Enhanced DNA dendrimers and methods for using the same

Abstract

The embodiments provide compositions comprising DNA dendrimers, targeted molecules, therapeutic agents, adapter molecules, supporting molecules, or combinations thereof. Pharmaceutical compositions, kits, and methods for using and producing them are also provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 518,457, filed on August 9, 2023, which is hereby incorporated by reference in its entirety.

[0002] Reference to Electronically Submitted Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML copy created on August 2, 2024, is named "COB - 015WO_SL" and has a size of 51,965 bytes.

[0003] This embodiment relates to DNA dendrimers and other molecules linked to or encoding a targeting molecule, a cargo molecule, an adapter molecule, a support molecule, or a combination thereof, as well as methods of using and generating them.

Background Art

[0004] In many research, diagnostic, and therapeutic applications, the targets of molecules such as reporter probes, small molecule drugs, peptides, or nucleic acids are present inside cells. For nucleic acids that require transcription and / or translation within the target cell, the nucleic acids must be safely transported into the cell nucleus. However, targeting molecules to selected cell types, achieving intracellular delivery into the cytosol, and subsequent trafficking to desired intracellular compartments such as the nucleus are challenging goals. Furthermore, therapeutic nucleic acid cargo must maintain a safe state protected from degradation and be expressed in the nucleus in a non - excessive or insufficient amount. Therefore, there is a need for compositions that can reliably deliver molecules such as therapeutic nucleic acids to the nucleus of target cells without degrading them and have the ability to optimize expression. This disclosure addresses such needs and others.

Summary of the Invention

[0005] In some embodiments, compositions are provided that comprise a DNA dendrimer, a targeting moiety, and an adapter molecule-cargo polynucleotide complex.

[0006] In some embodiments, the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding at least one molecule of interest. In some embodiments, the cargo polynucleotide is a nicked cyclic polynucleotide, a linear polynucleotide having closed 5′ and 3′ ends, a linear polynucleotide having open 5′ and 3′ ends, or a linear polynucleotide having one open end and one closed end, wherein the one open end and the one closed end may be located at either the 5′ or 3′ of the polynucleotide.

[0007] In some embodiments, the cargo polynucleotide further comprises at least one DNA targeting sequence (DTS).

[0008] In some embodiments, the cargo polynucleotide further includes at least one nuclear localization signal (NLS).

[0009] In some embodiments, the cargo polynucleotide comprises at least one DTS and at least one NLS.

[0010] In some embodiments, the adapter molecule includes a DNA dendrimer-binding sequence (DBS) and a cargo-binding region (CBR). In some embodiments, the DBS includes a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 4, 10, 11, 22, or 23.

[0011] In some embodiments, the CBR is linked or conjugated to the cargo polynucleotide by DNA ligation. In some embodiments, the CBR is linked or conjugated to the cargo polynucleotide by chemical coupling.

[0012] In some embodiments, the adapter molecule further comprises at least one NLS. In some embodiments, the at least one NLS comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 1, 2, 15, or 43.

[0013] In some embodiments, at least one NLS further includes at least one spacer located before or after the at least one NLS. In some embodiments, the at least one spacer includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to either SEQ ID NO: 5 or 29.

[0014] In some embodiments, the adapter molecule further comprises at least one DTS.

[0015] In some embodiments, the adapter molecule further comprises a cell-permeable peptide sequence (CPP). In some embodiments, the CPP comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 6.

[0016] In some embodiments, the adapter molecule further includes at least one cleavage site. In some embodiments, the cleavage site is a val-cit linker.

[0017] In some embodiments, the adapter molecule further comprises at least one flexible linker. In some embodiments, the at least one flexible linker is polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PGLA), (GG)n, (GGGGS)n, or (GGGGA)n, where each n is independently 1 to 5.

[0018] In some embodiments, the composition further comprises a support molecule. In some embodiments, the support molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the PEG molecule is PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

[0019] In some embodiments, the supporting molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 19. In some embodiments, the supporting molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the supporting molecule. In some embodiments, the PEG molecule is PEG2000.

[0020] In some embodiments, the supporting molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 19, and PEG2000 conjugated at the N-terminus or C-terminus of SEQ ID NO: 19. In some embodiments, PEG2000 is conjugated at the N-terminus of SEQ ID NO: 19. In some embodiments, PEG2000 is conjugated at the C-terminus of SEQ ID NO: 19.

[0021] In some embodiments, the supporting molecule includes the sequence of sequence number 30.

