WO2024059686A2

WO2024059686A2 - Darpin backbones and rigidified electron microscopy imaging scaffolds

Info

Publication number: WO2024059686A2
Application number: PCT/US2023/074156
Authority: WO
Inventors: Todd Yeates; Roger CASTELLS GRAELLS; Kyle MEADOR
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2022-09-15
Filing date: 2023-09-14
Publication date: 2024-03-21
Anticipated expiration: 2025-03-15
Also published as: WO2024059686A3

Abstract

Disclosed herein are DARPin backbones that form multimer and self-assembling protein cages that comprise proteins, which comprise DARPin backbones, fused to subunit proteins of the self-assembling protein cages and methods of making and using thereof.

Description

DARPIN BACKBONES AND RIGIDIFIED ELECTRON MICROSCOPY IMAGING SCAFFOLDS [0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the benefit of U.S. Patent Application No.63/406,976, filed September 15, 2022, which is herein incorporated by reference in its entirety. [0003] REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB [0004] The content of the XML file of the sequence listing named “20230905_034044_244WO1_ST26” which is 159,140 bytes in size was created on September 5, 2023 and electronically submitted via Patent Center herewith the application is incorporated herein by reference in its entirety. [0005] ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT [0006] This invention was made with Government support under GM129854, awarded by the National Institutes of Health and under DE-FC02-02ER63421, awarded by the Department of Energy. The Government has certain rights in the invention. [0007] BACKGROUND OF THE INVENTION [0008] 1. FIELD OF THE INVENTION [0009] The field generally relates to DARPins and cryo-electron microscopy. [0010] 2. DESCRIPTION OF THE RELATED ART [0011] Cryo-electron microscopy (cryo-EM) is a rapidly expanding method for determining the atomic structures of large molecular assemblies. It is, however, not suitable for determining the structures of small-to-medium sized molecules. A size of about 40-50 kDa. For comparison, the average eukaryotic protein chain is about 35 kDa in mass, while bacterial proteins are generally smaller. [0012] The idea of binding a small protein of interest (“cargo”) to a larger carrier (“scaffold”) to make it large enough to image using cryo-EM goes back many years. Nanobodies, antibody fragments, and DARPins have been employed as scaffolds with limited resolution, e.g., 3.8 Å for a 27 kDa cargo protein; 2.49 Å for a cargo protein of about 250 kDa; 3.03 Å for a 57 kDa cargo protein; about 2.7 Å for a cargo protein of about 52 kDa. For proteins smaller than 50 kDa, the finest resolution achieved thus far in the prior art is about 3.5 Å for a 22 kDa cargo protein. [0013] Unfortunately, a resolution of 3 Å is generally accepted as being the necessary threshold for robust structure analysis at the atomic level. [0014] SUMMARY OF THE INVENTION [0015] In some embodiments, the invention is directed to a protein comprising (1) X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N (SEQ ID NO: 1) wherein X1 is Y, Q, I or D, preferably Y, Q, or I, more preferably, Y or Q, most preferably Y; X2 is D or K, preferably D; X3 is D or Y, preferably D; X4 is Q, E, or A, preferably Q or E, more preferably Q; X5 is A or D, preferably A; X6 is A or W, preferably A; X7 is K or L, preferably K; and wherein when X5 is D then X6 is A, and when X6 is W then X7 is L; (2) H-X8-E-I-V-X9-V-L-L-X10-R-G-X11-D-X12-X13 (SEQ ID NO: 2) wherein X8 is L or P, preferably L; X9 is E or K, preferably E; X10 is K, L, or S, preferably K; X11 is A or W, preferably A; X12 is I or V, preferably I; and X13 is N or D, preferably N; and (3) H-L-E-I-X14-E-X15-L-L-X16-X17-G-A-D-V-N (SEQ ID NO: 3) wherein X14 is V or A, preferably V; X15 is L or K, preferably L; X16 is R or W, preferably R; X17 is W, C, or Y, preferably W or C, more preferably W; and wherein X16 is R or one of X16 and X17 is W, preferably when X16 is W then X17 is Y, preferably when X15 is K then X16 is R and X17 is C. [0016] In some embodiments, the invention is directed to a protein comprising or consisting of K-K-L-L-E-A-X-X-X-X-X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N- X-X-X-X-X-X-X-X-P-L-X-L-A-X-X-X-X-H-X8-E-I-V-X9-V-L-L-X10-R-G-X11-D- X12-X13-X-X-X-X-X-X-X-X-P-L-H-L-A-X-X-X-X-H-L-E-I-X14-E-X15-L-L-X16- X17-G-A-D-V-N (SEQ ID NO: 4) wherein X1 is Y, Q, I or D, preferably Y, Q, or I, more preferably, Y or Q, most preferably Y; X2 is D or K, preferably D; X3 is D or Y, preferably D; X4 is Q, E, or A, preferably Q or E, more preferably Q; X5 is A or D, preferably A; X6 is A or W, preferably A; X7 is K or L, preferably K; X8 is L or P, preferably L; X9 is E or K, preferably E; X10 is K, L, or S, preferably K; X11 is A or W, preferably A; X12 is I or V, preferably I; X13 is N or D, preferably N; X14 is V or A, preferably V; X15 is L or K, preferably L; X16 is R or W, preferably R; X17 is W, C, or Y, preferably W or C, more preferably W; and each X is independently any amino acid residue; and wherein when X5 is D then X6 is A, when X6 is W then X7 is L, X16 is R or one of X16 and X17 is W, preferably when X16 is W then X17 is Y, preferably when X15 is K then X16 is R and X17 is C. In some embodiments, the protein comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 51 to SEQ ID NO: 71. In some embodiments, the protein comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 17. [0017] In some embodiments, the invention is directed to a fusion protein comprising or consisting of the protein as described above, e.g., having SEQ ID NO: 4, linked to a subunit protein of a self-assembling protein cage. In some embodiments, the fusion protein comprises a protein having SEQ ID NO: 4 fused to a subunit protein of a self- assembling protein cage, said subunit protein comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the protein is fused to the subunit protein via a peptide linker. In some embodiments, the peptide linker is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid residues long. In some embodiments, the peptide linker is not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long. In some embodiments, the peptide linker (a) is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid residues long, and (b) not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long. In some embodiments, the peptide linker joins the N-terminus of the protein to the C-terminus of the subunit protein. In some embodiments, the peptide linker forms an alpha-helical structure. In some embodiments, the peptide linker has an amino acid sequence comprising KEELD (SEQ ID NO: 37), PAPAP (SEQ ID NO: 38), KEELD (SEQ ID NO: 39), KDELD (SEQ ID NO: 40), RDERN (SEQ ID NO: 41), an (E(A)3K)n motif, an (E4(R/K)4)n motif, or an (AP)n motif. In some embodiments, the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a subunit sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 36. In some embodiments, the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the fusion protein comprises or consists of a sequence selected from the group consisting of SEQ NO: 42 to SEQ ID NO: 50. [0018] In some embodiments, the invention is directed to a protein particle comprising multiple copies of a fusion protein as described herein, e.g., a fusion protein comprising or consisting of the protein as described above, e.g., having SEQ ID NO: 4, linked to a subunit protein of a self-assembling protein cage. In some embodiments, the fusion protein comprises a protein having SEQ ID NO: 4 fused to a subunit protein of a self- assembling protein cage, said subunit protein comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the protein is fused to the subunit protein via a peptide linker. In some embodiments, the peptide linker (a) is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid residues long. In some embodiments, the peptide linker is not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long. In some embodiments, the peptide linker joins the N-terminus of the protein to the C-terminus of the subunit protein. In some embodiments, the peptide linker forms an alpha-helical structure. In some embodiments, the peptide linker has an amino acid sequence comprising KEELD (SEQ ID NO: 37), PAPAP (SEQ ID NO: 38), KEELD (SEQ ID NO: 39), KDELD (SEQ ID NO: 40), RDERN (SEQ ID NO: 41), an (E(A)₃K)n motif, an (E₄(R/K)₄)n motif, or an (AP)n motif. In some embodiments, the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a subunit sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 36. In some embodiments, the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the fusion protein comprises or consists of a sequence selected from the group consisting of SEQ NO: 42 to SEQ ID NO: 50. In some embodiments, the protein particle further comprises one or more additional subunit proteins of the self-assembling protein cage, wherein the one or more additional subunit proteins lack a DARPin backbone having SEQ ID NO: 4. In some embodiments, the one or more additional subunit proteins comprise or consist of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a subunit sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 36. In some embodiments, the one or more additional subunit proteins comprise or consist of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the one or more additional subunit proteins have an amino acid sequence that differs from subunit protein that is fused to a DARPin backbone having SEQ ID NO: 4. [0019] In some embodiments, the present invention is directed to a method of assaying the structure of a target protein of interest which comprises binding the target protein to the protein particle as described herein to result in a complex, and obtaining an image of the complex. In some embodiments, the target protein is bound to a moiety such as a solid substrate (e.g., a bead), another protein, or a chemical (e.g., a drug). In some embodiments, the image is obtained by cryo-electron microscopy. In some embodiments, the methods comprise assaying the structure of the target protein of interest when bound to a given ligand (e.g., another protein or chemical such as a drug of interest), which comprise forming a complex comprising the given ligand bound to the target protein and the fusion protein bound to the given ligand and/or the target protein; and obtaining a cryo-electron micrograph of the complex. In some embodiments, the methods further comprise comparing the cryo-electron micrograph to a reference cryo- electron micrograph, said reference cryo-electron micrograph is of (a) a complex comprising the fusion protein bound to the target protein, said complex excludes the presence of the given ligand or another ligand bound thereto; (b) a complex comprising (i) the given ligand bound to a mutant of the target protein and (ii) the fusion protein bound to the given ligand and/or the mutant, said mutant having at least one amino acid substitution, deletion, or addition as compared to the target protein; or (c) a complex comprising (i) a second ligand bound to the target protein and (ii) the fusion protein bound to the given ligand and/or the target protein, wherein said second ligand is different the given ligand. In some embodiments, the fusion protein is provided in the form of a REMIS cage as described herein. [0020] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description explain the principles of the invention. [0021] DESCRIPTION OF THE DRAWINGS [0022] This invention is further understood by reference to the drawings wherein: [0023] Figure 1: Schematically shows the structure of a REMIS cage. [0024] Figure 2: Schematically shows the structure of a prior art cage protein having DARPins attached thereto, which DARPins lack a DARPin backbone comprising SEQ ID NO: 4. [0025] Figure 3: Cryo-EM micrograph of imaging scaffold bound to KRas (left), 2D classes from the selected particles (right). [0026] Figure 4: Cryo-EM imaging of KRas bound to the drug molecule AMG510 while bound to the REMIS cage RCG-33. The molecular model is shown on the left. A surface rendering of the 3-D cryo-EM density map that formed the basis for the molecular model is shown on the right. For clarity, only the KRas region (and not the REMIS cage region) of the cryo-EM image is shown. [0027] Figure 5: 2D classes for the imaging scaffold bound to MBP [0028] DETAILED DESCRIPTION OF THE INVENTION [0029] Disclosed herein are DARPin backbones. DARPins having DARPin backbones as described herein are capable of forming multimers when in close proximity. Also disclosed herein are self-assembling protein cages which comprise the DARPin backbones. Adjacent DARPin backbones on the protein cages form multimers and thereby “rigidify” the protein cage structure. As exemplified herein, when used as a scaffold for Cyro-EM imaging, the rigidified protein cages enable 3 Å resolution imaging of proteins smaller than 20 kDa. Thus, the rigidified protein cages are referred to herein as Rigid Electron Microscopy Imaging Scaffold cages (“REMIS cages”) and the fusion proteins that comprise a DARPin backbone fused to a subunit of a self- assembling protein cage are referred to herein as “REMIS subunits”. [0030] The experimental results provided herein demonstrate that REMIS cages enable cyro-EM imaging at a resolution of about 3Å for proteins smaller than 20 kDa. The exemplified REMIS cages present 12 copies of the target cargo, thereby tending to provide EM images with very large numbers of individual views of the cargo protein. The high symmetry of the REMIS cage means that an individual particle (REMIS cage plus its bound target cargo) presents the target cargo in 12 different orientations, thereby strongly mitigating preferred orientation effects that challenge many cryo-EM studies. [0031] DARPIN BACKBONES [0032] DARPin backbones have an amino acid sequence that comprises (1) X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N (SEQ ID NO: 1) wherein X1 is Y, Q, I or D, preferably Y, Q, or I, more preferably, Y or Q, most preferably Y; X2 is D or K, preferably D; X3 is D or Y, preferably D; X4 is Q, E, or A, preferably Q or E, more preferably Q; X5 is A or D, preferably A; X6 is A or W, preferably A; X7 is K or L, preferably K; and wherein when X5 is D then X6 is A, and when X6 is W then X7 is L; (2) H-X8-E-I-V-X9-V-L-L-X10-R-G-X11-D-X12-X13 (SEQ ID NO: 2) wherein X8 is L or P, preferably L; X9 is E or K, preferably E; X10 is K, L, or S, preferably K; X11 is A or W, preferably A; X12 is I or V, preferably I; and X13 is N or D, preferably N; and (3) H-L-E-I-X14-E-X15-L-L-X16-X17-G-A-D-V-N (SEQ ID NO: 3) wherein X14 is V or A, preferably V; X15 is L or K, preferably L; X16 is R or W, preferably R; X17 is W, C, or Y, preferably W or C, more preferably W; and wherein X16 is R or one of X16 and X17 is W, preferably when X16 is W then X17 is Y, preferably when X15 is K then X16 is R and X17 is C. [0033] It is noted that (a) amino acid residues X5, X6, 9L of SEQ ID NO: 1, X7, 11R of SEQ ID NO: 2, X15, X16, and X17 are key in the formation of multimers, (b) amino acid residues 1Y and 4Q of SEQ ID NO: 1, and 15I of SEQ ID NO: 2 improve multimer formation and/or multimer stability or rigidity, and (c) amino acid residues X8 and X14 are based on variability at corresponding locations in known DARPins, and are not expected to disrupt multimer formation, stability, or rigidity. [0034] DARPin Backbone Formula: [0035] In some embodiments, the DARPin backbones have an amino acid sequence that comprises or consists of K-K-L-L-E-A-X-X-X-X-X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N-X-X-X-X-X- X-X-X-P-L-X-L-A-X-X-X-X-H-X8-E-I-V-X9-V-L-L-X10-R-G-X11-D-X12-X13-X-X- X-X-X-X-X-X-P-L-H-L-A-X-X-X-X-H-L-E-I-X14-E-X15-L-L-X16-X17-G-A-D-V-N (SEQ ID NO: 4) wherein X1 is Y, Q, I or D, preferably Y, Q, or I, more preferably, Y or Q, most preferably Y; X2 is D or K, preferably D; X3 is D or Y, preferably D; X4 is Q, E, or A, preferably Q or E, more preferably Q; X5 is A or D, preferably A; X6 is A or W, preferably A; X7 is K or L, preferably K; X8 is L or P, preferably L; X9 is E or K, preferably E; X10 is K, L, or S, preferably K; X11 is A or W, preferably A; X12 is I or V, preferably I; X13 is N or D, preferably N; X14 is V or A, preferably V; X15 is L or K, preferably L; X16 is R or W, preferably R; X17 is W, C, or Y, preferably W or C, more preferably W; and each X is independently any amino acid residue; and wherein when X5 is D then X6 is A, when X6 is W then X7 is L, X16 is R or one of X16 and X17 is W, preferably when X16 is W then X17 is Y, preferably when X15 is K then X16 is R and X17 is C, and wherein amino acid residues