Compositions and methods comprising light-and-magnetoresponsive protein domains

Abstract

Provided herein are compositions comprising light-and-magnetoresponsive protein domain, such as engineered light-oxygen-voltage-sensing (LOV) domains, and methods of using the same. The light-and magnetoresponsive proteins domains may be used to form compositions such as fusion proteins comprising heterologous protein components and bioluminescent protein domains with activity that is dependent on the presence of light, e.g., light emitted by a reaction catalyzed by the bioluminescent protein domain, and on the presence of a magnetic field.

Description

COMPOSITIONS AND METHODS COMPRISING LIGHT-AND- MAGNETORESPONSIVE PROTEIN DOMAINSRELATED APPLICATIONS

[0001] This application claims the priority and benefit of U. S. Provisional Application No. 63 / 722,465, filed on November 19, 2024, and U. S. Provisional Application No. 63 / 867,555, filed on August 20, 2025, the contents of each of which are incorporated herein in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (NOBS_001_02WO_SeqList_ST26,xml; Size: 100,996 bytes; and Date of Creation: November 18, 2025) are herein incorporated by reference in their entirety.BACKGROUND

[0003] Recombinant proteins are highly successful and vital therapeutic tools for treating serious conditions including cancer and autoimmune disease. Unfortunately, proteins may have unintended consequences because the activity of a protein in the body may be both effective as well as toxic. The beneficial and toxic effects of a protein do not always occur in the same location. For example, a therapeutic protein may be effective for treating cancer when it is active in one organ or cell type in the body, but toxic when it is active in another location. Therefore, there exists a need for means to control the activity of proteins in order to improve their efficacy and safety.SUMMARY

[0004] Provided herein are methods of using a light-and-magnetoresponsive composition, comprising: (a) providing a composition comprising (i) a bioluminescent protein domain; and (ii) a light-and-magnetoresponsive protein domain; (b) exposing the composition to a substrate, wherein the bioluminescent protein domain is capable of catalyzing a reaction of the substrate, wherein the reaction emits light; and (c) exposing1323198441the composition to a magnetic field. In some embodiments, the magnetic field is a resonant magnetic field. In some embodiments, the magnetic field is generated by a wearable device.

[0005] Also provided herein is an engineered LOV domain comprising an induced dimer (iLID) amino acid substitution. In some embodiments, the engineered LOV domain comprises the SsrA peptide sequence of AANDENYF (SEQ ID NO: 84). In some embodiments, the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 39 and 40.

[0006] Also provided herein is engineered LOV domain, wherein the LOV domain is truncated. In some embodiments, the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 88-95.

[0007] Also provided herein is an engineered LOV domain comprising an amino acid residue selected from the group consisting of 45R, 91F, 100Y, 116R, 117L, 118H, 119G, 122E, 1241, 125A, 130N, 134F, 135Q, 137A, 141D, and 143F.

[0008] Also provided herein is an engineered LOV domain comprising an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 26 and 48

[0009] Also provided herein is a fusion protein comprising an engineered LOV domain and a bioluminescent protein domain, wherein the engineered LOV domain is inserted within the bioluminescent protein domain.BRIEF DESCRIPTION OF THE DRAWINGS10010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will323198441be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0011] FIG. 1 show’s the effect of magnetic fields on the luminescence of E. col colonies expressing a NanoBiT™-MagLOV fusion protein, as described in Example 10. The x-axis represents time in arbitrary units, and the y-axis represents the level of luminescence. Gray shading indicates times when the magnetic field was applied. Individual lines show results for separate, genetically identical colonies on the same plate.

[0012] FIG.2 shows results from an enzyme-linked immunosorbent assay (ELISA) measuring binding of a luciferase-mCherry optobody fusion protein to mCherry. The x-axis indicates the concentration of mCherry in nM, and the y-axis indicates the optical density (OD), The samples tested under dark conditions are labeled “AK-D-1” and “AK-D-2,” and the samples tested under light conditions are labeled “AK-L-1” and “AK-L-2.” Illumination was delivered by a Thorlabs light emitting diode (LED) at 75% max power.

[0013] FIG.3 shows results from a luciferase activated ELISA measuring binding of a luciferase-mCherry optobody fusion protein to mCherry. The x-axis indicates the concentration of mCherry in nM, and the y-axis indicates the optical density (OD).

[0014] FIG.4 shows the level of RFP detected in a plasma membrane region or a cytoplasmic membrane region of an illuminated HeLa cell over time. The x-axis represents time in arbitrary units, and the y-axis represents the level of RFP fluorescence in the indicated region of the cell.

[0015] FIG. 5 shows the level of RFP detected in a plasma membrane region or a cytoplasmic membrane region of an illuminated HeLa cell over time. The x-axis represents time in arbitrary units, and the y-axis represents the level of RFP fluorescence in the indicated region of the cell.10016] FIG.6 shows the level of RFP detected in a plasma membrane region or a cytoplasmic membrane region of two illuminated HeLa cells over time. The x-axis represents time in arbitrary units, and the y-axis represents the level of RFP fluorescence in the indicated region of the ceils.

[0017] FIGS. 7A-7C show results from an ELISA measuring binding of an anti-Myc antibody to Myc-tagged Mag-iLID or iLID constructs bound to SspB in light, dark, or3323198441light and magnetic field conditions, as described in Example 14. In each figure, the x-axis indicates the concentration of anti-Myc antibody in nM, and the y-axis indicates the OD.

[0018] FIG. 7 A shows the results for Mag-iLID.

[0019] FIG. 7B shows the results for the control iLID

[0020] FIG. 7C shows the results for the control iLID.

[0021] FIGS. 8A-8D show results from an ELISA measuring binding of an anti-Myc antibody to Myc-tagged of anti-mCherry antibodies fused to improved MagLOV or Mag- iLID to mCherry in light, dark, or light and magnetic field conditions, as described in Example 15, In each figure, the x-axis indicates the concentration of anti-Myc antibody to Myc-tagged anti-mCherry antibodies in nM, and the y-axis indicates the OD, scaled such that a 0 signal is the plate reader signal when there is no antibody present, and 1 is the saturated asymptote of a best fit curve to the data.

[0022] FIG. 8A shows results for a non-magnetoresponsive optobody with a wildtype AsLOV2 insertion as the AK74 insertion loop site (SEQ ID NO: 87).

[0023] FIG. SB shows results for an anti-Cherry antibody fused to the MagLOV domain of SEQ ID NO. 71.

[0024] FIG. 8C shows results for an improved MagLOV sequence with iLID mutations of SEQ ID NO: 26, added to the AK74 insertion site of the Lam8 anti-mCherry nanobody (SEQ ID NO: 44).

[0025] FIG. 8D shows results for an improved MagLOV with iLID amino acid substitutions as shown in SEQ ID NO. 40, inserted into the AK74 loop insertion site of an anti-mCherry antibody (SEQ ID NO: 68).

[0026] FIG.9 shows a bioluminescence powered magnetoresponsive effect without external light. In the presence of 200 uM fluorofurimazine, A MagLuc fused to iLID actuates the binding of iLID to a SspB antigen in response as the luminescence of MagLuc changes. Both constructs were expressed in E. coli. When CFast and NFast come towards each other, they complete a fluorescent protein and generate a new red fluorescent signal. In periods where the magnet is active as indicated with the bar, the production of light from Magluc is reduced, and therefore the iLID to sspb binding is reduced, resulting in less fluorescence signal. This fluorescence trace comes from a representative cell expressing both plasmids.4323198441

[0027] FIG. 10 shows results from an ELISA measuring binding of an anti-Myc antibody to Myc-tagged anti-GFP antibodies fused to improved MagLOV with iLID amino acid substitutions with SEQ ID No. 40 to GFP in light, dark, or light and magnetic field conditions, as described in Example 17. The x-axis indicates the concentration of antibody in nM, and the y-axis indicates the OD, scaled such that a 0 signal is the plate reader signal when there is no antibody present, and 1 is the saturated asymptote of a best fit curve to the data,

[0028] FIGS. 11A-11C show luminescence modulation over time of fusion proteins with TurboLuc fused to the improved MagLOV variant of SEQ ID NO: 26 exposed to an appropriate luciferase substrate. Luminescence traces were collected from imaged E. coli grown on a petri dish expressing the indicated TurboL uc-MagLOV fusion protein. In each of FIGS. 11A-11C, at left is shown mean- corrected luminescence modulation normed around 1. In the middle, a bandpass filter at 515 nanometers is provided to show the contribution in the typical MagLOV fluorescence range. At right is the raw trace, with the 515 nm data collected at the end after a filter is robotically placed between the sample and the camera.

[0029] FIG. 11A shows results for a split TurboLuc with an improved MagLOV domain added in between the two components (SEQ ID NO: 73).

[0030] FIG. 11B shows results for a non-split TurboLuc with a C-terminal fusion to the improved MagLOV domain (SEQ ID NO: 74).

[0031] FIG. 11C shows results for a split TurboLuc variant with an improved MagLOV domain fused to the C-terminus, linking both of the split components of TurboLuc into one single construct.

[0032] FIGS. 12A-12C show Renilla luciferase variants fused to the improved MagLOV of SEQ ID NO: 26. Outputs are shown as raw traces of luminescence measurements of Renilla luciferase exposed to a luciferase substrate, with a magnet moving into proximity to and from the plate every five frames.

[0033] FIG. 12A shows the raw luminescence output of an imaged E. coli colony when a magnet is repeatedly triggered every’ five frames. The E. coli are expressing SEQ ID NO: 76, which is an engineered Renilla luciferase commonly referred to as RLuc8-535, fused to the SEQ ID NO: 26 improved MagLOV domain.5323198441

[0034] FIG. 12B shows the same luminescence data, but for a mutated SEQ ID NO: 77, which is a mutant variant of SEQ ID NO: 76 with enhanced magnetoresponse.

[0035] FIG. 12C shows an unmodified RLuc8-535 Renilla luciferase that is not fused to a magnetoresponsive domain (SEQ ID NO: 86).

[0036] FIG. 13 shows results from an ELISA of an anti-mCherry antibody with an improved MagLOV domain (SEQ ID NO: 40) fused in at the GG15 loop insertion site, under dark, light, and light plus magnet conditions. The x-axis indicates the concentration of anti-Myc antibody to Myc-tagged anti-mCherry antibodies in nM, and the y-axis indicates the OD.

[0037] FIG. 14 shows the normalized luminescence response of a bacterial colony over time, after the addition of coelenterazine for the fusion protein of SEQ ID NO: 66. Black circular markers represent mean-corrected intensity measurements at discrete time frames, wherein each data point represents the colony's raw luminescence intensity normalized by colony mean intensity over time, thereby removing photobleaching effects as well as accounting for the decline or rise in oxygen or luciferase substrate concentration that may occur over the course of imaging independently from the magnetic modulation. The continuous black line represents a driven exponential response model fitted to said data points, wherein the model is defined parameters as explained in Example 16.DETAILED DESCRIPTIONIntroduction

[0038] Provided herein are compositions comprising (a) a bioluminescent protein domain, and (b) a light-and-magnetoresponsive protein domain, and methods of using the same. In some embodiments, the light-and-magnetoresponsive protein domain is sensitive to light emitted by a reaction catalyzed by the bioluminescent protein domain. In some embodiments, the light-and-magnetoresponsive protein domain is sensitive to exposure to a magnetic field. Also provided herein are compositions comprising (a) a light-and-magnetoresponsive protein domain and (b) a heterologous protein component.

[0039] Described herein is the use of light-and-magnetoresponsive protein domains to form protein compositions that are sensitive to magnetic fields. As described in Example63231984411, a light-and-magnetoresponsive protein domain may be fused to a heterologous protein component such as human interleukin-2 so that activity of the heterologous protein component is made dependent on a change by the light-and-magnetoresponsive protein domain.

[0040] The compositions described herein may comprise both a light-and- magnetoresponsive protein domain and a bioluminescent domain. As described in Example 3, such compositions may be expected to be sensitive both to a magnetic field as well as light emitted by a reaction catalyzed by the composition’s bioluminescent domain. This is believed to allow for the magnet field and / or light-dependent control of the activity of a heterologous protein component.

[0041] Without wishing to be bound by theory, it is believed that such compositions may allow for the modulation of the activity of the heterologous protein component, so that it is only activated or deactivated upon exposure to a magnetic field and emission of light from reactions catalyzed by the bioluminescent domain, e.g,, upon exposure to a substrate such as a luciferin. Such compositions may be useful for controlling the activity of the heterologous protein component because its activation or deactivation is controlled by the application of a magnetic field and / or the substrate of the bioluminescent domain. In the context of a dark environment without naturally occurring light, a substrate of the bioluminescent protein domain may be administered so that, upon emission of light by the bioluminescent protein domain and exposure to a magnetic field, the activity of the heterologous protein component is increased or decreased. In particular, the emission of light and the presence of the magnetic field together can function as an “AND gate,” so the composition’s activity is dependent on both the presence of the light and the magnetic field. This is believed to allow for highly specific control of the composition’s activity, including allowing for control of the timing of activity and the precise location of activity.

[0042] While exemplary embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the inventions claimed herein. It should be understood that various alternatives to the embodiments described herein may be employed in practicing323198441the embodiments of the disclosure. It is intended that the claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.Definitions

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention,

[0045] The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having8323198441sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.Light-and-magnetoresponsive protein domains

[0046] Provided herein are compositions comprising light-and-magnetoresponsive protein domains, and methods of using the same. Light-and-magnetoresponsive protein domains are sensitive to exposure to of at least one particular wavelength of light (or a range of wavelengths of light) as well as exposure to a magnetic field. Without wishing to be bound by theory, light-and-magnetoresponsive protein domains are believed to undergo changes such as conformational changes upon exposure to light and / or exposure to a magnetic field. This change in the light-and-magnetoresponsive protein domain may mediate, in turn, a change in the overall composition. For example, if a light-and- magnetoresponsive protein domain is fused to a heterologous protein component, then exposure to light and / or a magnetic field may induce a conformational change in the fusion protein that activates or deactivates a heterologous protein component. In some embodiments, conformational changes include unfolding, tilting, rotating and multimerizing (e.g., dimerizing, trimerizing), or a combination of any of the foregoing (e.g., unfolding and multimerizing). In some embodiments, the conformational change is an allosteric change. In some embodiments, the conformational change induces multimerization (e.g., dimerization, trimerization) of the composition. In some embodiments, the change in the light-and-magnetoresponsive protein domain is not a conformational change.

[0047] In some embodiments, the light-and-magnetoresponsive protein domain is sensitive to a magnetic field with a strength of about 0.01 millitesla (mT) to about 1000 mT. In some embodiments, the light-and-magnetoresponsive protein domain is sensitive to a magnetic field with a strength of about 0.01 mT, about 0.1 mT, about 1 mT, about 10 mT, about 100 mT, or about 1000 mT. In some embodiments, the light-and-magnetoresponsive protein domain is sensitive to magnetic field of about 5 mT to about 25 mT. In some embodiments, the light-and-magnetoresponsive protein domain is9323198441sensitive to magnetic field of about 5 mT, about 10 mT, about 15 mT, about 20 mT, or about 25 mT.

[0048] In some embodiments, the light-and-magnetoresponsive protein domain is sensitive to visible light (e.g., from about 400-nm to 700-nm light). In particular embodiments, the light-and-magnetoresponsive protein domain is sensitive to blue light (e.g., from about 380-nm to about 500-nm light, in particular, about 450-nm light), red light (e.g., from about 620-nm to about 750-nm light) or far-red light (e.g., from about 710-nm to about 850-nm light). In some embodiments, the light-and-magnetoresponsive protein domain is sensitive to infrared light (e.g., from greater than 700-nm to about 1-mm light).

[0049] Light-and-magnetoresponsive protein domains may be characterized by their Xmax, which is the wavelength at which the light-and-magnetoresponsive protein domain absorbs the most light. In some embodiments, the light-and-magnetoresponsive protein domain has amaxof about 400 nm to 700 nm. In some embodiments, the light-and-magnetoresponsive protein domain has a Xmax of about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 480 nm, about 490 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 610 nm, about 620 nm, about 630 nm, about 640 nm, about 650 nm, about 660 nm, about 670 nm, about 680 nm, about 690 nm, or about 700 nm. In some embodiments, the light-and-magnetoresponsive protein domain has a max of about 450 nm.

[0050] In some embodiments, the light-and-magnetoresponsive protein domain is not an electromagnetic perceptive gene (EPG). In some embodiments, the light-and-magnetoresponsive protein domain is not a wild-type electromagnetic perceptive gene (EPG).

[0051] In some embodiments, the light-and-magnetoresponsive protein domain comprises a light-oxygen-voltage-sensing (LOV) domain. LOV domains are protein sensors that bind a cofactor, such as a flavin chromophore. LOV domains are typically understood as having two distinct conformational shapes. In the default dark state, the J alpha helix is docked to the rest of the LOV domain. In the bright state, a transient covalent bond is formed between the C450 and a flavin mononucleotide (FMN) cofactor,10323198441resulting in a destabilization of the J alpha helix and a broader conformational change in the protein it is bound to. The C-terminal Ja helix of AsL0V2, in particular, undocks and unfolds upon excitation with blue light (e.g., λmax=450 nm), resulting in a substantial increase in the distance between the N- and C-termini of AsL0V2, which are typically within less than 10 A of one another in the absence of light. As described, for example, in U. S. Patent No. US11746131B2, wild-type LOV domains have been used to make the activities of various proteins light-dependent.

[0052] In some embodiments, the light-and-magnetoresponsive protein domain comprises an engineered LOV domain. An exemplary engineered LOV domain is described in Hayward, R. F. et al, (2024), Magnetic control of the brightness of fluorescent proteins (vO. O.l). Zenodo, (see word wide web:doi. org / 10.5281 / zenodo.11406498),

[0053] In some embodiments, the engineered LOV domain is derived from Avena saliva Phototropin 1. In some embodiments, the engineered LO V domain is derived from the LOV 2 domain from Avena saliva Phototropin 1, herein referred to as “AsLOV2”.

[0054] In some embodiments, the engineered LOV domain is an engineered AsLOV2 domain. In some embodiments, the engineered LOV domain comprises one or more mutations relative to the wild-type AsLOV2 ammo acid sequence. In some embodiments, the engineered LOV domain comprises a C450P substitution relative to the wild-type AsLOV2 amino acid sequence. In some embodiments, the engineered LOV domain comprises a D540M substitution relative to the wild-type AsLOV2 amino acid sequence. In some embodiments, the engineered LOV domain comprises a Q513K substitution relative to the wild-type AsLOV2 ammo acid sequence. In some embodiments, the engineered LOV domain comprises a L396V substitution relative to the wild-type AsLOV2 amino acid sequence. In some embodiments, the engineered LOV domain comprises a G528K substitution relative to the wild-type AsLOV2 ammo acid sequence. In some embodiments, the engineered LOV domain comprises C450P, D540M, Q513K, L396V, and G528K substitutions relative to the wild-type AsLOV2 amino acid sequence.

[0055] Exemplary amino acid sequences of light-and-magnetoresponsive protein domains are provided in Table 1.11323198441Table 1. Exemplary light-and-magnetoresponsive protein domain amino acid sequences _SEQAmino Acid SequenceID NO LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNPRFLQGPE1 TDRAT VRKI RDAI DNQTE VTVQL 1 NYTKS GKKFWNL FHVQPMRDQKGDVQYFI G VKL D G T E H VRDA AE RE KVML I KK T AE N I ME AAKE L LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNPRFLQGPETDRA2 TVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFH’VQPMRDQKGDVQYFIGVKLD GTEHVRDAAEREKVML I KKTAENIMEAA MLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNPRFLQGP26 E T DRAT VRKI RDAI DNQT E VTVQL I NYTKS GKKFWNL FHVQPMRDQKGDVQYF I G VKL D G T E H VRD AAE R I KVML INK T AAN I D E AAKE L LATTLERIEKNFVITDPRLPDNPI IFASDSFLQLTEYSREEILGRNPRFLQGPE48 TDRAT VRKI RDAI DNQTE VTVQL I NYTKS GKKFWNL FHVQPMRDQKGDVQYFI GVKL DG T E HVRDAAE RI KVML I NKT AAN I DE AAKE L

[0056] In some embodiments, the engineered LOV domain comprises an ammo acid sequence selected from the group consisting of SEQ ID NOS: 1, 26, and 48, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered LOV domain comprises an amino acid sequence selected from the group of SEQ ID NOS: 1, 26, and 48. In some embodiments, the engineered LOV domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 26, and 48 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0057] In some embodiments, the engineered LOV domain comprises an ammo acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered LOV domain comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered LOV domain comprises the ammo acid sequence of SEQ ID NO: 1 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.12323198441

[0058] Also described herein are engineered LOV domains with improved characteristics. As described in Example 14, a directed evolution campaign was carried out which resulted in an improved LOV domain variant.

