luminescent phospholipids).

[0011] In some embodiments, the bioluminescent protein or components of the multi-element bioluminescent complex are inserted into internal positions within the capture agent. In some embodiments, the position within the capture agent is selected to enhance the efficiency of bioluminescent activation of the catalyst by providing greater proximity or a preferred conformation.

[0012] In some embodiments, the components of the bioluminescent protein or multi-elemental bioluminescent complex are cyclically replaced.

[0013] Since bioluminescent proteins, or components of a bioluminescent complex (or its fusion with other components of the system herein), can be expressed in cells or delivered into cells, such systems provide the generation of light for initiating photocatalytic activity within cells.

[0014] In some embodiments, components of the system herein (e.g., bioluminescent proteins, or components of bioluminescent complexes) are fused to proteins / peptides that result in the specific localization of the components within the cell. For example, the localized protein / peptide may localize within a cellular compartment, bind to a specific protein, bind to DNA or RNA, or bind to a specific nucleic acid sequence. The localization of the components within the cell, or the binding of the components to a specific cellular component, similarly localizes the subsequent photocatalytic activation chemistry (e.g., cage decaching via bond cleavage, removal of protecting groups, or conformational change of a photoswitch). In some embodiments, by localizing the system to a specific cellular target (e.g., a protein, nucleic acid sequence, etc.), the activated molecule can interact with or act on the cellular target.

[0015] The systems and methods described herein provide bioluminescence-induced catalytic activation of molecular entities (e.g., caged molecules, photoswitchable molecules, etc.) in a proximity-dependent manner, offering novel functional biological tools for studying dynamic environments and molecular processes in physiologically relevant contexts, including living cells, complex cell models, and model organisms. The techniques described herein utilize non-invasive, intrinsic light sources to activate photosensitive catalysts, which can further participate in the localized activation of molecular entities that can interact with the environment, be detected, and interact with adjacent molecules / biomolecules. The systems described herein can be utilized for a wide range of spatiotemporally controlled phenotypic, proteomic, and genomic analyses. [Brief explanation of the drawing]

[0016] [Figure 1] This is a diagram illustrating a bioluminescence-induced photocatalytic system utilizing bioluminescent proteins (NanoLuc) and photosensitive photocatalysts. The photosensitive photocatalyst, upon absorbing light, is involved in the activation of caged or photoswitchable molecules, which can then participate in interactions with biomacromolecules in their surrounding environment. [Figure 2] A-C. Exemplary structures of activatable molecules: (A) Caged molecules that are de-caged via photo-induced photocatalytic cleavage of a photodissociable protecting group, (B) Caged molecules that are de-caged via photo-induced photocatalytic abstraction (oxidation) of hydrogen, (C) Photoswitchable molecules that can undergo conformational changes. [Figure 3] Illustration of a photosensitive catalyst modified to enable bioconjugation and subsequent access to a bioluminescent light source. Exemplary catalysts include iridium-based catalysts, ruthenium-based catalysts, and rose bengal (organic photosensitizer). R represents the binding motif, and linker Q represents the bioconjugation motif. Exemplary bioconjugation motifs include 2-pyridinecarboxaldehyde (PCA) and 2-cyanobenzothiazole (CBT) linkers for direct bioconjugation, and chloroalkanes for indirect conjugation via binding to a HaloTag fusion. [Figure 4] An exemplary linker Q designed for indirect conjugation via binding to a HaloTag fusion. Haloalkanes of various lengths are designed for attachment to components of the system described herein (e.g., attachment to a photocatalyst). [Figure 5] Exemplary photocatalyst molecular structure bound to a HALOTAG substrate. [Figure 6](A) Diagram of a system for localizing a haloalkane-binding photocatalyst, added extracellularly or assembled intracellularly, into a cell along with a bioluminescent complex component (LgBiT) genetically fused to a modified dehalogenase (HALOTAG). (B) Fluorescence experiment demonstrating the intracellular localization of LgBiT-HaloTag fusions labeled with a fluorescent haloalkane ligand. (C) Experiment demonstrating the binding reaction rate of an extracellularly added haloalkane-binding Ir photocatalyst to LgBiT-HaloTag fusions localized to different intracellular compartments. The results show complete binding within 60 minutes. [Figure 7] A diagram illustrating a system that enables bioluminescence-induced spatiotemporal decaching of caged effector molecules in intact cells. The complementarity of HiBiT, genetically fused to the target protein, and LgBiT, genetically fused to a HaloTag and linked to a catalyst, allows for the localization of the target catalyst, light source, and protein, enabling localized photocatalytic decaching of the caged effector molecule for subsequent manipulation of the target site. [Figure 8] A diagram illustrating a system for bioluminescence-induced spatiotemporal turn-on of photoswitchable molecules in intact cells. Complementary HiBiT genetically fused to the target protein and LgBiT genetically fused to a HaloTag and linked to a catalyst enables localization of the target catalyst, light source, and protein, allowing for localized, reversible conformational switching and turn-on of the activity of effector molecules against the target. [Figure 9]A-C. (A) Exemplary activatable molecules containing azide quenching fluorescent dyes. (B) Illustration of a system for bioluminescence-induced spatiotemporal turn-on fluorescence. Complementarity between HiBiT genetically fused to the target protein and LgBiT genetically fused to the HaloTag and linked to the catalyst enables localization of the target catalyst, light source, and site. (C) Illustration of a system for bioluminescence-induced spatiotemporal fluorescence turn-on in the nucleus. Electroporation of a nuclear protein complex containing a dCas9-NanoLuc-HaloTag fusion linked to gRNA and the catalyst enables localization of the target catalyst, light source, and site. [Figure 10] (A) Illustration of a system enabling bioluminescence-induced spatiotemporal molecular cage decaching of caged fluorophores for subsequent detection of nucleic acids in intact cells. Caged fluorophores conjugated to antisense oligonucleotides are targeted to specific nucleic acids. The photocatalytic system is localized to the proximal nucleic acid sequence by either (B) gRNA conjugated to a dCas9-NanoLuc-HaloTag fusion linked to the catalyst, or (C) an antisense oligonucleotide conjugated to HiBiT. Complementarity with LgBiT, which is genetically fused to the HaloTag and linked to the catalyst, enables the localization of the desired catalyst, light source, and nucleic acid. Upon treatment with flimazine, the bioluminescent complex emits light, which induces photocatalytic cage decaching of the proximal fluorophores. [Figure 11] A-D. Effects of chloroalkane length on catalyst energy transfer efficiency, cell permeability, and HaloTag binding reaction rate. (A) Structures of modifiable Ir catalysts and their derivatives further conjugated with chloroalkanes of different lengths. (B) Physiological properties of Ir catalyst conjugates and their effects on the energy transfer efficiency from NanoLuc to Ir catalyst. (C) Effects of chloroalkane length on the HaloTag binding reaction rate of chloroalkane-catalyst conjugates in either cell lysates or (D) inside living cells. [Figure 12]A-D. Effects of chloroalkane length on catalyst energy transfer efficiency, cell permeability, and HaloTag binding reaction rate. (A) Structures of modifiable Ru catalysts and their derivatives further conjugated with chloroalkanes of different lengths. (B) Physiological properties of Ru catalyst conjugates and their effects on the energy transfer efficiency from NanoLuc to Ru catalyst. (C) Effects of chloroalkane length on the HaloTag binding reaction rate of chloroalkane-catalyst conjugates in living cells, either in the cytosol or (D) the nucleus. [Figure 13] A-C. Optimization of bioluminescent photocatalytic complexes comprising a bioluminescent energy donor, a chloroalkane-catalyst conjugate, and a HaloTag providing means for inducing proximity between the two. (A) Schemes of HT178-cpNLuc-179 chimeras or complementary-based HT178-cpmLgBiT-179 chimeras comprising a cyclically substituted NanoLuc (i.e., cpNLuc) or a cyclically substituted mutant LgBiT (i.e., cpmLgBiT) inserted into a surface loop (between residues 178-179) of the HaloTag adjacent to the ligand interaction site. (B) NanoLuc-HaloTag fusions and chimeras complemented by HiBiT peptides, and (C) LgBiT-HaloTag fusions and chimeras for BRET efficiency to the bound HaloTag TMR-fluorescent ligand. [Figure 14] A-D Bioluminescence-induced cage release of azide-quenched coumarin. (A) Structure of aminocaged coumarin PBI-8977. (B) Fluorescence imaging of HeLa cells expressing the HT178-cpNLuc-179 chimera and treated with increasing concentrations of PBI-8977 and fluoroflimazine in the presence and absence of Ru-8974. (C) Image density measurement showing catalyst-dependent turn-on fluorescence. [Figure 15]A-D Bioluminescence-induced cage release of azido-quenched ethidium bromide (EMA). (A) Structure of EMA. (B) Fluorescence imaging of HeLa cells expressing the HT178-cpNLuc-179 chimera and treated with increasing concentrations of EMA and fluoroflimazine in the presence and absence of either Ru-8974 or Ir-9049. (C) Image density measurements showing catalyst-dependent turn-on fluorescence. [Figure 16] A-B Bioluminescent-induced release of signaling molecules from caging transition metal complexes in biochemical settings. (A) Illustration illustrating bioluminescent-induced release of serotonin from the [Ru2+(bpy)2]2 caging complex. (B) Fluorescence scan of caged or 45-minute-exposed [Ru(bpy)2(PMe3)(5HT)]2+. [Figure 17] A-C. Bioluminescence-induced cage release during Ru-catalyzed p-azidobenzyl reduction. (A) Illustration illustrating the bioluminescence-induced photocatalytic cleavage of p-azidobenzyl-luciferin during Ru-catalyzed azido reduction that releases luciferin. (B) Firefly (FFLY) luminescence during incubation of p-azidobenzyl-luciferin pre-incubated with Ru-8974-bound and unbound purified HT178-cpNLuc-179 in the presence and absence of fluoroflimazine. (C) Increased magnification of FFLY luminescence during bioluminescence or LED-induced photocatalytic cage release of p-azidobenzyl-luciferin. [Figure 18] A. Catalytic excitation of o-nitrobenzyl and subsequent bioluminescence-induced cage release during photolysis. (A) Illustration illustrating bioluminescence-induced photocatalytic cleavage of o-nitrobenzyl-F-luciferin releasing F-luciferin. (B) FFLY emission during incubation of purified HT178-cpNLuc-179 pre-incubated with Ru-8974 in and out of fluoroflimazine, and (C) magnification of FFLY emission during bioluminescence or LED-induced photocatalytic cage release of o-nitrobenzyl-F-luciferin. [Figure 19]Bioluminescence-induced photocatalytic cage release upon excitation of coumarin derivatives. Illustrated diagram illustrating bioluminescence-induced photocatalytic photodegradation of coumarin-4-methyl upon excitation, releasing ibrutinib from 6-bromo-7-hydroxycoumarin-4-methyl-ibrutinib. [Figure 20] Bioluminescence-induced isomerization of azobenzene. This illustration illustrates the bioluminescence-induced catalytic photoisomerization of azobenzene, where, upon excitation, azobenzene possesses a significantly lower energy barrier to rotation around the N=N double bond, allowing for conformational changes that affect either the distance and / or orientation of R1 and R2 relative to each other. Such spatiotemporally controlled conformational changes can be used to dramatically increase the affinity of effector molecules to biomolecular targets.

[0017] definition While any methods and materials similar to or equivalent to those described herein may be used when implementing or testing the embodiments described herein, several preferred methods, compositions, apparatus, and materials are described herein. However, before describing the materials and methods, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or procedures described herein, as specific molecules, compositions, methodologies, or procedures may change according to routine experimentation and optimization. It should also be understood that the terms used in the description are intended solely to describe specific versions or embodiments and are not intended to limit the scope of the embodiments described herein.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of any conflict, however, the definitions provided herein shall prevail. Accordingly, in the context of the embodiments described herein, the following definitions apply:

[0019] In this specification and in the accompanying claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context explicitly indicates otherwise. For example, "a peptide" refers to one or more peptides and their equivalents known to those skilled in the art.

[0020] As used herein, the term "and / or" includes any combination of the listed items, including any of the individually listed items. For example, "A, B, and / or C" encompasses A, B, C, AB, AC, BC, and ABC, each of which should be considered separately described by stating "A, B, and / or C".

[0021] Where used herein, the term “contains” and its linguistic variations indicate the presence of the described features, elements, or steps of a method, etc., without excluding the presence of additional features, elements, or steps of a method, etc. In contrast, the term “consisting of” and its linguistic variations indicate the presence of the described features, elements, or steps of a method, etc., and exclude any undescribed features, elements, or steps of a method, etc., except for impurities that are usually present. The phrase “consisting essentially of” indicates any further features, elements, or steps of a method, etc., that do not substantially affect the basic properties of the described features, elements, or steps of a method, etc., as well as any further features, elements, or steps of a method, etc., that do not substantially affect the basic properties of the composition, system, or method. Many of the embodiments described herein are described using the open term “contains.” Such embodiments include a plurality of closed "consisting of" and / or "essentially consisting of" embodiments, which may be claimed or described using such alternative language.

[0022] As used herein, the term “system” refers to a group of devices, reagents, compositions, etc., that are collectively grouped together for a desired function or purpose. The components of a system may reside in a single reaction mixture, cell, container, etc., or they may be maintained separately, for example, for later combination to achieve a desired function or purpose.

[0023] Where used herein, the term “substantially” means that the described properties, parameters, and / or values ​​do not need to be exactly realized, but deviations or deviations, including, for example, tolerances, measurement errors, measurement accuracy limits, and other factors known in the art, may occur in amounts that do not interfere with the effect of the property intended to be provided. A substantially non-existent (e.g., substantially non-luminescent) property or feature may be in noise, in the background, below the detection capability of the assay used, or a fraction of a significant property (e.g., the luminescence intensity of a bioluminescent protein or bioluminescent complex) (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%, <0.0000001%).

[0024] As used herein, the term “luminescence” refers to the emission of light by a substance as a result of a chemical reaction ("chemiluminescence") or an enzymatic reaction ("bioluminescence").

[0025] As used herein, the term “bioluminescence” refers to the generation and emission of light by reactions catalyzed or enabled by enzymes, proteins, protein complexes, or other biomolecules (e.g., bioluminescent complexes). In a typical embodiment, a substrate for a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted into an unstable form by the bioluminescent entity; the substrate then emits light.

[0026] As used herein, the term “luminescent photic” refers to a chemical moiety or compound that can be placed into an excited electronic state (for example, by a chemical or enzymatic reaction) and emit light when returning to its electronic ground state.

[0027] As used herein, the term “imidazopyrazine luminescent phosphophore” means, in addition to what is disclosed in WO2003 / 040100, U.S. Patent No. 12 / 056,073 (paragraph

[0086] ), U.S. Patent No. 8,669,103, and U.S. Provisional Application No. 63 / 379,573 (their disclosures are incorporated herein by reference in their entirety), “natural coelenterazine,” as well as its synthesis (e.g., derivatives or variants) and natural analogues (frimagin). This refers to a genus of luminescent phosphophosphores that include flimazine analogs (e.g., fluoroflimazine), including coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine.

[0028] As used herein, the term “coelenterazine” refers to a naturally occurring ("natural") imidazopyrazine having the following structure: [ka]

[0029] As used herein, the term "flimazine" refers to a coelenterazine derivative having the following structure: [ka]

[0030] As used herein, the term "fluoroflimazine" refers to a flimazine derivative having the following structure: [ka] (U.S. Patent Application No. 16 / 548,214, incorporated in its entirety by reference).

[0031] As used herein, the term "luciferin" refers to a compound having the following structure: [ka]

[0032] As used herein, the term “bioluminescent resonance energy transfer” (“BRET”) refers to a distance-dependent interaction in which energy is transferred from a donor bioluminescent protein / complex and substrate to an acceptor molecule without the emission of photons. The efficiency of BRET depends on the inverse sixth power of the intermolecular distance and is useful over distances equivalent to the dimensions of the biomolecule (e.g., within 30–80 Å depending on the degree of spectral overlap).

[0033] As used herein, the term “Oplophorus luciferase” (“OgLuc”) refers to a luminescent polypeptide having significant sequence identity, structural conservation, and / or functional activity of the luciferase produced by and derived from the deep-sea shrimp Opplophorus gracilirostris. Specifically, an OgLuc polypeptide refers to a luminescent polypeptide having significant sequence identity, structural conservation, and / or functional activity of the 19 kDa mature subunit of the Opplophorus luciferase protein complex, such as SEQ ID NO: 1 (NANOLUC), comprising 10 β-chains (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10), and producing luminescence using a substrate such as coelenterazine or a derivative or analog of coelenterazine.

[0034] As used herein, the term “complementary” refers to the properties of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that can hybridize with each other, dimerize, or otherwise form a complex. For example, “complementary peptides and polypeptides” can come together to form a single complex. Complementary elements may require assistance (facilitation) to form a complex (e.g., from the interacting elements), such as placing the element in the appropriate conformation for complementarity, jointly determining the position of the complementary elements, lowering the interaction energy for complementarity, and overcoming low affinity for each other.

[0035] As used herein, the term “complex” refers to an assembly or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with each other. In one embodiment, “in contact” or more specifically “direct contact” means that two or more molecules are close enough that attractive non-covalent interactions, such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions, dominate the interaction of the molecules. In such embodiments, a complex of molecules (e.g., peptides and polypeptides) is formed under assay conditions such that the complex is thermodynamically favorable (e.g., compared to the non-aggregated or non-complexed states of its constituent molecules). As used herein, the term “complex” refers to an assembly of two or more molecules (e.g., peptides, polypeptides, or combinations thereof) unless otherwise specified.