of DARPin loop regions (underlined) may be selected to bind a target of interest. [0036] It is noted that (a) amino acid residues X5, X6, 19L, X7, 54R, X15, X16, and X17 of SEQ ID NO: 4 are key in the formation of multimers, (b) amino acid residues 11Y, 14Q, and 58I of SEQ ID NO: 4 improve multimer formation and/or multimer stability or rigidity, and (c) amino acid residues X8 and X14 of SEQ ID NO: 4 are based on variability at corresponding locations in known DARPins, and are not expected to disrupt multimer formation, stability, or rigidity. [0037] Exemplary DARPins: [0038] Exemplary DARPins having a DARPin backbone include DARPin against GFP: KKLLEAARAGYDDQVAALLAKGADVNAADDVGVTPLHLAAQRGHLEIVEVLLKRGADINAADLWG QTPLHLAATAGHLEIVELLLRWGADVNARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKF GKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 5) DARPin against GFP: KKLLEAARAGDKYAVDALLAKGADVNAADDVGVTPLHLAAQRGHLEIVEVLLKRGWDINAADLWG QTPLHLAATAGHLEIVELLLWYGADVNARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKF GKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 6) DARPin against GFP: KKLLEAARAGYDDKVAWLLALGADVNAADDVGVTPLHLAAQRGHLEIVEVLLKRGADINAADLWG QTPLHLAATAGHLEIVEKLLRCGADVNARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKF GKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 7) DARPin against GFP: KKLLEAARAGIDDAVAALLAKGADVNAADDVGVTPLHLAAQRGHLEIVKVLLLRGADINAADLWG QTPLHLAATAGHLEIVELLLRCGADVNARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKF GKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 8) DARPin against GFP: KKLLEAARAGIDDAVDWLLALGADVNAADDVGVTPLHLAAQRGHLEIVKVLLSRGADINAADLWG QTPLHLAATAGHLEIVELLLRCGADVNARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKF GKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 9) DARPin against MBP: KKLLEAARAGQDDEVAALLAKGADVNAADNTGTTPLHLAAYSGHLEIVEVLLKRGADVDASDVFG YTPLHLAAYWGHLEIVELLLRWGADVNAMDSDGMTPLHLAAKWGYLEIVEVLLKHGADVNAQDKF GKTAFDISIDNGNEDLAEILQKLN (SEQ ID NO: 10) DARPin against MBP: KKLLEAARAGQDDEVAALLAKGADVNAADNTGTTPLHLAAYSGHLEIVEVLLKRGADIDASDVFG YTPLHLAAYWGHLEIVELLLRWGADVNAMDSDGMTPLHLAAKWGYLEIVEVLLKHGADVNAQDKF GKTAFDISIDNGNEDLAEILQKLN (SEQ ID NO: 11) DARPin against KRas: KKLLEAARAGQDDEVAALLAKGADVNANDSAGHTPLHLAAKRGHLEIVEVLLKRGADINAMDNTG FTPLHLAALRGHLEIVELLLRWGADVNAQDRTGRTPLHLAAKLGHLEIVEVLLKNGADVNAQDKF GKTAFDISIDNGNEDLAEILQKL (SEQ ID NO: 12) DARPin against KRas: KKLLEAARAGQDDEVAALLAKGADVNAHDTFGFTPLHLAALYGHLEIVEVLLKRGADINADDSYG RTPLHLAAMRGHLEIVELLLRWGADVNAADEEGRTPLHLAAKRGHLEIVEVLLKNGADVNAQDKF GKTAFDISIDNGNEDLAEILQKL (SEQ ID NO: 13) DARPin against HER2-ECD: KKLLEAARAGX1X2X3X4VX5X6LLAX7GADVNAKDEYGLTPLYLATAHGHX8EIVX9VLLX10R GX11DX12X13AVDAIGFTPLHLAAFIGHLEIX14EX15LLX16X17GADVNAQDKFGKTAFDIS IGNGNEDLAEILQKLN (SEQ ID NO: 14)* DARPin against AcrB: KKLLEAARAGX1X2X3X4VX5X6LLAX7GADVNARDFTGWTPLHLAAHFGHX8EIVX9VLLX10R GX11DX12X13AKDSLGVTPLHLAARRGHLEIX14EX15LLX16X17GADVNASDSHGFTPLHLA AKRGHLEIVEVLLKNGADVNAQDKFGKTAFDISIDNGNEDLAEILQKLN (SEQ ID NO: 15)* DARPin against Caspase: KKLLEATRAGX1X2X3X4VX5X6LLAX7GADVNAMDDAGVTPLHLAAKRGHX8EIVX9VLLX10R GX11DX12X13ARDIWGRTPLHLAATVGHLEIX14EX15LLX16X17GADVNAQDKFGKTAFDIS IDNGNEDLAEILQKLN (SEQ ID NO: 16)* DARPin against IL-13: KKLLEAARAGX1X2X3X4VX5X6LLAX7GADVNARDSYGSTPLHLAAREGHX8EIVX9VLLX10R GX11DX12X13AADFIGDTPLHLAAYRGHLEIX14EX15LLX16X17GADVNASDITGETPLHLA AQIGHLEIVEVLLKHGADVNAQDKFGKTPADIAADNGHEDIAEVLQKLN (SEQ ID NO: 17)* * For SEQ ID NOs: 14−17, X1 to X17 are as indicated for SEQ ID NO: 4. [0039] Proteins comprising a DARPin backbone will typically comprise between 4 and 6 tandem copies of the 33 amino acid motif (“repeat unit”) that identifies the protein as belonging to the DARPin protein family. In each repeat unit, the amino acid residues of the loop regions are variable and may be selected based on the intended cargo protein to be bound thereby. See, e.g., Plückthun (2015). Proteins comprising a DARPin backbone may comprise “capping repeats”, i.e., the first and last repeat units that are typical of DARPins. [0040] REMIS SUBUNITS [0041] A protein having a DARPin backbone may be recombinantly linked to a subunit of a self-assembling protein cage (“protein cage”) to result in a REMIS subunit. Subunits of protein cages include those described in US8969521, US9066870, US9630994, US10248758, US10501733, US20200397886, US20210163540, and WO2020/220044 and the following: Example, Subunit B: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKR (SEQ ID NO: 18) Cage Protein T33-51, Subunit A: MRITTKVGDKGSTRLFGGEEVWKDDPIIEANGTLDELTSFIGEAKHYVDEEMKGILEEIQNDIYK IMGEIGSKGKIEGISEERIKWLAGLIERYSEMVNKLSFVLPGGTLESAKLDVCRTIARRAERKVA TVLREFGIGTLAAIYLALLSRLLFLLARVIEIEKNKLKEVRS (SEQ ID NO: 19) Cage Protein T33-51, Subunit B: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRLNIPEKIWSIHRVSLE (SEQ ID NO: 20) Cage Protein T33-31, Subunit A: MVRGIRGAITVEEDTPAAILAATIELLLKMLEANGIQSYEELAAVIFTVTEDLTSAFPAEAARLI GMHRVPLLSAREVPVPGSLPRVIRVLALWNTDTPQDRVRHVYLNEAVRLRPDLESAQLE (SEQ ID NO: 21) Cage Protein T33-31, Subunit B: MEEVVLITVPSALVAVKIAHALVEERLAACVNIVPGLTSIYREEGSVVSDHELLLLVKTTTDAFP KLKERVKELHPYEVPEIVALPIAEGNREYLDWLRENTGLE (SEQ ID NO: 22) Cage Protein T33-21, Subunit A: MRITTKVGDKGSTRLFGGEEVWKDSPIIEANGTLDELTSFIGEAKHYVDEEMKGILEEIQNDIYK IMGEIGSKGKIEGISEERIAWLLKLILRYMEMVNLKSFVLPGGTLESAKLDVCRTIARRALRKVL TVTREFGIGAEAAAYLLALSDLLFLLARVIEIEKNKLKEVRS (SEQ ID NO: 23) Cage Protein T33-21, Subunit B: MPHLVIEATANLRLETSPGELLEQANKALFASGQFGEADIKSRFVTLEAYRQGTAAVERAYLHAC LSILDGRDIATRTLLGASLCAVLAEAVAGGGEEGVQVSVEVREMERLSYAKRVVARQRLE (SEQ ID NO: 24) Cage Protein T33-28, Subunit A: MESVNTSFLSPSLVTIRDFDNGQFAVLRIGRTGFPADKGDIDLCLDKMIGVRAAQIFLGDDTEDG FKGPHIRIRCVDIDDKHTYNAMVYVDLIVGTGASEVERETAEEEAKLALRVALQVDIADEHSCVT QFEMKLREELLSSDSFHPDKDEYYKDFL (SEQ ID NO: 25) Cage Protein T33-28, Subunit B: MPVIQTFVSTPLDHHKRLLLAIIYRIVTRVVLGKPEDLVMMTFHDSTPMHFFGSTDPVACVRVEA LGGYGPSEPEKVTSIVTAAITAVCGIVADRIFVLYFSPLHCGWNGTNFLE (SEQ ID NO: 26) Cage Protein I53-50, Subunit A: MKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAII GAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKL FPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKA FVEKIRGCTE (SEQ ID NO: 27) Cage Protein I53-50, Subunit B: MNQHSHKDYETVRIAVVRARWHAEIVDACVSAFEAAMADIGGDRFAVDVFDVPGAYEIPLHARTL AETGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLSTGVPVLSAVLTPHRYRDSDAHTLLF LALFAVKGMEAARACVEILAAREKIAAGSLE (SEQ ID NO: 28) Cage Protein I53-47, Subunit A: MPIFTLNTNIKATDVPSDFLSLTSRLVGLILSKPGSYVAVHINTDQQLSFGGSTNPAAFGTLMSI GGIEPSKNRDHSAVLFDHLNAMLGIPKNRMYIHFVNLNGDDVGWNGTTF (SEQ ID NO: 29) Cage Protein I53-47, Subunit B: MNQHSHKDYETVRIAVVRARWHADIVDACVEAFEIAMAAIGGDRFAVDVFDVPGAYEIPLHARTL AETGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLSTGVPVLSAVLTPHRYRDSAEHHRFF AAHFAVKGVEAARACIEILAAREKIAAGSLE (SEQ ID NO: 30) Cage Protein I53-40, Subunit A: MTKKVGIVDTTFARVDMASAAILTLKMESPNIKIIRKTVPGIKDLPVACKKLLEEEGCDIVMALG MPGKAEKDKVCAHEASLGLMLAQLMTNKHIIEVFVHEDEAKDDAELKILAARRAIEHALNVYYLL FKPEYLTRMAGKGLRQGFEDAGPARE (SEQ ID NO: 31) Cage Protein I53-40, Subunit B: MSTINNQLKALKVIPVIAIDNAEDIIPLGKVLAENGLPAAEITFRSSAAVKAIMLLRSAQPEMLI GAGTILNGVQALAAKEAGATFVVSPGFNPNTVRACQIIGIDIVPGVNNPSTVEAALEMGLTTLKF FPAEASGGISMVKSLVGPYGDIRLMPTGGITPSNIDNYLAIPQVLACGGTWMVDKKLVTNGEWDE IARLTREIVEQVNPGSLE (SEQ ID NO: 32) Cage Protein I52-32, Subunit A: MKYDGSKLRIGILHARWNLEIIAALVAGAIKRLQEFGVKAENIIIETVPGSFELPYGSKLFVEKQ KRLGKPLDAIIPIGVLIKGSTMHFEYICDSTTHQLMKLNFELGIPVIFGVLTCLTDEQAEARAGL IEGKMHNHGEDWGAAAVEMATKFNLE (SEQ ID NO: 33) Cage Protein I52-32, Subunit B: MGMKEKFVLIITHGDFGKGLLSGAEVIIGKQENVHTVGLNLGDNIEKVAKEVMRIIIAKLAEDKE IIIVVDLFGGSPFNIALEMMKTFDVKVITGINMPMLVELLTSINVYDTTELLENISKIGKDGIKV IEKSSLKM (SEQ ID NO: 34) Cage Protein I32-28, Subunit A: MGDDARIAAIGDVDELNSQIGVLLAEPLPDDVRAALSAIQHDLFDLGGELCIPGHAAITEDHLLR LALWLVHYNGQLPPLEEFILPGGARGAALAHVCRTVCRRAERSIKALGASEPLNIAPAAYVNLLS DLLFVLARVLNRAAGGADVLWDRTRAH (SEQ ID NO: 35) Cage Protein I32-28, Subunit B: MILSAEQSFTLRHPHGQAAALAFVREPAAALAGVQRLRGLDSDGEQVWGELLVRVPLLGEVDLPF RSEIVRTPQGAELRPLTLTGERAWVAVSGQATAAEGGEMAFAFQFQAHLATPEAEGEGGAAFEVM VQAAAGVTLLLVAMALPQGLAAGLPPALE (SEQ ID NO: 36) [0042] DARPin backbones as described above may be recombinantly linked to a subunit of a protein cage directly or indirectly via a peptide linker. The peptide linker will typically be a sequence that is predicted to favor an alpha-helical conformation in accordance with rules and patterns in the art. See, e.g., Padilla et al. (2001). [0043] In some