[0022] In some embodiments, the targeting moiety is an antibody, a naturally occurring ligand or functional derivative thereof for a receptor, a vitamin, a hormone, a small molecular mimetic of a naturally occurring ligand, a peptide, a polypeptide, a peptide mimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, any other molecule that specifically binds to a cell surface molecule and induces endocytosis of the binding moiety, or any combination thereof.

[0023] In some embodiments, a composition is provided which comprises a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety. In some embodiments, the support molecule is as shown herein. In some embodiments, the targeting moiety is as shown herein.

[0024] In some embodiments, the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding at least one molecule of interest. In some embodiments, the cargo polynucleotide optionally comprises a DBS. In some embodiments, the cargo polynucleotide is a complete circular polynucleotide (e.g., a plasmid), a nicked circular polynucleotide, a linear polynucleotide having closed 5′ and 3′ ends, a linear polynucleotide having open 5′ and 3′ ends, or a linear polynucleotide having one open end and one closed end, where the one open end and one closed end can be present at either the 5′ or 3′ of the cargo polynucleotide.

[0025] In some embodiments, the cargo polynucleotide further comprises at least one DTS.

[0026] In some embodiments, the cargo polynucleotide further comprises at least one NLS.

[0027] In some embodiments, the cargo polynucleotide comprises at least one DTS and at least one NLS.

[0028] In some embodiments, the cargo polynucleotide comprises a DBS, and the DBS comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NO: 3, 4, 10, 11, 22, or 23.

[0029] In some embodiments, the support molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the PEG molecule is PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

[0030] In some embodiments, the support molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 19. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the PEG molecule is PEG2000.

[0031] In some embodiments, the supporting molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 19, and PEG2000 conjugated at the N-terminus or C-terminus of SEQ ID NO: 19. In some embodiments, PEG2000 is conjugated at the N-terminus of SEQ ID NO: 19. In some embodiments, PEG2000 is conjugated at the C-terminus of SEQ ID NO: 19.

[0032] In some embodiments, the supporting molecule includes the sequence of sequence number 30.

[0033] In some embodiments, any of the compositions shown herein may contain a nitrogen / phosphate (N / P) ratio within a set threshold. In some embodiments, the N / P ratio is 0.5 to 10. In some embodiments, the N / P ratio is 2 to 5.

[0034] In some embodiments, pharmaceutical compositions are provided, comprising a composition as shown herein and a pharmaceutically acceptable carrier.

[0035] In some embodiments, a method is provided for delivering a molecule of interest to the nucleus of a target cell, the method comprising contacting the target cell with a composition or pharmaceutical composition as shown herein, wherein the targeting portion binds to the target cell.

[0036] In some embodiments, a method is provided for treating a disease, which involves administering a composition or pharmaceutical composition as shown herein to a target to treat the disease, wherein the targeting portion binds to target cells.

[0037] In some embodiments, a plasmid is provided which comprises at least two restriction enzyme recognition sites, at least one promoter, at least one coding sequence for at least one molecule of interest, and optionally, a plasmid backbone containing a DBS. In some embodiments, the plasmid can form a cargo polynucleotide having a variety of structures, depending on whether the plasmid has one or more 5' and 3' ends, or neither. In some embodiments, the plasmid is a complete circular polynucleotide, a nicked circular nucleotide, a linear nucleotide with closed 5' and 3' ends, a linear nucleotide with open 5' and 3' ends, or a linear nucleotide with one open end and one closed end.

[0038] In some embodiments, the plasmid further comprises at least one DTS.

[0039] In some embodiments, the plasmid further comprises at least one NLS.

[0040] In some embodiments, the plasmid comprises at least one DTS and at least one NLS.

[0041] In some embodiments, the DBS exists and includes nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to any one of sequence numbers 3, 4, 10, 11, 22, or 23.

[0042] In some embodiments, methods are provided for producing compositions such as those shown herein, which include contacting a plasmid as shown herein with one or more restriction enzymes that cleave at restriction enzyme recognition sites in order to form a nicked cyclic polynucleotide, a linear polynucleotide having closed 5' and 3' ends, a linear polynucleotide having open 5' and 3' ends, or a linear polynucleotide having one open end and one closed end; ligating a cargo polynucleotide to an adapter molecule to form an adapter molecule-cargo polynucleotide complex; and ligating the adapter molecule-cargo polynucleotide complex and a targeting moiety to a DNA dendrimer to form a composition.

[0043] In some embodiments, methods are provided for producing compositions such as those shown herein, which include contacting a plasmid as shown herein, comprising a DNA dendrimer-binding sequence (DBS), with one or more restriction enzymes that cleave at restriction enzyme recognition sites, in order to form a nicked cyclic polynucleotide, a linear polynucleotide having closed 5' and 3' ends, a linear polynucleotide having open 5' and 3' ends, or a linear polynucleotide having one open end and one closed end; and ligating the cargo polynucleotide and the targeting moiety to a DNA dendrimer to form a composition.