[0059] In some embodiments, the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the engineered LOV domain comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the engineered LOV domain comprises the amino acid sequence of SEQ ID NO: 26 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0060] In some embodiments, the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the engineered LOV domain comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the engineered LOV domain comprises the ammo acid sequence of SEQ ID NO: 48 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0061] In some embodiments, the engineered LOV domain comprises an ammo acid residue selected from the group consisting of 45R, 91F, 100Y, 116R, 117L, 118H, 119G, 122E, 1241, 125A, BON, 134F, 135Q, 137A, 141D, and 143F, or a combination thereof, according to the numbering of amino acid residues in SEQ ID NO: 48. In some embodiments, the engineered LO V domain comprises each of the ammo acid residues 45R, 91F, 100Y, 116R, 117L, 118H, 119G, 122E, 1241, 125A, BON, 134F, 135Q, 137A, 141D, and 143F.Truncated LOV domains

[0062] In some embodiments, the engineered LOV domain is a truncated LOV domain. In some embodiments, the engineered LOV domain is a truncated AsLOV2 domain. In some embodiments, a truncated LOV domain lacks a human nuclear export sequence. Truncated LOV domains are believed to be advantageous for improving coupling of a 13323198441LOV domain to a heterologous protein component. Further, removal of a human nuclear export signal is believed to be advantageous for the proper functioning of the composition, since light may unexpectedly trigger nuclear export (see Gil, A. A., et al. Nat Commun 11, 4044 (2020)). Exemplary truncated LOV domains are described in Examples 1 and 16. In some embodiments, the truncated LOV domain is equal to or less than 130, 125, 120, 115, 110, 105, 100, 95, or 90 amino acid residues in length.

[0063] In some embodiments, the truncated LOV domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 88-95, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the truncated LOV domain comprises an ammo acid sequence selected from the group of SEQ ID NOS: 2 and 88-95. In some embodiments, the truncated LOV domain comprises an ammo acid sequence selected from the group consisting of SEQ ID NOS: 2 and 88-95 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0064] In some embodiments, the truncated LOV domain comprises an ammo acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 2 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ammo acid substitutions.

[0065] In some embodiments, the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 88 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.14323198441

[0066] In some embodiments, the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 89 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0067] In some embodiments, the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 90, In some embodiments, the truncated LOV domain comprises the ammo acid sequence of SEQ ID NO: 90. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 90 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ammo acid substitutions.

[0068] In some embodiments, the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the truncated LOV domain comprises the ammo acid sequence of SEQ ID NO: 91. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 91 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ammo acid substitutions.

[0069] In some embodiments, the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the15323198441truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 92 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0070] In some embodiments, the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 93 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ammo acid substitutions.

[0071] In some embodiments, the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 94, In some embodiments, the truncated LOV domain comprises the ammo acid sequence of SEQ ID NO: 94. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 94 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ammo acid substitutions.

[0072] In some embodiments, the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the truncated LOV domain comprises the ammo acid sequence of SEQ ID NO: 95. In some embodiments, the truncated LOV domain comprises the amino acid sequence of SEQ ID NO: 95 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ammo acid substitutions.Engineered LOV domains with induced dimer (iLID) amino acid substitutions

[0073] Also described herein are engineered LO V domains with induced dimer (iLID) amino acid substitutions. In some embodiments, the iLID amino acid substitution comprises insertion of an N residue after AMO. In some embodiments, the iLID amino acid substitution comprises insertion of NY residues after El 42. In some embodiments,16323198441the engineered LOV domain comprises the SsrA peptide sequence of AANDENYF (SEQ ID NO: 84).

[0074] In some embodiments, the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the engineered LOV domain comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the engineered LOV domain comprises the amino acid sequence of SEQ ID NO: 39 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ammo acid substitutions.

[0075] In some embodiments, the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the engineered LOV domain comprises the ammo acid sequence of SEQ ID NO: 40. In some embodiments, the engineered LOV domain comprises the amino acid sequence of SEQ ID NO: 40 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ammo acid substitutions.Bioluminescent protein domains

[0076] Provided herein are compositions comprising bioluminescent protein domains. In general, bioluminescent protein domains are capable of catalyzing reactions that emit light. Any protein domain that catalyzes a reaction that emits light may be considered a bioluminescent protein domain for use in the present disclosure.

[0077] In some embodiments, in a composition comprising a bioluminescent protein domain and a light-and-magnetoresponsive protein domain, as described above, the bioluminescent protein domain is capable of catalyzing a reaction that emits light, which induces a change in the light-and-magnetoresponsive protein domain. In some embodiments, the change in the light-and-magnetoresponsive protein domain is a conformational change.17323198441NOBS-001 / 02WG 356250-2007

[0078] In some embodiments, the bioluminescent protein domain is capable of catalyzing a reaction that emits light, wherein the luminescence spectrum of the emitted light overlaps with the activation spectrum of the light-and-magnetoresponsive protein.

[0079] Typically, a bioluminescent domain is selected for pairing with a light-and-magnetoresponsive protein domain based on an overlap between the spectrum of wavelengths light emitted by the bioluminescent domain and the excitation spectrum of the light-and-magnetoresponsive protein domain. The more closely the peak emission wavelength and the excitation wavelength match, the more efficient the induction of the conformational system is expected to be. For example, since LOV domains typically show a Amax of 450 nm, then, in some embodiments in which the composition comprises a LOV domain, it is advantageous to select a bioluminescent domain that catalyzes a reaction that emits light with an emissions peak close to 450 nm is used,

[0080] Various types of bioluminescent protein domains are known in the art. Exemplary bioluminescent protein domains are described below.

[0081] In some embodiments, the bioluminescent protein domain is a luciferase domain. Luciferase domains are derived from luciferase enzymes. Luciferase enzymes typically catalyze the oxidation of a luciferin substrate and emit light. In some embodiments, the luciferase domain is an engineered luciferase domain. In some embodiments, the luciferase domain is a wild-type luciferase domain. The emissions spectra of various luciferase domains are known in the art (see, e.g., world wide web: goldbio.com / articles / article / How-Different-Luciferin-Luciferase-Systems-Used-In-Biotechnology).

[0082] In some embodiments, the luciferase domain is a NanoLuc®, TurboLuc, firefly (e.g., Pholinus pyrails) luciferase, Remlla reniformis luciferase, copepod (e.g., Metridia longa) luciferase, bacterial (e.g., Vibrio fischeri, Vibrio haw eyi, or Vibrio harveyi) luciferase, or dinoflagellate luciferase domain.

[0083] In some embodiments, the luciferase domain is a NanoLuc® luciferase domain. NanoLuc® luciferase is a highly sensitive and stable luciferase, with a small size (England CG. Bioconjug Chem. 2016 May 18;27(5): 1175-1187). NanoLuc® when paired with the substrate furimazine typically exhibits an emission maximum of 460 nm. In some embodiments, the luciferase domain comprises an amino acid sequence having at18323198441least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the luciferase domain comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the luciferase domain comprises the amino acid sequence of SEQ ID NO: 3 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0084] In some embodiments, the luciferase domain is a firefly luciferase domain, e.g., a luciferase domain derived from a Photinus pyralis protein. In some embodiments, the luciferase domain comprises an ammo acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the ammo acid sequence of SEQ ID NO: 6, In some embodiments, the luciferase domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the luciferase domain comprises the amino acid sequence of SEQ ID NO: 6 comprising I, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0085] In some embodiments, the luciferase domain is a click beetle luciferase domain, e.g., a luciferase domain derived from a Caribbean Pyrophonis plagiophthalamus protein. In some embodiments, the luciferase domain comprises an ammo acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the ammo acid sequence of SEQ ID NO: 7. In some embodiments, the luciferase domain comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the luciferase domain comprises the ammo acid sequence of SEQ ID NO: 7 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0086] In some embodiments, the luciferase domain is a split luciferase domain. As used herein, a split luciferase domain refers to a domain derived from a luciferase protein that has been split into two or more fragments. In the case that a luciferase protein is split into two fragments, both fragments are generally required to emit light. In some19323198441embodiments, a luciferase protein is split into first split luciferase domain and a second split luciferase domain. In some embodiments, the bioluminescent protein domain comprises a first split luciferase domain derived from a first species, and a second split luciferase domain derived from a second species.

[0087] In some embodiments, the composition comprises a NanoBit™ luciferase domain. NanoBiT™ is a two-subunit system based on NanoLuc® luciferase. In some embodiments, the composition comprises a NanoBit™ LgBiT luciferase subunit and a NanoBit™ SmBiT luciferase subunit. In some embodiments, the first split luciferase domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the ammo acid sequence of SEQ ID NO: 4. In some embodiments, the first split luciferase domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first split luciferase domain comprises the amino acid sequence of SEQ ID NO: 4 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the second split luciferase domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the ammo acid sequence of SEQ ID NO: 5. In some embodiments, the second split luciferase domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the second split luciferase domain comprises the amino acid sequence of SEQ ID NO: 5 comprising one or more conservative amino acid substitutions.

[0088] In some embodiments, the bioluminescent protein domain is an engineered luciferase domain. Engineered luciferase domains are known in the art, and include mutant versions of luciferase (e.g., with ammo acid mutations that enhance luminescence, or shift the emissions spectrum), as well as fusion proteins comprising luciferase. In some embodiments, the bioluminescent protein domain is a spectrum-shifted luciferase. In some embodiments, the bioluminescent protein domain is Antares, a fusion of the luciferase NanoLuc® to the orange fluorescent protein CyOFP. In some embodiments, the bioluminescent protein domain is Nano-lantern, Renilla luciferase (RLuc) variant20323198441fused to the yellow fluorescent protein Venus. In some embodiments, the bioluminescent protein domain is a luciferase domain, wherein the luciferase domain is covalently linked to a fluorescent protein domain.

[0089] In some embodiments, the bioluminescent protein domain is a photoprotein domain. Like luciferase enzymes, photoproteins are capable of catalyzing a reaction of a luciferin substrate, wherein the reaction emits light. However, unlike luciferase enzymes, photoproteins do not display enzyme kinetics. Instead, when mixed with luciferin, photoproteins display luminescence proportional to the amount of the photoprotein. Like luciferase domains, photoprotem domains exhibit a range of emissions spectra.Accordingly, a photoprotein protein is typically selected for forming a composition with a light- and-magnetoresponsive protein domain based on an overlap of their emission and excitation spectra. For example, aequorin is a bioluminescent protein, isolated from the hydromedusan Aequorea victoria, which emits blue light upon binding Ca2+; it has an emissions peak of about 470 nm. As another example, hydroid Obelia. geniculate obelin has an emissions peak of around 495 nm (Markova, Svetlana V et al. Biochemistry vol.41,7 (2002): 2227-36). Further photoprotem domains are known in the art. Photoproteins are further described in Shanfian S, et al. Biomed Pharmacother. 2018 May; 101:74-86.

[0090] In some embodiments, the photoprotein domain is a wild-type photoprotein domain. In some embodiments, the photoprotein domain is an engineered photoprotein domain. In some embodiments, the photoprotein domain is a spectral- shifted photoprotein domain.

[0091] In some embodiments, the photoprotein domain is selected from the group consisting of an obelin domain, an aequorin domain, and a clytin domain. In some embodiments, the photoprotein domain is derived from hydromedusan Aequorea aequorin), Halistaura (Mitrocoma) halistaurin, Phialidium (Clytia) phialidin, hydroid Obelia geniculate and Obelia longissima obelms, and ctenophore Mnemiopsis and Beroe mnemiopsin and beroin.

[0092] Luciferase and photoprotein domains are described, for example, in International Publication No. WO1998026277A2, which is hereby incorporated by reference in its entirety.21323198441

[0093] Amino acid sequences of exemplary bioluminescent protein domains are provided in Table 2.Table 2. Exemplary bioluminescent protein domain amino acid sequences SEQ TDDescription Amino Acid SequenceNO VFTL E D FVGDWRQT AG YNL DQVL EQGGVS S L FQNL GVS VT P IQRIVLSGENGLKIDIHVI I PYEGLSGDQMGQIEKI FKVVYNanoLu c®3 P V D D H H F K V I L H Y GT L VI D G VT P NM I D Y F G R P YE G I AV F D G luciferase K K I T VT GT L WN GN K 11 D E RL I N P D G S L L F RVT I N G VT G WRL CERILANanoBit™ MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVT LgBiT P I QRI VRS GENALKI D I HVI I P YEGL S ADQMAQI EEVFKW 4luciferase Y P VD D H H F K V I L P Y G T L V I D G VT P NML N Y F G R P Y E G I AV F D subunit GKKITVTGTLWNGNKI I DERL IT PDGSMLFRVT INS NanoBit™SmBiT5 VTGYRLFEEILluciferasesubunitMEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGT I A FT DAH I E VN I T YAE Y F EMS VRL AE AMKR YGL NT NH R I VVC S E N S L Q F FM P VL G A L F I G V A V A P A N D I Y N E R E L L N S M N I S Q PFirefly TVVFVSKKGLQKILNVQKKLPI IQKI I IMDSKTDYQGFQSM luciferase N- YT FVT SHL P PGFNE YDFVPE S FDRDKT I AL IMNS S G ST GLP terminus 6 KGVAL PHRT AC VRFS HARD P I FGNQ I I P DT A I L S VVP FHHG (Photinus F GM F T T L G Y L I C G F R V VLM Y R F E E E L F L R S L Q D Y K I Q S AL L pyralis) VPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVA K R F H L P G I RQ G Y G L T E T T S A I L I T P E G D D K P G AV G K W P F F EAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNA LIDKD MMKRE KNV I YG P E PL H P L E D KT AGEML F RAL RKH S HL P Q Al C-terminus of VDVFGDESLSYKEFFEATCLLAQSLHNCGYKMNDVVSICAE Click beetle NNKRF F I P 11 AAWYI GMI VAP VNE S Y I P DELC KVMG I S E PQ luciferase I VFC T KN I L NKVL E VQ S RT N F I KR 111 L DT VE N I H GC E S L P {Caribbean N F I S R Y S D GN I AN F K P L H Y D P VE Q VAA I L C S S GT T GL P K. GV MQTHQN I C VRL I HAL D P EAGT QL I PGVT VL VYVP F FHAFGFPyrophorusS INLGYFMVGLRVIMLRRFEQEAFLKAIQDYEVRS IVNVPAplagiophthala 7 I ILFLSKSPLVDKYDLSSLRELCCGAAPLAKEVAEIAVKRL mus) ammo NL P G I RC G F GL T E S T S AN I H S L G D E F K S G S L G RVT P LMAAK acids 395-542 I ADRET GKALGPNQVGELC I KGPMVS KGYVNNVKAT KEAI D with mutations DDGWLHSGDFGYYDEDEHFYVVDRYKELIKYKGSQVAPAEL F420I, G421A, E S I LL KNPC I RDVAVVG I P DL EAGEL P S AFVVI Q P GKE I TA E453S KE V YD YLAE RV S HT K YL RGGVRFVDS I P RNVT GK I T RKELLKQLLEKSSKLSubstrates of bioluminescent protein domains22323198441

[0094] The bioluminescent protein domains described herein are capable of catalyzing a reaction that emits light. In some embodiments, the reaction emits light upon oxidation of a substrate. Typically, the emission spectrum of a bioluminescent protein domain varies depending on the identity of the substrate used (see, e.g., Gaspar N, et al. J Photochem Photobiol B. 2021 Mar;216: 112128). Therefore, when using a bioluminescent protein domain to act on a light-and-magnetoresponsive protein as described herein, typically one would select a substrate and bioluminescent protein domain pair that together have an emission spectrum that overlaps with the excitation spectrum of the light-and-magnetoresponsive protein.

[0095] Various luciferins have been described in the art, for example, in International Publication No. WO1998026277A2, and Yao Z, et al. Curr Opin Chem Biol. 2018 Aug;45: 148-156. In some embodiments, the luciferin is selected from the group consisting of coelenterazine or an analog thereof, a firefly luciferin, a snail luciferin, a bacterial luciferin, a dinoflagellate luciferin, vargulin or an analog thereof, or a fungal luciferin. In some embodiments, the luciferin is a hydrofurimazine, a fluorofurim azine, a diphenylterazine, or an / XkaLumine. In some embodiments, the luciferin is a synthetic luciferin. In some embodiments, the luciferin is a naturally occurring luciferin. In some embodiments, one or more distinct luciferins are used.

[0096] In some embodiments, the luciferin is coelenterazine. In some embodiments, the luciferin is an analog of coelenterazine. Coelenterazine analogs are described, for example, in International Publication No. WO2014036482 A2.

[0097] In some embodiments, the luciferin is furimazme. In some embodiments, the bioluminescent protein domain is a NanoLuc® luciferase, and the luciferin is furimazme.

[0098] In some embodiments, the bioluminescent protein domain catalyzes a reaction that emits light with a wavelength capable of inducing a change in a light-and-magnetoresponsive protein domain as described herein.Heterologous Protein Components

[0099] In some embodiments, the compositions provided herein further comprises a heterologous protein component. “Heterologous protein component,” as used herein, refers to a protein or peptide component that is different from the protein or peptide of the23323198441light-and-magnetoresponsive protein domain (whether or not derived from the same organism as the peptide or protein of the light-and-magnetoresponsive protein domain). Typically, the heterologous protein component is not derived from the same organism as the peptide or protein of the light-responsive domain.

[0100] As described above, in some embodiments, the compositions comprising light-and-magnetoresponsive protein domains described herein are capable of undergoing changes, e.g., conformational in response to exposure to light and / or magnetic fields. In some embodiments, the change mediates, in turn, a change in the overall composition. The overall change may activate or deactivate the heterologous protein component. For example, a conformational change may expose a binding site on the heterologous protein component, thus allowing it to bind a ligand. In another example, a conformational change may mask a binding site, thus blocking ligand binding. In this way, activity of the heterologous protein component may be made dependent on the presence of light and a magnetic field,

[0101] In some embodiments, the heterologous protein component is a selected from the group consisting of a cytokine, an antibody or a fragment thereof, a synthetic binding protein, a chimeric antigen receptor (CAR), a CRISPR-Cas protein, a channelrhodopsin, a transcription factor, an antibody-drug conjugate, and an antibody-radioligand conjugate.

[0102] Examples of heterologous protein components include nanobodies, monobodies, minibodies, antibodies or fragments thereof, growth factors, designed ankyrin repeat proteins (DARPins) (e.g., E40, pE59), antimicrobial peptides (e.g., LL-37, IDR-1018, IDR-1019, pexiganan, Bac2A, W3), enzymes and therapeutic proteins or peptides. In some embodiments, the heterologous protein component is an anticalin. In some embodiments, the heterologous protein component is selected from a nano body, a monobody, an antibody or fragment thereof, a DARPin, or an anticalin.

[0103] In some embodiments, the heterologous protein component is a therapeutic protein or peptide. A “therapeutic protein or peptide” is a heterologous protein component useful as a therapeutic agent. Some therapeutic proteins or peptides can bind a cell surface target, such as a cell surface protein or receptor. In some embodiments, the therapeutic protein or peptide is a cytokine (e.g., an interferon), peptide hormone (e.g., insulin), growth factor, (e.g., an epidermal growth factor, a fibroblast growth factor, and24323198441platelet-derived growth factor). Examples of growth factors which bind and activate a receptor tyrosine kinase include epidermal growth factor and fibroblast growth factor-7 (also known as keratinocyte growth factor). Other nonlimiting examples of therapeutic proteins and peptides include insulin, monoclonal antibodies (e.g., trastuzumab, ipilimumab, infliximab, adalimumab), circulating receptor fusion proteins (e.g., etanercept), thrombolytic proteins (e.g., tissue plasminogen activator), and natural product toxins (e.g., conotoxin).

[0104] In some embodiments, the heterologous protein component comprises a cytokine. In some embodiments, the cytokine is human interleukin-2.

[0105] In some embodiments, the heterologous protein component comprises an antibody. The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0106] In some embodiments, the heterologous protein component comprises an antibody fragment. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen- combining sites and is still capable of cross-linking antigen.

[0107] In some embodiments, a light and magnetoresponsive protein domain is inserted into the AK74 insertion loop site of an antibody or fragment thereof. In some embodiments, a truncated MagLOV domain is inserted into the AK74 insertion loop site of an antibody or fragment thereof.

[0108] In some embodiments, any one of the antibodies or fragments thereof is conjugated to a drug or to a radioligand.25323198441

[0109] In some embodiments, the heterologous protein component comprises a nanobody (e.g., which binds and inhibits a cell surface receptor, such as a receptor tyrosine kinase). Typically, nanobodies are small binding proteins formed from the single variable domain of camelid antibodies.

[0110] In some embodiments, the heterologous protein component comprises a synthetic binding protein, such as an affibody, an anticalin, a monobody, a minibody, or a DARPin.

[0111] In some embodiments, the heterologous protein component comprises a monobody. Monobodies, examples of which are known in the art, are synthetic binding proteins that are constructed using a fibronectin type III domain (FN3) as a molecular scaffold. Monobodies are an alternative to antibodies and nanobodies for creating a target-binding protein,

[0112] In some embodiments, the heterologous protein component comprises a DARPin.

[0113] In some embodiments, the heterologous protein component comprises an anticalin. An anticalin is an artificial protein able to bind to antigens, proteins or small molecules. Anticalins are derived from human lipocalms.

[0114] In some embodiments, the heterologous protein component comprises a chimeric antigen receptor (CAR). In some embodiments, the heterologous protein component comprises a T cell receptor (TCR).

[0115] In some embodiments, the heterologous protein component comprises an enzyme. In some embodiments, the heterologous protein component comprises a nuclease. In some embodiments, the heterologous protein component comprises a protease.