embodiments, the peptide linker is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 and not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long. In some embodiments, the peptide linker comprises an amino acid sequence or amino acid motif that forms an alpha-helical structure. In some embodiments, the amino acid sequence of the peptide linker comprises KEELD (SEQ ID NO: 37), PAPAP (SEQ ID NO: 38), KEELD (SEQ ID NO: 39), KDELD (SEQ ID NO: 40), RDERN (SEQ ID NO: 41), (E(A)₃K)n motif, (E4(R/K)4)n motif, or (AP)n motif. In some embodiments, the amino acid sequence of the subunit has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 18 to SEQ ID NO: 36. [0044] In some embodiments, REMIS subunits comprise (A) a protein having a DARPin backbone that (1) comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, or (2) comprises or consists of SEQ ID NO: 4, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, and each X is independently any amino acid; (B) a peptide linker that is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 and not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long, said peptide linker preferably forms an alpha-helical structure and/or has an amino acid sequence comprising KEELD (SEQ ID NO: 37), PAPAP (SEQ ID NO: 38), KEELD (SEQ ID NO: 39), KDELD (SEQ ID NO: 40), RDERN (SEQ ID NO: 41), (E(A)₃K)n motif, (E₄(R/K)₄)n motif, or (AP)n motif; and (C) a subunit having an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 18 to SEQ ID NO: 36. In some embodiments, the peptide linker joins the N- terminus of the DARPin backbone to the C-terminus of the subunit. In some embodiments, the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17. [0045] In some embodiments, REMIS subunits comprise (A) a protein having a DARPin backbone that (1) comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, or (2) comprises or consists of SEQ ID NO: 4, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, and each X is independently any amino acid; (B) a peptide linker that comprises or consists of KEELD (SEQ ID NO: 32), and (C) a subunit having an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, wherein the peptide linker joins the N-terminus of the DARPin backbone to the C- terminus of the subunit. In some embodiments, the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17. [0046] In some embodiments, REMIS subunits comprise (A) a protein having a DARPin backbone that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; (B) a peptide linker that comprises or consists of KEELD (SEQ ID NO: 32), and (C) a subunit having an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, wherein the peptide linker joins the N-terminus of the DARPin backbone to the C- terminus of the subunit. In some embodiments, the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17. [0047] In some embodiments, the REMIS subunit comprises or consists of one of the following: RCG-10, Subunit B, which binds GFP: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRKEELDKKLLEAARAGYDDQVAALLAKGADVNA ADDVGVTPLHLAAQRGHLEIVEVLLKRGADINAADLWGQTPLHLAATAGHLEIVELLLRWGADVN ARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 42) RCG-5, Subunit B, which binds GFP: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRKEELDKKLLEAARAGDKYAVDALLAKGADVNA ADDVGVTPLHLAAQRGHLEIVEVLLKRGWDINAADLWGQTPLHLAATAGHLEIVELLLWYGADVN ARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 43) RCG-8, Subunit B, which binds GFP: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRKEELDKKLLEAARAGYDDKVAWLLALGADVNA ADDVGVTPLHLAAQRGHLEIVEVLLKRGADINAADLWGQTPLHLAATAGHLEIVEKLLRCGADVN ARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 44) RCG-13, Subunit B, which binds GFP: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRKDELDKKLLEAARAGIDDAVAALLAKGADVNA ADDVGVTPLHLAAQRGHLEIVKVLLLRGADINAADLWGQTPLHLAATAGHLEIVELLLRCGADVN ARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 45) RCG-14, Subunit B, which binds GFP: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRRDERNKKLLEAARAGIDDAVDWLLALGADVNA ADDVGVTPLHLAAQRGHLEIVKVLLSRGADINAADLWGQTPLHLAATAGHLEIVELLLRCGADVN ARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 46) RCG-34, Subunit B, which binds MBP: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRKEELDKKLLEAARAGQDDEVAALLAKGADVNA ADNTGTTPLHLAAYSGHLEIVEVLLKRGADVDASDVFGYTPLHLAAYWGHLEIVELLLRWGADVN AMDSDGMTPLHLAAKWGYLEIVEVLLKHGADVNAQDKFGKTAFDISIDNGNEDLAEILQKLN (SEQ ID NO: 47) RCG-35, Subunit B, which binds MBP: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRKEELDKKLLEAARAGQDDEVAALLAKGADVNA ADNTGTTPLHLAAYSGHLEIVEVLLKRGADIDASDVFGYTPLHLAAYWGHLEIVELLLRWGADVN AMDSDGMTPLHLAAKWGYLEIVEVLLKHGADVNAQDKFGKTAFDISIDNGNEDLAEILQKLN (SEQ ID NO: 48) RCG-32, Subunit B, which binds KRas: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRKEELDKKLLEAARAGQDDEVAALLAKGADVNA NDSAGHTPLHLAAKRGHLEIVEVLLKRGADINAMDNTGFTPLHLAALRGHLEIVELLLRWGADVN AQDRTGRTPLHLAAKLGHLEIVEVLLKNGADVNAQDKFGKTAFDISIDNGNEDLAEILQKL (SEQ ID NO: 49) RCG-33, Subunit B, which binds KRas: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRKEELDKKLLEAARAGQDDEVAALLAKGADVNA HDTFGFTPLHLAALYGHLEIVEVLLKRGADINADDSYGRTPLHLAAMRGHLEIVELLLRWGADVN AADEEGRTPLHLAAKRGHLEIVEVLLKNGADVNAQDKFGKTAFDISIDNGNEDLAEILQKL (SEQ ID NO: 50) [0048] REMIS CAGES [0049] REMIS cages comprise multiple copies of a REMIS subunit as described herein. In some embodiments, REMIS cages also comprise multiple copies of an additional subunit, which additional subunit does not contain a protein having a DARPin backbone linked thereto. In some embodiments, the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 18 to SEQ ID NO: 36. In some embodiments, the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17. [0050] In some embodiments, the REMIS cages comprise or consist of (I) a REMIS subunit that comprises or consists of (A) a protein having a DARPin backbone that (1) comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, or (2) comprises or consists of SEQ ID NO: 4, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, and each X is independently any amino acid; (B) a peptide linker that is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 and not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long, said peptide linker preferably forms an alpha-helical structure and/or has an amino acid sequence comprising KEELD (SEQ ID NO: 37), PAPAP (SEQ ID NO: 38), KEELD (SEQ ID NO: 39), KDELD (SEQ ID NO: 40), RDERN (SEQ ID NO: 41), (E(A)3K)n motif, (E4(R/K)4)n motif, or (AP)n motif; and (C) a subunit having an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 18 to SEQ ID NO: 36, wherein the peptide linker joins the N-terminus of the DARPin backbone to the C-terminus of the subunit; and; and (II) an additional subunit that comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 18 to SEQ ID NO: 36. In some embodiments, the sequence of the additional subunit is different from that of the subunit of the REMIS subunit. In some embodiments, the subunit of the REMIS subunit and the additional subunit are counterparts, i.e., subunit A and subunit B, of a known self-assembling protein cage. In some embodiments, the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17. [0051] In some embodiments, the REMIS cages comprise or consist of (I) a REMIS subunit that comprises or consists of (A) a protein having a DARPin backbone that (1) comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, or (2) comprises or consists of SEQ ID NO: 4, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, and each X is independently any amino acid; (B) a peptide linker that comprises or consists of KEELD (SEQ ID NO: 32), and (C) a subunit having an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, wherein the peptide linker joins the N-terminus of the DARPin backbone to the C- terminus of the subunit; and (II) an additional subunit that comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 18 to SEQ ID NO: 36. In some embodiments, the sequence of the additional subunit is different from that of the subunit of the REMIS subunit. In some embodiments, the subunit of the REMIS subunit and the additional subunit are counterparts, i.e., subunit A and subunit B, of a known self-assembling protein cage. In some embodiments, the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17. [0052] In some embodiments, the REMIS cages comprise or consist of (I) a