[0044] In some embodiments, a method is provided for producing compositions as shown herein, which includes contacting an uncut circular plasmid as shown herein, DNA dendrimers linked to a targeting region, and a support molecule that can condense the sizes of the cargo polynucleotides and DNA dendrimers, such that the cargo polynucleotides and DNA dendrimers associate with each other to form a composition.

[0045] In some embodiments, a kit is provided which comprises a composition as shown herein, a pharmaceutical composition as shown herein, a plasmid as shown herein, or any combination thereof. [Brief explanation of the drawing]

[0046] [Figure 1] The diagram shows a plasmid map and individual topologies that can be altered by contacting or not contacting a plasmid with a specific restriction enzyme. [Figure 2] This shows the GFP fluorescence percentage for various polynucleotide topologies. [Figure 3] The graph shows the number of GFP-positive cells per unit area for various polynucleotide topologies. [Figure 4] This shows the relative average fluorescence of polynucleotides containing DNA targeting sequences (DTS) compared to polynucleotides without DTS in CHO-K1 cells. [Figure 5] This shows the relative average fluorescence of polynucleotides containing DNA targeting sequences (DTS) compared to polynucleotides without DTS in A427 cells. [Figure 6] This shows the relative average fluorescence of polynucleotides containing DNA targeting sequences (DTS) compared to polynucleotides without DTS in C2C12 cells. [Figure 7] This shows the relative integral fluorescence of polynucleotides containing DNA targeting sequences (DTS) compared to polynucleotides without DTS in C2C12 cells. [Figure 8] The relative peak fluorescence of polynucleotides containing DNA targeting sequences (DTS) compared to polynucleotides without DTS in C2C12 cells is shown. [Figure 9]Bright-field and GFP imaging of HepG2 cells 48 hours after transfection using Gen1 A control plasmid (Figure 9A), Gen1 B control plasmid (Figure 9B), Gen3 a(-) plasmid (Figure 9C), and Gen3 c(-) plasmid (Figure 9D) are shown. [Figure 10] This shows the total fluorescence signal (cumulative density) of HepG2 cells transfected with various plasmids after 48 hours. [Figure 11] The results of a cell titer glo assay using 0.2 μg of plasmid per well are shown. [Figure 12] The results of a cell titer glo assay using 1 μg of plasmid per well are shown. [Figure 13] (a) shows the ability of the various support molecules provided herein to complex with nucleotide cargoes. (b) shows the ability of the various support molecules provided herein to protect nucleotide cargoes from the degradation of DNAse 1. [Figure 14] (a) shows the ability of the various support molecules provided herein to complex with nucleotide cargoes. (b) shows the ability of the various support molecules provided herein to protect nucleotide cargoes from the degradation of DNAse 1. [Figure 15] (a) shows the ability of the various support molecules provided herein to complex with nucleotide cargoes. (b) shows the ability of the various support molecules provided herein to protect nucleotide cargoes from the degradation of DNAse 1. [Figure 16] (a) shows the ability of various support molecule blends provided herein to complex with nucleotide cargoes. (b) shows the ability of various support molecule blends provided herein to protect nucleotide cargoes from DNAse 1 degradation. [Modes for carrying out the invention]

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Similar or equivalent methods and materials may be used in carrying out or testing, but preferred methods and materials are described herein. In case of any conflict, including definitions, the specification of the invention shall prevail. In addition, materials, methods, and examples are merely illustrative and not intended to limit the invention. Other features and effects of the embodiments described herein will become apparent from the modes for carrying out the invention herein and the claims.

[0048] Techniques and procedures for recombinant operations, including nucleic acid and peptide synthesis, are typically carried out in accordance with conventional methods in the art and various general references provided throughout this specification (e.g., Sambrook et al, 2001, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., eds., 2005, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY; and Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC).

[0049] The term "approximately" or "about" means a value within ±10% of the number it is used with. Therefore, approximately 50% means a range of 45% to 55%.

[0050] Unless otherwise specified in the context, the singular forms "a," "an," and "the" include plural references.

[0051] As used herein, the terms “comprise,” “have,” “include,” and their cognates mean “including but not limited to.” Various compositions, methods, and devices are described using the word “comprising” (which is to be interpreted as “including but not limited to”) various components or steps, but such compositions, methods, and devices may either “consist essentially of” or “consist of” such components or steps.