[0116] In some embodiments, the heterologous protein component comprises a CRISPR / Cas protein. In some embodiments, the heterologous protein component comprises a RuvC domain.

[0117] In some embodiments, the heterologous protein component comprises a Class 2 Type V Cas protein. In some embodiments, the heterologous protein component comprises a Cas 12 protein. In some embodiments, the Cas 12 protein is Cas 12a, Cas 12b, Cas 12c, CasY, or Casl2e. In some embodiments, the heterologous protein component comprises a catalytically dead Cas 12 protein.

[0118] In some embodiments, the heterologous protein component comprises a Class 2 Type II Cas protein. In some embodiments, the heterologous protein component26323198441comprises Cas9. In some embodiments, the heterologous protein component comprises a catalytically dead Cas9.

[0119] In some embodiments, the heterologous protein component comprises a transcription factor, e.g., a sequence-specific DNA-binding protein that modulates transcriptional expression.

[0120] In some embodiments, the heterologous protein component is a protein tag. In some embodiments, the protein tag is a SsrA tag, a Myc tag, or a His tag.Exemplary Compositions

[0121] Exemplary compositions comprising light-and-magnetoresponsive protein domains are described herein.

[0122] In some embodiments, a heterologous protein component as described herein is covalently linked to a light-and-magnetoresponsive protein domain.

[0123] In some embodiments, the heterologous protein component and the light-and-magnetoresponsive protein domain are covalently linked to form a fusion protein. The term “fusion protein” refers to a synthetic, semi-synthetic or recombinant single protein molecule that comprises all or a portion of two or more different proteins and / or peptides. The fusion can be an N-terminal fusion (e.g., with respect to the heterologous protein component), a C-terminal fusion (e.g., with respect to the heterologous protein component) or an internal fusion (e.g., with respect to the light-and-magnetoresponsive protein domain and / or the heterologous protein component). In some embodiments, the fusion protein is an internal fusion protein, and the light-and-magnetoresponsive protein domain (e g., an engineered LOV domain) is inserted into the heterologous protein component.

[0124] In some embodiments, a bioluminescent protein domain is covalently linked to a heterologous protein component. In some embodiments, a bioluminescent protein domain is covalently linked to a heterologous protein component to form a fusion protein,

[0125] In some embodiments, a bioluminescent protein domain is covalently linked to a light-and-magnetoresponsive protein domain. In some embodiments, a bioluminescent protein domain is covalently linked to a light-and-magnetoresponsive protein domain to form a fusion protein.27323198441

[0126] In some embodiments, a bioluminescent protein domain is covalently linked to a heterologous protein component and a light-and-magnetoresponsive protein domain. In some embodiments, a bioluminescent protein domain is covalently linked to a heterologous protein component and a light-and-magnetoresponsive protein domain to form a fusion protein.

[0127] In some embodiments, provided herein is fusion protein comprising, from N- to C-terminus, a first split luciferase domain, a light-and-magnetoresponsive protein domain, and a second split luciferase domain. In some embodiments, provided herein is fusion protein comprising, from N- to C-terminus, an N-terminal split luciferase domain, a light-and-magnetoresponsive protein domain, and a C -terminal split luciferase domain.

[0128] In some embodiments, provided herein is fusion protein comprising, from N- to C-terminus, a first split luciferase domain, a light-and-magnetoresponsive protein domain, a second split luciferase domain, and a heterologous protein component. In some embodiments, provided herein is fusion protein comprising, from N- to C-terminus, an N-terminal split luciferase domain, a light-and-magnetoresponsive protein domain, a C-terminal split luciferase domain, and a heterologous protein component.

[0129] In some embodiments, provided herein is fusion protein comprising, from N- to C-terminus, a heterologous protein component, a first split luciferase domain, a light-and-magnetoresponsive protein domain, and a second split luciferase domain. In some embodiments, provided herein is fusion protein comprising, from N- to C-terminus, a heterologous protein component, an N-terminal luciferase domain, a light-and- magnetoresponsive protein domain, and a C-terminal split luciferase domain.

[0130] In some embodiments, provided herein is fusion protein comprising a bioluminescent protein domain, a heterologous protein component, and a light-and- magnetoresponsive protein domain, wherein the bioluminescent protein domain is internally fused within the heterologous protein component. In some embodiments, the bioluminescent protein domain is fused within a loop region of the heterologous protein component.

[0131] In some embodiments, provided herein is fusion protein comprising one light- and-magnetoresponsive protein domain. In some embodiments, provided herein is fusion protein comprising two light-and-magnetoresponsive protein domains.28323198441

[0132] In some embodiments, a light-and-magnetoresponsive protein domain can be fused to a heterologous protein component and / or a bioluminescent protein domain via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 ammo acids and 25 amino acids in length. These linkers are generally produced by using synthetic, linker encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used.

[0133] In some embodiments, a flexible peptide is used as a linker. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. Example linker polypeptides include glycine polymers (G)n, glycme-serine polymer (including, for example, (GS)n, GSGGSn (SEQ ID NO: 11), GGSGGSn (SEQ ID NO: 12), and GGGSn (SEQ ID NO: 13), where n is an integer of at least one), glycme-alanine polymers, alanme-serine polymers, glycine-proline polymers, proline polymers and proline-alanine polymers. Example linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 14), GGSGG (SEQ ID NO: 15), GSGSG (SEQ ID NO: 16), GSGGG (SEQ ID NO: 17), GGGSG (SEQ ID NO: 18), GSSSG (SEQ ID NO: 19), GGSGGGSGG (SEQ ID NO: 36), GPGP (SEQ ID NO: 20), GGP, PPP, PPAPPA (SEQ ID NO: 21), PPPGPPP (SEQ ID NO: 22) and the like. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 23). In some embodiments, the linker comprises the amino acid sequence of GGGSGGGSGGGSGGGS (SEQ ID NO: 10). In some embodiments, the linker comprises the ammo acid sequence of GDPLVQCGGIALAATM (SEQ ID NO: 28).

[0134] In some embodiments, a rigid peptide is used as a linker. In some embodiments, the linker is an alpha-helix forming linker. In some embodiments, the linker is a prolinerich sequence. Examples of rigid peptide linkers include, but are not limited to, (EAAAK)n (SEQ ID NO: 24) and (XP)n. In some embodiments, a rigid peptide linker is used to fuse a light-and-magnetoresponsive domain to a bioluminescent domain. In some29323198441embodiments, a rigid peptide linker is used to fuse a light-and-magnetoresponsive domain to a heterologous protein component. Rigid peptide linkers may be advantageous for coupling a conformational change in a light-and-magnetoresponsive domain to a heterologous protein component.

[0135] The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.

[0136] In some embodiments, the fusion protein comprises a cell -penetrating peptide component, allowing for intracellular delivery of the fusion protein. Fusion proteins comprising a cell-penetrating peptide component can be used to contact the outside of a cell, penetrate the cell, and then bind an intracellular target determined by the heterologous protein component. A “cell-penetrating peptide” is a short peptide that facilitates cellular intake / uptake of a molecular component with which it is associated. The cell-penetrating peptide can be associated with the fusion proteins described herein either through chemical linkage via covalent bonds or through non-covalent interactions. The function of the cell-penetrating peptide is to deliver the fusion protein into cells (e.g., through endocytosis). Examples of suitable cell penetrating peptides for the fusion proteins described herein are the Twin-argmine translocation (Tat) pathway signal sequence (InterPro Accession: IPR006311) and polyarginine sequences.

[0137] Fusion proteins of the invention can be produced recombinantly or synthetically, using routine methods and reagents that are well known in the art. For example, a fusion protein of the invention can be produced recombinantly in a suitable host cell (e.g., bacteria, yeast, insect cells, mammalian cells) according to methods known in the art. See, e.g., Current Protocols in Molecular Biology, Second Edition, Ausubel etal. eds., John Wiley & Sons, 1992; and Molecular Cloning: A Laboratory Manual, 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. For example, a nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein described herein can be introduced and expressed in suitable host cell (e.g., E. coll), and the expressed fusion protein can be isolated / purified from the host cell (e.g., in inclusion bodies) using routine methods and readily available reagents. For example, DNA fragments coding for30323198441different protein sequences (e.g., a light-and-magnetoresponsive protein domain, a heterologous protein component, and / or a bioluminescent protein domain) can be ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of nucleic acid fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments that can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992).Compositions comprising a magnetically-controlled bioluminescent proteindomain

[0138] Provided herein is a fusion protein comprising a bioluminescent protein domain fused to a light-and-magnetoresponsive protein domain. Any one of the bioluminescent protein domains and light-and-magnetoresponsive protein domains may be used. In some embodiments, the fusion protein is capable of catalyzing a reaction that emits light upon exposure to a magnetic field and a substrate, e.g., a luciferin. A light-and-magnetoresponsive protein domain may be fused to the N- or C-terminus of a bioluminescent protein domain, or it may be inserted internally into the bioluminescent protein domain amino acid sequence. In some embodiments, the engineered LOV domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 26, and 48, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered LOV domain comprises an amino acid sequence selected from the group of SEQ ID NOS: 1, 26, and 48. In some embodiments, the engineered LOV domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 26, and 48 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the bioluminescent protein domain is a luciferase domain. In some embodiments, the bioluminescent protein domain is a split luciferase domain.31323198441NOBS-001 / 02WG 356250-2007

[0139] As one example, as described in Example 3, a luciferase enzyme fused to a light-and-magnetoresponsive protein domain (MagLOV) was designed. The resulting magnetically-controlled luciferase may be used in compositions comprising light- responsive protein domains, so that activation of the luciferase is controlled by the presence of a magnetic field. Thus, upon exposure to a magnetic field and a suitable substrate, the magnetically-controlled luciferase catalyzes a reaction that emits light, which can activate a light-responsive protein.

[0140] In particular embodiments, a truncated LOV domain as described herein is fused to a bioluminescent protein domain. Fusion of a truncated LOV domain fused to a bioluminescent protein domain is believed to be particularly advantageous because the smaller overall protein is expected to be easier to express and deliver to a subject, and have fewer exposed immunogenic epitopes, A truncated LOV domain may be fused to the N- or C-terminus of a bioluminescent protein domain, or it may be inserted internally into the bioluminescent protein domain amino acid sequence. In some embodiments, the truncated LOV domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 88-95, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the truncated LOV domain comprises an amino acid sequence selected from the group of SEQ ID NOS: 2 and 88-95. In some embodiments, the truncated LOV domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 88-95 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the bioluminescent protein domain is a luciferase domain. In some embodiments, the bioluminescent protein domain is a split luciferase domain.

[0141] In some embodiments, fusion protein comprising a bioluminescent protein domain and a truncated LOV domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 50-55 and 57-67, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the fusion protein32323198441comprises an amino acid sequence selected from the group of SEQ ID NOS: 50-55 and 57-67. In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 50-55 and 57-67 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.

[0142] Also provided herein is a composition comprising a bioluminescent protein domain and a light-and-magnetoresponsive protein domain as described herein, wherein the bioluminescent protein domain and the light-and-magnetoresponsive protein domain are not covalently linked as a fusion protein. Again, any one of the bioluminescent protein domains and light-and-magnetoresponsive protein domains described herein may be used. In some embodiments, the composition is capable of catalyzing a reaction that emits light upon exposure to a magnetic field and catalysis of a substrate, e.g. a luciferin.

[0143] In some embodiments, emission of light is capable of activating, exciting, or inducing a change in a light-responsive protein. Various light-responsive proteins are known in the art and include, but are not limited to: CRY2, channelrhodopsins (e.g. ChRl and ChR2 derived from Chlamydomonas reinhardtii), PYP (derived from Halorhodospira halophila), heterodimerization / homodimerization domains (e.g., PixD + PixE, VVD, and pMag+nMag), photocleavable domains (e.g., PhoCl), light-inducible nuclear import domains (e.g., LEXY, and LINuS), engineered photocontrolled enzymes (e.g., LINX and LINuS), and LOV domains (e.g., AsLOV, BcLOV4, and cpLOV). In some embodiments, the light-responsive protein is a variant of an AsLOV domain. In some embodiments, the light-responsive protein is a slow-recovery variant of an AsLOV domain, e.g., a variant with a N414A, V416L, N414L, N538E, G5228E, or L496I amino acid substitution relative to a wild-type AsLOV domain.

[0144] In some embodiments, provided herein are compositions comprising: (a) a first fusion protein comprising a bioluminescent protein domains fused to a light-and-magnetoresponsive protein domain; and (b) a second fusion protein comprising a light- responsive protein and a heterologous protein component. In some embodiments, the heterologous protein component of the second fusion protein is an antibody.

[0145] The light-responsive protein may itself be covalently linked to any one of the heterologous protein components described herein. As a result, activity of the33323198441heterologous protein components may be made dependent on the light-responsive protein.Compositions with improved light-induced dimer (iLID) amino acid substitutions

[0146] Provided herein are compositions comprising an engineered LOV domain comprising induced dimer (iLID) amino acid substitutions, as described above. As described in Example 14, an engineered LOV domain comprising iLID amino acid substitutions is also referred to herein as “Mag-iLID” or a Mag-iLID domain. In some embodiments, a Mag-iLID domain is fused to a protein tag, such as an SsrA peptide. Fusion to an SsrA peptide allows the Mag-iLID domain to bind to SspB, the heterodimerization pair for SsrA. The presence of Mag-iLID controls the binding so that the binding of the Mag-iLID-SsrA fusion construct’s binding to SspB depends on the presence of a magnetic field and / or light. In this way, Mag-iLID may be used to create a controllable binding pair that only binds in the presence of both a magnetic field and light. In embodiments further comprising a bioluminescent protein domain, its binding will depend on the presence of a magnetic field and administration of a substrate of the bioluminescent domain,

[0147] In some embodiments, a Mag-iLID-SsrA fusion construct is fused to a first bioluminescent protein domain, and SspB is fused to a second bioluminescent protein domain. In some embodiments, a Mag-iLID-SsrA fusion construct is fused to a first bioluminescent protein domain and an antibody, and SspB is fused to a second bioluminescent protein domain. In some embodiments, a Mag-iLID-SsrA fusion construct is fused to a first bioluminescent protein domain, and SspB is fused to a second bioluminescent protein domain and an antibody. In some embodiments, a Mag-iLID-SsrA fusion construct is fused to an antibody, and SspB is fused to a bioluminescent protein domain. In such embodiments, binding of the antibody to its target is dependent on the presence of light and / or a magnetic field.

[0148] In some embodiments, a Mag-iLID-SsrA fusion construct or SspB is engineered to be expressed on the surface of a CAR-T cell, or the surface of a particle such as a lipid nanoparticle (LNP), a virus-like particle (VLP), or a viral particle. When the respective Mag-iLID-SsrA fusion construct and SspB binding partners meet, their binding affinity to each other will change depending on the presence of light and / or a magnetic field. In 34323198441embodiments further comprising a bioluminescent protein domain, binding of the Mag-iLID-SsrA fusion construct to SspB will depend on the presence of a magnetic field and administration of a substrate of the bioluminescent domain.

[0149] In some embodiments, a cell is engineered to express surface-presenting proteins fused to SspB, or otherwise coat the cell with SspB. This is believed to allow for the targeted delivery of a therapy fused to a Mag-iLID-SsrA fusion construct to the coated cell, in a way that is dependent on the presence of a magnetic field and / or light. In embodiments further comprising a bioluminescent protein domain, binding of the Mag-iLID-SsrA fusion construct to the SspB-coated cell will depend on the presence of a magnetic field and administration of a substrate of the bioluminescent domain.

[0150] In some embodiments, a Mag-iLID-SsrA fusion construct or SspB is fused to a DNA-binding domain to allow for the controllable recruitment of transcription factor effector domains to the DNA-binding domain. For example, if Mag-iLID-SsrA is fused to a DNA-binding domain, SspB is fused to a protein domain that promotes the activation or repression of transcription, or vice versa. This allows for the modulation of transcription in a way that is dependent on the presence of light and / or a magnetic field. In embodiments further comprising a bioluminescent protein domain, modulation of transcription depends on the presence of a magnetic field and administration of a substrate of the bioluminescent domain.Nucleic Acids and Vectors

[0151] Also provided herein are nucleic acids encoding the compositions described herein. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, such as mRNA. In some embodiments, the nucleic acid is a plasmid.

[0152] Provided herein are expression cassettes encoding the bioluminescent protein domains, light-and-magnetoresponsive protein domains, and / or heterologous protein components as described herein. Expression of the coding sequences may be driven by a promoter. The coding sequences may be codon-optimized, e.g., codon- optimized for expression in a human subject.

[0153] Also provided herein are plasmids encoding the compositions described herein. The plasmids may comprise one or more expression cassettes, for expressing the35323198441bioluminescent protein domains, light-and-magnetoresponsive protein domains, and / or heterologous protein components, as well as additional protein products. In some embodiments, the plasmid further comprises an expression cassette for a reporter gene such as a gene encoding a fluorescent protein.

[0154] Also provided herein are vectors comprising the nucleic acids described herein. In some embodiments, the vector is a viral vector. Examples of viral vectors include adeno-associated viral (AAV) vectors, adenoviral vectors, retroviral vectors, and lentiviral vectors.Cells

[0155] Also provided herein are one or more cells comprising any one of the nucleic acids, vectors, or compositions described herein.

[0156] In some embodiments provided herein is a cell comprising any one of the compositions or fusion proteins described herein.

[0157] In some embodiments, provided herein is a cell comprising a component of the compositions described herein. In some embodiments, the cell comprises a light-and-magnetoresponsive protein domain. In some embodiments, the cell comprises a light-and-magnetoresponsive protein domain covalently linked to a heterologous protein component. In some embodiments, the cell comprises a fusion protein comprising the light-and-magnetoresponsive protein domain fused to a heterologous protein component. In some embodiments, the cell comprises a light-and-magnetoresponsive protein domain covalently linked to a bioluminescent protein domain and a heterologous protein component. In some embodiments, the cell comprises a fusion protein comprising the light-and-magnetoresponsive protein domain fused to a bioluminescent protein domain and heterologous protein component.

[0158] In some embodiments, provided herein is a first cell comprising a first component of the compositions described herein, and a second cell comprising a second component of the compositions described herein. In some embodiments, a first cell comprises a light-and-magnetoresponsive protein domain, or a light-and-magnetoresponsive protein domain covalently linked to a heterologous protein component, and a second cell comprises a bioluminescent protein domain.36323198441

[0159] A cell that can comprise the compositions of the present disclosure can be any of a variety of cells, including, e.g., in vitro cells; in vivo cells; ex vivo cells; primary cells; cells of an immortalized cell line; cancer cells; animal cells; plant cells; algal cells; fungal cells; etc. In some embodiments, the one or more cells are eukaryotic cells, prokaryotic cells, or cells from a multicellular organism (e.g., a cell line) cultured as a unicellular entity. In some embodiments, the one or more cells are mammalian, bacterial, yeast, insect, or fungal cells. In some embodiments, the one or more cells are human cells.

[0160] In some embodiments, the one or more cells are in vitro cells (e.g., established cultured cell line including, but not limited to HEK293 cells, HEK293T cells, HEK293-F cells, Lenti-X 293T cells, BHK cells, HepG2 cells, Saos-2 cells, HuH7 cells, A549 cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER. C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS, WI38 cells, MRC5 cells, HeLa, HT1080 cells, or CHO cells). A cell can be an ex vivo cell (cultured cell from an individual).

[0161] Further suitable cells may include, in some embodiments, a stem cell (e g. an embryonic stem (ES) cell, an induced pluripotent stem (IPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic stem cell, a neuron progenitor cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a retinal cell, a cancer cell, a T-cell, a B-cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplanted expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, fibroblasts, osteoblasts, chondrocytes, exogenous cell, endogenous cell, stem cell, hematopoietic stem cell, bone-marrow derived progenitor cell, myocardial cell, skeletal cell, fetal cell, undifferentiated cell, multi-potent progenitor cell, unipotent progenitor cell, a monocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, and a post-natal stem cell.

[0162] In some embodiments, the one or more cells are immune cells. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the37323198441immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg). In some cases, the cell expresses a chimeric antigen receptor (CAR).

[0163] In some embodiments, the one or more cells are stem cells. Stem cells may include, for example, adult stem cells. Adult stem cells can also be referred to as somatic stem cells. In some embodiments, the stem cell is a hematopoietic stem cell (HSC), neural stem cell or a mesenchymal stem cell. In other embodiments, the stem cell is a mesenchymal stem ceil (MSC).Methods

[0164] Provided herein are methods of using the compositions described herein. In some embodiments, provided herein is a method of using the compositions described herein comprising a light-and-magnetoresponsive domain, a bioluminescent protein domain, and a heterologous protein component. In some embodiments, the method comprises (a) providing the composition, (b) exposing the composition to a substrate, wherein the bioluminescent protein domain is capable of catalyzing a reaction of the substrate, w’herein the reaction emits light, and (c) exposing the composition with a magnetic field. In some embodiments, the heterologous protein component is activated upon exposure to the substrate and the magnetic field. In some embodiments, the heterologous protein component is deactivated upon exposure to the substrate and the magnetic field.