REMIS subunit that comprises or consists of (A) a protein having a DARPin backbone that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; (B) a peptide linker that comprises or consists of KEELD (SEQ ID NO: 32), and (C) a subunit having an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, wherein the peptide linker joins the N-terminus of the DARPin backbone to the C-terminus of the subunit; and (II) an additional subunit that comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 18 to SEQ ID NO: 36. In some embodiments, the sequence of the additional subunit is different from that of the subunit of the REMIS subunit. In some embodiments, the subunit of the REMIS subunit and the additional subunit are counterparts, i.e., subunit A and subunit B, of a known self- assembling protein cage. In some embodiments, the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17. [0053] The subunits (the REMIS subunit and, if present, the additional subunit) are typically present in 12, 24, or 60 copies in the assembled REMIS cage, according to the symmetries of Platonic solids. In its assembled form, a REMIS cage would typically be in the range of about 400 kDa up to about 3 MDa. [0054] As provided herein, the DARPin backbones of REMIS subunits protrude from REMIS cages like “arms”. The protruding arms form a multimer, thereby resulting in a more rigid framework compared to prior cage structures lacking a DARPin backbone. A REMIS cage is schematically shown in Figure 1 as solid shapes and ribbon diagrams. Figure 2 schematically shows the “prior art”, i.e., protein cages having DARpins that lack a DARPin backbone as described herein. As shown in Figure 2, the DARpins do not bind each other. [0055] DARPin backbones, REMIS subunits, and REMIS cages described herein may be made using methods known in the art including chemical synthesis, biosynthesis or in vitro synthesis using recombinant DNA methods, and solid phase synthesis. See, e.g., Kelly & Winkler (1990) Genetic Engineering Principles and Methods, vol. 12, J․K․ Setlow ed., Plenum Press, NY, pp 1-19; Merrifield (1964) J Amer Chem Soc 85:2149; Houghten (1985) PNAS USA 82:5131-5135; and Stewart & Young (1984) Solid Phase Peptide Synthesis, 2ed. Pierce, Rockford, IL, which are herein incorporated by reference. DARPin backbones, REMIS subunits, and REMIS cages may be purified using protein purification techniques known in the art such as reverse phase high- performance liquid chromatography (HPLC), ion-exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See, e.g., Olsnes and Pihl (1973) Biochem 12(16):3121-3126; and Scopes (1982) Protein Purification, Springer-Verlag, NY, which are herein incorporated by reference. Polynucleotides that encode the DARPin backbones, REMIS subunits, and REMIS cages described herein are also contemplated herein. In some embodiments, the DARPin backbones, REMIS subunits, and REMIS cages are isolated. [0056] In some embodiments, the DARPin backbones and REMIS subunits are substantially purified. As used herein, a “substantially purified” compound refers to a compound that is removed from its natural environment and/or is at least about 60% free, preferably about 75% free, and more preferably about 90% free, and most preferably about 95-100% free from other macromolecular components or compounds with which the compound is associated with in nature or from its synthesis. [0057] In some embodiments, a protein comprising a DARPin backbone specifically binds the given target it was designed to bind. As used herein, a protein comprising a DARPin backbone “specifically binds” its given target if it reacts or associates more frequently, more rapidly, with greater duration, and/or with greater binding affinity with the given target than it does with a given alternative, and/or indiscriminate binding that gives rise to non-specific binding and/or background binding. As used herein, “non- specific binding” and “background binding” refer to an interaction that is not dependent on the presence of a specific structure (e.g., a given epitope). [0058] The following examples are intended to illustrate but not to limit the invention. [0059] EXAMPLES [0060] Cloning and Protein Expression [0061] The C-termini of subunit B from the T33-51 cage (SEQ ID NO: 18) was genetically fused to the N-termini of the DARPin sequence DARP14-3G124Mut5 (SEQ ID NO: 40). Nucleic acid molecules comprising amino acid substitutions at desired locations in the backbone of the DARPin sequence were cloned into pET-22b and pSAM vectors. The superfolder GFP V206A (sfGFP V206A) vector was obtained from (Liu et al., 2019). DNA manipulations were carried in Escherichia coli XL2 cells (Agilent). The proteins were expressed in E. coli BL21(DE3) cells (New England Biolabs) in Terrific Broth at 18 °C overnight upon 1 mM IPTG induction at OD 1.0. [0062] Upon collection of the cells, pellets were resuspended in resuspension buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0) supplemented with benzonase nuclease, 1 mM PMSF, protease inhibitor cocktail EDTA-free (Thermo Scientific) and 0.1% LDAO and lysed using an EmulsiFlex C3 homogenizer (Avestin). The cell lysate was cleared by centrifugation at 20,000 xg for 20 minutes at 4 °C, the resulting supernatant was recovered and centrifuged at 10,000 xg for 10 minutes at 4 °C and then loaded onto a HisTrap column (GE Healthcare) pre-equilibrated with the same resuspension buffer. The imaging scaffold was eluted with a linear gradient to 300 mM imidazole. Upon elution, 5 mM EDTA and 5 mM BME were added immediately. The eluted protein was concentrated using Amicon Ultra-15100 kDa MWCO for the imaging scaffold and 3 kDa MWCO for the GFP. The concentrated protein was further purified by size exclusion chromatography using a Superose 6 Increase column, eluted with 20 mM Tris pH 8.0, 100 mM NaCl, 5 mM BME, 5 mM EDTA. Fractions were analyzed by SDS-PAGE and negative stain EM for the presence of the imaging scaffold. [0063] KRas G12V and KRas G13C proteins were prepared using methods in the art. [0064] Negative Stain EM [0065] The concentration of a 3.5 μl sample of fresh Supersoe 6 Increase eluent was adjusted to about 100 μg/ml, applied to glow-discharged for one minute and blotted away. After three washes with filtered MiliQ water, the grid was stained with 2% uranyl acetate for 30 seconds. Images were taken on a Tecnai T12, a T20 or a TF20. [0066] Cryo-EM Data Collection [0067] Concentrated imaging scaffolds (1-10 mg/ml) were mixed with the GFP cargo to a molar ratio of 1:2 and diluted to a final concentration of 0.5-0.7 mg/ml. The final buffer composition was 20 mM Tris pH 8.0, 100 mM NaCl. Quantifoil 300 mesh R2/2 copper grids were glow discharged for 30 seconds at 15 mA using a PELCO easiGLow. 3.5 μl of the sample was applied to the grid and then blotted and plunge-frozen into liquid nitrogen-cooled liquid ethane using a Vitrobot Mark IV (FEI). Cryo-EM data were collected on a Gatan K3 Summit direct electron detector on a Titan Krios (FEI). Images were recorded with Leginon (add ref) and SerialEM (add ref) with a pixel size of 1.1 Å for designs 5, 8, 10, 13, 14, 33 (G13C), 34 datasets and 0.856 Å for design 33 (G12V) dataset, over a defocus range of -2.5 to -0.4 µm. [0068] Cryo-EM Data Processing and Model Building [0069] Motion correction, CTF correction, particle picking and 2D classification were performed in cryoSPARC v.3.2 using methods in the art. [0070] An initial set of particles was automatically picked using a blob-picker. The extracted particles were 2D classified and then an ab initio reconstruction was generated. The model was used to obtain a 3D reconstruction enforcing T symmetry. The 3D structure was used to generate 2D projections of the particles and then used to repick the particles from the images using a template picker. The picked particles were extracted from the micrographs and then 3D reconstructed enforcing T symmetry. These were further refined using focused 3D classification and local refinements. [0071] The models were subjected to multiple rounds of refinement with Coot (Emsley et al., 2010) and PHENIX (Afonine et al., 2018) [0072] CYRO-EM IMAGING Five different REMIS cages were made based on the original T33-51 protein cage. The original T33-51 protein cage comprises subunit A (SEQ ID NO: 19) and subunit B (SEQ ID