[0052] As used herein, the term “antibody” refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. Antibodies may be intact immunoglobulins derived from natural or recombinant sources, or they may be the immunoreactive portion of intact immunoglobulins. Antibodies useful in the present invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, and F(ab)2, as well as single-chain antibodies (scFv), camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0053] As used herein, “complementary” refers to the broad concept of subunit sequence complementarity between two nucleic acids, for example, between two DNA molecules. Nucleic acids are considered complementary at these positions if the nucleotide positions in both molecules are occupied by nucleotides that can normally form base pairs with each other. Thus, two nucleic acids are substantially complementary if at least about 50%, preferably at least about 60%, and more preferably at least about 80% of the corresponding positions in each molecule are occupied by nucleotides that normally form base pairs with each other (e.g., A:T and G:C nucleotide pairs).

[0054] As used herein, “DNA-based carrier” refers to a delivery system containing a deoxyribonucleic acid molecule. A non-exclusive example of a DNA-based carrier is a DNA dendrimer. Other DNA-based carriers include double-stranded DNA, single-stranded DNA, and single-stranded hairpin DNA, or multimers thereof.

[0055] As used herein, “DNA dendrimer” or “dendrimer” refers to a matrix of polynucleotides exhibiting branching formed by the sequential or generational addition of branching layers to or from a core molecule such as an initiation monomer.

[0056] As used herein, “starting monomer” is a polynucleotide compound that helps generate the nucleus for dendrimer formation.

[0057] As used herein, “extended monomer” is a polynucleotide compound that can be bound to and / or to an initial monomer during the construction of a dendrimer. The extended monomers form layers of the dendrimer. The first layer of the dendrimer is the layer of extended monomers closest to the initial monomer. The outer layer is the layer furthest from the initial monomer and forms the surface of the dendrimer. Extended monomers are also referred to in the art as matrix monomers, matrix extended monomers, and matrix polynucleotide monomers.

[0058] As used herein, the terms “substitute,” “substituted,” “mutate,” or “mutated” refer to altering, deleting, or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to create a variant of that sequence.

[0059] The term "polynucleotide" or "nucleic acid molecule" refers to a molecule containing a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemical reactions. Double-stranded and single-stranded DNA and RNA are non-exclusive examples of polynucleotides.

[0060] The terms “polypeptide” or “protein” refer to a molecule comprising at least two amino acid residues linked by a peptide bond to form a polypeptide. In some embodiments, the term “peptide” may also be used.

[0061] As used herein, the term “variant” means a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, including substitution, insertion, or deletion.

[0062] Conventional notation for representing polynucleotide sequences is used herein. That is, the left end of a single-stranded polynucleotide sequence is called the 5' end, and the leftward direction of a double-stranded polynucleotide sequence is called the 5' direction.

[0063] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence that controls or promotes the expression of a gene product operably ligated to a promoter / regulatory sequence. In some examples, this sequence may be a core promoter sequence, and in other examples, this sequence may also include enhancer sequences and other regulatory elements that control or promote the expression of a gene product. A promoter / regulatory sequence may, for example, cause a gene product to be expressed in an inducible manner. An “inducible” promoter is a nucleotide sequence that, when operably ligated to a polynucleotide encoding or defining a gene product, produces a gene product only if a corresponding (e.g., binding) inducer is substantially present.

[0064] As used herein, “targeting moiety” refers to a molecule that binds to a molecule present on the cell surface of a target cell.

[0065] As used herein, “targeted DNA-based carrier” and “DNA-based carrier containing a targeted moiety” refer to a composition comprising a DNA-based carrier and a targeted moiety. The targeted moiety may be linked to the DNA-based carrier directly or via a linker. Alternatively, the targeted moiety may be linked to another molecule, such as a cargo molecule or a second non-nucleic acid carrier in the composition. The DNA-based carrier is targeted by being present in the same composition together with the targeted cargo or targeted secondary carrier.

[0066] As used herein, the term “vector” refers to a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. It will be understood that a vector may be a naked nucleic acid or a nucleic acid that has formed a complex with a protein or lipid. Examples of vectors include, but are not limited to, replicons (e.g., RNA replicons, bacteriophages) to which DNA fragments may be bound and replicated. Thus, examples of vectors include, but are not limited to, RNA, autonomously self-replicating circular or linear DNA or RNA (i.e., “plasmids”), encompassing both expression plasmids and non-expression plasmids. Where a recombinant microorganism or cell culture is described as harboring an “expression vector”, the expression vector includes both extrachromosomal circular and linear DNA and DNA incorporated into the host chromosome(s). Where a vector is held by a host cell, the vector may be stably replicated by the cell during mitosis as an autonomous structure, or it may be incorporated into the host genome.