[0165] In some embodiments, provided herein is a method of using the compositions described herein comprising a light-and-magnetoresponsive domain, and a heterologous protein component. In some embodiments, the compositions for use in this method do not comprise a bioluminescent protein domain. In some embodiments, the method comprises (a) providing the composition, and (b) exposing the composition to a magnetic field. In some embodiments, the heterologous protein component is activated upon exposure to the magnetic field. In some embodiments, the heterologous protein component is deactivated upon exposure to the magnetic field.

[0166] In some embodiments, the composition is first administered to a subject. The composition may be administered by subcutaneous, intradermal, intraneural, intranodal, intramedullary, intramuscular, intralumbar, intrathecal, subarachnoid, intraventricular, intracapsular, intravenous, intralymphatical, intravitreal, subretinal, or intraperitoneal38323198441routes. In some embodiments the subject is a human. In some embodiments, the subject is in need of treatment of a cancer, an autoimmune disorder, or an infectious disease.Following administration of the composition, the subject may be exposed to a magnetic field and / or administered a substrate of the bioluminescent protein domain, as described in further detail below.Exposing to a Magnetic Field

[0167] In some embodiments, the methods described herein comprise a step of exposing the compositions described herein to a magnetic field. Exposure to a magnetic field allows for the control of the activation or deactivation of the compositions described herein, so that the compositions are active or deactivated only in the location and at the time that a magnetic field is applied. As described in further detail below, this may be paired with administration of a substrate of a bioluminescent protein domain to allow for a high level of control over the compositions’ activity. In some embodiments, the magnetic field modulates the activity of a bioluminescent protein domain, and the light emitted by the reaction catalyzed by the bioluminescent protein domain modulates the activity of an optogenetic protein domain.

[0168] In some embodiments, the magnetic field is a resonant magnetic field. In some embodiments, a resonant magnetic field differentially induces activity in a plane perpendicular to the direction of the static field, where the position of that plane is defined by its relative position to the magnetic field sources, the resonant frequency of applied radio waves, and the strength of an applied static field. In some embodiments, a resonant field is induced by a magnetic resonance imaging (MRI) machine.

[0169] In some embodiments, the magnetic field is a static magnetic field. In some embodiments, the magnetic field is an alternating magnetic field.

[0170] In some embodiments, the magnetic field is generated by a permanent magnet. In some embodiments, the magnetic field is generated by an electromagnet placed nearby to create differential field strengths across the patient or other substrate.

[0171] In some embodiments, the magnetic field is generated by one or more magnets. In some embodiments, the magnetic field is generated by one or more magnets configured as a Halbach array. In some embodiments, the magnetic field is generated by one or more magnets configured as a Halbach array manufactured from powdered ferrite mixed in a39323198441flexible binder that was exposed to a Halbach magnetization field pattern as it was extruded. In some embodiments, a Halbach array enables directional control over the magnetic field, such that the magnetic field is delivered to a targeted region.

[0172] A magnetic field may be generated by a wearable device. A wearable device allows for the generation of a magnetic field in a subject who has been administered a composition as described herein. The wearable device should be placed on the body of a subject in physical proximity to where the composition should be activated or deactivated. For example, if a composition as described herein is designed to be activated in the lungs, then a subject would wear a wearable device with a magnet near the lungs. In some embodiments, the wearable device comprises magnets attached to or embedded within a brace or other material to secure them in place. In some embodiments, the wearable device comprises magnets attached to or embedded within a brace that is placed on the body of a subject, such as a belt, an arm brace, a vest, bracelet, anklet, leg sleeve, or sock. In some embodiments, multiple magnets are arranged so as to create a zero point. In some embodiments, magnets are placed on movable arms to modulate the position of the field.

[0173] In some embodiments, a magnetic field is generated by a mix of electromagnets and permanent magnets. In some embodiments, a magnetic field is generated by one or more electromagnets. In some embodiments, the electromagnets are driven by alternating current. In some embodiments, the magnets are magnetic resonance (MR)-safe magnets.

[0174] In some embodiments, the magnets are implantable. In some embodiments, the magnets are implanted at locations near the disease tissue. In some embodiments, the magnets are implanted in locations which anticipate likely sites of metastasis of disease or emergence of disease. In some embodiments, the magnetic field strength and / or positioning is modulated by a computer.

[0175] In some embodiments, the magnetic field is generated by a handheld magnet. In some embodiments, the handheld magnet is selected from the group consisting of a rare earth (e.g., neodymium or samarium-cobalt), ferrite, or alnico magnet. In some embodiments the magnets are rare-earth non-ferromagnetic magnets.

[0176] In some embodiments, the magnet generates a magnetic field of about 0.01 mT to about 1000 mT. In some embodiments, the magnet generates a magnetic field of about403231984410.01 mT, about 0.1 mT, about 1 mT, about 10 mT, about 100 mT, or about 1000 mT. In some embodiments, the magnet generates a magnetic field of about 5 mT to about 25 mT. In some embodiments, the magnet generates a magnetic field of about 5 mT, about 10 mT, about 15 mT, about 20 mT, or about 25 mT.

[0177] In some embodiments, the magnetic field is applied for about 1, 5, 10, 20, 30, 40, 50, or 60 seconds or more. In some embodiments, the magnetic field is applied for about 1, 5, 10, 20, 30, 40, 50, or 60 minutes or more. In some embodiments, the magnetic field is applied for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more. In some embodiments, the magnetic field is applied for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days or more.Exposure to a substrate of the bioluminescent protein domain

[0178] In some embodiments, the methods described herein comprise a step of exposing the composition to a substrate of the bioluminescent protein domain. This allows the bioluminescent protein domain to catalyze a reaction that emits light, which in turn acts on the light-and-magnetoresponsive domain. Exposure to the substrate adds an additional layer of control to the activity of the composition described herein because it can function as an “AND gate,” meaning that the composition’s activity is dependent on both the presence of the light and the magnetic field.

[0179] Any one of the substrates described herein may be used in the methods described herein. In some embodiments, the substrate is a luciferin. In some embodiments, the luciferin is selected from the group consisting of coelenterazine or an analog thereof, a firefly luciferin, a snail luciferin, a bacterial luciferin, a dinoflagellate luciferin, vargulin or an analog thereof, or a fungal luciferin. In some embodiments, the luciferin is furimazine. In some embodiments, the luciferin is a synthetic luciferin. In some embodiments, the luciferin is a naturally-occurring luciferin.

[0180] In some embodiments, the substrate is administered to a subject. The substrate may be administered by a subcutaneous, intradermal, intraneural, mtranodal, intramedullary, intramuscular, intralumbar, intrathecal, subarachnoid, intraventricular, intracapsular, intravenous, intralymphatical, intravitreal, subretinal, or intraperitoneal route.41323198441

[0181] In some embodiments, a subject is first administered a composition comprising a bioluminescent domain as described herein. Second, the subject is administered a substrate of the bioluminescent protein domain. Third, the subject is exposed to a magnetic field as described above. The magnetic field is applied only m the location that the composition is designed to be activated or deactivated. Thus the activity of the composition is highly controlled based on the need for both the substrate and the magnetic field to be present in the same location and at the same time.Activation and deactivation of a heterologous protein component

[0182] As described above, the compositions described herein are sensitive to exposure to light and / or magnetic fields. In some embodiments, a exposure to light and / or a magnetic field activates or deactivates the heterologous protein component. Accordingly, in some embodiments of the methods described herein, a heterologous protein component is activated upon exposure to a substrate of a bioluminescent protein and a magnetic field. Activated as used herein means that the activity of the heterologous protein component is increased relative to a baseline.

[0183] In some embodiments of the methods described herein, a heterologous protein component is deactivated upon exposure to a substrate of a bioluminescent protein and a magnetic field. In some embodiments of the methods described herein, a heterologous protein component is activated or deactivated upon exposure to a magnetic field. Deactivated as used herein means that the activity of the heterologous protein component is decreased relative to a baseline.

[0184] Typically, the baseline that is used for reference to determine whether a heterologous protein component is activated or deactivated is the activity level of the heterologous protein component before exposure to a substrate of the bioluminescent domain and exposure to a magnetic field.

[0185] “Activity,” as used herein with respect to heterologous protein components, encompasses, for example, metabolic activity, enzymatic activity, binding activity and therapeutic (e.g., a biopharmaceutical) activity. In some embodiments, the activity is a binding activity, for example, the ability to bind to a target protein m an in vitro binding activity assay or an in vivo extracellular or intracellular binding activity assay. In some embodiments of the compositions described herein, binding activity is altered upon42323198441exposure of the composition to a substrate of the bioluminescent domain and exposure to a magnetic field that induce a change in the composition and alters the interaction of the heterologous protein component with the target protein. Typically, binding activity of the compositions described herein is mediated by the heterologous protein component of the composition, e.g., a fusion protein, which is made available for binding, or obstructed from binding, depending on the state of the composition.

[0186] “Binding activity” is observed when a heterologous protein component described herein binds to a target molecule, such as a protein, nucleic acid (e.g., deoxyribonucleic acid (DNA), ribonucleic acid (RNA)), carbohydrate, lipid, organic small molecule, etc. A heterologous protein component “binds to” a target molecule if the dissociation constant (Ka) of the interaction between the two species is less than about 10 pM less than about 1 pM or less than about 100 nM. Decreased binding (indicated by a larger Ka value) corresponds to decreased binding activity. Increased binding (indicated by a smaller Ka value) corresponds to increased binding activity. In some embodiments, exposure of the composition to the substrate of the bioluminescent domain increases its binding activity. In some embodiments, exposure of the composition with a magnetic field increases its binding activity. In some embodiments, exposure of the composition to the substrate of the bioluminescent domain decreases its binding activity. In some embodiments, exposure of the composition with a magnetic field decreases its binding activity. In some embodiments, the conformational change induced by exposing a composition described herein to a substrate of the bioluminescent domain and a magnetic field results in an increase or decrease of activity (e.g., binding activity) of at least twofold, at least five-fold, at least ten-fold, and more preferably at least fifty-fold. In some embodiments, the composition does not exhibit any measurable degree of activity in at least one conformational state.ENUMERATED EMBODIMENTS

[0187] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure. The present disclosure is exemplified by the numbered embodiments set forth below.43323198441Set I

[0188] Embodiment 1-1. A composition comprising: (a) a bioluminescent protein domain; and (b) a light-and-magnetoresponsive protein domain.

[0189] Embodiment 1-2. The composition of embodiment I- 1, wherein the light-and- magnetoresponsive protein domain is capable of responding to light emitted by a reaction catalyzed by the bioluminescent protein domain and is capable of responding to a magnetic field.

[0190] Embodiment 1-3. The composition of embodiment 1-1 or embodiment 1-2, wherein the light-and-magnetoresponsive protein domain comprises a light-oxygenvoltage-sensing (LOV) domain,

[0191] Embodiment 1-4, The composition of embodiment 1-3, wherein the LOV domain is an engineered LOV domain.

[0192] Embodiment 1-5. The composition of embodiment 1-4, wherein the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: I -2 and 26.

[0193] Embodiment 1-6. The composition of embodiment 1-5, wherein the engineered LOV domain comprises an ammo acid sequence selected from the group consisting of SEQ ID NOS: 1-2 and 26.

[0194] Embodiment 1-7. The composition of any one of embodiments 1-1 - 1-6, wherein the bioluminescent protein domain comprises a luciferase domain.

[0195] Embodiment 1-8. The composition of embodiment 1-7, wherein the luciferase domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 3-7.44323198441

[0196] Embodiment 1-9. The composition of embodiment 1-8, wherein the luciferase domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 3-7.

[0197] Embodiment I- 10. The composition of embodiment 1-8, wherein the luciferase domain is a split luciferase domain.

[0198] Embodiment 1-11. The composition of any one of embodiments 1-1 - 1-6, wherein the bioluminescent protein domain comprises a photoprotein domain.

[0199] Embodiment 1-12. The composition of embodiment 1-11, wherein the photoprotein domain is selected from the group consisting of an obelin domain, an aequorin domain, and a clytin domain.

[0200] Embodiment 1-13. The composition of any one of embodiments 1-1 - 1-12, wherein the composition further comprises a heterologous protein component,

[0201] Embodiment 1-14. The composition of embodiment 13, wherein the heterologous protein component is a selected from the group consisting of a cytokine, an antibody or a fragment thereof, a synthetic binding protein, a chimeric antigen receptor (CAR), a CRISPR-Cas protein, a channelrhodopsm, a transcription factor, a DNA-binding domain, an antibody-drug conjugate, and an antibody -radioligand conjugate.

[0202] Embodiment 1-15. The composition of embodiment 1-13 or embodiment 1-14, wherein the heterologous protein component is covalently linked to the light-and-magnetoresponsive protein domain.

[0203] Embodiment 1-16. The composition of embodiment 1-15, wherein the heterologous protein component and the light-and-magnetoresponsive protein domain are covalently linked to form a fusion protein.

[0204] Embodiment 1-17. The composition of embodiment 1-15 or embodiment 1-16, wherein the bioluminescent protein domain is covalently linked to: (a) the heterologous protein component; (b) the light-and-magnetoresponsive protein domain; or (c) the heterologous protein component and the light-and-magnetoresponsive protein domain.

[0205] Embodiment 1-18. The composition of embodiment 1-17, w’herein the heterologous protein component, the bioluminescent protein domain, and the light-and- magnetoresponsive protein domain are covalently linked to form a fusion protein, w’herein the bioluminescent protein domain is fused to: (a) the heterologous protein45323198441component; (b) the light-and-magnetoresponsive protein domain; or (c) the heterologous protein component and the light-and-magnetoresponsive protein domain.

[0206] Embodiment 1-19. The composition of embodiment 1-18, wherein the fusion protein comprises, from N- to C-terminus, a first split luciferase domain, the light-and-magnetoresponsive protein domain, and a second split luciferase domain.

[0207] Embodiment 1-20. The composition of embodiment 1-18, wherein the fusion protein comprises, from N- to C-terminus, a first split luciferase domain, the light-and-magnetoresponsive protein domain, a second split luciferase domain, and the heterologous protein component.

[0208] Embodiment 1-21. The composition of any one of embodiments 1-1 - 1-20, wherein the biolum in escent protein domain is capable of catalyzing a reaction of a substrate, wherein the reaction emits light.

[0209] Embodiment 1-22. The composition of embodiment 1-21, wherein the substrate is a luciferin.

[0210] Embodiment 1-23. The composition of embodiment 1-22, wherein the luciferin is selected from the group consisting of coelenterazine or an analog thereof, a firefly luciferin, a snail luciferin, a bacterial luciferin, a dinoflagellate luciferin, vargulin or an analog thereof, or a fungal luciferin.

[0211] Embodiment 1-24. The composition of any one of embodiments 1-1 - 1-23, wherein the composition comprises two light-and-magnetoresponsive protein domains.

[0212] Embodiment 1-25. A composition comprising: (a) a light-and-magnetoresponsive protein domain; and (b) a heterologous protein component.

[0213] Embodiment 1-26. The composition of embodiment 1-25, wherein the light-and- magnetoresponsive protein domain comprises a LOV domain.

[0214] Embodiment 1-27. The composition of embodiment 1-26, wherein the LOV domain is an engineered LOV domain.

[0215] Embodiment 1-28. The composition of embodiment 1-27, wherein the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%,46323198441sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-2 and 26.

[0216] Embodiment 1-29. The composition of embodiment 1-28, wherein the engineered LOV domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-2 and 26.

[0217] Embodiment 1-30. The composition of any one of embodiments 1-25 - 1-29, wherein the heterologous protein component is a selected from the group consisting of a cytokine, an antibody or a fragment thereof, a synthetic binding protein, a chimeric antigen receptor (CAR), a CRISPR-Cas protein, a channelrhodopsin, a transcription factor, a DNA-bmdmg domain, an antibody-drug conjugate, and an antibody-radioligand conjugate.

[0218] Embodiment 1-31. The composition of any one of embodiments 1-25 - 1-30, wherein the heterologous protein component is covalently linked to the light-and-magnetoresponsive protein domain.

[0219] Embodiment 1-32. The composition of embodiment 1-31, wherein the heterologous protein component and the light-and-inagnetoresponsive protein domain are covalently linked to form a fusion protein.

[0220] Embodiment 1-33. One or more nucleic acids encoding the composition of any one of embodiments 1-1 - 1-32.

[0221] Embodiment 1-34. One or more cells comprising the one or more nucleic acids of embodiment 1-33, or the composition of any one of embodiments 1-1 - 1-32.

[0222] Embodiment 1-35. A method of using a light-and-magnetoresponsive composition, comprising: (a) providing the composition of any one of embodiments 1-1 - 1-24; (b) exposing the composition to a substrate, wherein the bioluminescent protein domain is capable of catalyzing a reaction of the substrate, wherein the reaction emits light; and (c) exposing the composition to a magnetic field.

[0223] Embodiment 1-36. The method of embodiment 1-35, wherein the heterologous protein component is activated upon exposure to the substrate and the magnetic field.

[0224] Embodiment 1-37. The method of embodiment 1-35, wherein the heterologous protein component is deactivated upon exposure to the substrate and the magnetic field.47323198441

[0225] Embodiment 1-38. A method of using a light-and-magnetoresponsive composition, comprising: (a) providing the composition of any one of embodiments 1-1 -1-32; and (b) exposing the composition to a magnetic field.

[0226] Embodiment 1-39. The method of embodiment 1-38, wherein the heterologous protein component is activated upon exposure to the magnetic field.

[0227] Embodiment 1-40. The method of embodiment 1-38, wherein the heterologous protein component is deactivated upon exposure to the magnetic field.[022S] The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.EXAMPLESExample 1: Design of a magnetically sensitive interleukin-2 (IL-2)MagLOV

[0229] MagLOV is an AsLOV2 variant with amino acid substitutions that make the protein magnetoresponsive as described by Hayward, R. F. et al. (see word wide web: doi.org / 10.5281 / zenodo.l 1406498). These mutations are C450P, D540M, Q513K, L396V, and G528K, relative to the wild-type AsLOV2 sequence of SEQ ID NO: 8, below:FLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGP ETDRATXTFKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGD VQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAAKEL(SEQ ID NO: 8).

[0230] Hayward et al. showed that the mutations made the MagLOV protein less fluorescent in the presence of a 10 mT magnetic field. Further, removing the magnetic field increased the fluorescence of the sample by roughly -75% in about 10 seconds.

[0231] The light-oxygen-voltage-sensing (LOV) domain is typically modeled as having two distinct conformational shapes. In the default dark state, the J alpha helix is docked to the rest of the LOV domain. In the bright state, a transient covalent bond is formed between the C450 and a flavin mononucleotide (FMN) cofactor, resulting in a destabilization of the J alpha helix and a broader conformational change in the protein it48323198441is bound to. Accordingly, it is believed that fusing the MagLOV domain to human interleukin-2 (IL- 2) can make IL-2 activity' dependent on conformational changes in the MagLOV domain, and thus, sensitive to the presence of a magnetic field.IL-2-MagLOV fusion protein design

[0232] A fusion protein was designed with the human IL-2 sequence fused to MagLOV such that IL-2 activity is modulated by a magnetic field. The amino acid sequence of IL-2 used is provided as SEQ ID NO: 9:PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT ELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTF MCEYADETATIVEFLNRWTFSQSIISTLT (SEQ ID NO: 9).

[0233] The N and C-termini of the LOV domain were truncated to form the amino acid sequence of SEQ ID NO: 2, Without wishing to be bound by theory, truncation is believed to improve the conformational coupling of the LOV domain on the IL-2 structure (see Gil, A. A., et al. Optogenetic control of protein binding using light-switchable nanobodies, Nat Commun 11, 4044 (2020)). Truncation also removed a human nuclear export sequence from the LOV domain.

[0234] Pseudobright and standard truncated MagLOV domain mutant sequences were inserted into each position in the IL-2 sequence, to generate a list of pairs of fusion protein sequences. Each position of LOV domain insertion had one wildtype version, and one pseudobright version. Pseudobright mutations emulate the conformational change of the protein in its light-induced states. The amino acid sequence of the pseudobright MagLOV sequence is provided as SEQ ID NO: 25:LATrLERIEKNFVITOPRLPDNPllFASDSFLQLTEYSREEILGRNPRFLQGPE TDRATVRKIRD DNQTEVWQLINYTKSGKKFAVNLFHVQPMRDQKGDV QYFIGVKLDGTEHVRDAAEREKVMLEKKTEENIMEAAKEL (SEQ ID NO: 25).The two mutations that make SEQ ID NO: 25 pseudobright are underlined and bolded above (see Gil, A. A., et al. Nat Commun 11, 4044 (2020)).Alphafold modeling method

[0235] For each pair, both sequences were modeled m Alphafold using co labfold, with the multiple sequence alignment mode MMseqs2 (Uniref + Environmental), as a complex49323198441with the IL-2 receptor A and the IL-2 receptor B. The IL-2 receptor A and IL-2 receptor B complex was modeled by inserting a linker of sequence GGGSGGGSGGGSGGGS (SEQ ID NO: 10) between the C-terminus of the A receptor and the N-terminus of the B receptor.