NO: 20) in a stoichiometry of A₁₂B₁₂. The REMIS cages contained a REMIS subunit comprising SEQ ID NO: 18 (instead of SEQ ID NO: 20) and a DARPin backbone (i.e., SEQ ID NO: 4) linked thereto via a peptide linker (i.e., SEQ ID NO: 37) in the same stoichiometry. Specifically, the REMIS cages were RCG-10, RCG-5, RCG- 8, RCG-13, and RCG-14, and their REMIS subunits were SEQ ID NO: 42 to SEQ ID NO: 46, respectively. Each REMIS cage assembled according to their elution volumes in size exclusion chromatography and bound their intended target (“target cargo”). [0073] To analyze the structures of the REMIS cages, a cryo-EM dataset was collected for each. Each REMIS cage was loaded with its target cargo, then subjected to identical specimen preparation by vitrification and data collection on a Titan Krios. For each design, particles were selected from the collected images and subjected to 3D image reconstruction, initially enforcing T symmetry. Analysis of the 3D reconstructions after truncation of each dataset to the same number of particles revealed different degrees of variability in the protruding DARpins. [0074] Following initial processing in symmetry T, to obtain the highest resolution images of the cargo, the particle symmetry was relaxed and a series of focused 3D classifications and image optimizations were performed using methods in the art. RCG- 10 was used as the reference scaffold upon which DARPin sequence changes could be made to present other proteins of interest. [0075] To test REMIS cages comprising DARPin backbones, which bind other target cargo, the oncogenic protein KRas (19.4 kDa) and MBP were selected as the target cargo and the loop regions of SEQ ID NO: 4 were substituted accordingly. As before, target cargo was added to REMIS cages and then purified by SEC prior to cryo-EM analysis. Three KRas mutants were studied: G12V, G12C, and G13C. The KRas mutants contained GDP as a bound ligand and were bound by RCG-33 (SEQ ID NO: 50). The DARPin represented by SEQ ID NO: 49 binds KRas including the KRas mutants. Following similar data processing as before, an overall resolution of about 2.4 Å for the entire particle, i.e., REMIS cage plus KRas cargo, was obtained and the structural differences between the mutants were observable. See Figure 3. Thus, cyro-EM imaging using the REMIS cages can be used to characterize the structures of proteins as small as about 19.4 kDa and also identify and/or distinguish proteins having single amino acid differences from one another. [0076] In one experiment on KRas mutant G12C, the drug molecule AMG510 (also known as sotorasib) was bound to KRas, and cryo-EM imaging revealed the conformation of the drug molecule while bound to KRas. See Figure 4. The resolution of REMIS cage plus MBP cargo was similar. See Figure 5. Therefore, cryo-EM imaging using the REMIS cages can be used to elucidate the conformation of protein- ligand interactions, i.e., the structure of the target protein when bound to a given ligand of interest. Because structural differences between proteins having single amino acid differences and protein-ligand interactions are observable via cryo-EM imaging using the REMIS cages, protein-ligand (e.g., drug) interactions between target protein and a given ligand of interest can be assayed and/or characterized by comparing the cryo-EM image of the complex comprising (i) the given ligand bound to the target protein and (ii) the REMIS cage bound to the target protein and/or the given ligand. In some embodiments, the reference cryo-electron micrograph is of (a) a complex comprising the fusion protein bound to the target protein, said complex excludes the presence of the given ligand or another ligand bound thereto; (b) a complex comprising (i) the given ligand bound to a mutant of the target protein and (ii) the fusion protein bound to the given ligand and/or the mutant, said mutant having at least one amino acid substitution, deletion, or addition as compared to the target protein; or (c) a complex comprising (i) a second ligand bound to the target protein and (ii) the fusion protein bound to the given ligand and/or the target protein, wherein said second ligand is different the given ligand. Differences between the cryo-EM image and one or more reference cryo-electron micrographs can then be used to characterize the protein-ligand interaction between a target protein and given ligand, identify the target protein or a ligand that binds the target protein, distinguish the target protein from other proteins, or distinguish a ligand bound to the target protein from other ligands that also bind the target protein. [0077] Structural analysis indicates that the key amino acid residues for multimer formation between DARPins comprising a DARPin backbone are at positions 16, 17, 19, 21, 54, 83, 86, and 87 of SEQ ID NO: 4 and amino acid residues a positions 11Y, 14Q, and 58I of SEQ ID NO: 4 improve the formation and/or structural integrity of multimers. [0078] REMIS cages having SEQ ID NO: 4, wherein X5 is A, X6 is A, X7 is K, X15 is L, X16 is R, and X17 is W were found to be more rigid than those where: ^ X5 is D, X6 is A, X7 is K, X15 is L, X16 is W, and X17 is Y; ^ X5 is A, X6 is W, X7 is L, X15 is K, X16 is R, and X17 is C; ^ X5 is A, X6 is A, X7 is K, X15 is L, X16 is R, and X17 is C; and ^ X5 is D, X6 is W, X7 is L, X15 is L, X16 is R, and X17 is C. [0079] Thus, in some embodiments, DARPin backbones, REMIS subunits, and REMIS cages comprise or consist of: SEQ ID NO: 51: KKLLEAXXXXX1X2X3X4VAALLAKGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX1 1DX12X13XXXXXXXXPLHLAXXXXHLEIX14ELLLRWGADVN; SEQ ID NO: 52: KKLLEAXXXXX1X2X3X4VDALLAKGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX1 1DX12X13XXXXXXXXPLHLAXXXXHLEIX14ELLLWYGADVN; SEQ ID NO: 53: KKLLEAXXXXX1X2X3X4VAWLLALGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX1 1DX12X13XXXXXXXXPLHLAXXXXHLEIX14EKLLRCGADVN; SEQ ID NO: 54: KKLLEAXXXXX1X2X3X4VAALLAKGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX1 1DX12X13XXXXXXXXPLHLAXXXXHLEIX14ELLLRCGADVN; or SEQ ID NO: 55: KKLLEAXXXXX1X2X3X4VDWLLALGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX1 1DX12X13XXXXXXXXPLHLAXXXXHLEIX14ELLLRCGADVN; wherein X1 is Y, Q, I or D, preferably Y, Q, or I, more preferably, Y or Q, most preferably Y X2 is D or K, preferably D X3 is D or Y, preferably D X4 is Q, E, or A, preferably Q or E, more preferably Q X8 is L or P, preferably L X9 is E or K, preferably E X10 is K, L, or S, preferably K X11 is A or W, preferably A X12 is I or V, preferably I X13 is N or D, preferably N X14 is V or A, preferably V, and each X is independently any amino acid. [0080] REMIS cages having SEQ ID NO: 4, wherein X1 is Y, X4 is Q, X5 is A, X6 is A, X7 is K, X12 is I, X15 is L, X16 is R, and X17 is W were found to be more rigid than those where: ^ X1 is D, X4 is A, X5 is D, X6 is A, X7 is K, X12 is I, X15 is L, X16 is W, and X17 is Y; ^ X1 is Y, X4 is K, X5 is A, X6 is W, X7 is L, X12 is I, X15 is K, X16 is R, and X17 is C; ^ X1 is I, X4 is A, X5 is A, X6 is A, X7 is K, X12 is I, X15 is L, X16 is R, and X17 is C; ^ X1 is I, X4 is A, X5 is D, X6 is W, X7 is L, X12 is I, X15 is L, X16 is R, and X17 is C; ^ X1 is Q, X4 is E, X5 is A, X6 is A, X7 is K, X12 is V, X15 is L, X16 is R, and X17 is W; and ^ X1 is Q, X4 is E, X5 is A, X6 is A, X7 is K, X12 is I, X15 is L, X16 is R, and X17 is W. [0081] Thus, in some embodiments, DARPin backbones, REMIS subunits, and REMIS cages comprise or consist of: SEQ ID NO: 56: KKLLEAXXXXYX2X3QVAALLAKGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX11D IX13XXXXXXXXPLHLAXXXXHLEIX14ELLLRWGADVN; SEQ ID NO: 57: KKLLEAXXXXDX2X3AVDALLAKGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX11D IX13XXXXXXXXPLHLAXXXXHLEIX14ELLLWYGADVN; SEQ ID NO: 58: KKLLEAXXXXYX2X3KVAWLLALGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX11D IX13XXXXXXXXPLHLAXXXXHLEIX14EKLLRCGADVN; SEQ ID NO: 59: KKLLEAXXXXIX2X3AVAALLAKGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX11D IX13XXXXXXXXPLHLAXXXXHLEIX14ELLLRCGADVN; SEQ ID NO: 60: KKLLEAXXXXIX2X3AVDWLLALGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX11D IX13XXXXXXXXPLHLAXXXXHLEIX14ELLLRCGADVN; SEQ ID NO: 61: KKLLEAXXXXQX2X3EVAALLAKGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX11D VX13XXXXXXXXPLHLAXXXXHLEIX14ELLLRWGADVN; or SEQ ID NO: 62: KKLLEAXXXXQX2X3EVAALLAKGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX11D IX13XXXXXXXXPLHLAXXXXHLEIX14ELLLRWGADVN; wherein X2 is D or K, preferably D X3 is D or Y, preferably D X8 is L or P, preferably L X9 is E or K, preferably E X10 is K, L, or S, preferably K X11 is A or W, preferably A X13 is N or D, preferably N X14 is V or A, preferably V, and each X is independently any amino acid. [0082] REMIS cages having SEQ ID NO: 4, wherein X1 is Y, X2 is D, X3 is D, X4 is Q, X5 is A, X6 is A, X7 is K, X8 is L, X9 is E, X10 is K, X11 is A, X12 is I, X13 is N, X14 is V, X15 is L, X16 is R, and X17 is W were found to be more rigid than those where: ^ X1 is D, X2 is K, X3 is Y, X4 is A, X5 is D, X6 is A, X7 is K, X8 is L, X9 is E, X10 is K, X11 is W, X12 is I, X13 is N, X14 is V, X15 is L, X16 is W, and X17 is Y; ^ X1 is Y, X2 is D, X3 is D, X4 is K, X5 is A, X6 is W, X7 is L, X8 is L, X9 is E, X10 is K, X11 is A, X12 is I, X13 is N, X14 is V, X15 is K, X16 is R, and X17 is C; ^ X1 is I, X2 is D, X3 is D, X4 is A, X5 is A, X6 is A, X7 is K, X8 is L, X9 is K, X10 is L, X11 is A, X12 is I, X13 is N, X14 is V, X15 is L, X16 is R, and X17 is C; ^ X1 is I, X2 is D, X3 is D, X4 is A, X5 is D, X6 is W, X7 is L, X8 is L, X9 is K, X10 is S, X11 is A, X12 is I, X13 is N, X14 is V, X15 is L, X16 is R, and X17 is C; ^ X1 is Q, X2 is D, X3 is D, X4 is E, X5 is A, X6 is A, X7 is K, X8 is L, X9 is E, X10 is K, X11 is A, X12 is V, X13 is D, X14 is V, X15 is L, X16 is R, and X17 is W; ^ X1 is Q, X2 is D, X3 is D, X4 is E, X5 is A, X6 is A, X7 is K, X8 is L, X9 is E, X10 is K, X11 is A, X12 is I, X13 is D, X14 is V, X15 is L, X16 is R, and X17 is W; ^ X1 is Q, X2 is D, X3 is D, X4 is E, X5 is A, X6 is A, X7 is K, X8 is L, X9 is E, X10 is K, X11 is A, X12 is I, X13 is N, X14 is V, X15 is L, X16 is R, and X17 is W; and ^ X1 is Q, X2 is D, X3 is D, X4 is E, X5 is A, X6 is A, X7 is K, X8 is L, X9 is E, X10 is K, X11 is A, X12 is I, X13 is N, X14 is V, X15 is L, X16 is R, and X17 is W. [0083] Thus, in some embodiments, DARPin backbones, REMIS subunits, and REMIS cages comprise or consist of: SEQ ID NO: 63: KKLLEAXXXXYDDQVAALLAKGADVNXXXXXXXXPLXLAXXXXHLEIVEVLLKRGADINXXXXXX XXPLHLAXXXXHLEIVELLLRWGADVN; SEQ ID NO: 64: KKLLEAXXXXDKYAVDALLAKGADVNXXXXXXXXPLXLAXXXXHLEIVEVLLKRGWDINXXXXXX XXPLHLAXXXXHLEIVELLLWYGADVN; SEQ ID NO: 65: KKLLEAXXXXYDDKVAWLLALGADVNXXXXXXXXPLXLAXXXXHLEIVEVLLKRGADINXXXXXX XXPLHLAXXXXHLEIVEKLLRCGADVN; SEQ ID NO: 66: KKLLEAXXXXIDDAVAALLAKGADVNXXXXXXXXPLXLAXXXXHLEIVKVLLLRGADINXXXXXX XXPLHLAXXXXHLEIVELLLRCGADVN; SEQ ID NO: 67: KKLLEAXXXXIDDAVDWLLALGADVNXXXXXXXXPLXLAXXXXHLEIVKVLLSRGADINXXXXXX XXPLHLAXXXXHLEIVELLLRCGADVN; SEQ ID NO: 68: KKLLEAXXXXQDDEVAALLAKGADVNXXXXXXXXPLXLAXXXXHLEIVEVLLKRGADVDXXXXXX XXPLHLAXXXXHLEIVELLLRWGADVN; SEQ ID NO: 69: KKLLEAXXXXQDDEVAALLAKGADVNXXXXXXXXPLXLAXXXXHLEIVEVLLKRGADIDXXXXXX XXPLHLAXXXXHLEIVELLLRWGADVN; SEQ ID NO: 70: KKLLEAXXXXQDDEVAALLAKGADVNXXXXXXXXPLXLAXXXXHLEIVEVLLKRGADINXXXXXX XXPLHLAXXXXHLEIVELLLRWGADVN; or SEQ ID NO: 71: KKLLEAXXXXQDDEVAALLAKGADVNXXXXXXXXPLXLAXXXXHLEIVEVLLKRGADINXXXXXX XXPLHLAXXXXHLEIVELLLRWGADVN; wherein each X is independently any amino acid. [0084] REMIS cages having SEQ ID NO: 63 were found to be the most rigid. Thus, in some embodiments, preferred DARPin backbones, REMIS subunits, and REMIS cages comprise or consist of SEQ ID NO: 63. [0085] REFERENCES [0086] The following references are herein incorporated by reference in their entirety with the exception that, should the scope and meaning of a term conflict with a definition explicitly set forth herein, the definition explicitly set forth herein controls: Afonine et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D Struct Biol.2018 Jun 1;74(Pt 6):531-544. doi: 10.1107/S2059798318006551. Epub 2018 May 30. Cannon et al. Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering. Protein Sci.2020 Apr;29(4):919-929. doi: 10.1002/pro.3802. Epub 2019 Dec 26. Coscia et al. Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein. Sci Rep.2016 Aug 3;6:30909. doi: 10.1038/srep30909. D'Imprima et al. Protein denaturation at the air-water interface and how to prevent it. Elife.2019 Apr 1;8:e42747. doi: 10.7554/eLife.42747. Emsley et al. Acta Crystallogr D Biol Crystallogr.2010 Apr;66(Pt 4):486-501. doi: 10.1107/S0907444910007493. Epub 2010 Mar 24. Henderson R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q Rev Biophys.1995 May;28(2):171-93. doi: 10.1017/s003358350000305x. Herzik et al. High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM. Nat Commun.2019 Mar 4;10(1):1032. doi: 10.1038/s41467-019- 08991-8. Kratz et al. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proc Natl Acad Sci U S A.1999 Mar 2;96(5):1915-20. doi: 10.1073/pnas.96.5.1915. Liu et al. Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system. Proc Natl Acad Sci U S A.2018 Mar 27;115(13):3362-3367. doi: 10.1073/pnas.1718825115. Epub 2018 Mar 5. Liu et al. A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold. Nat Commun.2019 Apr 23;10(1):1864. doi: 10.1038/s41467-019-09836-0. Noble et al. Routine single particle CryoEM sample and grid characterization by tomography. Elife.2018 May 29;7:e34257. doi: 10.7554/eLife.34257. Padilla J et al. Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc Natl Acad Sci U S A.2001 Feb 27;98(5):2217-21. doi: 10.1073/pnas.041614998. Epub 2001 Feb 20. Plückthun A. Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol.2015;55:489-511. doi: 10.1146/annurev-pharmtox-010611-134654. Punjani A et al. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods.2017 Mar;14(3):290-296. doi: 10.1038/nmeth.4169. Epub 2017 Feb 6. Yao et al. Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM. Structure.2019 Jul 2;27(7):1148-1155.e3. doi: 10.1016/j.str.2019.04.003. Epub 2019 May 9. PMID: 31080120; PMCID: PMC6610650. Yeates et al. Development of imaging scaffolds for cryo-electron microscopy. Curr Opin Struct Biol.2020 Feb;60:142-149. doi: 10.1016/j.sbi.2020.01.012. Epub 2020 Feb 14. [0087] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. [0088] The use of the singular can include the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” can include plural referents unless the context clearly dictates otherwise. [0089] As used herein, “and/or” means “and” or “or”. For example, “A and/or B” means “A, B, or both A and B” and “A, B, C, and/or D” means “A, B, C, D, or a combination thereof” and said “A, B, C, D, or a combination thereof” means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D). [0090] As used herein, the phrase “one or more of”, e.g., “one or more of A, B, and/or C” means “one or more of A”, “one or more of B”, “one or more of C”, “one or more of A and one or more of B”, “one or more of B and one or more of C”, “one or more of A and one or more of C” and “one or more of A, one or more of B, and one or more of C”. [0091] The phrase “comprises or consists of A” is used as a tool to avoid excess page and translation fees and means that in some embodiments the given thing at issue: comprises A or consists of A. For example, the sentence “In some embodiments, the composition comprises or consists of A” is to be interpreted as if written as the following two separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition consists of A.” [0092] Similarly, a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself. For example, the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C.” As another example, the sentence “In some embodiments, the composition comprises at least A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C.” [0093] As used herein, the term “sample” is used in its broadest sense and includes specimens and cultures obtained from any source, as well as biological samples and environmental samples. [0094] As used herein, an “isolated” compound refers to a compound that is isolated from its native environment. For example, an isolated polynucleotide is a one which does not have the bases normally flanking the 5’ end and/or the 3’ end of the polynucleotide as it is found in nature. As another example, an isolated polypeptide is a one which does not have its native amino acids, which correspond to the full-length polypeptide, flanking the N-terminus, C-terminus, or both. [0095] As used herein, the terms “protein”, “polypeptide” and “peptide” are used interchangeably to refer to two or more amino acids linked