[0067] As used herein, “promoter” means a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of nucleic acids in cells. A promoter may include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also include distal enhancer or repressor elements that may be located thousands of base pairs away from the transcription start site. Such promoter-enhancers may, for example, modify tissue specificity or transduction efficiency. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can regulate the expression of gene components constitutively, differentially to the cell, tissue, or organ in which expression occurs, differentially to the developmental stage in which expression occurs, or in response to external stimuli, such as physiological stress, pathogens, metal ions, or inducers.

[0068] As used herein, the term “topology” refers to various structural arrangements of any vector, plasmid, or polynucleotide disclosed herein. For example, a polynucleotide may have a cyclic topology, such as a plasmid, in which the polynucleotide does not have a 5' or 3' end. Polynucleotides disclosed herein may also have a linear topology, in which the polynucleotide has both a 5' and a 3' end. Polynucleotides having a linear topology may have one or both ends of the polynucleotide covalently closed. In some embodiments, certain restriction enzymes, such as TelN protelomerase, can covalently close the DNA ends.

[0069] composition A. DNA-based carriers In some embodiments, the compositions disclosed herein include DNA-based carriers. These DNA-based carriers include, but are not limited to, DNA dendrimers, double-stranded linear DNA, single-stranded linear DNA, and single-stranded hairpin DNA, which are formulated as monomolecular structures or as several units of conformations cross-linked with each other (e.g., multimers). In some embodiments, the compositions include DNA dendrimers. DNA dendrimers are flexible, branched, spherical particles formed from DNA monomers hybridized with each other (typically about 130–150 nm in diameter, but can be designed to other sizes). Each DNA monomer consists of two polynucleotide chains sharing a central region of complementary sequences that hybridize the two strands, forming a double-stranded region and leaving four single-stranded polynucleotide terminal portions. These terminal sequences are complementary to each other and can therefore hybridize (layer-wise) to the terminal sequences of other DNA monomers. Optionally, DNA dendrimers include DNA chains cross-linked by covalent bonds.

[0070] The double-stranded region formed by the complementary sequence region between the first oligonucleotide and the second oligonucleotide is approximately 4 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is approximately 5 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is approximately 6 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is approximately 7 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is approximately 8 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is approximately 9 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is approximately 10 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is approximately 15 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is approximately 20 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is approximately 25 to approximately 2000 base pairs long. In some embodiments, the double-stranded region is about 30 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 35 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 40 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 45 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 50 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 60 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 70 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 80 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 90 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 100 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 200 to about 2000 base pairs long. In some embodiments, the double-stranded region is about 300 to 2000 base pairs long. In some embodiments, the double-stranded region is about 400 to 2000 base pairs long. In some embodiments, the double-stranded region is about 500 to 2000 base pairs long. In some embodiments, the double-stranded region is about 1000 to 2000 base pairs long. In some embodiments, the double-stranded region is about 1500 to 2000 base pairs long.

[0071] In some embodiments, the double-stranded region is approximately 4 to 1500 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 1000 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 500 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 400 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 300 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 200 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 100 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 90 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 80 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 70 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 60 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 4 to 45 bases long. In some embodiments, the double-stranded region is approximately 4 to 40 bases long. In some embodiments, the double-stranded region is approximately 4 to 35 bases long. In some embodiments, the double-stranded region is approximately 4 to 30 bases long. In some embodiments, the double-stranded region is approximately 4 to 25 bases long. In some embodiments, the double-stranded region is approximately 4 to 20 bases long. In some embodiments, the double-stranded region is approximately 4 to 15 bases long. In some embodiments, the double-stranded region is approximately 4 to 10 bases long. In some embodiments, the double-stranded region is approximately 4 to 9 bases long. In some embodiments, the double-stranded region is approximately 4 to 8 bases long. In some embodiments, the double-stranded region is approximately 4 to 7 bases long. In some embodiments, the double-stranded region is approximately 4 to 6 bases long. In some embodiments, the double-stranded region is approximately 4 to 5 bases long.