[0036] As used herein, the terms “capture protein” or “capture agent” mean a protein or other molecular element that forms a stable covalent bond with its substrate, ligand, or other molecule upon interaction. A capture protein may be a receptor that forms a covalent bond with its ligand, or an enzyme that forms a covalent bond with its substrate. An example of a capture protein suitable for use in embodiments of the present invention is the HALOTAG protein described in U.S. Patent No. 7,425,436 (which is incorporated herein by reference in its entirety).

[0037] As used herein, the terms “capture ligand,” “capture moiety,” or “capture element” refer to ligands, substrates, etc., that form a covalent bond with a capture protein during interaction with the capture protein. An example of a capture ligand suitable for use in embodiments of the present invention is the HALOTAG ligand, described, for example, in U.S. Patent No. 7,425,436 (which is incorporated herein by reference in its entirety). The moiety found to be suitable for use as a HALOTAG ligand is the haloalkane (HA) group (e.g., the chloroalkane (CA) group). In embodiments described herein that specify an HA or CA capture ligand, other suitable capture ligands may be substituted unless otherwise specified.

[0038] As used herein, the term “activatable molecule” refers to a molecule that can be converted by a catalyst from an activatable form (e.g., an inactive or inert form by a blocking group or inert conformation of the “cage”) to an activated form (e.g., a non-blocking, decaking, or activated conformer). In some embodiments, the activatable molecule incorporates a photoresponsive moiety that limits its activity.

[0039] As used herein, the term “caged molecule” refers to a molecule that has been made inactive (e.g., chemically inactive, biologically inactive, undetectable, etc.) by chemical modification (e.g., blocking group) so that the molecule is structurally or sterically non-functional. The conversion of a caged molecule to an uncaged activated molecule involves photocatalytic uncaged of the activatable molecule (e.g., cleavage of blocking groups) and the release of the active form from the molecule (e.g., capable of interacting with a binding partner, detectable, etc.).

[0040] As used herein, the terms “photoswitch” or “photoswitchable molecule” refer to a compound that can assume both active and inactive (or activatable) conformations. A conformational change, rather than a chemical change, results in the photo-switching of the molecule from inactive to active. A reversible change in structural geometry, initiated by bioluminescence-induced photocatalysis, activates the molecule.

[0041] As used herein, the term “cell target” refers to a cellular entity (e.g., intracellular or surface-exposed) that can interact with an activatable molecule (e.g., a caged molecule, a photoswitch) (e.g., a molecule, a cellular compartment, a complex, etc.). Cell targets can be biomacromolecules such as proteins, polypeptides, nucleic acids (e.g., DNA or RNA), lipids, polysaccharides, or complexes with polypeptides containing any of these. A cell target may consist of multiple components, subunits, or polypeptides; for example, a cell target is a protein complex. Examples of cell targets may include receptors or enzymes.

[0042] As used herein, the term “bioactive agent” generally means a substance that is physiologically or pharmacologically active, or suitable for detection. In some embodiments, the bioactive agent is a potentially therapeutic compound (e.g., small molecules, peptides, nucleic acids, etc.) or a drug-like molecule. The bioactive agents for use in the embodiments described herein are not limited by size or structure.

[0043] As used herein, the term “photocatalyst” refers to a molecule that, upon absorbing light of a suitable wavelength, is capable of activating an adjacent activatable molecule(s) through either an energy transfer or electron transfer event, thereby converting the activatable molecule(s) to an activated state and / or reducing its activation energy and / or increasing the rate of a chemical reaction. In some embodiments, the excited photocatalyst may regenerate itself after each energy transfer or electron transfer event, thereby repeatedly participating in the activation of an adjacent activatable molecule. In some embodiments, a photocatalyst that, upon absorbing light of a suitable wavelength, is capable of participating in an energy transfer event with oxygen to generate a reactive species (e.g., proton, singlet oxygen, etc.) is referred to as a “photosensitizer.” Some embodiments of this specification described in conjunction with photocatalysts may encompass or be limited to photosensitizers.

[0044] As used herein, the term “low molecular weight” refers to organic compounds with low molecular weight (e.g., <2000 daltons, <1000 daltons, <500 daltons) and dimensions on the order of 1 nm (e.g., length, width, diameter, etc.). Larger structures such as peptides, proteins, and nucleic acids are not low molecular weight, although the monomers that make them up (ribo or deoxyribonucleotides, amino acids, etc.) are considered low molecular weight.

[0045] As used herein, the term "cell permeable" refers to a compound or portion that can effectively pass through a cell membrane that has not been synthetically permeableized.

[0046] Definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are as defined in Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 thSpecified according to the Ed. (endpaper), specific functional groups are generally defined as described therein. In addition, for general principles of organic chemistry, as well as specific functional parts and reactivity, see Sorrell, Organic Chemistry, 2 nd edition,University Science Books,Sausalito,2006;Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition,John Wiley & Sons,Inc.,New York,2013;Larock,Comprehensive Organic Transformations,3 rd Edition,John Wiley & Sons,Inc.,New York,2018;Carruthers,Some Modern Methods of Organic Synthesis,3 rd This information is provided in Edition, Cambridge University Press, Cambridge, 1987, and the entirety of each of those contents is incorporated herein by reference.

[0047] As used herein, the term “physiological conditions” encompasses any conditions suitable for living cells, such as aqueous conditions including temperature, pH, salinity, and chemical composition, which are primarily suitable for living cells.

[0048] As used herein, the terms “conjugated” and “conjugation” refer to a covalent bond between two molecular entities (e.g., post-synthesis and / or during synthesis). The conjugated entities may be peptides or proteins “fused” by a peptide bond, or may include other molecular entities (e.g., nucleic acids, small molecules, etc.) linked directly or by a suitable linker.

[0049] The term "binding domain" refers to a domain that specifically binds to a given antigen or epitope, independently of other epitope or antigen-binding domains. Examples of binding domains include antibodies, antibody fragments, receptor domains that bind to target ligands, proteins that bind to immunoglobulins (e.g., protein A, protein G, protein A / G, protein L, protein M), binding domains of immunoglobulin-binding proteins (e.g., protein A, protein G, protein A / G, protein L, protein M), oligonucleotide probes, peptide nucleic acids, DARPin, antikalin, nanobodies, aptamers, affimers, purified proteins (the analyte itself or proteins that bind to the analyte), and analyte-binding domains of proteins. Table A provides a list of exemplary binding domains that may be used individually or in various combinations in the methods, systems, and assays (e.g., immunoassays) described herein. [Table 1]

[0050] As used herein, the term “antibody” refers to the entire antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab', and F(ab')2, variable light chain, variable heavy chain, Fv, etc.). Antibodies may be polyclonal or monoclonal or recombinant antibodies, chimeric antibodies, humanized antibodies, human antibodies, etc. As used herein, when an antibody or other entity “specifically recognizes” or “specifically binds” to an antigen or epitope, the antibody or other entity preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds to the antigen or epitope with substantially higher affinity than other entities that do not present the antigen or epitope. In this context, “substantially higher affinity” means an affinity high enough to enable the detection of the antigen or epitope, distinguishable from the entity using the desired assay or measuring instrument. Typically, it is expressed as binding affinity, with a binding constant (K a ) but at least 10 7 M -1 (For example, >107 M -1 、 > 10 8 M -1 、 > 10 9 M -1 、 > 10 10 M -1 、 > 10 11 M -1 、 > 10 12 M -1 、 > 10 13 M -1 and so on) is meant. In certain such embodiments, an antibody can bind to different antigens as long as the different antigens contain their specific epitopes. In certain cases, for example, homologous proteins from different species may contain the same epitope.

[0051] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that includes at least a portion of the antigen-binding region or variable region. Antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, scFv, Fd, variable light chain, variable heavy chain, diabody, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, for example, Hudson et al. (2003) Nat. Med. 9:129-134 (which is incorporated herein by reference in its entirety). In certain embodiments, antibody fragments are generated by enzymatic or chemical cleavage of intact antibodies (e.g., papain and pepsin digestion of antibodies) produced by recombinant DNA techniques or chemical polypeptide synthesis. For example, a "Fab" fragment includes one light chain and the C H1 and the variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab’" fragment includes one light chain, and C H1 domain and C H2It contains one heavy chain including an additional constant region extending between the domains. Interchain disulfide bonds can be formed between the two heavy chains of the Fab' fragment to form the "F(ab')2" molecule. The "Fv" fragment contains variable regions from both the heavy and light chains but lacks a constant region. The single-chain Fv(scFv) fragment contains heavy and light chain variable regions linked by a flexible linker, forming a single polypeptide chain together with the antigen-binding region. Exemplary single-chain antibodies are discussed in detail in WO88 / 01649 and U.S. Patents 4,946,778 and 5,260,203 (these are incorporated herein by reference in their entirety). In certain cases, a single variable region (e.g., a heavy chain variable region or a light chain variable region) may have the ability to recognize and bind an antigen. Other antibody fragments will be understood by those skilled in the art.

[0052] As used herein, the terms “biomolecules” or “biological molecules” mean molecules and ions present in living organisms that are essential for biological processes such as cell division, morphogenesis, or development. Biomolecules include large macromolecules (or polyanions) such as proteins, carbohydrates, lipids, and nucleic acids, as well as smaller molecules such as primary metabolites, secondary metabolites, and natural products. A more general name for this class of substances is biomolecules. Biomolecules are usually endogenous, but they can also be exogenous. For example, drugs can be naturally occurring products, semi-synthetic (biopharmaceuticals), or fully synthetic.

[0053] As used herein, the term "alkyl" refers to a group of 1 to 30 carbon atoms, for example, 1 to 16 carbon atoms (C1-C1). 16 Alkyl), 1 to 14 carbon atoms (C1-C 14 Alkyl), 1 to 12 carbon atoms (C1-C 12 Alkyl), 1 to 10 carbon atoms (C1-C 10 Alkyl), 1-8 carbon atoms (C1-C8 alkyl), 1-6 carbon atoms (C1-C6 alkyl), 1-4 carbon atoms (C1-C4 alkyl), 6-20 carbon atoms (C6-C20 Alkyl, or 8-14 carbon atoms (C8-C 14 This refers to a linear or branched saturated hydrocarbon chain containing alkyl. Typical examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0054] As used herein, the term "amino" means the -NH2 group.

[0055] As used herein, the term “haloalkyl” means an alkyl group in which at least one hydrogen atom (e.g., one, two, three, four, five, six, seven, or eight hydrogen atoms) is substituted with a halogen, as defined herein.

[0056] As used herein, the term “heteroalkyl” means an alkyl group in which one or more carbon atoms (and any associated hydrogen atoms) are independently substituted with a heteroatomic group such as -NR-, -O-, -S-, -S(O)-, -S(O)2-, etc., as defined herein, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclyl, each of which may be optionally substituted. For example, one, two, or three carbon atoms may be independently substituted with the same or different heteroatomic groups. Examples of heteroalkyl groups include, but are not limited to, -OCH3, -CH2OCH3, -SCH3, -CH2SCH3, -NRCH3, and -CH2NRCH3, where R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which may be optionally substituted. Heteroalkyl groups also include groups in which the alkyl carbon atom is oxidized (i.e., -C(O)-). [Modes for carrying out the invention]

[0057] This specification provides systems, methods, and compositions for bioluminescence-induced photocatalytic activation of molecular entities in a proximity-dependent manner, which can be operated within biological systems. In particular, this specification provides activatable molecular entities, systems thereof, including bioluminescent proteins or complexes, their luminescent substrates, photocatalysts, and activatable molecular entities, which incorporate photoresponsive moieties that limit their activity, as well as methods for catalytically activating activatable molecular entities via bioluminescence-induced catalysis.

[0058] The need to study dynamic microenvironments, signaling pathways, and molecular processes in physiologically relevant contexts necessitates novel functional biological tools that enable such analyses non-destructively in living cells and complex models. Proximity-dependent bioluminescence-induced catalytic activation of molecular entities provides a solution to this need by utilizing a non-invasive, intrinsic light source to activate molecular entities (e.g., molecules incorporating photosensitive moieties that limit their activity, including photodissociable protecting groups (photocages), photoswitches, etc.) in biological systems for subsequent activity (e.g., detection, interaction with biomacromolecules, etc.). Components of such photocatalytic systems include a bioluminescent light source (e.g., luciferase (e.g., NanoLuc) or bioluminescent complex (e.g., NanoBiT, NanoTrip, etc.)) and a pair of (1) a photosensitive catalyst (transition metal or organic dye catalyst) and (2) an activatable molecule. When a luminescent substrate is added, the bioluminescent entity (e.g., NanoBiT, NanoTrip, NanoLuc, etc.) generates light that induces localized photocatalytic activation of activatable molecules. These activated molecules can be utilized for a wide range of spatiotemporalally controlled phenotypic, proteomic, and genomic analyses, including the detection, activation, inactivation, and degradation of proximal proteins and nucleic acids, as well as the exploration and modification of biological processes.

[0059] The advantages of using bioluminescence as a light source rather than total light emission (e.g., LED or laser) include the use of a mild, minimally destructive endogenous light source, reduced phototoxicity, efficient light delivery to induce catalysis in intact cells and complex models, local and conditional (+substrate) light delivery for higher spatiotemporal resolution to catalytic activation and downstream chemistry, and the ability to link the light source to target molecules and / or other components of the system (e.g., photocatalysts).

[0060] In some embodiments, the present invention provides a system comprising one or more of the following: a structurally complementary component of a bioluminescent protein or bioluminescent complex; a luminescent phose (the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phose through interaction with the luminescent phose); a photocatalyst (the photocatalyst is activated upon absorption of light of a first wavelength); and an activatable molecule (the activatable molecule is converted into an activating molecule upon proximity to an activated photocatalyst).

[0061] In some embodiments, components of a bioluminescent protein or bioluminescent complex are linked to a photocatalyst. In some embodiments, linking the photocatalyst to a light source provides adequate proximity for activating the photocatalyst.

[0062] Bioluminescent proteins or complexes This disclosure includes bioluminescent polypeptides, bioluminescent complexes, and materials and methods related to their components. Specifically, light emitted from a bioluminescent protein or complex (or from a luminescent phore acted upon by a bioluminescent protein or complex) is used to activate a photocatalyst.

[0063] NanoLuc In some embodiments, the systems and methods described herein include a bioluminescent protein. In some embodiments, the bioluminescent protein is a luciferase enzyme. Suitable luciferase enzymes include those selected from the following group: Photinus pyralis or North American firefly luciferase, Luciola cruciata or Japanese firefly or Genji firefly luciferase, Luciola italic or Italian firefly luciferase, Luciola lateralis or Japanese firefly or Heike luciferase, N. nambi luciferase, Luciola mingrelica or Eastern European firefly luciferase, Photuris pennsylvanica or Pennsylvania firefly luciferase, Pyrophorus plagiophthalamus or click beetle luciferase, Phrixothrix hirtus or railroad worm luciferase, Renilla reniformis or wild-type Renilla luciferase, Renilla reniformis Rluc8 mutant Renilla luciferase, Renilla reniformis green-emitting Renilla luciferase, Gaussia Gaussia princeps wild-type luciferase, Gaussia princeps Gaussia-Dura luciferase, Cypridina noctiluca or Cypridina luciferase, Cypridina hilgendorfii or Cypridina or Vargula luciferase, Metridia longa or Metridia luciferase, TurboLuc (Auld et al. Biochemistry 2018, 57, 31, 4700-4706: the whole is incorporated by reference), nanolanthanum (Suzuki et al.Nature Communications volume 7, article number: 13718 (2016); the entire article is incorporated by reference), and Oplophorus luciferases (e.g., Oplophorus gracilirostris (OgLuc luciferase), Oplophorus grimaldii, Oplophorus spinicauda, ​​Oplophorus foliaceus, Oplophorus noraezeelandiae, Oplophorus typus, Oplophorus noraezelandiae, or Oplophorus spinous).

[0064] In some embodiments, the bioluminescent protein is the luciferase of Oplophorus gracilirostris, NanoLuc® luciferase (Promega Corporation, U.S. Patent Nos. 8,557,970 and 8,669,103; these are incorporated herein by reference in their entirety). PCT applications PCT / US2010 / 033449, U.S. Patent Nos. 8,557,970, PCT applications PCT / 2011 / 059018 and 8,669,103 (each of which is incorporated herein by reference in its entirety for any purpose) describe compositions and methods comprising bioluminescent polypeptides. Such polypeptides may find applications in the embodiments herein and may be used in conjunction with the compositions, assays, apparatus, systems, and methods described herein. In some embodiments, the compositions, assays, apparatus, systems, and methods provided herein include a bioluminescent polypeptide having sequence identity with SEQ ID NO: 1, or at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or in between) with SEQ ID NO: 1. In some embodiments, any of the aforementioned bioluminescent proteins are conjugated to one or more other components of the assays and systems described herein (e.g., fused, chemically conjugated, etc.) (e.g., fused to a HALOTAG protein).

[0065] In some embodiments, the bioluminescent protein is a cyclically substituted version of a native or modified bioluminescent protein (see, for example, U.S. Patent No. 10,774,364; the whole is incorporated by reference).