[0236] For each sequence, Alphafold returned a predicted aligned error (PAE) matrix showing the interactions between each amino acid in the complex with each other. From this matrix, a mean iPAE was calculated by taking the mean score of each PAE interactions in the interaction regions of the PAE graph, where that interaction region is the set of amino acid interactions between the IL-2-MagLOV fusion protein and the IL-2 receptor A and B complex. A high mean iPAE score implied a greater level of uncertainty about the relative ammo acid positions, implying a reduction in binding affinity. For each pair of pseudobright and wildtype sequences, a ratio was calculated by¬ dividing the mean iP AE of the pseudobright by the mean iPAE of the wildtype sequence. This ratio correlates with the expected differential in binding occurring, where a ratio >1 implies that light reduces binding affinity, whereas a ratio <1 implies that light increases binding affinity. Sequence pairs with the highest absolute value of the mean iPAE pseudobright / wildtype ratio identify insertion sites and sequence variants which are likely to be ideal locations to place the MagLOV domain.Alphafold modeling results

[0237] The resulting structures predicted by Alphafold were consistent with reported LOV insertion positions with predictable effects on antigen binding (see He L, et al. Design of Smart Antibody Mimetics with Photosensitive Switches, Adv Biol (Weinh).2021 May;5(5)). Broadly, insertion sites in loop regions facing towards the antigen binding site are believed to increase binding upon light activation by masking the antigen binding site in the dark state and shifting away from the antigen binding site upon activation with light to allow binding to the antigen. Insertion sites in loops facing away from the antigen binding are more likely to function to reduce binding with light, due to the destabilization of the J alpha helix propagating into broader structural changes which effect antigen binding.

[0238] The MagLOV sequence used in this example increases the proportion of bright- state formation in the presence of a magnetic field. Depending on the magnetic field50323198441strategy used, sequence pairs with either a ratio <1 or a ratio >1 are preferred. If the goal is to increase binding in the presence of a magnetic field, then sequences with a ratio of <1 are preferred. This would be true if, for example, the magnetic field was administered via a vest with sown in permanent magnets, and the target of interest was proximal to the magnets held close to the skin of the treated patient. If the goal is to reduce binding in the presence of a magnetic field, then sequences with a ratio of >1 are preferred. This would be true if, for example, the magnetic field was administered by placing two or more opposed permanent magnets or electromagnets such that the north poles are facing each other, or alternately, the south poles are facing each other, such that a zero point is established in the center where there is no added magnetic field, where m comparison there is an elevated magnetic field in the tissue surrounding that zero point. If an alternate LOV domain sequence was used such that the magnetic field reduced the proportion of bright-state structures, then the inverse of the above would be preferred.Laboratory assays

[0239] The ability of the IL-2-MagLOV fusion proteins to bind IL-2 receptors is assessed. The IL-2 receptors are bound to membranes or beads. It is determined whether IL-2 binding is sensitive to the presence of a magnetic field.

[0240] Expression of the IL-2-MagLOV fusion proteins is assessed.Example 2: Fusion of a magnetically sensitive LOV fusion protein domain to a luciferase domain

[0241] In many therapeutic applications, the co-administration of light and magnetic field to modulate binding properties is hampered by the challenges of delivering light to the necessary tissue. This can be resolved by building off of Example 1 by directly combining a magnetically sensitive LOV domain with a protein domain that itself emits light.

[0242] Specifically, a luciferase such as NanoLuc is fused to the N-terminus of the magnetically sensitive IL-2 described in Example 1 via a flexible amino acid linker sequence.

[0243] By coadministering a luciferase substrate such as luciferin alongside this luciferase-IL2-MagLOV fusion protein, light is emitted proximal to the MagLOV51323198441domain. The enables the actuation of the protein using only external magnetic fields and the light emitted by the luciferase itself.

[0244] Similar effects are achieved by the co-administration of luciferase that is not directly fused to the LOV domain, or by the introduction of a range of phosphorescent, chemiluminescent, or phosphorescent compounds, which are or are not bound to the magnetically controllable protein.Example 3: Magnetically controlled luciferase enzyme design

[0245] The following example describes the design of an enzyme that is engineered to be magnetically controlled. Specifically, luciferase is chosen as an exemplary enzyme, because modulated luciferase activity can be used to drive light-controlled optogenetic systems such as those that use the wildtype AsLOV sequence. This example demonstrates how to improve an enzyme by making its effect modulated by magnetic fields.

[0246] First, a position is identified for the incorporation of a MagLOV domain within a luciferase enzyme. The C-terminus of the Click beetle luciferase (Caribbean Pyrophorus plagiophthalamus) amino acids 395-542 with mutations F420I, G421A, E453S (SEQ ID NO: 7) is used, as well as the N-terminus of the firefly luciferase Photinus pyra.lis) amino acids 1-415 (SEQ ID NO: 6). Internally to the luciferase protein, the MagLOV sequence is placed in place of the AsLOV (A. saliva Phototropin 1, amino acids 404-546) that has been previously demonstrated (see M, Hatton, et al., Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein, Proc. Natl. Acad. Sci. U. S. A. 110 (23) 9332-9337), Positioning the MagLOV sequence in place of the AsLOV is believed to enable magnetic modulation of the light-controlled conformational and activity changes previously shown with AsLOV.

[0247] In the default dark state, the LOV domain interacts with the J-alpha helix, causing complementation of the luciferase N and C-terminal fragments. Upon light activation, the LOV domain dissociates, increasing light production. The introduction of MagLOV is expected to mean that an increased magnetic field results in reduced luciferase activity, reducing light production.52323198441

[0248] Because the activity of nearby luciferase will produce more light to induce conformational change in nearby Luciferase-MagLOV fusion proteins, there is expected to be a local cooperativity effects due to feedback loops between local luciferase photon production and local MagLOV photoexcitation. Normally, magnetic field strength decreases with the cube of the distance from the magnetic field source. With cooperativity, the magnetic field strength is expected to fall off non-linearly with a rate greater than the cube of the distance, enhancing the ability to tightly control spatial localization of the luciferase activity.Example 4: Magnetically controlled protein design with cooperativity

[0249] Two magnetically sensitive (e.g., MagLOV) domains are engineered into the structure of a protein such as an enzyme or antibody. These two magnetically sensitive domains are placed in series or inserted in different sites of the source protein sequence. It is believed that the presence of two magnetically sensitive domains will introduce cooperativity into the activation of the enzyme or antibody.

[0250] Cooperativity may be beneficial in cases in which it is desired that the enzyme or antibody is active in one region of the body, but not another. Without wishing to be bound by theory, with cooperativity, an S-shaped response curve may be observed such that there is a non-linear relationship between the magnetic field strength at the position of the desired activation and the activity level. The non-linearity that cooperativity induces makes it possible to design a magnetic field to achieve a clean separation of activity vs. no activity across relatively short distances.Example 5: Using magnets to purify a magnetoresponsive antibody

[0251] Beads coated in an antigen are used to purify a magnetoresponsive antibody.

[0252] Alternatively, beads are coated with a magnetoresponsive antibody to capture the antibody’s target protein in a reversible fashion.Example 6: Designing magnetically sensitive scFv, nanobody, monobody, and IgG antibodies using Alphafold

[0253] The method described in Example 1 to model designs of IL-2-MagLOV fusion proteins is applied to fusion proteins of MagLO V and scFv, nanobody, monobody, or IgG antibodies.53323198441

[0254] Fusion protein sequences are designed with the MagLOV domain fused to the antibody at a variety of positions, and, in particular in loop regions. In some sequences, MagLOV is inserted into the antibodies fragment in loops in close physical proximity to the CDR regions.

[0255] Alphafold is used to model the MagLOV -antibody fusion proteins and calculate predicted aligned error (PAE) for each.

[0256] Experiments are performed to assess whether binding of the MagLOV-antibody fusion proteins to the antigen of the antibody is sensitive to exposure to a magnetic field.Example 7: Magnetic Actuation of Optically Modulated CAR-T

[0257] CAR-T systems modulated by light have been demonstrated through in vivo mouse models (see, e.g., Nguyen, N. T., Huang, K., Zeng, H. et al. Nano-optogenetic engineering of CAR T cells for precision immunotherapy with enhanced safety. Nat. Nanotechnol. 16, 1424-1434 (2021); see also International Publication No.WO2021113116A1). Modulating these systems with magnetic fields instead of merely light would allow control deeper into the body, enabling therapeutic use in humans. Using the magnetically actuatable luciferase as described in Example 3, the photoactivatable CAR-T system is altered to be controlled by magnetic fields in addition to light.

[0258] Specifically, a magnetically actuatable luciferase is co-expressed and free-floating in the cell. Magnetic stimulation acts on the magnetically sensitive LOV domain, modulating the activity of the luciferase. Modulation in the luciferase activity changes the amount of light emitted, which can then in turn modulate the activity of the co-expressed receptor dimerization pair.

[0259] Alternately, the magnetically-inducible luciferase construct is fused to either of the dimerization pairs in a location that does not interfere with its binding activity. Fusing the luciferase to the B / D dimerization component may improve efficient energy transfer from the luciferase to the light-inducible LOV2 or CRY2 domain. In both cases, the magnetically actuatable luciferase sequence as described in Example 3 can be incorporated for expression into the packaged virus sequence for integration into Jurkat T Cells.54323198441NOBS-001 / 02WG 356250-2007

[0260] To develop the T cells, HEK293T cells are used to package an engineered chimeric antigen receptor with the retroviral vector pMSGVl and a vector encoding the amphotropic envelope glycoprotein RD114, using iMFectin DNA transfection reagent. Supernatant containing the packaged viruses is collected at 48 and 72 hours post transfection.

[0261] Before transfection, the virus-containing supernatants are concentrated using an Amicon Ultra- 15 Centrifugal Filter. Before performing T cell transduction, human primary CD8 positive T cells are activated for 48 hours using Dynabeads Human T- Activator CD3 / CD28 system at a bead to cell ratio of 1:1. Using manufacturer’s instructions, retroviral transduction is performed on the primary T cells using RetroNectin and the collected viral supernatant.

[0262] For transfection, Jurkat T cells are grown to a concentration of 105cells per mL then co-incubated with 2 mL of the 0.45 uM filtered virus supernatant and 10 ug per mL polybrene in a well of a 12 well plate. After adding the cells, supernatant, and polybrene, plates are centrifuged at 2000g for 2 hours at 32 degrees C, and then incubated for another 2 hours at 37 degrees C. The viral supernatant is then removed and replaced with fresh RPMI medium supplemented with 10% FBS, 100 U per L penicillin, and 100 ug per mL streptomycin. Then the process, starting with the coincubation of virus supernatant, is repeated three more times to increase the efficiency of the transduction.

[0263] Experiments are performed to assess whether activity of the T cells is sensitive to light and magnetic fields.Magnetic Actuation of a Light-and-Magnetoresponsive CAR-T

[0264] Starting from the optically-controlled CAR-T system as described above, an alternate implementation of a magnetically modulated CAR-T is based on the second Opto-CAR-T system described in Nguyen, N. T., et al., which utilizes the AsLOV2-based iLID system (Nano-optogenetic engineering of CAR T cells for precision immunotherapy with enhanced safety. Nat. Nanotechnol. 16, 1424-1434 (2021)).

[0265] A construct is built as described in Nguyen et al., substituting in the iLID for Mag-iLID as described in Example 14, and adding a luciferase sequence such as NanoLuc to the genetic payload expressed within the CAR-T. Alternately, the luciferase is a magnetically inducible luciferase. The luciferase is fused onto the Mag-iLID55323198441construct to maximize energy delivery to the Mag-iLID; or, the luciferase is expressed separately, free-floating, or initially expressed and then cleaved.

[0266] To modulate the activity of the CAR-T, the appropriate luciferase substrate is provided (e g. coelenterazine for NanoLuc). To then further modulate the CAR-T activity, a magnetic field is added or removed, which will change the light¬ responsiveness of the Mag-iLID, and also the light-production of the magnetically modulated luciferase if that is used.

[0267] When delivered to a patient, a magnet is placed near a site where you want to modulate activity. If the CAR-T is engineered to increase activity near the magnetic field, then the magnet should be where you want the CAR-T to be more active (e.g. in an oncology setting, near cancer cells). If the CAR-T is engineered to reduce activity in the magnetic field, then place the magnet where you want to reduce activity, for example in an oncology setting, at a site of anticipated on-target'' off-tumor activity.

[0268] In a therapeutic or research setting, this may be engineered ex vivo as an allogenic or autologous cell therapy. Or, alternatively, it is delivered as an in vivo CAR-T gene therapy, delivering a genetic payload to T cells without first removing them from the patient.Example 8: Luciferase -MagLOV fusion to N-terminus of channelrhod opsin

[0269] The Lucif erase -MagLOV fusion protein described in Example 3 is fused to the N-terminus of a Volvox channelrhodopsin, to modulate the effect of the channelrhodopsin protein (see Murphy, Emily F et al. NeuroImage vol, 301 (2024): 120882).

[0270] Channelrhodopsin activity is assessed to determine whether the channelrhodopsin is sensitive to the presence of a magnetic field.Example 9: Design of a Magnetically Inducible Cas9 system

[0271] A MagLOV domain is fused to a Cas9 protein. The MagLOV domain is used in place of a light-sensitive domain as described in Mathony, J., et al. (2020). Optogenetics and CRISPR: A New Relationship Built to Last. In: Niopek, D. (eds) Photoswitching Proteins, Methods in Molecular Biology, vol 2173. Humana, New York, NY.

[0272] Cas9 activity is assessed. In particular, whether the Cas9 activity is sensitive to the presence of a magnetic field is assessed.56323198441Example 10: Magnetically controlled luciferase enzyme

[0273] NanoBiT™ luciferase was fused to MagLOV to generate a magnetically controlled luciferase enzyme.Materials and Methods

[0274] A plasmid was cloned with an expression construct fusing the NanoBiT™ LgBiT and SmBiT subunits to MagLOV. The amino acid sequence of the fusion protein encoded by the construct is provided below.Table 3. MagLOV-NanoBiT™ amino acid sequence, from N- to C-terminus Component of SEQID Amino Acid SequenceFusion Protein NO MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVT P I QRI VRS GENALKI DI HVI I P YEGL S ADQMAQ I EEVFKVVLgBiT YPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFD 4G K K I T VT GT L WN G N K I I D E RL I T P D G S ML F RVT I N S Linker GS - ML AT T L E R I E K N F V I T D P RL P D N P 11 FA S D S F L Q L T E Y S R E MagLOV EILGRNPRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTV KSGKKFWNLFHVQPMRDQKGDVQYFIGVKLDGTEHVRDAAE 26 ariant 1RI KVML I NKT AANI DEAAKELLinker GGSGGGSGG 36SmBit VTGYRLFEEIL 5

[0275] The coding sequence was cloned into a plasmid under control of a constitutive promoter.Bacterial Transformation and Streaking (Day 1 )

[0276] A plasmid encoding the NanoBiT™-MagLOV fusion was transformed into Escherichia coli BL21 cells using a heat shock method. Specifically, 1 gL of purified plasmid DNA was added to 55 gL of homemade chemically competent BL21 cells. The transformation mixture was incubated on ice for 30 minutes, subjected to heat shock at 42°C for 45 seconds, then recovered for 1 hour at 37°C with shaking in SOC medium. Transformed cultures were plated on LB agar supplemented with carbenicillin and incubated overnight at 37°C.

[0277] Following overnight incubation, bacterial colonies were streaked onto fresh LB agar plates containing carbenicillin. Three to four constructs were streaked per plate. Plates were incubated overnight at 37°C57323198441Post-Incubation Preparation (Day 2)

[0278] After incubation, plates were left at room temperature throughout the day. This step allowed bacterial colonies to grow larger and slightly dry out, which helped prevent colony displacement during substrate addition in the imaging step.Imaging (Day 3)

[0279] A fresh fluorofurimazine working solution was prepared in the following order to prevent precipitation, with gentle mixing between each addition:500 pL dimethyl sulfoxide (DMSO)3 pL fluorofurimazine (from a 100 mM stock in DMSO; working aliquots stored at -20°C; main stock at -80°C)5 mL of PEG 8000 in PBS (prepared by dissolving 10 g PEG in 50 mL PBS) 1 mL PBSThe diluted fluorofurimazine solution was pipetted directly onto individual bacterial colonies. Plates were placed onto the imaging system, ensuring even distribution of liquid across the surface to prevent pooling on one side,

[0280] Luminescence was measured from single E. coli colonies over time. A permanent magnet was moved every 25 seconds while measuring luminescence. The traces were plotted, as shown in FIG. 1.Results

[0281] Luminescence traces were assessed for periodic changes indicative of magnetic field effects. As shown in FIG. 1, the level of luminescence decreased during times when the magnetic field was applied. Therefore, the luciferase’s activity was sensitive to the presence of magnetic fields.Example 11. Light activation of an optobody fused to luciferase

[0282] Luciferase was fused to a LaM8 mCherry-targeted nanobody with AsLOV2 inserted into the AK74 position (“mCherry optobody”). The ability of the nanobody to bind mCherry was measured in the presence or absence of light using enzyme-linked immunosorbent assays (ELISAs).Materials and Methods58323198441

[0283] The amino acid sequence of the luciferase-mCherry optobody fusion protein is provided in Table 4.Table 4. Luciferase-mCherry optobody fusion protein amino acid sequence, from N-to C-terminusComponent of Fusion SEQ ID Amino Acid SequenceProtein NO N -terminal methionine M - residueFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGV S VT P I QRI VL S GENGL K I D I HVI I P YE GL S GDQMGQNanoLuc® luciferase I EKT FKVVYP VDDHHFKVILHYGTLVI DGVT PNMT D 27YFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLIN P D G S L L F R VT I N G VT G W RL C E R I L ALinker G D P L VQ C G G 1 AL A AT M 28 mCherry-targeted - AQVQL VE S GGGL VQAGG SL RL SC AVS GRP F S E YNL G optobody, i.e., a LaM8 WFRQAP GKE RE FVAR1 RS S GT T VYT D S VKGRF SAS R mCherry targeted nanobody D NAGLERIEKNFVITDPRLPDNPI IFASDSFLQLTEwith AsLOV2 inserted into Y S REEP L G RNC R F L Q G P E T D R AT VRK I R DA I D NQ T EVT VQL I NYT KS GKKFWNLFHLQPMRDQKGDVQ YF I G 29 the / XK74 position(AsLOV2 domain VQLDGTEHVRDAAEREGVML I KKTAENI DEAAGKNM corresponding to SEQ ID GYLQLNSLEPEDTAVYYCAMSRVDTDSPAFYDYWGQ GTQVTVSTPRS NO: 30 is underlined)Sequence for ribosome GT GGPT LL KE GKRT GGGVE 31displayLuciferase-activated enzyme-linked immunosorbent assay (ELISA )

[0284] Two replicates were performed per sample per condition. Two conditions were tested: a “light” condition in which furimazine was added, and a “dark” condition in which furimazine was not added.

[0285] The luciferase-activated ELISA was performed by following these steps:Streptavidin binding plate wells were washed 1-3 times with phosphate-buffered saline (PBS). -200 nM Biotinylated mCherry (diluted with PBS) was added. This was incubated at room temperature for 2 hours on shaking platform, then washed three times with PBS. 3% bovine serum albumin (BSA) was prepared in PBS, and 200 pL was added to each well. This was incubated at room temperature for 1.5 hours on shaking platform, then washed three times with PBST (PBS + 0.05% Tween-20). 2x serial dilutions of samples were made by using the stock proteins + PBST. 50-100 pL / well of dilutions were added to the plate. Furimazine was added to the wells in the “light” condition; the59323198441final concentration of Furimazine in well was -100 pM (i.e., 2 iiL was added to each well of a 2.5 mM stock). This was incubated at room temperature for 5 minutes with mixing, then washed three times with PBST. 1:400 dilution of anti-Myc HRP conjugate were prepared in PBST, then 50-100 pL / well was added. Furimazine was added to the light conditions again, and incubated at room temperature for 10 minutes with mixing, then washed three times with PBST, then 1-2 times with PBS. 100 µL of tetramethylbenzidine was added (TMB) per well, and incubated 5-15 minutes with some mixing. 100 pL of 0.5M sulfuric acid was added to stop the reaction. The plates were imaged on a plate reader to measure the optical density (OD) within 30 minutes of adding sulfuric acid. Results of the luciferase-activated ELISA are provided in FIG. 3,

[0286] In a separate experiment, ELIS As were performed as described above, except that illumination was delivered by a Thorlabs light emitting diode (LED) at 75% max power, rather than by luciferase. In this experiment, no Furimazine was added. The results of the LED-illuminated ELISA are provided in FIG. 2,Results

[0287] FIG. 2 shows results from an ELISA assay for luciferase-mCherry optobody fusion protein binding to mCherry in the presence and absence of light emitted from an LED. In the dark, the optobody bound its target more weakly than in the light.