[0072] In some embodiments, the double-stranded region is about 5 to about 200 base pairs long. In some embodiments, the double-stranded region is about 6 to about 200 base pairs long. In some embodiments, the double-stranded region is about 7 to about 200 base pairs long. In some embodiments, the double-stranded region is about 8 to about 200 base pairs long. In some embodiments, the double-stranded region is about 9 to about 200 base pairs long. In some embodiments, the double-stranded region is about 10 to about 200 base pairs long. In some embodiments, the double-stranded region is about 11 to about 200 base pairs long. In some embodiments, the double-stranded region is about 12 to about 200 base pairs long. In some embodiments, the double-stranded region is about 13 to about 200 base pairs long. In some embodiments, the double-stranded region is about 14 to about 200 base pairs long. In some embodiments, the double-stranded region is about 15 to about 200 base pairs long. In some embodiments, the double-stranded region is about 20 to 200 base pairs long. In some embodiments, the double-stranded region is about 25 to 200 base pairs long. In some embodiments, the double-stranded region is about 30 to 200 base pairs long. In some embodiments, the double-stranded region is about 35 to 200 base pairs long. In some embodiments, the double-stranded region is about 40 to 200 base pairs long. In some embodiments, the double-stranded region is about 45 to 200 base pairs long. In some embodiments, the double-stranded region is about 50 to 200 base pairs long. In some embodiments, the double-stranded region is about 60 to 200 base pairs long. In some embodiments, the double-stranded region is about 70 to 200 base pairs long. In some embodiments, the double-stranded region is about 80 to 200 base pairs long. In some embodiments, the double-stranded region is about 90 to 200 base pairs long. In some embodiments, the double-stranded region is about 100 to about 200 base pairs long. In some embodiments, the double-stranded region is about 125 to about 200 base pairs long. In some embodiments, the double-stranded region is about 150 to about 200 base pairs long. In some embodiments, the double-stranded region is about 175 to about 200 base pairs long.

[0073] In some embodiments, the double-stranded region is approximately 5 to 175 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 150 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 125 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 100 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 90 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 80 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 70 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 60 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 45 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 40 base pairs long. In some embodiments, the double-stranded region is approximately 5 to 35 base pairs long. In some embodiments, the double-stranded region is about 5 to 30 base pairs long. In some embodiments, the double-stranded region is about 5 to 25 base pairs long. In some embodiments, the double-stranded region is about 5 to 20 base pairs long. In some embodiments, the double-stranded region is about 5 to 15 base pairs long. In some embodiments, the double-stranded region is about 5 to 14 base pairs long. In some embodiments, the double-stranded region is about 5 to 13 base pairs long. In some embodiments, the double-stranded region is about 5 to 12 base pairs long. In some embodiments, the double-stranded region is about 5 to 11 base pairs long. In some embodiments, the double-stranded region is about 5 to 10 base pairs long. In some embodiments, the double-stranded region is about 5 to 9 base pairs long. In some embodiments, the double-stranded region is about 5 to 8 base pairs long. In some embodiments, the double-stranded region is about 5 to 7 base pairs long. In some embodiments, the double-stranded region is about 5 to 6 base pairs long.

[0074] In some embodiments, the double-stranded region is approximately 25 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 26 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 27 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 28 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 29 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 30 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 31 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 32 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 33 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 34 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 35 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 36 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 37 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 38 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 39 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 40 to 50 base pairs long. In some embodiments, the double-stranded region is approximately 45 to 50 base pairs long.

[0075] In some embodiments, the double-stranded region is approximately 25 to 45 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 40 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 39 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 38 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 37 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 36 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 34 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 33 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 32 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 31 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 30 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 29 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 28 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 27 base pairs long. In some embodiments, the double-stranded region is approximately 25 to 26 base pairs long.

[0076] In some embodiments, the double-stranded region is approximately 25 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 26 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 27 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 28 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 29 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 30 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 31 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 32 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 33 to 35 base pairs long. In some embodiments, the double-stranded region is approximately 34 to 35 base pairs long.