[0066] In some embodiments, the systems and methods herein include a bioluminescent complex (e.g., two or more components (e.g., peptides and / or polypeptides) that are combined by structural complementation to form a complex capable of activating a luminescent phose to emit light). In some embodiments, the luminescent phose emits significantly more light in the presence of the bioluminescent complex than in the presence of any one of the components alone. In some embodiments, the bioluminescent complex is formed from fragments of a luciferase enzyme (e.g., peptides and / or polypeptides). In some embodiments, the bioluminescent complex is a cyclically substituted version of a natural or modified bioluminescent component (e.g., formed from two fragments of a cyclically substituted luciferase) (see, for example, U.S. Patent No. 10,774,364; the whole is incorporated by reference).

[0067] PCT applications PCT / US14 / 26354, PCT / US19 / 036844, and PCT / US20 / 62499, U.S. Patent No. 9,797,889, U.S. Patent Application No. 16 / 439,565, and U.S. Publication No. 2021 / 0262941 (each incorporated herein in whole by reference for all purposes) describe compositions and methods for constructing bioluminescent complexes, and such complexes, as well as their peptide and polypeptide components, may be used in combination with the assays and methods described herein, and applications may be found in the embodiments herein.

[0068] In some embodiments, peptide and polypeptide components are provided for the construction of a bioluminescent complex that can produce luminescence in the presence of a suitable substrate (e.g., coelenterazine or a coelenterazine analog (e.g., flimazine, fluoroflimazine, etc.)). In some embodiments, complementary polypeptides and peptides collectively span the length of the luciferase sequence (or >75%, >80%, >85%, >90%, >95%, or more) (or collectively contain at least 40% sequence identity to the luciferase sequence (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, or more)). In some embodiments, the “complementary” polypeptides and peptides each correspond to a portion of the luciferase sequence. They are distinct molecules. Due to structural complementarity, they come together to form a bioluminescent complex. Preferred luciferase sequences may include sequence number 1 or 2, or any of the full-length luciferase sequences listed above. In some embodiments, the bioluminescent complex includes the NANOBIT or NANOTRIP system (Promega; Madison, WI). In some embodiments, the peptide and / or polypeptide components of the bioluminescent complex collectively contain at least 60% sequence identity with sequence number 1 and / or sequence number 2 (e.g., >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >99%). In some embodiments, the peptide and / or polypeptide components of the bioluminescent complex include HIBIT (sequence number 3), SMBIT (sequence number 4), LGBIT (sequence number 5), LGTRIP (sequence number 6), and / or SMTRIP9 (sequence number 7).In some embodiments, the peptide and / or polypeptide components of the bioluminescent complex include at least 60% sequence identity (e.g., >60%, >65%, >70%, >75%, >80%, >85%, 90%, >95%, >99%) with HIBIT (SEQ ID NO: 3), SMBIT (SEQ ID NO: 4), LGBIT (SEQ ID NO: 5), LGTRIP (SEQ ID NO: 6), and / or SMTRIP9 (SEQ ID NO: 7).

[0069] In some embodiments, any of the aforementioned components of the bioluminescent complex is linked (e.g., fused, chemically bound, linked, etc.) to one or more other components of the assays and systems described herein (e.g., fused to the HALOTAG protein).

[0070] There are various features of bioluminescent complexes that are found to be used in the embodiments herein, which may offer advantages in specific applications. For example, a bioluminescent complex (e.g., a complex formed by the complementation of HiBiT / LgBiT) generates light only through the complementation of its component peptides / polypeptides. Therefore, by directly or indirectly conjugating (e.g., fusing, linking, etc.) one or more components of the bioluminescent complex to other components of the system (e.g., photocatalysts, activatable molecules, targets, etc.), proximity of those components to the bioluminescent complex during photogeneration is ensured. By linking two other components of the system to separate components of the bioluminescent complex, proximity of these components during photogeneration by the complex is ensured. In some embodiments, the use of a bioluminescent complex provides improved spatiotemporal resolution via conditional activation at specific sites, as the two components need to come together to form a complex.

[0071] In some embodiments, the bioluminescent protein or components of the multi-element bioluminescent complex are inserted into internal positions within the capture agent. In some embodiments, the position within the capture agent is selected to enhance the efficiency of bioluminescent activation of the catalyst by providing greater proximity or a preferred conformation.

[0072] In some embodiments, the components of the bioluminescent protein or multi-elemental bioluminescent complex are cyclically replaced.

[0073] Fluorescent phosphonate substrate In some embodiments, the systems and methods herein include a luminescent substrate that emits light through interaction with a bioluminescent protein and / or complex described herein. Suitable luminescent phosphodes for use with the bioluminescent protein or complex in the systems or methods will be understood. For example, firefly luciferin having the following structure: [ka] This is a luciferin found in many Lampyridae species and is a substrate for beetle luciferase.

[0074] Latia luciferin having the following structure: [ka] It originates from the freshwater snail Latia neritoides.

[0075] Bacterial luciferin with the following structure: [ka] It is used as a substrate for many bacterial luciferases.

[0076] Celenterazine with the following structure: [ka] It is found in radiolarians, ctenophores, cnidarians, squid, brittle stars, copepods, chaetognaths, fish, and shrimp, and is a luminescent substrate of the luciferases of these organisms. Variants and derivatives of coelenterazine, such as flimazine and fluoroflimazine, are used in embodiments herein (for example, together with bioluminescent proteins and complexes derived from Oplophorus).

[0077] Other luminescent substrates include the following: Dinoflagellates: [ka] Valgurin (cypridin luciferin): [ka] N.nambi: [ka]

[0078] The pairing of appropriate bioluminescent proteins or complexes with luminescent phores is well understood in the art. In certain embodiments, bioluminescent proteins utilizing imidazopyrazine luminescent phores such as coelenterazine, furimazine, or fluorofurimazine are provided in the systems or methods of this specification (U.S. Patent Application No. 16 / 548,214; the whole is incorporated by reference). In some embodiments, the systems or methods include (1) an Oplophorus-derived polypeptide (e.g., NANOLUC) or a component of an Oplophorus-derived bioluminescent complex (e.g., NANOBIT, NANOTRIP), and an imidazopyrazine luminescent phore (e.g., coelenterazine, furimazine, fluorofurimazine, etc.). In some embodiments, the systems and methods herein include imidazopyrazine luminescent phosphophores such as natural coelenterazine, furimazine, fluorofurimazine, coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine, in addition to those disclosed in WO2003 / 040100, U.S. Patent Application No. 12 / 056,073 (paragraph

[0086] ) and U.S. Patent No. 8,669,103 (these disclosures are incorporated herein by reference in their entirety).

[0079] In some embodiments, the luminescent phosphodiolus emits light through interaction with bioluminescent proteins or complexes. In some embodiments, the luminescent phosphodiolus emits light in the visible light spectrum (e.g., about 400 to about 700 nm (e.g., 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, or a range between these)). In some embodiments, the luminescent phosphodiolus emits light with wavelengths between 400 and 500 nm (e.g., 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, or a range between these).

[0080] photocatalyst In some embodiments, the systems and methods herein include a photocatalyst that can absorb light emitted from a bioluminescent phosphophore (by interaction with a bioluminescent protein or complex) and subsequently activate an adjacent activatable molecule. Any compound or part that can receive light energy emitted from a bioluminescent protein or complex activating phosphophore and subsequently participate in the activation of an activatable molecule may be utilized in embodiments herein. In some embodiments, the excited photocatalyst participates in the activation of an adjacent activatable molecule by any other suitable energy or electron transfer mechanism in which the adjacent activatable molecule is activated via Foerster resonance energy transfer, Dexter energy transfer, single electron transfer, or singlet oxygen generation and hydrogen abstraction from the activatable molecule (direct oxidation).

[0081] In some embodiments, the photocatalyst is iridium-based or ruthenium-based (Bevernaegie et al. 'A Roadmap Towards Visible Light Mediated Electron Transfer Chemistry with Iridium(III) Complexes.' ChemPhotoChem 2021, 5, 217., Day et al. Advances in Photocatalysis: A Microreview of Visible Light Mediated Ruthenium and Iridium Catalyzed Organic Transformations Org. Process Res. Dev. 2016, 20, 1156-1163; the whole is incorporated by reference). In some embodiments, the photocatalyst has the structure of formula (I) [ka] [In the formula, Each series of dashed lines ( [ka] ) indicates the presence or absence of a condensed 6-membered ring, M is a transition metal, m1, m2, m3, n1, n2, n3, p1, p2, and p3 are each independently 0, 1, or 2. R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c Each of these is independently selected from halo, alkyl, haloalkyl, amino, heteroalkyl, and group-linker-Q, where Q is a scavenging element. X 1a , X 1b , X 2a , X 2b , X 3a , and X 3bEach is independently selected from N and C, where X 1a and X 1b At least one of them is N, and X 2a and X 2b At least one of them is N, and X 3a and X 3b At least one of them is N, X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d Each is independently selected from CH and N, A is an anion, q is 0, 1, or 2.

[0082] In some embodiments, the photocatalyst comprises a transition metal selected from Ru and Ir.

[0083] In some embodiments, the photocatalyst is an iridium-based photocatalyst selected from the following: [ka] or derivatives thereof, the compound being functionalized with at least one group-linker-Q (wherein Q is a capture element).

[0084] In some embodiments, the photocatalyst is a ruthenium-based photocatalyst selected from the following: [ka] or derivatives thereof, the compound being functionalized with at least one group-linker-Q (wherein Q is a capture element).

[0085] In some embodiments, M is Ru. In some embodiments, M is Ir.

[0086] In some embodiments, m2, n2, and p2 are each 0, and each series of dashed lines represents the absence of a fused 6-membered ring, i.e., the compound has the following formula. [Chemical formula]

[0087] In some embodiments, X 1a is N, X 1b is C, X 2a is N, X 2b is C, X 3a is C, X 3b is N. In some embodiments, X 1a is N, X 1b is C, X 2a is N, X 2b is C, X 3a is N, X 3b is N.

[0088] In some embodiments, X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d are each CH. In some embodiments, X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d are each N.

[0089] In some embodiments, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c are each independently selected from fluoro, methyl, tert-butyl, trifluoromethyl, and group-linker-Q. In some embodiments, R 1a , R 1b , R1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c One or less of them is a group-linker-Q.

[0090] In some embodiments, the compound comprises one group “-linker-Q”, wherein Q is a capture element. In some embodiments, the capture element is an “affinity molecule” and the corresponding capture agent is an “acceptor” (e.g., a small molecule, protein, antibody, etc.) that selectively interacts with the affinity molecule. Examples of such pairs include an antigen as a capture element and an antibody as a capture agent, a small molecule as a capture element and a protein (e.g., streptavidin and biotin) having high affinity for the small molecule as a capture agent, and the like.

[0091] In some embodiments, Q is a substrate of a dehalogenase, e.g., haloalkane dehalogenase. Systems comprising a mutant hydrolase (e.g., mutant dehalogenase) that covalently binds to a substrate (e.g., haloalkyl substrate) are described, for example, in U.S. Patent Nos. 7,238,842, 7,425,436, 7,429,472, 7,867,726 (each of which is incorporated herein by reference in its entirety). For example, HALOTAG is a commercially available modified dehalogenase enzyme that forms a stable (e.g., covalent) bond (e.g., ester bond) with a haloalkyl substrate and is used in embodiments herein.

[0092] In some embodiments, Q has the formula -(CH2) n -Y, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and Y is a halogen (i.e., F, Cl, Br, or I). In some embodiments, n is 4, 5, 6, 7, or 8 and Y is Cl. In some embodiments, n is 6 and Y is Cl, such that Q has the formula -(CH2)6-Cl.

[0093] Linkers include various combinations of such groups, such as esters (-C(O)O-), amides (-C(O)NH-), carbamates (-NHC(O)O-), ureas (-NHC(O)NH-), phenylenes (e.g., 1,4-phenylene), linear or branched alkylenes, and / or oligo- and polyethylene glycols (-(CH2CH2O) x A linker having a -) bond or the like can be provided. In some embodiments, the linker may contain two or more atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 atoms, or 2 to 200 atoms in any range between them (e.g., 2 to 20, 5 to 10, 15 to 35, 25 to 100, etc.)). In some embodiments, the linker includes a combination of oligoethylene glycol bonds and carbamate bonds. In some embodiments, the linker is of the formula -O(CH2CH2O) z1 -C(O)NH-(CH2CH2O) z2 -C(O)NH-(CH2) z3 -(OCH2CH2) z4 The formula has O-, where z1, z2, z3, and z4 are each independently selected from 0, 1, 2, 3, 4, 5, and 6. For example, in some embodiments, the linker has a formula selected from the following: [ka]

[0094] In some embodiments, q is 0, 1, or 2. Those skilled in the art will recognize that the value of q depends on the selection of other variables and is chosen to balance the total charge in the remainder of the molecule. For example, if the total charge of the metal-based portion of the molecule is +1, in some embodiments q is 1 and A is a monovalent anion (e.g., a halide or hexafluorophosphate). In some embodiments, if the total charge of the metal-based portion of the molecule is +2, in some embodiments q is 2 and A is a monovalent anion (e.g., a halide or hexafluorophosphate).

[0095] Figure 5 shows an exemplary photocatalyst linked to a HALOTAG substrate. Alternative positions for linking to the photocatalyst, other photocatalysts, different linkers, and linker lengths are within the scope of this specification.

[0096] In some embodiments, the photocatalyst is an organic photo-redox catalyst. In some embodiments, the organic photo-redox catalyst is selected from quinones, pyrylium, acridinium, and xanthenes.

[0097] In some embodiments, the photocatalyst is a quinone-based organic photoredox catalyst selected from the following: [ka]

[0098] In some embodiments, the photocatalyst is a pyrylium-based organic photoredox catalyst selected from the following: [ka]

[0099] In some embodiments, the photocatalyst is an acridinium-based organic photoredox catalyst selected from the following: [ka]

[0100] In some embodiments, the photocatalyst is a xanthene-based organic photoredox catalyst selected from the following: [ka] In the formula, R, if present, represents a potential linker-Q binding site.

[0101] In some embodiments, any suitable position in the above-described photocatalytic structure can be used as the linker-Q binding site.

[0102] In some embodiments, the photocatalyst is a thiazine-based organic photoredox catalyst selected from the following: [ka] In the formula, R is the linking site of linker-Q. In some embodiments, R is an amine, carboxyl, tert-butyl, tert-butyl-methoxy, ether, hydroxyl, PEG, etc.

[0103] In some embodiments, the photocatalyst (e.g., quinone-based, pyrylium-based, acridinium-based, xanthene-based, or thiazine-based photoredox catalyst) is conjugated to linker-Q. In some embodiments, the linker (e.g., linker-Q) is bound to the photocatalyst at any suitable position on the photocatalytic structure. In some embodiments, suitable positions for binding the photocatalyst are understood in the art.

[0104] Activatable molecules In some embodiments, the systems and methods herein include activatable molecules that incorporate photoresponsive moieties that limit their activity, and which, when acted upon by an activated photocatalyst, are converted from activatable molecules to activated molecules. In some embodiments, the photocatalyst catalyzes the cleavage of bonds on the activatable molecule (e.g., releasing the activatable molecule from another entity, releasing a moiety from the activatable molecule). In some embodiments, the photocatalyst catalyzes the oxidation of the activatable molecule (i.e., hydrogen abstraction) to turn on its reactivity or release it from another entity. In some embodiments, the photocatalyst catalyzes a reversible conformational change that turns on the reactivity of the activatable molecule. In some embodiments, the photocatalyst catalyzes the formation of bonds to the activatable molecule (e.g., bonding the activatable molecule to another entity). The embodiments herein are not limited by the chemical mechanism of molecular activation.

[0105] In some embodiments, the photocatalyst facilitates energy transfer to the activatable molecule. In some embodiments, the photocatalyst transfers energy to the activatable molecule by a mechanism of Foerster resonance energy transfer, Dexter energy transfer, single-electron transfer, or any other suitable energy transfer mechanism. In some embodiments, the photocatalyst generates singlet oxygen for the direct oxidation of the activatable molecule.

[0106] In some embodiments, the activatable molecule is a caged compound. A caged compound is an activatable molecule that has been inactivated by chemical modification (e.g., chemically inactive, biologically inactive, undetectable, etc.). In some embodiments, the conversion of an activatable molecule to an active molecule involves decaching the activatable molecule. In some embodiments, an activated photocatalyst facilitates the decaching of the activatable molecule, resulting in the release of the active form from the molecule. The embodiments herein are not limited by the identity of the caged molecule, the caching modification, or the chemistry required for decaching. For example, activatable molecules may include photocaged fluorophores, photocaged probes, photocaged drugs, photocaged signaling molecules, photocaged neurotransmitters, photocaged crosslinkers, photocaged proteolytic targeting chimeras (PROTACs), photocaged gRNAs, photocaged nucleic acids (RNA or DNA), photocaged nucleotides, and the like.

[0107] Examples of bond cleavage that occurs in order to release photodissociable protecting groups and activating molecules include the following: [ka]

[0108] Other photodissociative protective chemistry is used in the embodiments described herein (Klan et al.). Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy (Chem. Rev. 2013, 113, 119-191; the entire work is incorporated by reference).

[0109] In some embodiments, the photocatalyst facilitates the abstraction of hydrogen from activatable molecules. Hydrogen abstraction is a chemical reaction in which a hydrogen free radical is abstracted from a substrate (activatable molecule) and taken up by the photocatalyst. In such a reaction, the photoactivation of the photocatalyst results in the loss of hydrogen free radicals, thereby activating the photocatalyst, which abstracts hydrogen from the activatable molecule and returns the photocatalyst to an inactive state. Through hydrogen abstraction, the activatable molecule is converted into an activated molecule.