[0288] FIG. 3 shows the results from a luciferase- activated ELISA, in which the reaction catalyzed by the fusion protein’s luciferase provided illumination in the Furimazine-treated samples. As shown in FIG. 3, treatment with Furimazine increased binding to mCherry. Without wishing to be bound by theory, this suggests that light emitted by the luciferase actuated binding of the optobody to mCherry.Example 12. Induction of nanobody-target co-localization under illumination

[0289] Experiments were performed to test whether light could control co-localization of a target and an mCherry optobody.Materials and Methods

[0290] In order to assess antibody binding, the AK74 opto-nanobody was tagged with a red fluorescent protein (mIRFP713). The amino acid sequence of the resulting fusion protein is provided in Table 5, below.60323198441Table 5. Luciferase-mCherry optobody-RFP fusion protein amino acid sequence, from N- to C-terminusComponent of SEQ ID Amino Acid SequenceFusion Protein NO N-terminal methionine M - FT L E DFVGDWRQT AG YNL DQVL EQGGVS S L FQNL GVS VT P I QR I VL S GE NGL K I D I HV 11 P YE GL S GDQMGQ I E NanoLuc® luciferase KT FKWYPVDDHHFKVT LH YGTLVI DGVT PNMI D YFG 27RPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGS L L F RVT I N GVT G WRL C E R I L ALinker GDPLVQCGGIALAATM 28 mCherry-targeted - optobody, i.e., a LaM8 AQ VQL VE S GG GL VQ AG G S L RL S C AVS GRP F S E YNL GVT mCherry targeted FRQAPGKEREFVARIRSSGTTVYTDSVKGRFSASRDN nanobody with AGL E RI E KNFVI T D P RL P DNP 11 FAS D S FLQLT E YS R; XsLOV2 fused into E E T L GRNC RFL Q GP E T D RAT VRK I RD A 1 DNQT E VT VQ29 the / XK74 position. L I N YTKS GKKFWNL FHLQPMRDQKGDVQYF I GVQLDGThe? XsLOV2 domain T E H VRD AAE RE GVML I KKT AE N I DE AAGKNMG YL QL N corresponding to SEQ SLEPEDTAVYYCAMSRVDTDSPAFYDYWGQGTQVTVSTP RS ID NO: 30 isunderlined.AE G S V A RQ P D L L T C D D E P I H 1 P G A I Q P H G L L L AL A A DMT I VAGS DNL PELT GLAI GAL I GRS AADVFDS ETHNR LT IALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQ L I F L E L E P P Q R D VA E P Q A F F RRT N S A T RR L Q A AE T L EmiRFP713 SACAAAAQEVRKITGFDRVMI YRFASDFSGEVIAEDR 32CAEVESKLGLHYPASTVPAQARRLYT INPVRI I PDIN YRPVPVT PDLNPVTGRP 1 DLS FAILRSVS PVHLEFMRN I GMH GT M S I S I L RG E RL WGL I VC H H RT P Y YVD L D G RQAC E L VAQ VL AWQ T G WE E

[0291] A plasmid expressing the fusion protein of Table 5 was co-transfected into a HeLa cells with a plasmid expressing a mCherry-CAAX fusion, as provided below (with the CAAX motif shown in bold, corresponding to SEQ ID NO: 33):MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGF. GEGRPYEGTQTAKLKVT KGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG VVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGA LKGETKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDTTSHNEDYTI VEQYERAEGRHSTGGMDELYKEFKKKKKKSKTKCVIM (SEQ ID NO: 34). The CA AX motif was added so that the mCherry would localize at the plasma membrane.Results61323198441NOBS-001 / 02WG 356250-2007

[0292] The luciferase- mCherry optobody-RFP fusion protein of Table 5 was expressed in HeLa cells in which the target (mCherry) was tethered to the plasma membrane. Upon addition of 25 uM furimazine (after the first 60 frames), the RFP signal concentrated on the plasma membrane, as shown in FIGS. 4-6. Accordingly, the luciferase-mCherry optobody-RFP fusion protein induced co-localization on the membrane in a light¬ dependent manner.Example 13. Exemplary construct sequences

[0293] An exemplary fusion protein of an mCherry’ -targeted nanobody to MagLOV- NanoBiT is provided in Table 6, below.Table 6. Exemplary fusion protein amino acid sequence, from N- to C-terminus Component of SEQ ID Amino Acid SequenceFusion Protein NO His tag MGSSHHHHHHHHSSGG 35 Myc tag EQKLISEEDL 37 MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLA VS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQLgBiT lEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNY 4 FGRPYEGIAVFDGKKIT VTGTLWNGNKI I DERL IT PD GSMLFRVTINSLinker GS - ML ATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTE YSREEILGRNPRFLQGPETD RAT VRK I RD A I DNQT EVMagLOV Variant 1 TVQL I NYTKS GKKFWNL FHVQPMRDQKGDVQYF I GVK 26 LDGTEHVRDAAERI KVML INKTAANI DEAAKELLinker GGSGGGSGG 36 SmBit VTGYRLFEEIL 5 Sequence forGTGGPTLLKEGKRTGGGVE 31 ribosome displayLinker GD P L V Q C GG I AL AAT M 28 mCherry-targeted - optobody, i.e., a AQ VQL VE S GGGL VQ AGG S L RL S C AVS GR P F S E YNL GWLaM8 mCherry FRQAP GKERE FVARI RS S GT T VYT D S VKGRFS AS RDN AGLERIEKNFVITDPRLPDNPI IFASDSFLQLTEYSRtargeted nanobodyEEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQwith AsLOV2 fused L I NYTKS GKKFWNL FHLQPMRDQKGDVQ YF I GVQLDG 29 into the AK74 T E H VRD AAE RE GVML I KKT AE N I D E AAGK NM G Y L Q L N position ( AsLOV2 SLEPEDTAVYYCAMSRVDTDSPAFYDYWGQGTQVTVS domain TPRScorresponding to62323198441SEQ ID NO: 30 isunderlined)Sequence forGTGGPTLLKEGKRTGGGVE 31ribosome display

[0294] An exemplary fusion of luciferase to an arui-mCherry nanobody with MagLOV inserted within the nanobody sequence is provided in Table 7.Table 7. Exemplary fusion protein amino acid sequence, from N- to C-terminus Component of SEQ ID Amino Acid SequenceFusion Protein NO His tag MGS S HHHHHHHH S S GG 35 Myc tag EQKLISEEDL 37 Glutamic acid E - residueFT L E DFVGDWRQT AG YNL DQVL EQGGVS S L FQNL GVS VT P I Q R I V L S G E N G L K I D I H V 11 P Y E G L S G D QM G Q I E K I F K V V Y P NanoLu c® V D D H H F K V I L H Y GT L VI D G VT P NM I D Y F G R P Y E G I AV F D G K 27 luciferase K I T VT GT L WN GN K 11 D E RL I N P D G S L L F RVT I N G VT G WRL CERILALinker GDPLVQCGGIALAATM 28 Anti-mCherrynanobody with AQ VQL VE S GG GL VQ AG G S L RL S C AVS GRP F S E YNL GWF RQ AP G K E RE F V AR I R S S GT T V YT D S V KG R F SA S RD N AML AT TLEMagLOVRIEKNFVITDPRLPDNPI I FASDSFLQLTEYSREEILGRNPdomainRFLQGPET DRAT VRKI RDAI DNQTEVT VQL INYT KS GKKFW 38 (MagLOV NLFHVQPMRDQKGDVQYFIGVKLDGTEHVRDAAERIKVMLIdomain of SEQ NKTAANIDEAAKELKNMGYLQLNSLEPEDTAVYYCAMSRVDID NO: 26 is T D S P AF YD YWGQGT QVT VS T P RSunderlined)Sequence forGT GGPT LL KE GKRT GGGVE 31ribosome display

[0295] Finally, an exemplary fusion of luciferase to MagLOV with improved light-induced dimer (iLID) amino acid substitutions is provided m Table 8.Table 8. Exemplary fusion protein amino acid sequence, from N- to C-terminus Component of SEQ ID Amino Acid SequenceFusion Protein NO His tag MGS S HHHHHHHH S S GG 35 Myc tag EQKLISEEDL 3763323198441WFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPI Q R I VR S G E N AL K I D I H V 11 P Y E GL S ADQMAQ I E E V F K W Y P LgBiT VD D H H F K V I L P Y GT L V I D G VT P NML It Y F G R P Y E G I AV F D G K K 4I T VT GT L WNGN K 11 D E RL I T P D G SML F RVT 1 N SLinker GS - ML A TTLERIEKNFVITDPRLPDNPI IFASDSFLQL T E Y S R E E I L G RN P R F L Q G P E T D RAT VRK I RD A I D NQ T E VT VQL I N YT K SMagLOV GKKFWNLFHVQPMRDQYGDVQYFTGVKLDGTERLHGAAERIA 39 VMLINKTAFQIAESsrA peptidewith iLID aminoAANDENYF 84 acidsubstitutionsLinker GGSGGGSGG 36 SmBiT VTGYRLFEEIL 5 Sequence for GT GGPT LL KE GKRT GGGVE 31ribosome displayExample 14. Binding of a heterodimerization domain induced by external light and a magnetic field

[0296] An improved MagLOV variant with improved light-induced dimer (iLID) ammo acid substitutions was generated. An experiment was performed to test whether binding of the Mag-iLID variant to its binding partner was affected by the presence of light and a magnetic field.Materials and MethodsDesign of magnetoresponsive heterodimerization domain

[0297] A directed evolution campaign was conducted to identify mutations in AsLOV2 which conferred enhanced magnetoresponse, by screening for enhanced luminescence of a luciferase- AsLOV2 fusion protein, similar to the construct described in Example 10. In general terms, this domain was developed by performing site-directed mutagenesis on luciferase AsLOV2 fusion proteins, and selecting the luciferase that showed the greatest change in luminescence when modulated with a magnet, and repeating the site-directed mutagenesis using that new starting point. After several rounds of this improvement, the mutations accumulated in the MagLOV domain were identified on the basis that they 64323198441likely conferred enhanced magnetoresponsive effect to fused proteins. The mutations identified in the MagLOV domain post-enrichment were believed to confer magnetoresponsive behavior. The amino acid substitutions in the improved MagLOV domain were as follows: W45R, L91F, K100Y, Hl 16R, VI 17L, R118H, DI 19G, G122E, E124I, R125A, K130N, E134F, N135Q, M137A, K141D, and L143F. The full-length sequence of the improved MagLOV domain sequence is provided in SEQ ID NO: 26.

[0298] These mutations were combined with iLID amino acid substitutions (after Al 40: insertion of N; and, after E142: insertion of NY) and fused to an SsrA peptide. iLID is an engineered variant of AsLOV2 described previously, preserving the light responsive domain, but with an enhanced binding region to SsrA’s binding partner, SspB.

[0299] By incorporating these magnetoresponsive mutations into iLID, a composition referred to herein as “Mag-iLID” was formed, comprising a binding region and a light-and-magnetoresponsive protein domain. This binding pair of Mag-iLID and SspB were expected to modulate binding affinity7differently in the dark, in the presence of light, or in the presence of light and a moderate strength magnetic field. The Mag-iLID sequence is provided in Table 9. Table 10, below, provides the ammo acid sequences of SspB and control iLID construct used in this experiment.Table 9, Mag-iLID amino acid sequence, from N- to C-terminusComponent ofAmmo Acid Sequence SEQ ID NO Fusion ProteinHis tag MGSSHHHHHHHHSSGG 35 Myc tag EQKLISEEDL 37 Linker GS - L AT T L E R I E KN F V I T D P RL P D N P 11 F A S D S FL Q L T E Y S RE MagLOV EILGRNPRFLQGPETDRAT VRKIRDAIDNQTEVTVQLINYva T K S G KK F WNL FH VQ PMRDQ YGD VQ Y F I GVKL D GT E RL H GA 40 riantAE R I A VML I N K T A F Q L A ESsrA peptidewith iLID aminoAANDENYF 84 acidsubstitutionsLinker GGSGGGSGG 3665323198441SmBiT VTGYRLFEEIL 5 Sequence for GT GG PT LL KE GKRT GGGVE 31 ribosome displayFull-length Mag- iLID sequence MGSSHHHHHHHHSSGGEQKLISEEDLGSLATTLERIEKNF (MagLOV VITDPRLPDNPIIFASDSFLQLTEYSREEILGRNPRFLQGvariant is PET DRAT VRKI RDAI DNQT EVT VQL I NYT KSGKKFWNL FH VQPMRDQYGDVQYFIGVKLDGTERLHGAAERIAVMLINKT 41 underlined)AFQIAEAANDENYFGGSGGGSGGVTGYRLFEEILGTGGPT(Construct No, LLKEGKRTGGGVE122)Table 10. Amino acid sequences of SspB and iLIDComponent Amino Acid Sequence SEQ ID NO SspBS S P KRP KLL RE YYDWLVDNS FT P YL VVDAT YL GVNVP VE YV KDGQTVLNLSASATGNLQLTNDFTQFNARFKGVSRELYI PM 42 (Construct No. GAAL A I YARE NGD GVMF E P E E I YD E L N I138)iLID (withposition ofMyc-tag MG S S H H H HH H H H S S G GEQKLISEEDLG SEFLATTLERIEKN underlined, and FVITDPRLPDNPIIFASDSFLQLTEYSREEILG RN C R F L Q GSsrA peptide PETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNVFHL 43 bolded) QPMRDYKGDVQYFIGVQLDGTERLHGAAEREAVMLIKKTAF QTAEAANDENYFGTGGPTLLKEGKRTGGGVE(Construct No.131)

[0300] As a negative control, unmodified iLID and SspB were also tested in parallel, with the expectation that the light condition vs light plus magnet condition results for iLID 1 plus SspB would be indistinct from each other.Protein Preparation

[0301] Plasmids encoding the Mag-iLID and iLID constructs were transformed into E. coli. The protein constructs were expressed in the bacteria and purified as follows. After three days of culturing in LB with appropriate antibiotics, the culture was placed into 50 mL Falcon tubes, and spun for ~ 1 hour at ~3-4 thousand xg. The pellet was then resuspended in ~ ImL Lysis Buffer (PBS with 300 (to 500) mM NaCl and 10 mM Imidazole). This solution was placed into Eppendorf tubes, and —150 uL BugBuster® Protein Extraction Reagent (lOx) was added. The tubes were placed on a moving tray for 66323198441NOBS-001 / 02WG 356250-20071 hour, then spun at 16k xg for 10-20 minutes. 50 uL of beads were added into Eppendorf tube, and the supernatant from the spin was added to the beads. This was placed on a moving tray for 1 hour, then washed with Lysis Buffer by spinning gently (<300xg), three times. The beads were resuspended in 50 uL Elution Buffer (PBS with 500 mM imidazole and 300 (to 500) mM NaCl). This was spun down, and the supernatant was harvested into fresh Eppendorf tubes. This was repeated three times, for a total of 150 uL in each tube. The tubes were spun to remove any remaining beads, and a column was used for buffer exchange to PBS.

[0302] SspB was Strep-tagged. To prepare the biotinylated SspB antigen, on day one, the avi-tagged construct was transformed into BL21(XDE3) pBirA Chemically Competent E. coll Cells (Amid Biosciences) and plated onto a LB agar plate with appropriate antibiotic selection and streptomycin. On day two, the plate was scraped and added to midi culture, -50-200 mL LB plus antibiotic for selection plus streptomycin. The culture was then incubated at 37°C, 220 rpm for a few hours. Finally, IPTG was added to 1 mM concentration, and Biotin added to 50 uM concentration, then incubated at 30C overnight. From day three forward, the antigen was prepared using the same method as was used to prepare the protein constructs as described above.ELISAs

[0303] ELISAs were performed to measure the binding of Mag-iLID and iLID for SspB in light, dark, and light plus magnetic field conditions. In these ELISAs, the heterodimerization pair of iLID-SspB or Mag-iLID-SspB is the “antigen,” which is detected by binding of a secondary antibody to the Myc-tag on the Mag-iLID or iLID construct.

[0304] Starting with a Pierce Streptavidin Coated High Capacity Plate (Thermo Fisher 15501), 200 nM of M138-MBP-SSPB-mCherry prepared proteins were added in 100 uL of PBS to each well, then put on a shaker at room temp for about 2 hours. Next, the target protein solution was removed, and the plate was washed 3x with PBS. Then, 100 uL of PBS with 3% BSA was added to each well. The plate was then put on a shaker at room temp for about 1 hour.

[0305] 3 strips were taken for each condition (dark, light, and light plus magnet). While running one condition, the remaining strips sat with the BSA solution until time to use in 67323198441the next conditions. Next, the BSA solution was removed. The plates were then washed 3x with 190 uL PBST (PBS with 0.05% Tween-20). The plate was placed on the shaker. The shaker was illuminated under the 445 nm LED at ~ 12 mW / cm2for light conditions, and placed under the same LED brightness with the addition of a roughly 20 mT magnetic plate beneath for the magnet plus light conditions. With the plate in place, samples were then added, with 50 uL per well of the serial dilutions of the protein of interest (Mag-iLID) or the control protein (iLID). The plate was then incubated at room temperature, shaking the plate at 1000 rpm for 10 minutes. Supernatant was then pipetted out of the wells, and the plate was washed 3x with PBST. Next, 100 uL of 0.1 ug / mL of anti-Myc HRP secondary antibody in PBST was added to each well. The plate was then incubated at room temp with plate shaking at 1000 rpm for 10 minutes. Next, the supernatant solution was removed by pipette, then the plate was washed 2x with PBST and then 2x with PBS. The plate was then removed from the shaker (and therefore also away from the LED beam or proximity from the magnet, depending on condition). Then, 100 uL / well of TMB substrate added. The plate was returned to the shaker for about 5 minutes at 1000 rpm at room temperature. Finally, 100 uL / well of 0.5 M sulfuric acid was added to stop the reaction. The A450 of the sample plate was then measured on plate reader.

[0306] This process was then repeated to collect duplicate data for all three conditions (dark, light, and light plus magnet). Once all duplicates were complete, ELISA titration curves were fit from this A450 plate reader data using standard techniques. The absolute and relative dissociation constants (Kd) of dark, light, and light plus magnet conditions was then be determined from fitting these curves.Results

[0307] The results of this experiment are provided in FIGS. 7A-7B.

[0308] iLID m the presence of light demonstrated an increased binding affinity (FIG.7B), whereas Mag-iLID demonstrated a reduced binding affinity in the presence of light (FIG. 7A). The presence of both external light and an external magnetic field further reduced the binding affinity of Mag-iLID relative to the dark state compared to external light alone (FIG. 7A). In comparison, the control iLID construct demonstrated the same level of binding affinity in both the light and the light plus magnet conditions (FIG. 7B).68323198441

[0309] Taken together, the presence of a magnetic field affected the binding of Mag-iLID to its target, but did not affect the binding of iLID to its target. These results confirm the magnetoresponsiveness of Mag-iLID binding to SspB.Example 15. Binding of a modified antibody induced by an external light and a magnetic field

[0310] Magnetoresponsive mCherry-targeted-optobodies with improved MagLOV domains or Mag-iLID were generated. An experiment was performed to test whether binding of the optobodies to mCherry was affected by the presence of light and a magnetic field.Materials and MethodsDesign of magnetoresponsive optobodies

[0311] Three antibody constructs were tested based on the mCherry-targeted LaM8 optobody. First, the improved MagLOV as described in Example 14 (SEQ ID NO: 26) was used to generate the construct provided in SEQ ID NO: 44. Second, the iLID mutations were also added to MagLOV to generate the construct provided in SEQ ID NO: 45. Such MagLOV-containing antibodies are referred to herein as “magbodies.”