[0077] In some embodiments, the double-stranded region is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, or greater than 200 bases, or any length between any of the listed values. In some embodiments, the double-stranded region is 4 bases long. In some embodiments, the double-stranded region is 5 bases long. In some embodiments, the double-stranded region is 6 bases long. In some embodiments, the double-stranded region is 7 nucleotides long. In some embodiments, the double-stranded region is 8 nucleotides long. In some embodiments, the double-stranded region is 9 nucleotides long. In some embodiments, the double-stranded region is 10 nucleotides long. In some embodiments, the double-stranded region is 11 nucleotides long. In some embodiments, the double-stranded region is 12 nucleotides long. In some embodiments, the double-stranded region is 13 nucleotides long. In some embodiments, the double-stranded region is 14 nucleotides long. In some embodiments, the double-stranded region is 15 nucleotides long. In some embodiments, the double-stranded region is 20 nucleotides long. In some embodiments, the double-stranded region is 21 nucleotides long. In some embodiments, the double-stranded region is 22 nucleotides long. In some embodiments, the double-stranded region is 23 nucleotides long. In some embodiments, the double-stranded region is 24 nucleotides long. In some embodiments, the double-stranded region is 25 nucleotides long. In some embodiments, the double-stranded region is 26 nucleotides long. In some embodiments, the double-stranded region is 27 nucleotides long. In some embodiments, the double-stranded region is 28 nucleotides long. In some embodiments, the double-stranded region is 29 nucleotides long. In some embodiments, the double-stranded region is 30 nucleotides long. In some embodiments, the double-stranded region is 31 nucleotides long. In some embodiments, the double-stranded region is 32 nucleotides long. In some embodiments, the double-stranded region is 33 nucleotides long. In some embodiments, the double-stranded region is 34 nucleotides long.In some embodiments, the double-stranded region is 35 base pairs long. In some embodiments, the double-stranded region is 36 base pairs long. In some embodiments, the double-stranded region is 37 base pairs long. In some embodiments, the double-stranded region is 38 base pairs long. In some embodiments, the double-stranded region is 39 base pairs long. In some embodiments, the double-stranded region is 40 base pairs long. In some embodiments, the double-stranded region is 41 base pairs long. In some embodiments, the double-stranded region is 42 base pairs long. In some embodiments, the double-stranded region is 43 base pairs long. In some embodiments, the double-stranded region is 44 base pairs long. In some embodiments, the double-stranded region is 45 base pairs long. In some embodiments, the double-stranded region is 46 base pairs long. In some embodiments, the double-stranded region is 47 base pairs long. In some embodiments, the double-stranded region is 48 base pairs long. In some embodiments, the double-stranded region is 49 base pairs long. In some embodiments, the double-stranded region is 50 base pairs long. In some embodiments, the double-stranded region is 55 base pairs long. In some embodiments, the double-stranded region is 60 base pairs long. In some embodiments, the double-stranded region is 65 base pairs long. In some embodiments, the double-stranded region is 70 base pairs long. In some embodiments, the double-stranded region is 75 base pairs long. In some embodiments, the double-stranded region is 80 base pairs long. In some embodiments, the double-stranded region is 85 base pairs long. In some embodiments, the double-stranded region is 90 base pairs long. In some embodiments, the double-stranded region is 95 base pairs long. In some embodiments, the double-stranded region is 100 base pairs long. In some embodiments, the double-stranded region is 125 base pairs long. In some embodiments, the double-stranded region is 150 base pairs long. In some embodiments, the double-stranded region is 175 base pairs long. In some embodiments, the double-stranded region is 200 base pairs long. In some embodiments, the double-stranded region is more than 200 base pairs long.

[0078] Each single-stranded arm is independently about 4 to 200 base pairs long. In some embodiments, each single-stranded arm is independently about 5 to 200 base pairs long. In some embodiments, each single-stranded arm is independently about 6 to 200 base pairs long. In some embodiments, each single-stranded arm is independently about 7 to 200 base pairs long. In some embodiments, each single-stranded arm is independently about 8 to 200 base pairs long. In some embodiments, each single-stranded arm is independently about 9 to 200 base pairs long. In some embodiments, each single-stranded arm is independently about 10 to 200 base pairs long. In some embodiments, each single-stranded arm is independently about 11 to 200 base pairs long. In some embodiments, each single-stranded arm is independently about 12 to 200 base pairs long. In some embodiments, each single-stranded arm is individually about 13 to about 200 base pairs long. In some embodiments, each single-stranded arm is individually about 14 to about 200 base pairs long. In some embodiments, each single-stranded arm is individually about 15 to about 200 base pairs long. In some embodiments, each single-stranded arm is individually about 20 to about 200 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to about 200 base pairs long. In some embodiments, each single-stranded arm is individually about 30 to about 200 base pairs long. In some embodiments, each single-stranded arm is individually about 35 to about 200 base pairs long. In some embodiments, each single-stranded arm is individually about 40 to about 200 base pairs long. In some embodiments, each single-stranded arm is individually about 45 to 200 base pairs long. In some embodiments, each single-stranded arm is individually about 50 to 200 base pairs long. In some embodiments, each single-stranded arm is individually about 60 to 200 base pairs long. In some embodiments, each single-stranded arm is individually about 70 to 200 base pairs long. In some embodiments, each single-stranded arm is individually about 80 to 200 base pairs long.In some embodiments, each single-stranded arm is individually about 90 to 200 base pairs long. In some embodiments, each single-stranded arm is individually about 100 to 200 base pairs long. In some embodiments, each single-stranded arm is individually about 125 to 200 base pairs long. In some embodiments, each single-stranded arm is individually about 150 to 200 base pairs long. In some embodiments, each single-stranded arm is individually about 175 to 200 base pairs long.