[0110] Examples of oxidation-driven cage de-cabling chemistry that leads to the release of active materials from molecules include the following: [ka]

[0111] Other oxidation-driven cage-de-cabling chemistry methods are used in the embodiments described herein.

[0112] In some embodiments, converting an activatable molecule into an activated molecule involves catalyzing a redox reaction using the activatable molecule as a substrate for the reaction. In such embodiments, the photocatalyst or photosensitizer absorbs light and is elevated to a redox-active or excited state. As a result, the photocatalyst or photosensitizer can catalyze the redox reaction and activate the activatable molecule.

[0113] In some embodiments, the activatable molecule is a photoswitchable molecule. In some embodiments, the photoswitchable molecule is a molecule that undergoes a reversible conformational change of its structural geometry upon exposure to light energy (e.g., of a specific wavelength), thereby turning on its activity (Huell, K. et al. In Vivo Photopharmacology Chem. Rev. 2018, 118, 10710-10747; the whole is incorporated by reference). Examples of activatable photoswitches and the activating molecules they convert include: [ka] [ka]

[0114] Other activatable / activated optical switches are used in the embodiments described herein.

[0115] Localization element (HALOTAG) In some embodiments, two or more components of the system herein are conjugated (e.g., linked, fused, etc.) to a molecular element that facilitates the localization of the components. In certain embodiments, a bioluminescent protein (or complex) and a photocatalyst are linked together, for example, via a molecular localization element that binds to the bioluminescent protein (or complex) and the photocatalyst, bringing them close enough to allow light from a luminescent phore interacting with the bioluminescent protein (or complex) to activate the photocatalyst.

[0116] In some embodiments, a bioluminescent protein or bioluminescent complex is fused to a first molecular entity, and a photocatalyst is conjugated to a second molecular entity, and the interaction of the first and second molecular entities brings the bioluminescent protein or bioluminescent complex close enough to the photocatalyst so that the light emitted by the luminescent phore through interaction with the bioluminescent protein or bioluminescent complex activates the photocatalyst. In some embodiments, the first molecular entity is a scavenger (scavenging protein), and the second molecular entity is a scavenging element.

[0117] In some embodiments, a bioluminescent protein or bioluminescent complex is fused to a modified dehalogenase capable of forming a covalent bond with its substrate, and the photocatalyst is conjugated to the dehalogenase substrate (see Figure 6A). In some embodiments, the binding of the modified dehalogenase to the dehalogenase substrate brings the bioluminescent protein or bioluminescent complex close enough to the photocatalyst so that the light emitted by the luminescent phore through interaction with the bioluminescent protein or bioluminescent complex activates the photocatalyst. In some embodiments, intracellular linkage of haloalkane conjugates to HALOTAGs fused to components of the system is possible, coupled with HaloTag's highly specific and rapid binding, by minimizing the effect on the cell permeability of the haloalkanes, thereby reducing the overall dependence of the components on cell permeability and allowing the system to be localized to specific cell compartments (Figures 6B-C).

[0118] In some embodiments, a commercially available HALOTAG system (Promega Corp.; Madison, WI) is used to link or ligate two or more components of the systems and methods described herein (e.g., a bioluminescent protein or bioluminescent complex and a photocatalyst). HALOTAG is a 297-residue self-labeled polypeptide (33 kDa) derived from a bacterial hydrolase (dehalogenase) enzyme, modified to covalently bond to its ligand haloalkane moiety. The HALOTAG ligand can be ligated to a solid surface (e.g., beads) or a functional group (e.g., a fluorophore), and the HALOTAG polypeptide can be fused to a variety of proteins of interest, enabling covalent bonding to the solid surface or functional group of the protein of interest.

[0119] HALOTAG polypeptides are hydrolases with genetically modified active sites that specifically bind to haloalkane ligands or chloroalkane linkers, increasing ligand binding rates (Pries et al. The Journal of Biological Chemistry. 270(18):10405-11, incorporated in whole by reference). The reaction forming the bond between the protein tag and the chloroalkane linker is rapid and essentially irreversible under physiological conditions (Waugh DS (June 2005). Trends in Biotechnology. 23(6):316-20; incorporated in whole by reference). In the native hydrolase enzyme, nucleophilic attack of the chloroalkane-reactive linker results in the substitution of the halogen with an amino acid residue, leading to the formation of an alkylenzyme covalent intermediate. This intermediate is then hydrolyzed by amino acid residues within the wild-type hydrolase (Chen et al. (February 2005) Current Opinion in Biotechnology. 16(1):35-40, incorporated in whole by reference). This would lead to the regeneration of the enzyme after the reaction. However, in the modified haloalkane dehalogenase HALOTAG, the reaction intermediate cannot be hydrolyzed due to the enzyme mutation and therefore cannot proceed through the second reaction. As a result, the intermediate persists as a stable covalent adduct without the associated reverse reaction (Marks et al. (August 2006) Nature Methods. 3(8):591-6, the whole of which is incorporated by reference).

[0120] HALOTAG fusion proteins can be expressed using standard recombinant protein expression techniques (Adams et al. (May 2002) Journal of the American Chemical Society. 124(21):6063-76; whole text incorporated by reference). HALOTAG polypeptides are relatively small proteins, and their reactions are exogenous to mammalian cells, thus avoiding interference from endogenous mammalian metabolic reactions (Naested et al. The Plant Journal. 18(5):571-6; whole text incorporated by reference). Once the fusion protein is expressed, a wide range of potential experimental fields exist, including enzyme assays, cell imaging, protein arrays, determination of intracellular localization, and many additional possibilities (Janssen DB (April 2004). Current Opinion in Chemical Biology. 8(2):150-9; whole text incorporated by reference).

[0121] Various HALOTAG ligands, functional groups, fusions, assays, modifications, uses, etc., are described in U.S. Patents No. 8,748,148, 9,593,316, 10,246,690, 8,742,086, 9,873,866, 10,604,745, U.S. Patent Application No. 2009 / 0253131, U.S. Patent Application No. 2010 / 0273186, 20130337539, U.S. Patent Application No. 2012 / 0258470, U.S. Patent Application No. 2012 / 0252048, U.S. Patent Application No. 2011 / 0201024, and U.S. Patent Application No. 2014 / 0322794, each of which is incorporated by reference as a whole.

[0122] In some embodiments, the capture protein described herein is a cyclically substituted modified dehalogenase or a split-modified dehalogenase.

[0123] In some embodiments, the capture protein herein is a modified dehalogenase having an insertion into its internal loop (e.g., a bioluminescent protein, a component of a bioluminescent complex, a cyclically substituted bioluminescent protein, a cyclically substituted component of a bioluminescent complex, an extended loop sequence, etc.).

[0124] In some embodiments, a first component of the system herein (e.g., a component of a bioluminescent protein or bioluminescent complex) is fused to a modified dehalogenase (e.g., HALOTAG or a variant thereof) (e.g., expressed as a fusion) or inserted into a surface loop of the modified dehalogenase, and a second component of the system herein (e.g., a photocatalyst) is linked to a dehalogenase substrate (e.g., a haloalkane) (e.g., directly or via a linker). For example, the structure of a photocatalyst linked to a dehalogenase substrate is P-linker-AX, where P is the photocatalyst and A is (CH2) 2-12Here, X is a halogen, and the linker is a linker portion that can link P to AX. In some embodiments, the linker is a polyatomic linear or branched chain containing C, N, S, or O, or a group containing one or more rings, e.g., saturated or unsaturated rings (e.g., one or more aryl rings, heteroaryl rings, or any combination thereof). In some embodiments, the linker includes combinations of -O(CH2)2-, -(CH2)O-, -CH2-, -NHC(O)O-, -OC(O)NH-, NHC(O)-, and -C(O)NH-. In some embodiments, the linker is 5 to 50 atoms long (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or in between). In some embodiments, the length of the linker for linking the photocatalyst allows for optimization of proximity and geometric shape (e.g., for efficient energy transfer). An exemplary linker-AX group is illustrated in Figure 3. In some embodiments, a first component of the system herein (e.g., a bioluminescent protein or component of a bioluminescent complex) is inserted into a modified dehalogenase (e.g., HALOTAG or a variant thereof) to increase proximity or provide a geometric shape favorable for energy transfer to a bound catalyst (e.g., expressed as an internal fusion) (see, for example, U.S. Provisional Application No. 63 / 338,369 (the whole is incorporated by reference)). In some embodiments, the site for insertion into the modified dehalogenase (e.g., HALOTAG or a variant thereof) is selected to provide optimal proximity and geometric shape for desired interactions between components while maintaining the function or activity of the modified dehalogenase (e.g., HALOTAG or a variant thereof) and the inserted component.

[0125] The scope of the embodiments herein is not limited by the type of linker available. Components and their parts may be directly linked (for example, the linker consists of a single covalent bond) or linked via a suitable linker. Embodiments are not limited to any particular linker group. A variety of linker groups are intended, and suitable linkers include alkyl groups, methylene carbon chains, ethers, polyethers, alkylamide linkers, peptide linkers, modified peptide linkers, poly(ethylene glycol) (PEG) linkers, streptavidin-biotin or avidin-biotin linkers, polyamino acids (e.g., polylysine), functionalized PEG, polysaccharides, glycosaminoglycans, dendritic polymers (WO93 / 06868, and Tomalia et al.) The linkers may include, but are not limited to, those described in al., Angew. Chem. Int. Ed. Engl. 29:138-175 (1990) (these are incorporated herein by reference in their entirety), PEG-chelate polymers (W94 / 08629, WO94 / 09056, and WO96 / 26754, these are incorporated herein by reference in their entirety), oligonucleotide linkers, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfides, or combinations thereof. In some embodiments, the linkers are cleavable (e.g., enzymatically (e.g., via a TEV protease site), chemically, photo-induced, etc.).

[0126] In some embodiments, a modified dehalogenase (e.g., HALOTAG) and a dehalogenase ligand (e.g., a haloalkane) are used to link any two components of the systems and methods described herein (i.e., not limited to linking components of a bioluminescent protein or bioluminescent complex to a photocatalyst). In other embodiments, components of a bioluminescent protein or bioluminescent complex are linked to a photocatalyst (or other components described herein) by a different mechanism.

[0127] In some embodiments, a first component of the system or method herein is linked (e.g., fused) to a capture agent (e.g., a capture protein), and a second component of the system or method is linked to a capture element. The binding of the capture element by the capture agent (e.g., a capture protein) results in colocalization of the first and second components. In some embodiments, the capture agent is a modified dehalogenase, and the capture element is a haloalkane. However, other capture agent / element pairs that may be used in embodiments herein include streptavidin / biotin, antibodies (or Ab fragments), and antigens, etc.

[0128] In other embodiments, the components of this specification are connected by chemical modification / conjugation, such as natural chemical ligation, Staudinger ligation, "traceless" Staudinger ligation, amide coupling, methods using activated esters, methods targeting lysine, tyrosine and cysteine ​​residues, imine bond formation (with and without orthoboric acid), boronic acid / diol interactions, disulfide bond formation, copper / copper free azide, diazo and tetrazine "click" chemical reactions, UV-enhanced thiolene conjugation, diaziline photolabeling, Diels-Alder cycloaddition, metathesis reactions, Suzuki cross-coupling, 2-cyanobenzothiazole (CBT) coupling, 2-pyridinecarboxaldehyde (PCA) coupling, and the like.

[0129] Targeting molecules and localization elements In some embodiments, the activating molecule interacts with (e.g., binds to) a target molecule (e.g., a cellular target, protein, nucleic acid), a chemical moiety, or a cellular compartment.

[0130] In some embodiments, a bioluminescent protein or complex is conjugated to a target binder, which can bind to a target molecule (e.g., a protein, nucleic acid, or other biomolecule (e.g., lipids, sugars, etc.)). In some embodiments, the target binder is a protein or peptide directly or indirectly fused to a component of the bioluminescent protein or complex. In some embodiments, the target molecule is a nucleic acid, and the target binder can bind specifically or nonspecificly to the nucleic acid. In some embodiments, the target binder is a wild-type or modified Cas protein (e.g., Cas9, dCas9, dCas12, dCas13, etc.), and the target molecule is a nucleic acid modified by CRISPR. In some embodiments, the system further includes a guide RNA (gRNA). In some embodiments, the target molecule is a target peptide or protein, and the target binder can bind to the target peptide or protein. In some embodiments, the target binder is a small molecule or nucleic acid directly or indirectly linked to a component of the bioluminescent protein or complex.

[0131] In a series of embodiments, bioluminescent proteins, components of bioluminescent complexes, photocatalysts, or activatable molecules are linked to specific ligands, nucleic acids, or targeted proteins (e.g., Cas9, dCas9, dCas12, dCas13, etc.). Exemplary targeted ligands include small molecules / drugs / signaling molecules that specifically bind to a target. In some embodiments, photocatalysts are linked to such small molecules / drugs / signaling molecules, thereby enabling the photocatalyst to localize with the target protein fused to HiBiT or NanoLuc. Other exemplary targeted proteins / ligands include antibodies, antibody fragments, protein A, the Ig-binding domain of protein A, protein G, the Ig-binding domain of protein G, protein A / G, the Ig-binding domain of protein A / G, protein L, the Ig-binding domain of protein L, protein M, the Ig-binding domain of protein M, oligonucleotide probes, peptide nucleic acids, DARPin, antikalin, nanobodies, aptamers, affimers, purified proteins, and analyte-binding domains of proteins. By linking the catalyst to the binding domain that recognizes the target protein, the catalyst can be localized to the target protein already fused to HiBiT or NanoLuc. In some embodiments, for example, an approach is used to increase the proximity of the functional moiety, which directly binds to the target protein, and the recognition moiety, to the activatable molecule, using a trifunctional molecule that includes a photoreactive moiety and a functional moiety that has a general affinity for nucleic acids.

[0132] In this case, the photocatalytic system localizes to the target protein through the complementarity of HiBiT fused to the target protein and the LgBiT-HaloTag-catalyst. Similarly, LgBiT fused to the target protein localizes the target protein to the HiBiT-HaloTag-photocatalytic system.

[0133] system In some embodiments, two or more (e.g., 2, 3, 4, or more) of the components of the system described herein are conjugated together. In some embodiments, one or more pairs of components of the system described herein are conjugated together. For example, the following pairs of components may be conjugated (e.g., linked by a linker, fused by a gene, etc.): bioluminescent protein and capture protein, photocatalyst and capture ligand, bioluminescent protein and photocatalyst, components of a bioluminescent complex and capture protein, components of a bioluminescent complex and photocatalyst, bioluminescent protein and target molecule, components of a bioluminescent complex and target molecule, bioluminescent protein and target binder (e.g., protein, antibody, antibody fragment, antibody binder, nucleic acid, small molecule ligand, etc.), components of a bioluminescent complex and target binder (e.g., protein, antibody, antibody fragment, antibody binder, nucleic acid, small molecule ligand, etc.), components of a bioluminescent complex and photocatalyst, components of a bioluminescent complex and capture ligand, activatable molecule and capture ligand, capture protein and target binder (e.g., protein, antibody, antibody fragment, antibody binder, nucleic acid, small molecule ligand, etc.), etc.

[0134] The components of the systems described herein may be delivered, combined, and / or manufactured in any suitable manner for a particular application. In embodiments in which the system is present within a cell, the components may be expressed intracellularly, exogenously added, and enabled to enter the cell (i.e., cell-permeable components), or delivered to the cell. Delivery of components to cells may occur in any suitable delivery vehicle, such as liposomes, micelles, nanoparticles, or viruses. In some embodiments, components are tagged to facilitate delivery into cells (e.g., linked to membrane transport motifs). In some embodiments, components are included to facilitate cell uptake and / or subsequent endosomal exit. Examples of such additional components include modified polyethyleneimine polymers and modified poly(amideamine) dendrimers for use in the delivery of biomolecules to cells (e.g., components that cannot passively enter cells but cannot be expressed intracellularly (e.g., LgBiT / photocatalytic direct conjugates)) (see U.S. Publication 2020 / 0399660, which is incorporated in whole by reference).

[0135] Exemplary combinations of system components within the scope of this specification include: The dipeptide containing HIBIT-TRIP9 fused to the target protein, the fusion of LGTRIP and HALOTAG, and the photocatalyst-HALOTAG linked to the haloalkyl HALOTAG ligand bind to the haloalkyl ligand. The high affinity of HIBIT-TRIP9 for LGTRIP forms a bioluminescent complex, localizing the photocatalytic system to the target protein, and bioluminescence induces photocatalysis. • A fusion of HIBIT and an antisense oligonucleotide or oligonucleotide probe, LGBIT fused to HALOTAG, and a photocatalyst linked to a haloalkyl HALOTAG ligand - HALOTAG binds to the haloalkyl ligand - hybridization of the oligonucleotide to the target nucleic acid sequence localizes the photocatalytic system to the target DNA / RNA, and bioluminescence induces photocatalysis. • SMBIT fused to an antibody against the target analyte, LGBIT / HALOTAG fused to a common immunoglobulin binding site, photocatalyst linked to a haloalkyl HALOTAG ligand, HALOTAG binding to the haloalkyl ligand - antibody binding localizes the photocatalytic system to the target analyte, and bioluminescence induces photocatalysis. • A fusion of HIBIT and Trip9 to an antisense oligonucleotide or oligonucleotide probe targeting the same DNA / RNA, LGTRIP fused to HALOTAG, and a photocatalyst-HALOTAG linked to a haloalkyl HALOTAG ligand binds to the haloalkyl ligand, and hybridization of the oligonucleotide to the target nucleic acid sequence localizes the photocatalytic system to the target DNA / RNA, and bioluminescence induces photocatalysis. • HiBiT fused to the target protein, LGBIT and HALOTAG fusion, photocatalyst linked to a haloalkyl HALOTAG ligand - HALOTAG binds to the haloalkyl ligand, and the high affinity of HIBIT for LGBIT forms a bioluminescent complex, localizing the photocatalytic system to the target protein, and bioluminescence induces photocatalysis. • dCas9 or dCas12g1 fused to both HaloTag and NanoLuc, and photocatalyst-HALOTAG linked to a haloalkyl HALOTAG ligand, are bound to the haloalkyl ligand. The gRNA targets the photocatalytic system to the desired DNA / RNA.