[0312] A control construct was also tested made of non-magnetoresponsive optobody, with a wildtype AsLOV2 insertion as the AK74 insertion loop site (SEQ ID NO: 87). The antibody sequences are provided in Table 11, below.Table 11. Amino acid sequences of antibody constructs testedSEQ IDDescription Amino Acid SequenceNO MGSSHHHHHHHHSSGGEQKLISEEDLEFTLEDFVGDWRQTAG YNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIH VI IPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI D G V T P N M I D Y F G R P Y E G I A V F D G K K I T V T G T L W N G N K 11 D E RN anoLuc-LaM8 - LINPDGSLLF RVT I NGVT GWRL C E R I L AGD P L VQC GG I AL AA AK74-MagLOV TMAQVQL VE S GGGL VQAGGS L RL S C AVS GRP F S E YNL GWFRQAP GKE RE FVARI RS S GT T VYT D S VKGRF S AS RDNAMLATTLE 44 ( SEQ ID NO: 26underlined) RIEKNFVITDPRLPDNPI IFASDSFLQLTEYSREEILGRNPR FLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNL FHVQPMRDQKGDVQ YF I GVKLDGT EHVRDAAERI KVML I NKT AAN I D E AAKE L KNMG YL QLN S L E P E DT AV Y YC. AMS RVDT DSPAF YD Y WGQGT QVT VST P RS GT GGPT LL KE GKRT GGGVE69323198441MGSSHHHHHHHHSSGGEQKLISEEDLEFTLEDFVGDWRQTAG YNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIH VI IPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI D G V T P N M I D Y F G R P Y E G I A V F D G K K I T V T G T L W N G N K 11 D E R LINPDGSLLF RVT I NGVT GWRL C E R I L AGD P L VQC GG I AL AAN anoLuc-LaM8 - TMAQVQL VE S GGGL VQAGGS L RL S C AVS GRP F S E YNL GWFRQ AK74 Mag- AP GKE RE FVARI RS S GT T VYT D S VKGRF S AS RDNAGL ERI EK 45 iLID NFVITDPRLPDNPI IFASDSFLQLTEYSREEILGRNPRFLQG PET DRAT VRK 1 RDAI DNQT E VT VQL I N YT KS GKKFWNL FHVQ PMRDQYGDVQYFIGVKLDGTERLHGAAERIAVMLINKTAFQI AEAAGKNMGYLQLNSLEPEDTAVYYCAMSRVDTDSPAFYDYW GQ GT Q VT VS T P R S GT G G P T L L K E G K RT G G G VE MAHHHHHHS SGGLNDI FEAQK. I EWHEHASMKI EEGKLVI WIN GDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATG D G P D 11 F WAH DRF GG YAQ S GT, L AE I T P D KAFQ D KL Y P FT WDA VRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKE LKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKD VGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETMyelin Basic AMT I NGP WAWSN I DT S KVNYGVT VL PT FKGQ P S KP FVGVL SA Protein (MBP)- GINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKS SspB-mCherry YEEELAKDPRIAATMENAQKGEIMPNTPQMSAFWYAVRTAVI / Xntigen (SspB NAAS G RQT VDEAL KDAQT NS S S NNNNNNNNNNL G I E GTT ENL 46 underlined., and Y F Q G S S S PKRPKLLRE YYDWLVDNS FT P YLVVDAT YLGVNVP mCherry VEYVKDGQIVLNLSASATGNLQLTNDFIQFNARFKGVSRELY I PMGAALAI YARENGDGVMFE PEE I YDELNI GGT VSKGEEDN italicized)MAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKL KVTKGGPL PEA WD PL SPQFMYGSKA YVKHPAD IPD YLKL S FP EGFKWERVPINFEDGGVVTVTQDSSLQPJGEFTYKVKLRGTNFP SDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGH YDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQY ERAEGRHS PGGPIDE L YK MGSSHHHHHHHHSSGGEQKLISEEDLEFTLEDFVGDWRQTAG YN L DQ VL E Q G GV S S L F Q NL G V S VT P I Q R I VL S G E N GL K I D I H VI IPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI D G V T P NM I D Y F G R P Y E G I A V F D G K K I T V T G T L W N G N K 11 D E R NanoLuc fused toLINPDGSLLF RVT I NGVT GWRL C E R I L AGD P L VQC GG I AL AA m Cherry targetedTMAQVQL VE S GGGL VQAGGS L RL S C AVS GRP F S E YNL GWFRQ optobody of SEQAP GKE RE FVARI RS S GT T VYT D S VKGRF SAS RDNAGL ERI EK 87 ID NO: 29N F V I T D P RL P DN P 11 F AS D S FL QL T E Y S RE E I L GRNC RFL QG P ET DRAT VRK I RDAI DNQT EVT VQL I N YT KS GKKFWNL FHLQ PMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENI DEAAGKNMGYLQLNSLEPEDTAVYYCAMSRVDTDSPAFYDYW G Q G T Q VT V S T P R S G T G G P T L L K E G K RT G G GV E MGSSHHHHHHHHSSGGEQKLISEEDLEFTLEDFVGDWRQTAGNanoLuc-LaM8- YNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIH AK74-MagLOV2 VI IPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI (MagLOV D G V T P N M I D Y F G R P Y E G I A V F D G K. K I T V T G T L W N G N K 11 D E R sequence of SEQ LINPDGSLLF RVT I NGVT GWRL C E R I L AGD P L VQC GG I AL AA 71 ID NO: 72 TMAQVQL VE S GGGL VQAGGS L RL S C AVS GRP F S E YNL GWFRQ underlined) AP GKE RE FVARI RS S GT T VYT D S VKGRF SAS RDNAMLATTLER I E KN F V I T D P RL P DN P 11 F A S D S F L Q L T E Y S RE E I L G WN P R70323198441FLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNL LHVQPMRDQKGDVQ YF I GVKLDGT EHVRDAAGRERVML I KKT AE N IME AAKE L KNMG YL QLN S L E P E DT A V Y YC AMS RVDT D S PAFYDYWGQGTQVTVSTPRSGTGGPTLLKEGKRTGGGVE

[0313] ELIS As were performed to test binding of the magnetoresponsive optobodies to antigen. Myelin Basic Protein (MBP)-SspB-mCherry fusion protein was used as the antigen, as provided in SEQ ID NO: 46. ELISAs were performed in dark, light, or light plus magnetic field conditions as described in Example 14. While the tested constructs included luciferase domains fused to the N-tenninus, the light for this experiment was provided by external illumination.Results

[0314] The results of the experiment are provided in FIGS. 8A-8D.

[0315] FIG. 8A shows a representative ELI SA curve of a control optobody with a wildtype AsLOV2 insertion as the ΔK74 insertion loop site (SEQ ID NO: 87). The estimated Kd in the dark condition was 245 nM; in light was 67.7 nM; and in the light plus magnetic field condition was 65.4 nM. This construct therefore shows that a wildtype optogenetic AsLOV2 optobody is essentially non-magnetoresponsive.

[0316] FIG. 8B shows a representative ELISA curve measuring binding of anti-Cherry antibodies fused to the MagLOV domain of SEQ ID NO: 71, under dark, dark plus magnet, light, and light plus magnet conditions. The Kd in the dark condition was estimated at 144 nM; in dark plus magnet was 132.6 nM; in light was 146.4 nM; and in light plus magnet was 297.2 nM. This shows that some constructs can preferentially respond to magnets without being as strongly modulated under light-only conditions.

[0317] FIG. 8C shows a representative ELISA curve of an improved MagLOV sequence with iLID mutations (SEQ ID NO: 26), added to the AK74 insertion site of the Lam8 anti-mCherry nanobody (SEQ ID NO: 44). The Kd was estimated to be 47 nM in the dark; 100.7 nM in the light; and 156.1 nM in the light plus magnetic field condition, showing that this construct is magnetoresponsive.

[0318] FIG. 8D shows a representative ELIS A curve comparing light versus light+magnet conditions for inserting the improved MagLOV with iLID amino acid substitutions as shown in SEQ ID NO: 40 inserted into the AK74 loop insertion site of an71323198441anti-mCherry antibody (SEQ ID NO: 68). The Kd shifted from an estimated 41.9 nM to 71 nM in the presence of a magnet.

[0319] Taken together, the results presented in FIGS. 8A-8D demonstrate that both mutants showed a distinct binding Kd in the presence of light plus magnet vs light vs dark, showing magnetoresponsive modulation of binding. Light plus magnet further reduced binding affinity compared to just light alone, demonstrating a magnetoresponsive control over binding affinity for the magbody. In comparison, the non-magnetoresponsive optobody demonstrates no change in binding when a magnet is present.Example 16, Enhanced induction of luminescence with magnetic field

[0320] Truncated MagLOV domains were generated, and tested for their ability to induce luminescence when fused to luciferase.Materials and Methods

[0321] Descriptions and amino acid sequences of the truncated MagLOV domain variants fused to luciferase are provided in Table 12, below. The “LOV CDS (bp)” column shows the intended size of the truncated LOV domain coding sequence, in basepairs.Table 12, Description and amino acid sequences of fusion proteins with truncated MagLOV domains inserted into luciferase _ _ _N- C- Mutations MagL LOV Fusion Protein Amino Acid FusionCDS terminal te rmin al compared Sequence SEQ OV (bp) deletion deletion to SEQ ID SEQ (bp) (bp) NO: 26 (T runcated MagLOV bolded) ID NO ID NO MGSSHHHHHHHHSSGGEQKL I SE E DLMVL T L E D FVG D WE Q T AA YNL D Q VL E Q G GV S S L L Q NLAVS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ I E E V F KVV Y P V D D HH F K V I L P YGTLVI DGVT PNMLNYFGRF3L P Y E G I A V F D G K K I T V T G T L W 330 15 50 88 87(LgBit) NGNKI IDERLITPDGSMLFR VTINSGSLERIEKNFVITDP RLPDNPIIFASDSFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DQKGDVQYF I GVKLDGT GG S72323198441LOV N- C- Mutations Fusion Protein Amino Acid Fusion MagL terminal terminal compared OV CDS deletion deletion to SEQ ID Sequence SEQ(bp) (Truncated MagLOV boided) ID NO SEQ (bp) NO: 26 ID NO (bp)GGGS GGVT GYRL FEE I LGT G GPTLLKEGKRTGGGVE MGSSHHHHHHHHSSGGEQKL I S E E D L M V F T L E D F V G D W E Q T AA YNL D Q VL E Q G GV S S L L Q NLAVS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL P Y GT L V I D G VT P NML N Y F G R P Y E G I A V F D G K K I T V T G T L W NGNKI IDERLITPDGSMLFR - 330 15 51 88 87VT INSGSLERIEKNFVITDP RLPDNPIIFASDSFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DQKGDVQYF I GVKLDGT GG S G G G S G GVT G YRL FEET L GT G G P T L L K E G K RT G G G V E MGSSHHHHHHHHSSGGEQKL I S E E D L M V F T L E D F V G D W E Q T A A YNL D Q VT., E Q G GV S S L L QNLAVS VT P I TRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ I E E V F KVV Y P V D D HH F K V I L P YGTLVI DGVT PNMLNYFGR P Y E G I A V F D G K K I T V T G T L W N GNK 11 D E RL I T P D G S ML F R - 330 15 87 52 88 VT INSGSLERIEKNFVITDP RLPDNPIIFASDSFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DQKGDVQYFIGVKLDGTGGS GGGS GGVT GYRL FEE I LGT G GPTLLKEGKRTGGGVE MGSSHHHHHHHHSSGGEQKL I S E E D L M V F T L E D F V G D W E Q T AA YNL D Q VL E Q G GV S S I, L QNLAVS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL F122S 380 36 15 53 89 P Y GT L V I D G VT P NML N Y F G R (LgBit)P Y E G I A V S D G K K I T V T G T L W N GNK 11 D E RL I T P DG SML F RVT I N S G S FVI TD PRLPDNP I IFASDSFLQLTEYSREEILG RNPRFLQGPETDRATVRKIR73323198441LOV N- C- Mutations Fusion Protein Amino Acid Fusion MagL terminal terminal compared OV CDS deletion deletion to SEQ ID Sequence SEQ(bp) (Truncated MagLOV boided) ID NO SEQ (bp) NO: 26 ID NO (bp)DAIDNQTE VTVQL INYTKS G KKFWNLFHVQPMRDQKGDVQ YFIGVKLDGTEHVRDAAERI KVMLIKRPLLT M G S S H H H H H H H H S S G G E Q K L I SEEDLMVFTLEDFVGDWEQ T A A YNL D Q VL E Q G GV S S L L Q NL AV S VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL P YGTLVI DGVT PNMLNYFGR P Y E G I A V F D G K K I T V T G T L W- 380 36 15 54 89 N GNK 11 D E RL IT P D G S ML F RVT INSGSFVITDPRLPDNPI IFASDSFLQLTEYSREEILG RNPRFLQGPETDRATVRKIR DAIDNQTE VTVQL INYTKS G KKFWNLFHVQPMRDQKGDVQ YFIGVKLDGTEHVRDAAERI KVMLIKRPLLT MGSSHHHHHHHHSSGGEQKL I S E E D LMV F T L E D F VG D WE Q T AA YNL D Q VL E Q G GV S S L L Q NLAVS VT P I QRI VRS KENAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL273 P YGTLVI DGVT PNMLNYFGR(shot P Y E G I A V F D G K K I T V T G T L W G50K 9055 72 87 test NGNKI IDERLITPDGSMLFR (LgBit)varia VT I N S G S 11 FASD S FLQLTEnt) YSREE ILGRNPRFLQGPETDRATVRKI RD Al DNQTE VTVQL INYTKS GKKFWNLFHVQPM RDQKGDVQYFIGVKLDGTGG S GGGS GGVT GYRI, FEE I LGT GGPTLLKEGKRTGGGVE MGSSHHHHHHHHSSGGEQKL I S E E D L M V F T L E D F V G D W E Q T AA YNL D Q VL E Q G GV S S L L Q NLAVS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL 57 333 12 - 87P Y GT L V I D G VT P NML N Y F G R P Y E G I A V F D G K K I T V T G T L W N GNK 11 D E RL I T P DG SML F RVT I N S GS H F RT YRE KL RDH G PETT74323198441LOV N- C- Mutations Fusion Protein Amino Acid Fusion MagL terminal terminal compared OV CDS deletion deletion to SEQ ID Sequence SEQ(bp) (Truncated MagLOV boided) ID NO SEQ (bp) NO: 26 ID NO (bp)MGS S HHHHHHHH S S GGEQKL I S E E D L M V F T L E D F V G D W E Q T AA YNL D Q VL E Q G GV S S L L Q NLAVS VT P I TRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ I E E V F K V V Y P V D D H H F K V I L P Y GT L V I D G VT P NML N Y F G R P Y E G I A V F D G K K I T V T G T L W N GNK 11 D E RL I T P DG SML F R 58 91 VTINSGSLERIEKNFVITDP RLPDNPI IWASDSFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DQKGDVQYF I GVKLDGT GG S G G G S G GVT G YRL F E E I L GT G GPTLLKEGKRTGGGVE M G S S H H H H H H H H S S G G E Q K L I SEEDLMVFTLEDFVGDWEQ T AA YNL D Q VL E 0 G GV S S L L QNL AV S VT P YQRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL P YGTLVI DGVT PNMLN YFGR P Y E G I A V F D G K K I T V T G T L W N G N K 11 D E R L I T P D G S M L F R 59 91 VTINSGSLERIEKNFVITDP RLPDNPI IWASDSFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DQKGDVQYFIGVKLDGTGGS GGGS GGVT G YRL FEE I L GT G GPTLLKEGKRTGGGVE MGS S HHHHHHHH S S GGEQKL I S E E D L M V F T L E D F V G D W E Q TAAYNLDQVLEQGGVS SLLQ NLAVS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ I E E V F K V V Y P V D D H H F K V I L P YGTLVI DGVT PNMLNYFGR60 88 P Y E G I A V F D G K K I T V T H T L WN G N K 11 D E R L I T P D G S M L Y R VTINSGSLERIEKNFVITDP RLPDNPI IFASDSFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR75323198441LOV N- C- Mutations Fusion Protein Amino Acid Fusion MagL terminal terminal compared OV CDS deletion deletion to SEQ ID Sequence SEQ(bp) (Truncated MagLOV boided) ID NO SEQ (bp) NO: 26 ID NO (bp)DQKGDVQYF I GVKLDGT GG S GGGS GGVT G YRL FEE I L GT G G P T L L K E G K RT G G G V E MGSSHHHHHHHHSSGGEQKL I S E E D LMV F T L E D F VG D WE Q TAAYNLDQVLEQGGVS SLLQ NLAVS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL P YGTLVI DGVT PNMLNYFGR P Y E G I A V F D G K K I T G T G T L W91 NGNEI IDERLITPDGSMLFR 61VT INSGSLERIEKNFVITDP RLPDNPI IWASDSFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DQKGDVQYF I GVKLDGT GG S GGGS GGVT GYRL FEE I LGT G GPTLLKEGKRTGGGVE MGSSHHHHHHHHSSGGEQKL I S E E D LMV F T L E D F VG D WE Q T AA YNL D Q VL E Q G GV S S L L Q NL AVS VT PITRIVRSGE NAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL P YGTLVI DGVT PNMLNYFGR P D E G I AVF D GKK I T VT GT L W62 92 NGNKI IDERLITPDGSMLFRVT INSGSLERIEKNFVITDP■£> T ‘ET’T T ’T'13’ VSREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKTGKKFWNLFHMQPMR DQKGDVQYF I GVKLDGTEHV RDAAERIKVMLIKRPLLT MGSSHHHHHHHHSSGGEQKL I S E E D L M V F T L E D F V G D W E Q T AA YNL D Q VL E Q G GV S S L L Q NLAVS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL63 90 P YGTLVI DGVT PNMLNT FGRP Y E G I A V F D G K K I T V T T T L W N GNK 11 D E RL I T P DG SML F RVT I N S G S 11 FASD S FLQLTEYSREE ILGRNPRFLQGPETD RATVRKI RD Al DNQTE VT VQ76323198441LOV N- C- Mutations Fusion Protein Amino Acid Fusion MagL terminal terminal compared OV CDS deletion deletion to SEQ ID Sequence SEQ(bp) (Truncated MagLOV boided) ID NO SEQ (bp) NO: 26 ID NO (bp)L INYTKS GKKFWNLFHVQPM RDQKGDVQYFIGVKLDGTGG S GGGS GGVT GYRL FEE I LGT GGPTLLKEGKRTGGGVE M G S S H H H H H H H H S S G G E Q K L I SEEDLMVFTLEDFVGDWEQ T A A YNL D Q VL E Q G GV S S L L Q NL AV S VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ IEEVFKVVYPVDDHHFKVIL P YGTLVI DGVT PNMLNYFGR P Y E G I A V F D G K K I T V T G T L W N GNK 11 D E RL IT P D G S ML F R 64 91 VTINSGSLERIEKNFVITDP RLPDNPI IWASDSFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DQKGDVQYFIGVKLDGTGGS GGGS GGVT GYRL FEE I L GT G G P T L L K E G K RT G G GV E MGS S HHHHHHHH S S GGEQKL I S E E D L M V F T L E D F V G D W E Q TAAYNLDQVLEQGGVS SLLQ NLAVS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ I E E V F K V V Y P V D D H H F K V I L P YGTLVI DGVT PNMLNYFGR P Y E G I A V F D G K K I T V T G T L W NGNKI IDERLITPDGSMI. FR 65 93 VTINSGSLERIEKNFVITDP RLPDNPI IWASDEFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DQKGDVQYF I GVKLDGT GG S G G G S G GVT G YRL F E E I L GT G G P T L L K E G K RT G G G V E M G S S H H H H H H H H S S G G E Q K L I S E E D LMV F T L E D F VG D WE Q T AA YNL D Q VL E 0 G GV S S L L QNL AV S VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ66 94 IEEVFKVVYPVDDHHFKVILP YGTLVI DGVT PNMLNYFGR P Y E G I A V F D G K K I T V T G T L W N G N K 11 D E R L I T P D G S M L F RVTINSGSLERIEKNFVITDP77323198441LOV N- C- Mutations Fusion Protein Amino Acid Fusion MagL terminal terminal compared OV CDS(b deletion deletion to SEQ ID Sequence SEQp) (Truncated MagLOV boided) ID NO SEQ (bp) NO: 26 ID NO (bp)RLPDNPI IWASDSFLQLTEY SREE I LGRNPRFLQGPE TAR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DPKGDVQYFIGVKLDGTGGS G G G S G G V T G YRL FEET L GT G G P T L L K E G K RT G G G V E MGSSHHHHHHHHSSGGEQKL I SEEDLMVFTLEDFVGDWEQ T A A YNL D Q VL E Q G GV S S L L Q NLAVS VT P I QRI VRS GENAL K I D I HVI I P YE GL S ADQMAQ PEEVE KVV Y P V D D HH F K V 1 LP YGTLVP DGVT PNMLNYFGR P Y E G T A V F D G K K 1 T V T G T W67 95 N GNK 11 D E RL 1 T P D G S ML F E< VTPNSGSLERIEKNFVITDP RLPDNPI IWASDSFLQLTEY SREE I LGRNPRFLQGPE TDR ATVRKIRDAIDNQTEVTVQL INYTKSGKKFWNLFHVQPMR DQKGDVQYFIGVKLDGTEHV RD AAERI KVML I K R P L L T

[0322] Luminescence was measured directly after the injection of coelenterazine. Video recordings were processed to segment each colony and aggregate the light produced by each, plotting that light over time. This data was then normalized to account for declining coelenterazine concentration.Results

[0323] The results of the luminescence measurements for the MagLOV-luciferase fusions using truncated MagLOV domains are provided in Table 13, below.Table 13. Luminescence measurements for MagLOV-luciferase fusionsSEQ tau up / Amplitude Period Peak / ID down R2 Round Phase Offset(%) trough (frames)NO (frames)50 10.44 2.77 / 1.43 20.4 0.14 0.9663 1.017 0.9697 2 51 10.22 5.37 / 2.69 20.33 0.14 0.9114 0.0851 0.9393 52 10.08 4.29 / 2.17 20.38 0.14 0.9571 0.0907 0.97177832319844153 10.18 3.09 / 1.90 20.34 0.14 0.9769 0.9750 0.9661 54 9.50 2.89 / 1.65 20.28 0.13 0.9850 0.0919 0.9734 55 9.46 3.67 / 1.86 20.45 0.15 0.9915 0.0884 0.9674 56 9.64 2.72 / 1.61 20.33 0.14 0.9951 0.0939 0.9707 57 9.34 3.35 / 1.73 20.44 0.14 0.9935 0.0887 0.9593 58 17.24 7.98 / 3.34 20.33 0.14 0.9149 0.1198 0.9733 59 10.65 5.09 / 2.78 20.41 0.14 0.9801 0.0901 0.9696 4 60 11.09 3.77 / 1.99 20.32 0.15 0.9746 0.1028 0.9739 61 11.10 3.75 / 1.80 20.34 0.15 0.9800 0.1033 0.9602 62 11.47 4.26 / 2.31 20.5 0.16 0.9767 0.1032 0.9575 5 63 14.45 4.57 / 2.03 20.35 0.14 0.9327 0.1281 0.9684 64 14.71 5.01 / 2.06 20.4 0.14 0.9714 0.1271 0.9717 64 16.08 9.40 / 2.88 20.34 0.15 0.9622 0.1042 0.9644 64 10.56 5.56 / 2.64 20.48 0.16 1.0298 0.0873 0.96546.16 / [data64 14.3 not 2.42 0.14 0.9625 0.1142 0.9694 6 collected]64 14.10 6.05 / 2.55 20.4 0.14 0.9744 0.1130 0.9758 65 15.3 6.73 / 2.64 20.39 0.14 0.9521 0.1174 0.9735 66 17.26 4.0 / 2.07 20.34 0.16 0.9428 0.1579 0.983867 17.18 5.08 / 2.24 20.39 0.17 0.9381 0.1473 0.98

[0324] As shown in Table 13, successive rounds of selection enhance the amplitude of the magnetic modulation. While we are selecting for increased amplitude, the tau up / down changes as well, modifying the dynamic response of these engineered luciferase constructs.