together. Groups or strings of amino acid abbreviations are used to represent peptides. Except when specifically indicated, peptides are indicated with the N-terminus on the left and the sequence is written from the N-terminus to the C-terminus. Except when specifically indicated, peptides are indicated with the N-terminus on the left and the sequences are written from the N-terminus to the C-terminus. Similarly, except when specifically indicated, nucleic acid sequences are indicated with the 5’ end on the left and the sequences are written from 5’ to 3’. [0096] As used herein, a given percentage of “sequence identity” refers to the percentage of nucleotides or amino acid residues that are the same between sequences, when compared and optimally aligned for maximum correspondence over a given comparison window, as measured by visual inspection or by a sequence comparison algorithm in the art, such as the BLAST algorithm, which is described in Altschul et al., (1990) J Mol Biol 215:403-410. Software for performing BLAST (e.g., BLASTP and BLASTN) analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The comparison window can exist over a given portion, e.g., a functional domain, or an arbitrarily selection a given number of contiguous nucleotides or amino acid residues of one or both sequences. Alternatively, the comparison window can exist over the full length of the sequences being compared. For purposes herein, where a given comparison window (e.g., over 80% of the given sequence) is not provided, the recited sequence identity is over 100% of the given sequence. Additionally, for the percentages of sequence identity of the proteins provided herein, the percentages are determined using BLASTP 2.8.0+, scoring matrix BLOSUM62, and the default parameters available at blast.ncbi.nlm.nih.gov/Blast.cgi. See also Altschul, et al., (1997) Nucleic Acids Res 25:3389-3402; and Altschul, et al., (2005) FEBS J 272:5101- 5109. [0097] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv Appl Math 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection. [0098] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated. [0099] Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

What is claimed is: 13. A fusion protein comprising a protein according to claim 2 fused to a subunit protein of a self-assembling protein cage, said subunit protein comprises or consists of a sequence having at least 95% sequence identity to SEQ ID NO: 18. 1. A protein comprising (1) X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N (SEQ ID NO: 1) wherein X1 is Y, Q, I or D, preferably Y, Q, or I, more preferably, Y or Q, most preferably Y; X2 is D or K, preferably D; X3 is D or Y, preferably D; X4 is Q, E, or A, preferably Q or E, more preferably Q; X5 is A or D, preferably A; X6 is A or W, preferably A; X7 is K or L, preferably K; and wherein when X5 is D then X6 is A, and when X6 is W then X7 is L; (2) H-X8-E-I-V-X9-V-L-L-X10-R-G-X11-D-X12-X13 (SEQ ID NO: 2) wherein X8 is L or P, preferably L; X9 is E or K, preferably E; X10 is K, L, or S, preferably K; X11 is A or W, preferably A; X12 is I or V, preferably I; and X13 is N or D, preferably N; and (3) H-L-E-I-X14-E-X15-L-L-X16-X17-G-A-D-V-N (SEQ ID NO: 3) wherein X14 is V or A, preferably V; X15 is L or K, preferably L; X16 is R or W, preferably R; X17 is W, C, or Y, preferably W or C, more preferably W; and wherein X16 is R or one of X16 and X17 is W, preferably when X16 is W then X17 is Y, preferably when X15 is K then X16 is R and X17 is C.

2. A protein comprising or consisting of K-K-L-L-E-A-X-X-X-X-X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N-X-X-X-X-X-X-X-X- P-L-X-L-A-X-X-X-X-H-X8-E-I-V-X9-V-L-L-X10-R-G-X11-D-X12-X13-X-X-X-X-X-X-X-X- P-L-H-L-A-X-X-X-X-H-L-E-I-X14-E-X15-L-L-X16-X17-G-A-D-V-N (SEQ ID NO: 4) wherein X1 is Y, Q, I or D, preferably Y, Q, or I, more preferably, Y or Q, most preferably Y; X2 is D or K, preferably D; X3 is D or Y, preferably D; X4 is Q, E, or A, preferably Q or E, more preferably Q; X5 is A or D, preferably A; X6 is A or W, preferably A; X7 is K or L, preferably K; X8 is L or P, preferably L; X9 is E or K, preferably E; X10 is K, L, or S, preferably K; X11 is A or W, preferably A; X12 is I or V, preferably I; X13 is N or D, preferably N; X14 is V or A, preferably V; X15 is L or K, preferably L; X16 is R or W, preferably R; X17 is W, C, or Y, preferably W or C, more preferably W; and each X is independently any amino acid residue; and wherein when X5 is D then X6 is A, when X6 is W then X7 is L, X16 is R or one of X16 and X17 is W, preferably when X16 is W then X17 is Y, preferably when X15 is K then X16 is R and X17 is C. 3. The protein according to claim 1 or claim 2, wherein the protein comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 51 to SEQ ID NO: 71.

4. A fusion protein comprising or consisting of the protein according to any one of claims 1 − 3 linked to a subunit protein of a self-assembling protein cage. 5. The fusion protein according to claim 4, wherein the protein is fused to the subunit protein via a peptide linker. 6. The fusion protein according to claim 5, wherein the peptide linker joins the N-terminus of the protein to the C-terminus of the subunit protein. 7. The fusion protein according to any one of claims 4 − 6, wherein the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a subunit sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 36. 8. The fusion protein according to any one of claims 4 − 7, wherein the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. 9. The fusion protein according to any one of claims 4 − 8, wherein the peptide linker is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid residues long. 10. The fusion protein according to any one of claims 4 − 9, wherein the peptide linker is not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long. 11. The fusion protein according to any one of claims 4 − 10, wherein the peptide linker forms an alpha-helical structure. 12. The fusion protein according to any one of claims 4 − 11, wherein the peptide linker has an amino acid sequence comprising KEELD (SEQ ID NO: 37), PAPAP (SEQ ID NO: 38), KEELD (SEQ ID NO: 39), KDELD (SEQ ID NO: 40), RDERN (SEQ ID NO: 41), an (E(A)3K)n motif, an (E4(R/K)4)n motif, or an (AP)n motif.

14. A protein particle comprising multiple copies of a fusion protein according to any one of claims 4 − 13. 15. The protein particle according to claim 14, and further comprising one or more additional subunit proteins of the self-assembling protein cage, wherein the one or more additional subunit proteins lack a DARPin backbone having SEQ ID NO: 4. 16. A method of assaying the structure of a target protein of interest, which comprises binding the target protein to the protein particle according to claim 14 or claim 15 to result in a complex, and obtaining an image of the complex. 17. The method according to 16, wherein the target protein is bound to a moiety such as a solid substrate (e.g., a bead), another protein, or a chemical (e.g., a drug). 18. The method according to claim 16 or claim 17, wherein the image is obtained by cryo- electron microscopy. 19. A method of assaying the structure of a target protein of interest when bound to a given ligand, which comprises forming a complex, which said complex comprises the given ligand bound to the target protein and the fusion protein according to any one of claims 4 − 12 bound to the given ligand and/or the target protein; and obtaining a cryo-electron image of the complex. 20. The method according to claim 19, which further comprises comparing the cryo-electron image to a reference cryo-electron micrograph, said reference cryo-electron micrograph is of (a) a complex comprising the fusion protein bound to the target protein, said complex excludes the presence of the given ligand or another ligand bound thereto; (b) a complex comprising (i) the given ligand bound to a mutant of the target protein and (ii) the fusion protein bound to the given ligand and/or the mutant, said mutant having at least one amino acid substitution, deletion, or addition as compared to the target protein; or (c) a complex comprising (i) a second ligand bound to the target protein and (ii) the fusion protein bound to the given ligand and/or the target protein, wherein said second ligand is different the given ligand.