[0136] Purpose In some embodiments, the systems and methods described herein are used to perform functional biological analyses that depend on the spatiotemporal activation of functional molecules, which are functional molecules incorporating photoresponsive moieties that limit their activity. A variety of applications are possible due to the advantages of the systems and methods described herein.

[0137] Exemplary applications include various bioluminescence-induced photocatalytic activations: 1) uncaching photocaged drugs for spatiotemporally controlled activation / inhibition of target proteins, activation / inhibition of populations of target proteins localized to specific cell compartments, release of lethal drugs, and targeted cell death; 2) uncaching photocaged signaling molecules for spatiotemporally controlled activation / inhibition of signaling pathways; 3) activation of photocaged / photoswitched PROTACs for spatiotemporally controlled degradation of target proteins or populations of target proteins localized to specific compartments; 4) activation of photocaged / photoswitched antisense RNA or siRNA for spatiotemporally controlled degradation or translation blockade of RNA; 5) uncaching fluorophores for spatiotemporally controlled detection of RNA and DNA, detection / separation of HiBiT-edited cells, detection of the transfer of HiBiT-tagged proteins to cell compartments expressing LgBiT-HaloTag-catalysts, etc.; and 6) other applications utilizing the systems, methods, and components of this specification are within the scope of this technology.

[0138] In some embodiments, provided herein are methods of activation dependent on the proximity of an activatable molecule within a cell, the method comprising bringing a cell into contact with a luminescent phost under conditions that allow the luminescent phost to enter the cell, the cell comprising: (a) a fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phost by interaction with the luminescent phost; (b) a conjugate of (A) a capture ligand and (B) a photocatalyst or photosensitizer, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of a first wavelength; and (c) an activatable molecule incorporating a photoresponsive moiety that limits its activity, which is converted into an active molecule when in proximity to the activated photocatalyst or photosensitizer, either through cleavage of a photodissociable protecting group and release of the active molecule, or a transient conformational change that turns on its activity.

[0139] In some embodiments, provided herein are methods for inducing activation dependent on the proximity of a functional molecule for subsequent interaction with a target molecule, the method comprising bringing a cell into contact with a luminescent phostium under conditions that allow the luminescent phostium to enter the cell, the cell comprising: (a) a fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phostium by interaction with the luminescent phostium; (b) a conjugate of (A) a capture ligand and (B) a photocatalyst or photosensitizer, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of a first wavelength; (c) an activatable molecule which is converted into an activating molecule when it is in proximity to the activated photocatalyst or photosensitizer; and (d) a target molecule which binds to and interacts with the target molecule when the activating molecule is in proximity to the target molecule. In some embodiments, the interaction between the activating molecule and the target molecule results in activation, inhibition, degradation, detection, etc.

[0140] In some embodiments, provided herein are methods of activation dependent on the proximity of an activatable molecule within a cell, the methods comprising: (a) expressing a fusion of a bioluminescent protein and a capture protein within a cell; (b) contacting a cell with a luminescent phostem under conditions that the luminescent phostem enters the cell, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phostem by interaction with the luminescent phostem; (c) contacting a cell with a conjugate of (i) a capture ligand and (ii) a photocatalyst or photosensitizer under conditions that the conjugate enters the cell, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of a first wavelength; and (d) contacting a cell with an activatable molecule, wherein the activatable molecule is converted into an activating molecule when it comes into proximity with an activated photocatalyst or photosensitizer.

[0141] In some embodiments, provided herein are methods for inducing activation dependent on the proximity of a functional molecule for subsequent interaction with a target molecule, the methods comprising: (a) expressing a fusion of a bioluminescent protein and a capture protein in a cell; (b) contacting the cell with a luminescent phore under conditions in which the luminescent phore enters the cell, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phore by interaction with the luminescent phore; (c) contacting the cell with a conjugate of (i) a capture ligand and (ii) a photocatalyst or photosensitizer under conditions in which the conjugate enters the cell, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of a first wavelength; and (d) contacting the cell with an activatable molecule, wherein the activatable molecule is converted into an activating molecule when it is in proximity to an activated photocatalyst or photosensitizer, and the activating molecule interacts with a target molecule when it is in proximity to the target molecule.

[0142] In some embodiments, provided herein are methods for activation dependent on the proximity of a photocatalyst or photosensitizer within a cell, the method comprising bringing a cell into contact with a luminescent phose under conditions that allow the luminescent phose to enter the cell, the cell comprising: (a) a fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phose through interaction with the luminescent phose; and (b) a conjugate of (A) a capture ligand and (B) a photocatalyst or photosensitizer, wherein the capture protein forms a covalent bond with the capture ligand through interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of a first wavelength.

[0143] In some embodiments, provided herein are methods for activation dependent on the proximity of a photocatalyst or photosensitizer within a cell, the methods comprising: (a) expressing a fusion of a bioluminescent protein and a capture protein within a cell; (b) contacting a cell with a luminescent phostem under conditions that allow the luminescent phostem to enter the cell, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phostem by interaction with the luminescent phostem; and (c) contacting a cell with a conjugate of (i) a capture ligand and (ii) a photocatalyst or photosensitizer under conditions that allow the conjugate to enter the cell, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of a first wavelength.

[0144] Figure 7 illustrates one exemplary general embodiment of the systems and methods described herein. In this embodiment, a first component of the bioluminescent complex (e.g., the LgBiT component of NanoBiT) is conjugated (e.g., fused) to a capture protein (e.g., HALOTAG). In some forms of this embodiment, the first component of the bioluminescent complex and the capture protein are expressed as a fusion in the cell. The photocatalyst is linked to a capture ligand (e.g., including a haloalkane). In some embodiments, the photocatalyst linked to the capture ligand can be added extracellularly and enter the cell (e.g., without permeabilizing the cell) to form a covalent bond with the capture protein. Exposure of the first component of the bioluminescent complex to a second component of the bioluminescent complex (e.g., HiBiT) and a suitable luminescent phore (e.g., flimazine, fluoroflimazine, etc.) results in the formation of an active bioluminescent complex and the emission of light. Exposure of the photocatalyst to light emitted from the bioluminescent complex activates the photocatalyst. The activated photocatalyst then participates in energy transfer events with photocaged molecules in its vicinity (e.g., molecules incorporating photodissociable protecting groups that limit their activity), inducing their photocatalytic decaching via the cleavage of the protecting groups. The released molecules are then available for interaction with and / or detection of biomacromolecules in their surrounding environment.

[0145] Figure 8 shows a similar exemplary general embodiment of the systems and methods illustrated in Figures 7 and 8, except that the activatable molecules incorporate a photoswitch that limits their activity. Upon binding with the activatable molecules, the molecules undergo a reversible conformational change, turning on their activity.

[0146] In the embodiments shown in Figures 7 and 8, a bioluminescent complex is used as the light source. In other embodiments, such as those illustrated in Figure 9b, a bioluminescent protein is used instead of the bioluminescent complex. The choice between the bioluminescent complex or the protein is determined based on the specific application. Where applicable, embodiments described for use with one bioluminescent entity herein may also be found for use with other bioluminescent entities herein or with other bioluminescent entities as understood in the art.

[0147] In some embodiments, the complementary use of HiBiT / LgBiT for forming bioluminescent complexes offers various advantages compared to other systems (e.g., those utilizing lasers or LEDs as light sources) or the systems of this specification that utilize bioluminescent proteins as light sources. For example, the use of a HiBiT / LgBiT complementary system (or other NanoBiT or NanoTrip-based complementary systems) as the primary light source, combined with a broad toolkit of photocatalytic / activatable molecules, offers several advantages within living cells or other biological systems. HiBiT is a small, minimally damaging tag suitable for tagging endogenous target proteins. Linking LgBiT to a photocatalyst (e.g., via HALOTAG or another capture system) provides, for example, the induction of proximity between the catalyst and the target protein tagged with HiBiT, the induction of proximity between the catalyst and the bioluminescent source (HiBiT / LgBiT), the generation of higher spatiotemporal resolution through conditional activation at a specific site (+flimazine) (HiBiT / LgBiT complementation), the utilization of a convenient approach to link the catalyst to the HaloTag-LgBiT fusion in a specific compartment expressing the fusion in a cell, either chloroalkane chemistry-biochemically or in a cellular manner, and a modality-independent approach to delivering the photocatalytic system to a site of interest (e.g., intracellular or extracellular). The use of bioluminescent complexes (or bioluminescent proteins) provides localized delivery of light of an appropriate wavelength (e.g., blue light) for catalytic activation within intact cells or other complex models. Other embodiments of this specification utilize SmBiT / LgBiT complementary systems, HiBiT / Trip9 / LgTrip complementary systems, or other complementary systems that require external complementation (e.g., facilitation) to form bioluminescent complexes. For example, since SmBiT and LgBiT do not form active bioluminescent complexes without facilitation, the use of such components in the systems / methods of this specification can be used with the requirement of additional localization events (e.g., binding of an element conjugated (directly or indirectly) to SmBiT to an element conjugated (directly or indirectly) to LgBiT) to generate light for activating the photocatalyst.

[0148] In other embodiments, the use of bioluminescent proteins offers various advantages compared to other systems (e.g., those utilizing lasers or LEDs as light sources) or the systems of this specification that utilize bioluminescent complexes as light sources. For example, bioluminescent proteins (e.g., NANOLUC) provide a single-entity light source that can be expressed in cells (e.g., alone or as a fusion with other components of the systems of this specification). In certain embodiments of this specification, the improved simplicity / efficiency of a single-entity light source is preferred over embodiments that require complements.

[0149] Figures 7 and 8 illustrate a system that enables bioluminescence-induced spatiotemporal photocatalytic activation of activatable molecules in intact cells for interaction with and / or detection of biomolecules in their surrounding environment.

[0150] Such embodiments may be used in the detection, activation, inactivation, and degradation of proximal proteins and nucleic acids, as well as in the exploration and modification of biological processes and signaling pathways.

[0151] Figure 9 illustrates a system for bioluminescence-induced spatiotemporal fluorescence turn-on of azide quenching fluorophores. The systems and methods illustrated in Figures 7 and 8, which utilize the complementarity between HiBiT genetically fused to the target protein and LgBiT genetically fused to a HaloTag and linked to a catalyst, are used herein to localize the light source, catalyst, and target site.

[0152] A similar system illustrated in Figure 9B is useful for targeting a photocatalytic system to a desired DNA locus. In such embodiments, a CRISPR enzyme conjugate (i.e., dCas-NanoLuc-HaloTag-catalyst) is used in combination with gRNA to target a specific DNA / RNA locus. Similar systems having other protein-binding or nucleic acid-binding proteins conjugated to components of this system are within the scope of this specification. In the CRISPR / dCas system, a guide RNA (gRNA) binds to a specific endogenous target DNA / RNA sequence within the cell. A mutant dCas9 enzyme binds to a complex of gRNA and the target sequence. In some embodiments, by ligating a bioluminescent protein (NanoLuc) or components of a bioluminescent complex to components of the CRISPR system, and by ligating the photocatalyst to a bioluminescent protein (NanoLuc) or components of a bioluminescent complex (e.g., directly or via HaloTag and HaloTag ligand), the photocatalyst can be activated near the target DNA / RNA. While Cas9 / dCas9 is the most commonly used enzyme in CRISPR, other Cas and dCas enzymes (e.g., dCas12, dCas13, etc.) can be used in the systems described herein to target nucleic acids.

[0153] Figure 10 illustrates a similar system for detecting nucleic acids within cells using a photocaged fluorophore conjugated to an antisense oligo that can further hybridize with a specific nucleic acid sequence. The bioluminescence-induced photocatalytic system can localize to a proximal nucleic acid sequence either through complementarity between a gRNA conjugated to a dCas9-NanoLuc-HaloTag fusion linked to the catalyst, or between an antisense oligo conjugated to HiBiT and LgBiT genetically fused to the HaloTag and linked to the catalyst. When the photocatalyst is exposed to light emitted from the bioluminescent enzyme / complex, the photocatalyst is activated, and this photocatalyst subsequently releases the fluorophore by participating in photocatalytic cage decaching via cleavage of a protecting group. [Examples]

[0154] Example 1 During the development of the bioluminescence-induced photocatalytic activation described herein, experiments were conducted to evaluate the effects of chloroalkane length and structure on iridium catalytic properties, including energy transfer efficiency (from NanoLuc to catalyst), binding reaction rate to HaloTag, and cell permeability (Figure 11). The structures of the modifiable catalyst Ir-8844 and its derivatives further conjugated with chloroalkanes of different lengths are shown in Figure 11A. The synthesis of these catalysts is described in Example 11.

[0155] The effects of chloroalkanes on the physiological and chemical properties of iridium catalysts and their ability to be activated by light emitted from NanoLuc (Figure 11B) were identified using the following analysis: a. Excitation and emission profiles: The excitation and emission spectra of 200 μM of catalyst in 100% DMSO were monitored using a SPARK multimode plate reader with the following settings: 380 nm excitation for emission scan, and 480 nm, 560 nm, or 600 nm emission for excitation scan. b. The luminescence energy (EmE) is calculated using Planck's equation λ Em It was calculated from that.

number

number

Number

Number

[0156] These analyses revealed that chloroalkanes increase the energy transfer efficiency from NanoLuc to the catalyst (2 - 7 fold) in a manner inversely correlated with the length of the chloroalkane. Since the chloroalkanes did not affect the emission energy of the catalyst (EmE), these results indicate that chloroalkanes increased the ability of the catalyst to absorb light in a manner inversely correlated with the length of the chloroalkane.

[0157] Chloroalkanes provide a means of inducing proximity between a catalyst and a bioluminescent light source via covalent binding of a chloroalkane-catalyst conjugate to a HaloTag genetically fused to the light source. The effect of chloroalkane length and structure on the binding reaction rate to HaloTag (Figure 11C) was evaluated by treating lysates prepared from cells expressing the HaloTag fusion protein with chloroalkane-catalyst conjugates at a final concentration of 2 μM. At incubation times of 0 to 120 minutes, fractions of each reaction (each containing a different chloroalkane-catalyst conjugate) were removed and treated with HaloTag TMR-fluorescent ligand (Promega) at a final concentration of 5 μM. This allowed binding of the fluorescent ligand to any unbound HaloTag fusion protein. Fractions at each time point were separated by SDS-PAGE and scanned with a Typhoon fluorescence imager (GE healthcare). Bands were quantified using ImageQuant (GE healthcare), and the binding reaction rate was determined as the percentage of binding over time compared to time zero when no chloroalkane-catalyst conjugate was added. All chloroalkane-catalyst conjugates showed similar binding reaction rates to HaloTag regardless of chloroalkane length, indicating that the effect of chloroalkane length on the binding reaction rate was minimal.

[0158] The effect of chloroalkane length and structure on the cell permeability of catalytic conjugates was further evaluated by the rate of their binding reaction to HaloTag intracellularly (Figure 11D). For this purpose, cells expressing HaloTag fusion proteins were treated with a chloroalkane-catalytic conjugate at a final concentration of 2 μM for 0–180 minutes, followed by treatment with a HaloTag TMR-fluorescent ligand at a final concentration of 5 μM for a further 15 minutes. This allowed the fluorescent ligand to bind to any unbound HaloTag fusion protein. The cells were then collected, lysed in surfactant lysis buffer, and analyzed at each time point as described above. This analysis revealed that shorter chloroalkanes minimized the effect on cell permeability and allowed for a faster binding reaction rate to HaloTag intracellularly.

[0159] Example 2 During the development of the bioluminescence-induced photocatalytic activation described herein, experiments were conducted to evaluate the effects of chloroalkane length and structure on ruthenium catalytic properties, including energy transfer efficiency (from NanoLuc to catalyst), binding reaction rate to HaloTag, and cell permeability (Figure 12). The structures of the modifiable catalyst Ru-8975 and its derivatives further conjugated with chloroalkanes of different lengths are shown in Figure 12A. The synthesis of these catalysts is described in Example 11.

[0160] The effects of chloroalkanes on the physiological and chemical properties of the ruthenium catalyst and its ability to be activated by light emitted from NanoLuc (Figure 11B) were identified using the analysis described in Example 1. These analyses revealed that the redshifted emission of the ruthenium catalyst resulted in a lower emission energy (EmE) compared to that of the ruthenium catalyst. Simultaneously, greater overlap between redshifted excitation and NanoLuc emission significantly increased the energy transfer efficiency from NanoLuc to the catalyst, and subsequently, the effect of chloroalkanes on energy transfer efficiency was significantly reduced (1 to 1.3 times).