[0325] FIG. 14 illustrates the normalized luminescence response of a bacterial colony over time, after the addition of coelenterazine for the fusion protein of SEQ ID NO: 66. In FIG. 14, the black circular markers represent mean-corrected intensity measurements at discrete time frames, wherein each data point represents the colony's raw luminescence intensity normalized by colony mean intensity over time, thereby removing photobleaching effects as well as accounting for the decline or rise in oxygen or luciferase substrate concentration that may occur over the course of imaging independently from the magnetic modulation. The continuous black line represents a driven exponential response model fitted to said data points, wherein the model is defined by a driven exponential function with the following fit parameters:79323198441An amplitude parameter defining the magnitude of modulation responseA tau up parameter defining the exponential rise time constant during magnetic field activationA tau down parameter defining the exponential decay time constant during magnetic field deactivationA period parameter defining the cycle duration of periodic stimulation, which should be set by the frequency with which the magnet is added and removed from the sample.A phase parameter defining temporal shift of the response relative to stimulation, which helps to adjust for the slight time differences for the moving magnet to arrive underneath each part of the plate.An offset parameter representing the baseline normalized intensity level, to account for any imperfections in the normalization against the mean intensity,

[0326] The shaded vertical bands indicating temporal windows of magnetic field activation, providing visual correlation between stimulus timing and luminescence response.Example 17. Binding of an antibody to GFP induced by a magnetic field and external light

[0327] An ELISA was performed as described in Example 14 with a VI Ill-based nanobody magbody targeting GFP. The amino acid sequence of the GFP-targeted magbody is provided in Table 14.

[0328] GFP was used as the antigen. Antigen and protein were expressed as described above. An external light source was used. Binding was measured in light, dark, and light plus magnetic field conditionsTable 14. Amino acid sequence of anti-GFP magbodyComponent Amino Acid Sequence SEQ ID NO Nanoluc - MGSSHHHHHHHHSSGGEQKLISEEDLEFTLEDFVGDWRQTAGYGFP-targeted N L D Q V L E Q G G V S S L F Q N L G V S VT P I Q R I V L 8 G E N G L K 1 D I H V IVHH IPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVMagbody TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINP 47 with DGSLLFRVTINGVTGWRLCERILAGDPLVQCGGIALAGSAQVQ MagLOV LVESGGGLVQAGGLERIEKNFVITDPRLPDNPTIFASDSFLQLinsertion TEYSREEILGRNPRFLQGPETDRATVRKIRDAIDNQTEVTVQL80323198441(MagLOV is INYTKSGKKFWNLFHVQPMRDQYGDVQYFIGVKLDGTERLHGA underlined) AER1AVMLINKTAFQ1AEAAGGSL RL S C AAS GRT YS I S AMGWFRQA P GKERB F V AG I S RS GGT T YYAD PVKGRFT I S RDNA KNTVY LQMNSLKPEDTAVYYCAARARGWTTFPAREIEYDYWGQGTQVTVGTGGPTLLKEGKRTGGGVEResults

[0329] As shown in FIG. 10, binding of the anti-GFP magbody to GFP differed between the light, dark, and light plus magnetic field conditions. Notably, the light and light plus magnet conditions did not produce identical levels of binding, which shows that the anti- GFP magbody was magnetoresponsive.

[0330] These results show that magbodies can be designed against multiple different targets. This sequence adds the MagLOV sequence in at a different insertion site, GG15, versus the AK74 insertion site, showing that the effect is not particular to a given insertion site, or target.Example 18. Magnetoresponsive Turboluc Variants

[0331] Magnetoresponsive Turboluc variants were generated by fusing to the improved MagLOV sequence of SEQ ID NO: 26 at the split site or at the C-terminus. Turboluc is an engineered variant of the naturally occurring Metndia pacifica luciferase, as previously described in the literature (Douglas S. Auld, et al., Biochemistry 201857 (31), 4700-4706).

[0332] FIGS. 11A-11C show luminescence outputs with and without magnet, both normalized and raw, and with a 515 filter to look at different parts of the luminescence spectra, FIG. 11A shows the results for the fusion protein provided in SEQ ID NO: 74, with Turboluc fused to SEQ 26 at its C-terminus.

[0333] FIG. 11B shows the results for the fusion protein provided in SEQ ID NO: 75, with split Turboluc fused together, and the improved MagLOV at its C-terminus.

[0334] FIG. 11C shows the results for the fusion protein provided in SEQ ID NO: 76, with a split Turboluc with the improved MagLOV inserted at the split site, showing that the split Turboluc variant can be made magnetoresponsive by fusing an improved MagLOV domain to the C-terminus and linking both of the split components of Turboluc into one single construct.81323198441Example 19. Renilla luciferase fused to improved MagLOV domain demonstrates magnetoresponsive luminescence induction of a non-NanoLuc luciferase

[0335] Renilla luciferase was fused to the improved MagLOV domain of SEQ ID NO: 26, as shown in Table 15, below.Table 15. Renilla luciferase-MagLOV fusion, from N- to C-terminusDescription Amino Acid Sequence SEQ ID NO MGSSHHHHHHHHTTSKVYDPEQRKRMITGPQWARCKQMN VL D S FI N Y Y D S E KHAE N AV I F L H GNAT S S Y L W R H WP H I E PVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELL NLPKKI IFVGHDWGSALAFHYAYEHQDRIKAIVHMESWD VIESWMGWPDIEEELALIKSEEGEKMVLENNFFVETLLPSRenillaKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGluciferase- GK P D WQ I VRN YN A YL RAS D DL P KL F I ESDPGFFS NAIVE 76 MagLOV fusion GAKKFPNTEFVKVKGLHFLQEDAPDEMGKYIKSFVERVLKN E Q G S L AT T L E R I E K N F V I T DPR L P D N P 11 F A S D S F I> Q L T EYSREEILGRNPRFLQGPETDRATVRKIRDAIDNQTEVTV QLTNYTKSGKKFWNLFHVQPMRDQKGDVQYFTGVKLDGTEHVR DAA E R I KVML I NKT AAN I D E AAKE LResults

[0336] The Renilla luciferase-MagLOV fusion produced a different level of luminescence in the presence of a magnetic field compared to the absence of a magnetic field. This demonstrates a magnetoresponsive effect on the luminescence of Renilla luciferase due to the fusion of a magnetoresponsive MagLOV domain. FIG. 12A shows the results for the fusion protein SEQ ID NO: 76. These results show the raw luminescence output over time, as the magnet turns off approximately every five frames. The initial decline from the starting peak shows the initial rapid decline in luminescence output, which will gradually recover if the luciferase is all owed to sit to recover.

[0337] FIG. 12B shows the same results for a construct that has been engineered, undergoing mutagenesis and selection, using the same methods described elsewhere for selecting and improving magnetoresponsive modulation in luciferases.

[0338] FIG 12C shows a control Renilla luciferase without a MagLOV fusion to demonstrate that no magnetoresponsive modulation of luminescence is detectable unless the improved MagLOV domain is added.Example 20. Alternate MagLOV insertion site in Magbody82323198441

[0339] An ELISA was performed as described in Example 14, using an alternative MagLOV insertion site (GG15 site) in a magbody. Antigen, protein, and luciferase were expressed as described in previous Examples. Biotinylated mCherry was applied to a streptavidin coated plate, as described in Example 14.

[0340] SEQ ID NO: 78 shows the sequence of the LaM8 nanobody with the gg site fused in.MGSSHHHHHHHHSSGGEQKLISEEDLGSAQVQLVESGGGLVQAGMLATrLERIEKN FVITDPRLPDNPTIFASDSFJ.. QJ.. TEYSREEILGRNPRFLQGPETDRATVRKIR. DAIDNQTF. VIT^QLINYTKSGKKFWNLFIIVQP^IRDQKGDVQYFIGVKLDGTEI-IVRDAAERIKVM LINKTAANIDEAAKELGSLRLSCAVSGRPFSEYNLGWFRQAPGKEREFVARIRSSGTT VYTDSVKGRFSASRDNAKNMGYLQLNSLEPEDTAVYYCAMSRVDTDSPAFYDYWG QGTQVTVSTPRSGTGGPTLLKEGKRTGGGVE (SEQ ID NO: 78).

[0341] Results are shown in FIG. 13. The estimated Kd in dark state was 52.3 nM, in the light state was 85 nM, and in light plus magnet was Kd 101.7 nM. The results show that the presence of a magnet modestly biased the Kd further than just light alone.Example 21. Method for identifying magnetically inducible changes in binding

[0342] It can be valuable to establish a protocol for enriching for magnetoresponsive binders. Briefly, magnetoresponsive binders can begin from an initial magbody design measured to demonstrate some magnetoresponsive effect. This initial starting point can be then diversified using site-directed or random mutagenesis. This diverse library can then be tagged using click-display protocols (e.g., as described in Yu Zeng, et al., Nucleic Acids Research, Volume 51, Issue 16, 8 September 2023, Page e8), which result in the fused library of DNA sequences attached to the proteins that they encode for.

[0343] Next, an antigen coated plate is prepared, where the antigen selected matches the intended target of the magbody. In alternating steps, the magbody library is added to the surface, while a magnetic field is applied to the plate, which is expected to cause magbodies which disproportionately increase binding in a magnetic field to adhere to the surface of the plate, while magbodies which disproportionately unbind in a magnetic field should relatively de-adhere to the plate. In this way, by switching the magnetic field on and off of the plate, a library enriched for the desired behavior will equilibrate into the supernatant in the plate. This supernatant can then be collected and subjected to83323198441additional magnetic field selection, to further enrich the library. Finally, these supernatant libraries can either be directly cloned and evaluated, or timepoints during this enrichment can be sequenced to identify how different sequences are responding to the enrichment selection pressure.

[0344] This protocol is expected to be very effective for engineering an enhanced magnetoresponsive effect. While many magnetoresponsive proteins can be engineered rationally, achieving magnetoresponsive effects that are easily detectable can sometimes require engineering using a method like this or other methods.Example 22. Engineering of a magnetoresponsive split fluorescent protein

[0345] Y-FAST (see M Plamont, et al., Proc. Natl. Acad. Sei. U. S. A. 113 (3) 497-502, (2016)) was fused to Mag-iLID and sspB. When Mag-iLID binds, the fluorescent signal from Y-FAST increased. This reveals real-time dynamics of the magnetic response on protein binding, and shows that Mag-iLID when fused to another protein can induce the co-localization of two protein domains when they are fused to Mag-iLID and sspb respectively.Materials and Methods

[0346] E. coli co-expressing a C-Fast tagged Magluc-iLID fusion and N-Fast microSSPB were grown on solid media under chloramphenicol and ampicillin selection overnight at 37C followed by one day at room temperature. An E. coli colony was resuspended in 50 ul of PBS with 50 uM tf-coral in a glass bottom 96-well plate pre-coated with poly-L-lysine. The tf-coral is a fluorogen that fluoresces in the red channel whenever the C-Fast and N-Fast tags are brought into proximity through binding of 1LID to SSPB (Tebo, A. G., Gautier, A. Nat Commun 10, 2822 (2019).). After at least a 20 minute incubation at room temperature, 50 ul of 400 uM fluorofurimazine in PBS was added to the well. A custom made electromagnet capable of delivering a 10 mT magnetic field was placed on top of the well, and turned on and off every 30 seconds. Samples were imaged on a Leica DMI8 microscope using a spectraX LED excitation source with a 575 / 25 nm filter, and imaged through the Leica triple dichroic and an emission 632 / 60 nm filter. Samples were imaged every 1 second, using 100 ms exposures.84323198441

[0347] The following protein constructs were tested. First, SEQ ID NO: 80 is the CFast-MycTag-MagLuc-iLID target sequence, where SEQ ID NO: 43 iLID is the lightinducible binding partner, and the MagLuc used is a SEQ ID NO: 82 variant of MagLuc using the improved MagLOV of SEQ ID NO: 26. Second, SEQ ID NO: 79 is the target NFAST-microsspB sequence where SEQ ID NO: 81 is the C-fast portion of the tf-coral split fluorescent protein, and SEQ ID NO: 83 is the micro sspB dimerization partner of iLID.

[0348] FIG.9 shows a bioluminescence powered magnetoresponsive effect without external light. In the presence of 200 uM fluorofurimazine, A MagLuc fused to iLID actuates the binding of iLID to a SspB antigen in response as the luminescence of MagLuc changes. Both constructs were expressed in E. coli. When CFast and NFast come towards each other, they complete a fluorescent protein and generate a new red fluorescent signal. In periods where the magnet is active as indicated with the bar, the production of light from Magluc is reduced, and therefore the iLID to sspb binding is reduced, resulting in less fluorescence signal. This fluorescence trace comes from a representative cell expressing both plasmids.

[0349] Instead of driving the binding by shining blue light on the iLID protein, fluorofurimazine, the substrate of the attached MagLuc protein was added. MagLuc luminescence decreased when a magnetic field was added, which in turn, decreased the red fluorescence / iLID binding. Note that the luminescence itself was undetectable at the short exposure times used to detect fluorescence / bmding, and that the red light used to excite fluorescence does not itself activate the iLID domain.85323198441

Claims

CLAIMS1. A method of using a light-and-magnetoresponsive composition, comprising:(a) providing a composition comprising(i) a bioluminescent protein domain; and(li) a light-and-magnetoresponsive protein domain;(b) exposing the composition to a substrate, wherein the bioluminescent protein domain is capable of catalyzing a reaction of the substrate, wherein the reaction emits light; and(c) exposing the composition to a magnetic field.

2. The method of claim 1, wherein the composition further comprises a heterologous protein component.

3. The method of claim 3, wherein the heterologous protein component is activated upon exposure to the substrate and the magnetic field.

4. The method of claim 3, wherein the heterologous protein component is deactivated upon exposure to the substrate and the magnetic field.

5. The method of any one of claims 1-4, w'herein the light-and-magnetoresponsive protein domain is a light-oxygen-voltage-sensing (LOV) domain.

6. The method of claim 5, wherein the LOV domain is an engineered LOV domain.

7. The method of claim 6, w'herein the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 26, and 48.863231984418. The method of claim 7, wherein the engineered LOV domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 26, and 48.

9. The method of claim 6, wherein the engineered LOV domains comprises an induced dimer (iLID) amino acid substitution.

10. The method of claim 9, wherein the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 39 and 40.

11. The method of claim 6, wherein the engineered LO V domain is a truncated LOV domain,12. The method of claim 11, wherein the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 88-95.

13. The method of any one of claims 1-12, wherein the biolummescent protein domain is a luciferase domain.

14. The method of claim 13, wherein the luciferase domain is a NanoLuc luciferase domain, a TurboLuc luciferase domain, a firefly luciferase domain, a copepod luciferase domain, a bacterial luciferase domain, or a dinoflagellate luciferase domain.8732319844115. The method of claim 13, wherein the luciferase domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 3-7.

16. The method of claim 15, wherein the luciferase domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 3-7.

17. The method of claim 13, wherein the luciferase domain is a split luciferase domain.

18. The method of any one of claims 1-12, wherein the bioluminescent protein domain is a photoprotein domain.

19. The method of claim 18, wherein the photoprotein domain is selected from the group consisting of an obelin domain, an aequorin domain, and a clytin domain.

20. The method of any one of claims 2-19, wherein the heterologous protein component is a selected from the group consisting of a cytokine, an antibody or a fragment thereof, a synthetic binding protein, a chimeric antigen receptor (CAR), a CRISPR-Cas protein, a channelrhodopsm, a transcription factor, a DN A-binding domain, an antibody-drug conjugate, and an antibody-radioligand conjugate.

21. The method of claim 20, wherein the heterologous protein component is covalently linked to the light-and-magnetoresponsive protein domain.

22. The method of claim 21, wherein the heterologous protein component and the light-and-magnetoresponsive protein domain are covalently linked to form a fusion protein.8832319844123. The method of claim 21 or claim 22, wherein the bioluminescent protein domain is covalently linked to:(a) the heterologous protein component;(b) the light-and-magnetoresponsive protein domain; or(c) the heterologous protein component and the light-and-magnetoresponsive protein domain.

24. The method of claim 23, wherein the heterologous protein component, the bioluminescent protein domain, and the light-and-magnetoresponsive protein domain are covalently linked to form a fusion protein, wherein the bioluminescent protein domain is fused to:(a) the heterologous protein component;(b) the light-and-magnetoresponsive protein domain; or(c) the heterologous protein component and the light-and-magnetoresponsive protein domain.

25. The method of claim 24, wherein the fusion protein comprises, from N- to C- terminus, a first split luciferase domain, the light-and-magnetoresponsive protein domain, and a second split luciferase domain.

26. The method of claim 24, wherein the fusion protein comprises, from N- to C- terminus, a first split luciferase domain, the light-and-magnetoresponsive protein domain, a second split luciferase domain, and the heterologous protein component.

27. The method of any one of claims 1 -26, wherein the substrate is a luciferin.

28. The method of claim 27, wherein the luciferin is selected from the group consisting of coelenterazine or an analog thereof, a firefly luciferin, a snail89323198441luciferin, a bacterial luciferin, a dinoflagellate luciferin, vargulin or an analog thereof, or a fungal luciferin.

29. The method of any one of claims 1-28, wherein the composition comprises two light-and-magnetoresponsive protein domains.

30. The method of any one of claims 1-29, wherein the composition and the substrate are administered to a subject.

31. The method of claim 30, wherein the subject is a human.

32. The method of any one of claims 1 -31, wherein the magnetic field is a resonant magnetic field,33. The method of any one of claims 1-31, wherein the magnetic field is generated by a wearable device.

34. An engineered LOV domain comprising an induced dimer (iLID) amino acid substitution, optionally wherein the engineered LOV domain comprises the SsrA peptide sequence of AANDENYF (SEQ ID NO: 84).

35. The engineered LOV domain of claim 34, wherein the engineered LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 39 and 40.

36. An engineered LOV domain, wherein the LOV domain is truncated.9032319844137. The engineered LOV domain of claim 36, wherein the truncated LOV domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 88-95.

38. An engineered LOV domain comprising an amino acid residue selected from the group consisting of 45R, 9 IF, 100Y, 116R, 117L, 118H, 119G, 122E, 1241, 125A, 130N, 134F, 135Q, 137A, 141D, and 143F.

39. An engineered LOV domain comprising an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 26 and 48.

40. The engineered LOV domain of any one of claims 34-39, wherein the engineered LOV domain is sensitive to light emitted by a reaction catalyzed by the bioluminescent protein domain and sensitive to a magnetic field.

41. A composition comprising the engineered LOV domain of any one of claims 34- 40 and a bioluminescent protein domain, optionally wherein the engineered LOV domain and the bioluminescent protein domain are covalently linked to form a fusion protein.

42. A fusion protein comprising an engineered LOV domain and a bioluminescent protein domain, wherein the engineered LOV domain is inserted within the bioluminescent protein domain.91323198441NOBS-001 / 02WG 356250-200743. The composition of claim 41 or the fusion protein of claim 42, wherein the bioluminescent protein domain is a luciferase domain.

44. The composition or fusion protein of claim 43, wherein the luciferase domain is a NanoLuc luciferase domain, a TurboLuc luciferase domain, a firefly luciferase domain, a copepod luciferase domain, a bacterial luciferase domain, or a dinoflagellate luciferase domain.

45. The composition or fusion protein of claim 43, wherein the luciferase domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 3-7.

46. The composition or fusion protein of claim 45, wherein the luciferase domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 3-7.

47. The composition or fusion protein of claim 43, wherein the luciferase domain is a split luciferase domain.

48. The composition of claim 41 or the fusion protein of claim 42, wherein the bioluminescent protein domain is a photoprotein domain.

49. The composition or fusion protein of claim 48, wherein the photoprotein domain is selected from the group consisting of an obelin domain, an aequorin domain, and a clytin domain.

50. A composition comprising the composition or fusion protein of any one of claims 34-49 and an antibody or a fragment thereof.9232319844151. The composition of claim 50, wherein the antibody or fragment thereof is fused to a light-and-magnetoresponsive protein domain.

52. One or more nucleic acids encoding the composition or fusion protein of any one of claims 34-51.

53. One or more cells comprising the one or more nucleic acids of claim 50, or the composition of any one of claims 34-51.93323198441

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