[0161] The effect of chloroalkane length and structure on the cell permeability of the catalytic conjugate was further evaluated by the rate of their binding reaction to HaloTag in the cytosol and nucleus of the cells described in Example 1 (Figures 11C and D). This analysis revealed that two relatively short chloroalkanes had similar effects on catalytic binding reaction rate and cell permeability.

[0162] Example 3 This embodiment describes further optimizations that provide means for inducing proximity between a bioluminescent photocatalytic complex comprising a bioluminescent energy donor, a chloroalkane-catalyst conjugate, and a HaloTag (Figure 13). To increase proximity, we designed a chimeric structure containing a cyclically substituted NanoLuc (e.g., cpNLuc at residue 67 / 68) or a cyclically substituted LgBiT mutant (e.g., cpmLgBiT at residue 67 / 68) incorporating four mutants E4D, Q42M, M106K, and T144D from LgTrip, inserted into the surface loop of the HaloTag (between residues 178-179) adjacent to the ligand interaction site (i.e., HT 178 -cpNLuc- 179 and HT 178 -cpmLgBIT- 179 (Figure 13A). First, NanoLuc-HaloTag and Chimera HT 178 -cpNLuc- 179 We compared the efficiency of bioluminescence resonance energy transfer (BRET) to the bound HaloTag TMR-fluorescent ligand between NanoLuc-HaloTag and HaloTag TMR-fluorescent ligand. For this purpose, we compared NanoLuc-HaloTag and HaloTag TMR-fluorescent ligands, both unconjugated and conjugated HT. 178 -cpNLuc- 179The solution was diluted to a final concentration of 6.6 nM in TBS + 0.01% BSA, and then treated with 10 times the amount of fluoroflimazine to a final concentration of 20 μM. After a 3-minute incubation, the raw emission (total RLU) or filtered emission of the donor (e.g., 450 nm / 8 nm BP) and acceptor (600 nm LP) was measured using a GloMax® Discover plate reader (Promega). The BRET ratio was further calculated for each sample by dividing the acceptor emission value by the donor emission value. HT 178 -cpNLuc- 179 Although it was a 10-fold dimer, it showed a 24-fold higher BRET efficiency (Figure 13B), indicating that the chimeric structure was able to induce higher proximity between the substrate binding site of NanoLuc and the bound fluorescent ligand, or to adopt a conformation favorable to energy transfer between the two, or both. Similar analyses were performed on LgBiT-HaloTag and chimeric HT. 178 -cpmLgBiT- 179 This study examined LgBiT-HaloTag and chimeric HT, which are either unconjugated or conjugated to HaloTag TMR-fluorescent ligands. 178 -cpmLgBiT- 179 The first solution was diluted to a final concentration of 13 nM in TBS + 0.01% BSA, supplemented with an equal volume of 130 nM HiBiT peptide for 30 minutes, and then treated with 10x fluoroflimazine at a final concentration of 20 μM. After a 3-minute incubation, the raw luminescence (total RLU) or BRET was measured as described above. The result was HT, a 100-fold dimer. 178 -cpmLgBiT- 179 Even then, it showed a 10-fold higher BRET efficiency (Figure 13B), indicating that the chimeric structure was able to induce greater proximity between the substrate binding site and the bound fluorescent ligand of the HiBiT / LgBiT complex, or adopt a conformation favorable to energy transfer between the two, or both.

[0163] Example 4 This embodiment demonstrates the feasibility of a bioluminescent photocatalyst complex assembled intracellularly to drive cage uncage of an azide quenching fluorophore (Figures 14 and 15). Figure 9 includes illustrations of the structure of an exemplary azide quenching fluorophore and the bioluminescence-induced fluorescence turn-on. Briefly, upon absorbing light, the excited catalyst participates in an energy transfer event with the azide quenching fluorophore, generating a nitrene through the removal of an N2 group, thereby restoring the fluorophore's fluorescence. HeLa cells were subjected to 50-fold dilution of HT in promoter-free carrier DNA. 178 -cpNLuc- 179 Transfect with a DNA construct encoding 2 × 10 5 Cells were plated into flasks at a concentration of cells / mL and incubated at 37°C in 5% CO2 for 16-18 hours. The following day, cells were identified and measured in 2x10⁶ units. 5 Cells / mL were replated into 24-well plates and incubated overnight at 37°C and 5% CO2. The following day, the plates were treated with either Ir-9049 catalyst, Ru-8974 catalyst, or chloroalkane-biotin (control) at a final concentration of 3 μM for 90 minutes to construct bioluminescent photocatalytic complexes within the cells. Cells were washed twice in HBSS buffer for 15 minutes each to remove excess unreacted catalyst or chloroalkane-biotin. After the final wash, the HBSS wash solution was replaced with Opti-MEM medium supplemented with 2% serum and 2–20 μM azide-quenched coumarin (PBI-8977, synthesized as in Example 11) or 2–20 μM azide-quenched ethidium bromide (ethidium bromide monoazide (EMA), Thermo Fisher). After 30 minutes of incubation, cells were treated with 20 μM fluoroflimazine for 45 minutes. After washing the cells twice, they were imaged using a BZ-X800 analyzer (Keyence) and image analysis was performed using cell profiler software. Images of cells treated with either PBI-8977 (Figure 14B-C) or EMA (Figure 15B-C) revealed a significant catalyst-dependent increase in fluorescence, demonstrating the ability of the bioluminescent photocatalytic complex to drive the cage uncaching of intracellular azide quenching fluorophores.

[0164] Example 5 This example shows a caging transition metal complex [Ru] in a biochemical setting. 2+ (bpy)2] 2 This demonstrates the feasibility of bioluminescence-induced release of signaling molecules from [Ru]. As illustrated in Figure 16A, the amino signaling molecule serotonin [Ru] 2+ (bpy)2] 2 It is caged through a coordination reaction with the core. Upon absorbing light, the excited ruthenium catalyst induces the oxidation-driven release of serotonin, while water molecules occupy the empty coordination sites.

[0165] Here, 6 nM NanoLuc (Nluc) enzyme and 5 μM fluoroflimazine were incubated in a white plate for 40 minutes in the presence and absence of 200 μM RuBi5 (Abcam). The reaction was then transferred to a black plate, and 200 μM RuBi5 (caged) was added to the control well. Fluorescence scanning using 260 nm and 450 nm excitation with a SPARK multimode plate reader (Tecan) revealed a bioluminescence-dependent increase in the 340 nm peak emission of serotonin. These results demonstrate that bioluminescence induces oxidation-driven release of caged molecules from the organometallic saging moiety.

[0166] Example 6 This example demonstrates the feasibility of bioluminescence-induced cage release during Ru-catalyzed p-azidobenzyl reduction in a biochemical setting (Figure 17). As shown in Figure 17A, luciferin is released by bioluminescence-induced ruthenium-catalyzed reduction of p-azidobenzyl caged luciferin, which is then available as a substrate for firefly (FFLY) luciferase. The synthesis of p-azidobenzyl caged luciferin is provided in Example 11. Briefly, it consists of TBS + 0.01% BSA + 1 mM NaAscorbate and 0-10 μM p-azidobenzyl-luciferin containing 60 nM HT linked to or unlinked to Ru-8974. 178 -cpNLuc- 179The reaction mixture containing the fluorine was incubated in a 96-well plate for 45 minutes with or without 20 μM fluoroflimazine. The residual NanoLuc bioluminescence signal was quenched by treatment with 10 mM competitive extracellular NanoLuc inhibitor and then diluted 5-fold in an FFLY detection plate containing FFLY luciferase, ATP, and Bright-Glo buffer. Bioluminescence was measured before and after dilution to the FFLY detection plate. The Ru-dependent increase in bioluminescence and FFLY-dependent bioluminescence demonstrates the ability of bioluminescence to drive photocatalytic cage uncage of p-azidobenzyl caged molecules. Furthermore, the higher efficiency of bioluminescence-driven cage uncage compared to LED further demonstrates the advantages of proximity-driven catalytic activation via BRET, which provides highly efficient light delivery to the site of interest.

[0167] Example 7 This example is in a biochemical setting. O - To demonstrate the feasibility of bioluminescence-induced cage release excitation and subsequent photodegradation of nitrobenzyl (Figure 18). As shown in Figure 18A, O - Bioluminescence-induced ruthenium-catalyzed excitation of nitrobenzyl-caged F-luciferin releases F-luciferin, which is then available as a substrate for FFLY luciferase. O - The synthesis of nitrobenzyl-caged F-luciferin is provided in Example 11. Briefly, it involves TBS + 0.01% BSA + 1 mM NaAscorbate and 0-10 μM O - 60 nM HT ligated to or unligated to Ru-8974 in nitrobenzyl-caged F-luciferin 178 -cpNLuc- 179The reaction mixture containing was incubated in a 96-well plate for 45 minutes with or without 20 μM fluoroflimazine. The residual NanoLuc bioluminescence signal was quenched by treatment with 10 mM competitive extracellular NanoLuc inhibitor, and then diluted 5-fold in an FFLY detection plate containing FFLY luciferase, ATP, and Bright-Glo buffer. Bioluminescence was measured before and after dilution to the FFLY detection plate. The Ru-dependent increase in bioluminescence and FFLY-dependent bioluminescence was observed. O - Demonstrates the ability of bioluminescence to drive photocatalytic cage decaching of nitrobenzyl caged molecules. Furthermore, the higher efficiency of bioluminescence-driven cage decaching compared to LEDs further demonstrates the advantages of proximity-driven catalytic activation via BRET, which provides highly efficient light delivery to the target site.

[0168] Example 8 The embodiment illustrated in Figure 19 depicts bioluminescence-induced photocatalytic cage release upon excitation of the cazing coumarin moiety. HT is coupled to the catalyst. 178 -cpNLuc- 179 When a bioluminescent complex containing is treated with fluoroflimazine, the excited catalyst participates in an energy transfer event with proximal coumarin-4-methyl-caged ibrutinib. The excited coumarin-4-methyl then undergoes further photolysis to release ibrutinib.

[0169] Example 9 The example shown in Figure 20 illustrates a bioluminescence-induced transient conformational change that turns on the reactivity of the effector molecule with the target biomolecule. HT linked to the catalyst 178 -cpNLuc- 179 When a bioluminescent complex containing is treated with fluoroflimazine, the excited catalyst participates in photosensitization of the azobenzene moiety, and when excited, the azobenzene moiety possesses a significantly lower energy barrier to rotation around the N=N double bond, allowing for subsequent conformational changes that affect either the distance and / or orientation of R1 and R2 relative to each other.

[0170] Example 10 In this example, the synthesis of the catalysts and activatable molecules described in this specification will be explained. [Table 2-1] [Table 2-2]

[0171] Synthesis of Ir catalyst: [Chemical formula] {Ir[dFCF3ppy]2Cl}2 is commercially available from Strem: www.strem.com / catalog / v / 77-0468 / 31 / iridium_870987-64-7, and {Ir[dFCF3(CO2H)ppy]2Cl}2 was synthesized according to the procedure reported in the literature: Science 367, 1091-1097 (2020).

[0172] GP1: The bi-Ir-Cl complex (0.1 mmol, 1.0 equivalent) was combined with AgOTf (53 mg, 0.2 mmol, 2.0 equivalents) in CH3CN (5 mL). This mixture was stirred overnight at room temperature in the dark. Next, the resulting suspension was filtered through Celite and concentrated. The residue was redissolved in DCM / MeOH (1 / 1, 10 mL), filtered through Celite, and concentrated to obtain intermediate 3 or 4 as a yellow film, which was used without further purification.

[0173] To a solution of intermediate 3 or 4 (0.1 mmol, 1.0 equivalent) in DCM / MeOH (1 / 1, 2 mL), bpy was added as a reactant (0.12 mmol, 1.2 equivalents). Then, the reaction mixture was stirred at room temperature for 16 hours. LC-MS indicated that intermediate 3 or 4 was completely converted. The solution was evaporated on Celite and purified by silica gel chromatography.

[0174] Ir-8844: 1H NMR (400 MHz,methylene chloride-d2) δ 8.48 (d, J = 8.7 Hz, 2H), 8.37 (d, J = 2.5 Hz, 2H), 8.07 (d, J = 8.9 Hz, 2H), 7.70 (d, J = 6.2 Hz, 4H), 7.12 - 6.98 (m, 2H), 6.73 - 6.57 (m, 2H), 5.73 (dd, J = 8.1, 2.2 Hz, 2H), 4.65 - 4.43 (m, 4H), 3.94 (d, J = 4.5 Hz, 4H), 3.79 - 3.42 (m, 16H). LRMS[M] + 1161.1.

[0175] bpy-1 synthesis [ka] Intermediate 8 was synthesized from commercially available starting material 7 according to the literature procedure: Science 367, 1091-1097 (2020).

[0176] Intermediate 9: NaH (60 wt%, 56 mg, 1.4 mmol, 2.0 equivalents) was added to a THF solution (7 mL) of intermediate 8 (200 mg, 0.7 mmol, 1.0 equivalent). The mixture was stirred at room temperature for 30 minutes. To the suspension, NaI (11 mg, 0.07 mmol, 0.1 equivalent) and 2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran (350 mg, 1.4 mmol, 2.0 equivalents) in DMF (3 mL) were added dropwise over 10 minutes. The mixture was then heated at 60°C for 48 hours. The reaction mixture was cooled and quenched with saturated NH4Cl aqueous solution (10 mL). The quenched reaction mixture was then concentrated under vacuum to remove the organic solvent and extracted with SiO2 (20 × 3 mL). The combined organic layers were washed with H2O (50 mL) and brine (50 mL), dried over Na2SO4, and concentrated to obtain the crude product, which was used in the next step without further purification.

[0177] bpy-1: Intermediate 9 (50 mg, 0.1 mmol, 1.0 equivalent) and TsOH ·H2O (19 mg, 0.1 mmol, 1.0 equivalent) was dissolved in MeOH (4 mL). The solution was stirred at room temperature for 2 hours. LC-MS showed complete conversion. The reaction mixture was concentrated on Celite, and the desired product was isolated using silica gel chromatography. 1 H NMR (400 MHz,chloroform-d) δ 8.64 (d, J = 5.1 Hz, 2H), 8.38 (d, J = 5.5 Hz, 2H), 7.41 (ddd, J = 19.4, 5.0, 2.2 Hz, 2H), 3.83 - 3.53 (m, 10H), 3.38 (t, J = 5.2 Hz, 2H), 3.13 (s, 3H), 1.59 (s, 6H), 1.56 (s, 6H). LRMS[M+H] + 419.5.

[0178] bpy-2 synthesis [ka] bpy-2: To a suspension of bpy10 (376 mg, 2.0 mmol, 1.0 equivalent) and K2CO3 (830 mg, 6.0 mmol, 3.0 equivalents) in DMF (5 mL), 2-(2-(2-chloroethoxy)ethoxy)ethane-1-ol (1.0 g, 6.0 mmol, 3.0 equivalents) was added dropwise over 5 minutes. The mixture was heated at 60°C for 20 hours, cooled to room temperature, diluted with ELISA (100 mL), and filtered through Celite. The filtrate was concentrated under vacuum to obtain the crude product. The desired product, bpy-2, was isolated by silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 8.46 (dd, J = 5.8, 1.8 Hz, 2H), 7.86 (d, J = 2.2 Hz, 2H), 7.05 (dt, J = 5.3, 2.3 Hz, 2H), 4.34 (t, J = 4.2 Hz, 4H), 3.92 (p, J = 2.2 Hz, 4H), 3.81 - 3.47 (m, 16H). LRMS[M+H] + 453.3.

[0179] Synthesis of Ir-8972 [ka] Intermediate 16: To a solution of bpy-2 (25 mg, 0.06 mmol, 1.0 equivalent) in THF (4 mL), pyridine (0.5 mL) and p-nitrophenyl chloroformate (15 mg, 0.07 mmol, 1.2 equivalents) were added. The solution was stirred overnight at room temperature. The reaction mixture was diluted with DCM (10 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain the crude product, intermediate 15, which was used in the next step without further purification.

[0180] To a solution of the crude product 15 (54 mg, 87 μmol, 1.0 equivalent) in ACN (2 mL), chloroalkaneamine reagent (15 mg, 92 μmol, 1.1 equivalents) and NEt3 (0.2 mL) were added. The solution was stirred overnight at room temperature and concentrated on Celite. The desired product was isolated using silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 8.47 (d, J = 5.8 Hz, 2H), 7.88 (d, J = 2.6 Hz, 2H), 7.07 (dd, J = 5.6, 2.6 Hz, 2H), 4.35 (t, J = 4.4 Hz, 4H), 4.17 (d, J = 5.0 Hz, 2H), 3.93 (q, J = 3.3 Hz, 4H), 3.77 - 3.64 (m, 12H), 3.61 - 3.43 (m, 12H), 3.30 -3.28 (m, 2H), 1.76 (t, J = 7.1 Hz, 2H), 1.66 - 1.30 (m, 6H). LRMS [M + H] + 702.3.

[0181] Ir-8972: Intermediate 3 (10.6 mg, 11 μmol, 1.0 equivalent) and Intermediate 16 (8 mg, 12 μmol, 1.1 equivalent) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred overnight at room temperature. The desired product was isolated by preparative HPLC using 0.1% TFA and ACN in H2O as the mobile phase.1 H NMR (400 MHz, methanol-d4) δ 8.56 (d, J = 9.0 Hz, 2H), 8.42 - 8.24 (m, 4H), 7.82 (d, J = 6.3 Hz, 4H), 7.25 (d, J = 6.8 Hz, 2H), 6.79 (t, J = 10.9 Hz, 2H), 5.77 (d, J = 8.1 Hz, 2H), 4.45 (d, J = 4.7 Hz, 4H), 4.07 (s, 2H), 3.90 (d, J = 4.7 Hz, 4H), 3.77 - 3.40 (m, 24H), 3.30 - 3.23 (d, J = 6.1 Hz, 2H), 1.78 - 1.66 (m, 2H), 1.56 (t, J = 7.2 Hz, 2H), 1.43 - 1.35 (m, 4H). LRMS[M] + 1410.4.

[0182] Synthesis of Ir-8973 [ka] Intermediate 17: To a solution of crude product 15 (30 mg, 49 μmol, 1.0 equivalent) in ACN (2 mL), chloroalkaneamine reagent (23 mg, 49 μmol, 1.0 equivalent) and NEt3 (0.2 mL) were added. The solution was stirred overnight at room temperature and concentrated on Celite. The desired product was isolated using silica gel chromatography. 1H NMR (400 MHz, methanol-d4) δ 8.47 (d, J = 5.8 Hz, 2H), 7.88 (t, J = 2.7 Hz, 2H), 7.06 (d, J = 5.7 Hz, 2H), 4.34 (d, J = 4.8 Hz, 4H), 4.16 (d, J = 4.9 Hz, 4H), 3.93 (h, J = 2.9 Hz, 4H), 3.82 - 3.44 (m, 38H), 3.33 - 3.25 (m, 2H), 1.77 (t, J = 7.3 Hz, 2H), 1.60 (t, J = 7.1 Hz, 2H), 1.44 (dq, J = 23.7, 7.7 Hz, 4H). LRMS [M + H] + 921.4.

[0183] Ir-8973: Intermediate 3 (4.3 mg, 4.6 μmol, 1.0 equivalent) and Intermediate 17 (4.2 mg, 4.6 μmol, 1.0 equivalent) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred overnight at room temperature. The desired product was isolated by silica column elution using DCM / MeOH as the eluent. 1 H NMR (400 MHz, methanol-d4) δ 8.56 (d, J = 8.9 Hz, 2H), 8.42 - 8.25 (m, 4H), 7.82 (d, J = 4.9 Hz, 4H), 7.25 (d, J = 6.4 Hz, 2H), 6.97 - 6.68 (m, 4H), 5.85 - 5.68 (m, 2H), 4.49 - 4.41 (m, 4H), 4.15 - 4.04 (m, 4H), 3.94 - 3.85 (m, 4H), 3.73 - 3.41 (m, 38H), 3.30 - 3.24 (m, 2H), 1.81 - 1.68 (m, 2H), 1.63 - 1.50 (m, 2H), 1.50 - 1.26 (m, 4H). LRMS[M+H] + 1410.4.

[0184] Synthesis of Ir-9049 [ka] To a 5 mL solution of intermediate 21:10 (94 mg, 0.5 mmol, 1.0 equivalent) in DMF, NaI (7.5 mg, 0.05 mmol, 0.1 equivalent), 1-chloro-2-(2-(2-methoxyethoxy)ethoxy)ethane (110 mg, 0.6 mmol, 1.2 equivalents), and K2CO3 (207 mg, 1.5 mmol, 3.0 equivalents) were added. The suspension was heated overnight at 60°C. After cooling, the mixture was diluted with SiO2 (50 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain the crude product. The desired product was isolated by silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 8.63 (d, J = 5.9 Hz, 1H), 8.46 (dd, J = 6.7, 2.1 Hz, 1H), 7.90 (s, 1H), 7.85 (s, 1H), 7.32 - 7.23 (m, 1H), 7.20 (d, J = 6.7 Hz, 1H), 4.43 (t, J = 4.6 Hz, 2H), 3.93 (p, J = 2.2 Hz, 2H), 3.76 - 3.47 (m, 8H), 3.35 (s, 3H). LRMS[M+H] + 335.4.

[0185] To a 5 mL solution of bpy-8:21 (25 mg, 75 μmol, 1.0 equivalent) in DMF, bromoethanol (47 mg, 374 μmol, 5.0 equivalents), NaI (1.2 mg, 7.5 μmol, 0.1 equivalent), and K2CO3 (31 mg, 224 μmol, 3.0 equivalents) were added. The mixture was stirred overnight at 60°C. After cooling, the mixture was diluted with HCl (50 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain the crude product. The desired product was isolated by silica gel chromatography. 1H NMR (400 MHz,chloroform-d) δ 8.66 - 8.44 (m, 2H), 8.13 - 7.75 (m, 2H), 7.00 - 6.85 (m, 2H), 6.79 (brs, 1H), 4.37 - 4.20 (m, 4H), 4.04 - 3.58 (m, 12H), 3.34 (s, 3H). LRMS[M+H] + 379.4.

[0186] bpy-8-CA: To a solution of bpy-8 (16 mg, 0.04 mmol, 1.0 equivalent) in THF (4 mL), pyridine (0.5 mL) and p-nitrophenyl chloroformate (10 mg, 0.05 mmol, 1.2 equivalents) were added. The solution was stirred overnight at room temperature. The reaction mixture was diluted with DCM (10 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was used in the next step without further purification.

[0187] To a solution of the crude product from the previous step in ACN (2 mL), chloroalkaneamine reagent (39 mg, 150 μmol, 3 equivalents) and NEt3 (0.2 mL) were added. The solution was stirred overnight at room temperature and concentrated on Celite. The desired product was isolated by silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 8.48- 8.46 (m, 2H), 7.95 - 7.74 (m, 2H), 7.17 - 6.92 (m, 2H), 4.47 - 4.25 (m, 6H), 3.95 - 3.42 (m, 20H), 3.35 (s, 3H), 3.29 - 3.17 (m, 2H), 1.77 - 1.50 (m, 2H), 1.63 - 1.50 (m, 2H), 1.47 - 1.21 (m, 4H). LRMS[M+H] + 628.3.

[0188] Ir-9049: Intermediate 3 (14 mg, 15 μmol, 1.0 equivalent) and bpy-8-CA (9.4 mg, 15 μmol, 1.0 equivalent) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred overnight at room temperature. The desired product was isolated by silica column chromatography using DCM / MeOH as the eluent. 1 H NMR (400 MHz, methanol-d4) δ 8.58 (d, J = 8.9 Hz, 2H), 8.39 (s, 2H), 8.33 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 6.9 Hz, 4H), 7.27 (s, 2H), 6.81 (t, J = 10.9 Hz, 2H), 5.79 (d, J = 8.0 Hz, 2H), 4.47 - 4.25 (m, 4H), 3.92- 3.78 (m, 2H), 3.75 - 3.58 (m, 20H), 3.32 (s, 3H), 1.78 - 1.70 (m, 2H), 1.63 - 1.50 (m, 2H), 1.47 - 1.37 (m, 4H). LRMS[M+H] + 1336.7.

[0189] Synthesis of Ir-9050 [ka] To a solution of 21 (25 mg, 75 μmol, 1.0 equivalent) in bpy-9:DMF (5 mL), 2-(2-(2-chloroethoxy)ethoxy)ethane-1-ol (63 mg, 374 μmol, 5.0 equivalents), NaI (1.2 mg, 7.5 μmol, 0.1 equivalent), and K2CO3 (31 mg, 224 μmol, 3.0 equivalents) were added. The mixture was stirred overnight at 60°C. After cooling, the mixture was diluted with SiO (50 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain the crude product. The desired product was isolated by silica gel chromatography. 1H NMR (400 MHz, methanol-d4) δ 8.60 - 8.35 (m, 2H), 7.99 - 7.55 (m, 2H), 7.15 - 6.94 (m, 2H), 4.38 - 4.27 (m, 4H), 3.90 (d, J = 4.7 Hz, 4H), 3.76 - 3.47 (m, 16H), 3.30 (s, 3H). LRMS[M+H] + 467.5.

[0190] To a solution of bpy-9 (16 mg, 0.35 mmol, 1.0 equivalent) in bpy-9-[(PEG)4]2-CA:THF (4 mL), pyridine (0.5 mL) and p-nitrophenyl chloroformate (8.3 mg, 0.04 mmol, 1.2 equivalents) were added. The solution was stirred overnight at room temperature. The reaction mixture was diluted with DCM (10 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was used in the next step without further purification.

[0191] To a solution of the crude product from the previous step in ACN (2 mL), chloroalkane reagent (54 mg, 150 μmol, 3 equivalents) and NEt3 (0.2 mL) were added. The solution was stirred overnight at room temperature and concentrated on Celite. The desired product was isolated by silica gel chromatography. LRMS [M + H] + 1154.6.

[0192] Ir-9050: Intermediate 3 (14 mg, 15 μmol, 1.0 equivalent) and bpy-9-CA (9.4 mg, 15 μmol, 1.0 equivalent) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred overnight at room temperature. The desired product was isolated by silica column chromatography using DCM / MeOH as the eluent. 1 H NMR (400 MHz, methanol-d4) δ 8.58 (d, J = 8.9 Hz, 2H), 8.39 (s, 2H), 8.33 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 6.9 Hz, 4H), 7.27 (s, 2H), 6.81 (t, J = 10.9 H

[0193] Synthesis of Ru catalyst: Ru-8975: The desired product was isolated as a diacetate. 1 H NMR (400 MHz, methanol-d4) δ 8.90 (d, J = 8.5 Hz, 1H), 8.83 - 8.70 (m, 4H), 8.31 (d, J = 8.5 Hz, 1H), 8.25 - 8.03 (m, 5H), 7.94 (dd, J = 12.4, 5.6 Hz, 2H), 7.78 (s, 1H), 7.75 - 7.62 (m, 3H), 7.60 - 7.48 (m, 3H), 7.36 (d, J = 6.4 Hz, 2H), 7.03 (s, 1H), 3.81 (t, J = 6.0 Hz, 2H), 3.59 (t, J = 7.1 Hz, 2H), 2.07 (t, J = 6.6 Hz, 2H), 1.90 (s, 6H). LRMS[M] 2+ / 2 333.6.

[0194] Synthesis of phenanthroline-1 [ka] Phenanthroline-1: Phenanthroline-11 (195 mg, 1.0 mmol, 1.0 equivalent) and 3-((tert-butyldimethylsilyl)oxy)propanal (188 mg, 1.0 mmol, 1.0 equivalent) were dissolved in ACN (10 mL), and concentrated H2SO4 (0.1 mL) was added. The solution was stirred at room temperature for 30 minutes, and then NaCNBH3 (94 mg, 1.5 mmol, 1.5 equivalents) was added all at once. The reaction mixture was then stirred at room temperature for a further 3 hours, and then quenched with saturated NaHCO3 aqueous solution (1 mL). The mixture was diluted with H2O (30 mL) and extracted with RINKAN (30 x 3 mL). The combined organic layer was washed with H2O (50 mL) and brine (50 mL), dried over Na2SO4, and concentrated under reduced pressure. The desired product 12 was purified by silica gel chromatography. 1H NMR (400 MHz, methanol-d4) δ 9.18 - 8.92 (m, 1H), 8.80 - 8.51 (m, 2H), 8.12 (d, J = 8.1 Hz, 1H), 7.81 - 7.45 (m, 2H), 6.75 (s, 1H), 3.89 (t, J = 6.0 Hz, 2H), 3.48 (t, J = 6.9 Hz, 2H), 2.18 - 1.88 (m, 2H), 0.92 (s, 9H), 0.09 (s, 6H). LRMS[M+H] + 368.5.

[0195] Phenanthroline intermediate 12 (120 mg, 0.33 mmol, 1.0 equivalent) was dissolved in an aqueous solution of MeOH / 6N HCl (1 / 1, 6 mL). The solution was stirred at room temperature for 6 hours. LC-MS showed that the conversion was complete. The desired product, phenanthroline-1, was isolated by silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 9.07 (d, J = 4.2 Hz, 1H), 8.68 (dd, J = 20.8, 6.3 Hz, 2H), 8.15 (d, J = 8.1 Hz, 1H), 7.74 (dd, J = 9.0, 4.1 Hz, 1H), 7.61 - 7.46 (m, 1H), 6.80 (s, 1H), 3.91 - 3.80 (m, 2H), 3.52 (t, J = 7.1 Hz, 2H), 2.14 - 1.99 (m, 2H). LRMS[M+H] + 254.3.

[0196] Synthesis of Ru-8974 [ka] Intermediate 19: Pyridine (0.5 mL) and p-nitrophenyl chloroformate (13 mg, 0.06 mmol, 1.0 equivalent) were added to a solution of phenanthroline-1 (16 mg, 0.06 mmol, 1.0 equivalent) in THF (4 mL). The solution was stirred overnight at room temperature. The reaction mixture was diluted with DCM (10 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain the crude product, intermediate 18, which was used in the next step without further purification.

[0197] To a solution of crude 18 (26 mg, 62 μmol, 1.0 equivalent) in ACN (2 mL), chloroalkane reagent (16 mg, 62 μmol, 1.0 equivalent) and NEt3 (0.2 mL) were added. The solution was stirred overnight at room temperature and concentrated on Celite. The desired product was isolated using silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 9.07 (d, J = 4.3 Hz, 1H), 8.71 (t, J = 7.6 Hz, 2H), 8.16 (d, J = 8.1 Hz, 1H), 7.75 (dd, J = 8.8, 4.1 Hz, 1H), 7.68 - 7.44 (m, 1H), 6.80 (s, 1H), 4.29 (t, J = 6.3 Hz, 2H), 3.60 - 3.25 (m, 14H), 2.25 - 2.15 (d, J = 6.6 Hz, 2H), 1.81 - 1.68 (m, 2H), 1.63 - 1.50 (m, 2H), 1.43 - 1.17 (m, 6H). LRMS [M + H] + 503.3.

[0198] Ru-8974:(bpy)2RuCl2 (7.0 mg, 14 μmol, 1.1 equivalent) and intermediate 19 (6.6 mg, 13 μmol, 1.0 equivalent) were dissolved in MeOH (2 mL). The solution was stirred overnight at 60°C. The desired product was isolated by silica column using DCM / MeOH as the eluent. 1H NMR (400 MHz, methanol-d4) δ 8.92 (d, J = 8.6 Hz, 1H), 8.81 - 8.60 (m, 4H), 8.32 (d, J = 8.4 Hz, 1H), 8.12 (dt, J = 28.6, 9.3 Hz, 5H), 7.94 (dd, J = 12.0, 5.6 Hz, 2H), 7.82 - 7.62 (m, 4H), 7.55 (p, J = 6.6, 5.9 Hz, 3H), 7.36 (d, J = 6.6 Hz, 2H), 7.04 (s, 1H), 4.36 - 4.15 (m, 2H), 3.67 - 3.41 (m, 14H), 3.30 - 3.17 (m, 2H), 2.25 - 2.15 (m, 2H), 1.81 - 1.68 (m, 2H), 1.63 - 1.50 (m, 2H), 1.43 - 1.17 (m, 6H). LRMS[M] 2+ / 2 458.1.

[0199] Synthesis of Ru-9003 [ka] To a solution of Ru-8975 (10 mg, 13 μmol, 1.0 equivalent) in Ru-9003:ACN (2 mL), pyridine (0.5 mL) and p-nitrophenyl chloroformate (7.7 mg, 39 μmol, 3.0 equivalents) were added. The solution was stirred overnight at room temperature. The reaction mixture was diluted with DCM (10 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain crude intermediate 20, which was used in the next step without further purification.

[0200] The crude intermediate 20 solution was redissolved in ACN (3 mL), and the chloroalkane intermediate (32 mg, 39 μmol, 3.0 equivalents) and NEt3 (0.2 mL) were added. The solution was stirred overnight at room temperature. The desired product was isolated by silica column using DCM / MeOH as the eluent. 1H NMR (400 MHz, メタノール-d4) δ 8.93 (d, J = 8.6 Hz, 1H), 8.81 - 8.66 (m, 6H), 8.33 (d, J = 8.3 Hz, 1H), 8.23 ​​- 8.05 (m, 7H), 7.95 (dd, J = 12.0, 5.6 Hz, 3H), 7.79 (t, J = 7.0 Hz, 1H), 7.68 (dd, J = 15.9, 9.3 Hz, 4H), 7.63 - 7.51 (m, 5H), 7.38 (d, J = 7.0 Hz, 3H), 7.05 (s, 1H), 4.34 - 4.22 (m, 2H), 4.19 - 4.05 (m, 2H), 3.82 - 3.42 (m, 22H), 3.30 - 3.17 (m, 2H), 2.25 - 2.15 (m, 2H), 1.80 - 1.66 (m, 2H), 1.66 - 1.52 (m, 2H), 1.45 - 1.19 (m, 4H). LRMS[M] 2+ / 2 545.8. z, 2H), 5.79 (d, J = 8.0 Hz, 2H), 4.49 - 4.41 (m, 4H), 4.15 - 4.04 (m, 4H), 3.94 - 3.85 (m, 4H), 3.79 - 3.47 (m, 57H), 3,24 - 3.15 (m, 2H), 1.90 - 1.67 (m, 2H), 1.67 - 1.47 (m, 2H), 1.46 - 1.28 (m, 4H). LRMS [M + H] + 1863.3.

[0201] Activation possible and molecular synthesis Synthesis of PBI-8977 (アジド matte クマリン)

change

[0202] Synthesis of PBI-7273 (p-azidobenzyl-luciferin) 6-((4-azidobenzyl)oxy)benzo[d]thiazole-2-carbonitrile [ka] To a solution of 6-hydroxybenzo[d]thiazole-2-carbonitrile (0.1 g, 0.5 mmol) in acetonitrile (30 ml), 1-azido-4-(bromomethyl)benzene (0.12 g, 0.57 mmol), potassium carbonate (0.16 mg, 1.13 mmol), and potassium iodide (0.19 mg, 1.13 mmol) were added. The mixture was refluxed overnight. After cooling, the solvent was evaporated, and the residue was extracted with ethyl acetate / water. The organic layer was collected, and the solvent was evaporated. The residue was purified by silica gel chromatography to obtain the product as a yellowish solid, which was used directly in the next step.

[0203] (S)-2-(6-((4-azidobenzyl)oxy)benzo[d]thiazole-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (PBI-7273) [ka] A solution of 6-((4-azidobenzyl)oxy)benzo[d]thiazole-2-carbonitrile (50 mg, 0.153 mmol) in acetonitrile was mixed with an aqueous solution of D-cysteine ​​(31 mg, 0.195 mmol). Triethylamine was added to adjust the pH of the reaction to 9. The resulting mixture was stirred for 20 minutes and purified directly by preparative HPLC (0.1% TFA aqueous solution and acetonitrile as eluent) to obtain a white powder product. MS: Calculated value: m / z = 412.05 [M+H] + ;Measurement value (ESI): m / z = 412.0 [M+H] + .1H NMR (400 MHz, DMSO-d6) δ: 13.20 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 4.0Hz, 1H), 7.54 (d, 2H), 7.28 (dd, J = 8.0 Hz, 4.0Hz, 1H), 7.17 (d, 2H), 5.43 (t, 1H), 5.21 (s, 2H), 3.74 (m, 2H).

[0204] Synthesis of PBI-4043 (o-nitrobenzyl-fluoro-luciferin) 5-Fluoro-6-((4-nitrobenzyl)oxy)benzo[d]thiazole-2-carbonitrile [ka] This compound was prepared in the same manner as 6-((4-azidobenzyl)oxy)benzo[d]thiazole-2-carbonitride and used directly in the following step.

[0205] (S)-2-(5-fluoro-6-((4-nitrobenzyl)oxy)benzo[d]thiazole-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (PBI-4043) [ka] This compound was prepared using the same method as PBI-7273. MS: Calculated value: m / z = 472.52 [M+H] + ;Measurement value (ESI): m / z = 472.10 [M+H] + .1H NMR (400 MHz, DMSO-d6) δ: 7.3-8.2 (m, 6H), 5.60 (s, 2H), 4.82 (m, 1H), 3.4-3.8 (m, 2H).

[0206] array SEQ ID NO: 1-NanoLuc-MKHHHHHHAIAMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAV Sequence ID 2 - Full Length NanoBiT-MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL Sequence ID 3-HiBiT-VSGWRLFKKIS Sequence ID 4-SmBiT-VTGYRLFEEIL Sequence ID 5-LgBiT-MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSHHHHHH Sequence ID 6-LgTrip-MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPD Sequence ID 7-SmTrip9-GSMLFRVTINS Sequence ID 8-HaloTag-MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG

Claims

1. It is a system, (a) Bioluminescent proteins, (b) A luminescent phosphatid, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phosphatid by interaction with the luminescent phosphatid, (c) A photocatalyst, wherein the photocatalyst is activated by exposure to light of the first wavelength, and (d) The system comprising an activatable molecule which is converted into an activated molecule when the activatable molecule is in close proximity to the activated photocatalyst.

2. It is a system, (a) Bioluminescent proteins, (b) A luminescent phosphat, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phosphat by interaction with the luminescent phosphat, and (c) The system comprising a photocatalyst, wherein the photocatalyst is activated by exposure to light of a first wavelength.

3. The system according to claim 1 or 2, wherein the bioluminescent protein is luciferase.

4. The system according to claim 3, wherein the luciferase is an Oplophorous polypeptide having at least 70% sequence identity with SEQ ID NO:

1.

5. The system according to claim 4, wherein the luciferase has 100% sequence identity with SEQ ID NO:

1.

6. The system according to claim 3, wherein the luciferase is a cyclically substituted variant of an Oplophorous polypeptide.

7. The system according to claim 6, wherein the luciferase comprises a first segment having at least 70% sequence identity with a first portion of SEQ ID NO: 1, and a second segment having at least 70% sequence identity with a second portion of SEQ ID NO:

1.

8. The system according to claim 7, wherein the first segment has 100% sequence identity with the first portion of sequence number 1, and the second segment has 100% sequence identity with the second portion of sequence number 1.

9. It is a system, (a) Structurally complementary components of the bioluminescent complex, (b) A luminescent phosphatid, wherein the bioluminescent complex catalyzes the emission of light of a first wavelength from the luminescent phosphatid by interaction with the luminescent phosphatid, (c) A photocatalyst, wherein the photocatalyst is activated by exposure to light of the first wavelength, and (d) The system comprising an activatable molecule which is converted into an activated molecule when the activatable molecule is in close proximity to the activated photocatalyst.

10. It is a system, (a) Structurally complementary components of the bioluminescent complex, (b) A luminescent phosphat, wherein the bioluminescent complex catalyzes the emission of light of a first wavelength from the luminescent phosphat by interaction with the luminescent phosphat, and (c) The system comprising a photocatalyst, wherein the photocatalyst is activated by exposure to light of a first wavelength.

11. The system according to claim 10, wherein the structurally complementary components include two or more peptides and / or polypeptides that can bind to each other to form an active bioluminescent complex.

12. The system according to claim 11, wherein the structurally complementary components collectively have at least 70% sequence identity with sequence number 2.

13. The system according to claim 12, wherein the structurally complementary component comprises a peptide having at least 70% sequence identity with SEQ ID NO: 3 or 4, and a polypeptide having at least 70% sequence identity with SEQ ID NO:

5.

14. The system according to claim 13, wherein the structurally complementary component comprises a peptide having 100% sequence identity with SEQ ID NO: 3 or 4, and a polypeptide having 100% sequence identity with SEQ ID NO:

5.

15. The system according to claim 12, wherein the structurally complementary component comprises a peptide having at least 70% sequence identity with SEQ ID NO: 3 or 4, a polypeptide having at least 70% sequence identity with SEQ ID NO: 6, and a peptide having at least 70% sequence identity with SEQ ID NO:

7.

16. The system according to claim 15, wherein the structurally complementary component comprises a peptide having 100% sequence identity with SEQ ID NO: 3 or 4, a polypeptide having 100% sequence identity with SEQ ID NO: 6, and a peptide having 100% sequence identity with SEQ ID NO:

7.

17. The system according to one of claims 13 to 16, wherein the polypeptide component is cyclically substituted.

18. The system according to one of claims 1 to 17, wherein the luminescent molecule is a luciferin or coelenterazine molecule.

19. The system according to claim 18, wherein the coelenterazine molecule is frimazine.

20. The system according to claim 19, wherein the coelenterazine molecule is flimazine or fluoroflimazine.

21. The system according to one of claims 1 to 20, wherein the photocatalyst is an iridium-based or ruthenium-based photocatalyst.

22. The aforementioned photocatalyst is defined by formula (I): 【Chemistry 1】 [In the formula, Each series of dashed lines ( 【Chemistry 2】 ) indicates the presence or absence of a condensed six-membered ring, M is a transition metal, m1, m2, m3, n1, n2, n3, p1, p2, and p3 are each independently 0, 1, or 2. R 1a 、 R 1b 、 R 1c 、 R 2a 、 R 2b 、 R 2c 、 R 3a 、 R 3b 、 and R 3c are each independently selected from halo, alkyl, haloalkyl, amino, heteroalkyl, and group-linker-Q, where Q is a capture element, X 1a , X 1b , X 2a , X 2b , X 3a , and X 3b Each is independently selected from N and C, where X 1a and X 1b At least one of them is N, and X 2a and X 2b At least one of them is N, and X 3a and X 3b At least one of them is N, X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d Each is independently selected from CH and N, A is an anion, q is 0, 1, or 2. The system according to claim 21, having the structure of [the specified structure].

23. The system according to claim 22, wherein the transition metal is selected from Ru and Ir.

24. The aforementioned photocatalyst is 【Transformation 3】 The system according to claim 23, wherein the iridium-based photocatalyst or a derivative thereof selected from the above, the compound being functionalized with at least one group-linker-Q, where Q is a scavenging element.

25. The aforementioned photocatalyst is 【Chemistry 4】 The system according to claim 23, wherein the ruthenium-based photocatalyst or a derivative thereof selected from the above, the compound being functionalized with at least one group-linker-Q, where Q is a scavenging element.

26. The aforementioned photocatalyst is given by formula: 【Transformation 5】 The system according to claim 22, having the following features.

27. R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c The system according to claim 22, wherein one of them is a base-linker-Q.

28. The system according to claim 27, wherein Q is a capture element.

29. The system according to claim 28, wherein Q is a haloalkane.

30. The linker is an ester (-C(O)O-), amide (-C(O)NH-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), phenylene (e.g., 1,4-phenylene), linear or branched alkylene, oligo- or polyethylene glycol (-(CH 2 CH 2 O) x The system according to claim 27, comprising -), or a combination thereof.

31. The linker is -O(CH 2 CH 2 O) z1 -C(O)NH-(CH 2 CH 2 O) z2 -C(O)NH-(CH 2 ) z3 - (OCH 2 CH 2 ) z4 The system according to claim 30, comprising O-, wherein z1, z2, z3, and z4 are each independently selected from 0, 1, 2, 3, 4, 5, and 6.

32. The aforementioned linker, 【Transformation 6】 The system according to claim 31, selected from the following.

33. The system according to one of claims 1 to 32, wherein the photocatalyst is an organic photoredox catalyst.

34. The system according to claim 33, wherein the organic photoredox catalyst is selected from quinone, pyrylium, acridinium, and xanthene.

35. The aforementioned organic photoredox catalyst 【Transformation 7】 The system according to claim 34, wherein the quinone is selected from the following.

36. The aforementioned organic photoredox catalyst 【Transformation 8】 The system according to claim 34, wherein the pyririum is selected from among.

37. The aforementioned organic photoredox catalyst 【Chemistry 9】 The system according to claim 34, wherein the acridinium is selected from the acridinium.

38. The aforementioned organic photoredox catalyst 【Chemistry 10】 The system according to claim 34, wherein xanthine is selected from.

39. The system according to claim 34, wherein the organic photoredox catalyst is a thiaidine-based organic photoredox catalyst.

40. The system according to one of claims 1 to 39, wherein the light of the first wavelength is within the visible light spectrum.

41. The system according to claim 40, wherein the light of the first wavelength is 400 to 500 nm.

42. The system according to claim 1 or 9, wherein the photocatalyst facilitates (i) energy transfer to the activatable molecule, (ii) hydrogen abstraction from the activatable molecule, or (iii) catalytic activity of a photoredox reaction.

43. The system according to claim 1 or 9, wherein the photocatalyst transfers energy to the activatable molecule by Foerster resonance energy transfer, Dexter energy transfer, single electron transfer, singlet oxygen, or a photocatalyst-driven conformational change.

44. The system according to one of claims 1 to 43, wherein the activatable molecule is a caged molecule or a photoswitchable molecule.

45. The system according to claim 44, wherein the activatable molecule is a caged molecule comprising a functional portion and a blocking portion.

46. The system according to claim 45, wherein the photocatalyst promotes the cleavage of the blocking portion from the functional portion, thereby activating the activatable molecule.

47. The blocking portion, 【Chemistry 11】 The system according to claim 44, selected from the above.

48. The system according to claim 44, wherein the blocking portion prevents the functional portion from binding to and / or being detected by a target.

49. The system according to claim 48, wherein the functional portion is a small molecule, a peptide, a protein, and / or a nucleic acid.

50. The system according to claim 44, wherein the activatable molecule is a photoswitchable molecule in an inactive conformation.

51. The system according to claim 50, wherein the photocatalyst promotes the conversion of the photoswitchable molecule from an inactive conformation to an active conformation, thereby activating the activatable molecule.

52. The system according to claim 51, wherein the photoswitchable molecule includes first and second functional portions connected to a photoswitch portion, and when the photoswitchable molecule is in the inactive conformation, the first and second functional portions are not in close proximity to interact and / or are not in a suitable orientation, and when the photoswitchable molecule is in the active conformation, the first and second functional portions are in close proximity to interact and / or are in a suitable orientation.

53. The aforementioned optical switch portion is in an inert conformation 【Chemistry 12】 and active conformation 【Chemistry 13】 Includes, in the formula, R 1 and R 2 The system according to claim 52, wherein the first and second functional parts are as described above.

54. The system according to claim 52, wherein the first and second functional portions are small molecule portions.

55. The system according to one of claims 1 to 54, wherein the components of the bioluminescent protein or the bioluminescent complex are fused to a first molecular entity, the photocatalyst or photosensitizer is linked to a second molecular entity, and the interaction between the first molecular entity and the second molecular entity brings the bioluminescent protein or bioluminescent complex close enough to the photocatalyst or photosensitizer so that the light emitted by the luminescent morphoform through interaction with the bioluminescent protein or bioluminescent complex activates the photocatalyst or photosensitizer.

56. The system according to claim 55, wherein the first molecular entity is a scavenger and the second molecular entity is a scavenging element.

57. The system according to claim 56, wherein the components of the bioluminescent protein or bioluminescent complex are fused to the N-terminus, C-terminus, or internal site within the capture agent.

58. The system according to claim 55, wherein the bioluminescent protein or the components of the bioluminescent complex are fused to a modified dehalogenase capable of forming a covalent bond with its substrate, or are inserted into the modified dehalogenase, and the photocatalyst or photosensitizer is linked to the dehalogenase substrate.

59. The system according to claim 58, wherein the binding of the modified dehalogenase to the dehalogenase substrate brings the bioluminescent protein or bioluminescent complex close enough to the photocatalyst or photosensitizer so that the light emitted by the luminescent phosphate through interaction with the bioluminescent protein or bioluminescent complex activates the photocatalyst or photosensitizer.

60. The system according to claim 58, wherein the modified dehalogenase has at least 70% sequence identity with SEQ ID NO:

8.

61. The system according to claim 60, wherein the modified dehalogenase has 100% sequence identity with SEQ ID NO:

8.

62. The system according to claim 58, wherein the dehalogenase substrate is a haloalkane.

63. The structure of the photocatalyst linked to the dehalogenase substrate is P-linker-AX, where P is the photocatalyst and A is (CH 2 ) 2-12 The system according to claim 58, wherein X is a halogen, and the linker is a linker portion capable of connecting P to A-X.

64. The system according to claim 63, wherein the linker is a polyatomic linear or branched chain containing C, N, S, or O, or a group containing one or more rings, for example, saturated or unsaturated rings, for example, one or more aryl rings, heteroaryl rings, or any combination thereof.

65. The linker is -O(CH 2 ) 2 - -(CH2)O-, -CH 2 The system according to claim 64, comprising combinations of -, -NHC(O)O-, -OC(O)NH-, NHC(O)-, and -C(O)NH-.

66. The system according to claim 64 or 65, wherein the linker has a length of 5 to 50 atoms.

67. A cell comprising the system described in one of claims 1 to 66.

68. A method of activation dependent on the proximity of molecules within a cell, comprising bringing a cell into contact with a luminescent phosi under conditions in which the luminescent phosi enters the cell, (a) A fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phosphatid by interaction with the luminescent phosphatid, (b) A conjugate of (A) a capture ligand and (B) a photocatalyst or photosensitizer, wherein the capture protein forms a covalent bond with the capture ligand through interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength, and (c) an activatable molecule, which is converted into an activated molecule when the activatable molecule is in close proximity to the activated photocatalyst or photosensitizer, The method, including the method described above.

69. A method of activation that depends on the proximity of activatable molecules within a cell, (a) Expressing a fusion of a bioluminescent protein and a capture protein within the cell, (b) The cells are brought into contact with the luminescent phosphatid under conditions that allow the luminescent phosphatid to enter the cells, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phosphatid by interaction with the luminescent phosphatid, (c) Contacting the cells with a conjugate of (i) a capture ligand and (ii) a photocatalyst or photosensitizer under conditions that allow the conjugate to enter the cells, wherein the capture protein forms a covalent bond with the capture ligand through interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength, and (d) The cells are brought into contact with an activatable molecule, wherein the activatable molecule is converted into an activating molecule when it is in close proximity to the activated photocatalyst or photosensitizer. The method, including the method described above.

70. A method of activation dependent on the proximity of a photocatalyst or photosensitizer within a cell, comprising bringing a cell into contact with a luminescent phosi under conditions in which the luminescent phosi enters the cell, (a) A fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phosphatid by interaction with the luminescent phosphatid, (b) A conjugate of (A) a capture ligand and (B) a photocatalyst or photosensitizer, wherein the capture protein forms a covalent bond with the capture ligand through interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength, The method, including the method described above.

71. A method of activation that depends on the proximity of a photocatalyst or photosensitizer within a cell, (a) Expressing a fusion of a bioluminescent protein and a capture protein within the cell, (b) The cells are brought into contact with the luminescent phosphatid under conditions in which the luminescent phosphatid enters the cells, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the luminescent phosphatid by interaction with the luminescent phosphatid, and (c) Contacting the cells with a conjugate of (i) a capture ligand and (ii) a photocatalyst or photosensitizer under conditions that allow the conjugate to enter the cells, wherein the capture protein forms a covalent bond with the capture ligand through interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength. The method, including the method described above.

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