[0079] In some embodiments, each single-stranded arm is individually about 5 to 175 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 150 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 125 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 100 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 90 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 80 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 70 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 60 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 45 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 40 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 30 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 25 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 20 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 15 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 14 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 13 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 12 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 11 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 10 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 9 base pairs long. In some embodiments, each single-stranded arm is individually about 5 to 8 base pairs long.In some embodiments, each single-stranded arm is independently about 5 to 7 base pairs long. In some embodiments, each single-stranded arm is independently about 5 to 6 base pairs long.

[0080] In some embodiments, each single-stranded arm is individually about 5 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 6 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 7 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 8 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 9 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 10 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 11 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 12 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 13 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 14 to about 50 base pairs long. In some embodiments, each single-stranded arm is individually about 15 to about 50 base pairs long. In some embodiments, each single-stranded arm is individually about 16 to about 50 base pairs long. In some embodiments, each single-stranded arm is individually about 17 to about 50 base pairs long. In some embodiments, each single-stranded arm is individually about 18 to about 50 base pairs long. In some embodiments, each single-stranded arm is individually about 19 to about 50 base pairs long. In some embodiments, each single-stranded arm is individually about 20 to about 50 base pairs long. In some embodiments, each single-stranded arm is individually about 21 to about 50 base pairs long. In some embodiments, each single-stranded arm is individually about 22 to about 50 base pairs long. In some embodiments, each single-stranded arm is individually about 23 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 24 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 30 to 50 base pairs long.In some embodiments, each single-stranded arm is individually about 35 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 40 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 45 to 50 base pairs long.

[0081] In some embodiments, each single-stranded arm is individually about 25 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 26 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 27 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 28 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 29 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 30 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 31 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 32 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 33 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 34 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 35 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 36 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 37 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 38 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 39 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 40 to 50 base pairs long. In some embodiments, each single-stranded arm is individually about 45 to 50 base pairs long.

[0082] In some embodiments, each single-stranded arm is individually about 25 to 45 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 40 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 39 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 38 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 37 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 36 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 34 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 33 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 32 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 31 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 30 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 29 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 28 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 27 base pairs long. In some embodiments, each single-stranded arm is individually about 25 to 26 base pairs long.

[0083] In some embodiments, each single-stranded arm is individually about 25 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 26 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 27 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 28 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 29 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 30 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 31 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 32 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 33 to 35 base pairs long. In some embodiments, each single-stranded arm is individually about 34 to 35 base pairs long.

[0084] In some embodiments, each single-stranded arm is individually about 12 to 18 base pairs long. In some embodiments, each single-stranded arm is individually about 13 to 18 base pairs long. In some embodiments, each single-stranded arm is individually about 14 to 18 base pairs long. In some embodiments, each single-stranded arm is individually about 15 to 18 base pairs long. In some embodiments, each single-stranded arm is individually about 16 to 18 base pairs long. In some embodiments, each single-stranded arm is individually about 17 to 18 base pairs long. In some embodiments, each single-stranded arm is individually about 12 to 17 base pairs long. In some embodiments, each single-stranded arm is individually about 12 to 16 base pairs long. In some embodiments, each single-stranded arm is individually about 12 to 15 base pairs long. In some embodiments, each single-stranded arm is individually about 12 to 14 base pairs long. In some embodiments, each single-stranded arm is individually about 12 to 13 base pairs long.

[0085] In some embodiments, each single-stranded arm is individually 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, or more than 200 bases, or any length between any of the listed values. In some embodiments, each single-stranded arm is individually 4 bases long. In some embodiments, each single-stranded arm is individually 5 nucleotides long. In some embodiments, each single-stranded arm is individually 6 nucleotides long. In some embodiments, each single-stranded arm is individually 7 nucleotides long. In some embodiments, each single-stranded arm is individually 8 nucleotides long. In some embodiments, each single-stranded arm is individually 9 nucleotides long. In some embodiments, each single-stranded arm is individually 10 nucleotides long. In some embodiments, each single-stranded arm is individually 11 nucleotides long. In some embodiments, each single-stranded arm is individually 12 nucleotides long. In some embodiments, each single-stranded arm is individually 13 nucleotides long. In some embodiments, each single-stranded arm is individually 14 nucleotides long. In some embodiments, each single-stranded arm is individually 15 nucleotides long. In some embodiments, each single-stranded arm is individually 16 base pairs long. In some embodiments, each single-stranded arm is individually 17 base pairs long. In some embodiments, each single-stranded arm is individually 18 base pairs long. In some embodiments, each single-stranded arm is individually 19 base pairs long. In some embodiments, each single-stranded arm is individually 20 base pairs long. In some embodiments, each single-stranded arm is individually 21 base pairs long. In some embodiments, each single-stranded arm is individually 22 base pairs long.In some embodiments, each single-stranded arm is individually 23 base pairs long. In some embodiments, each single-stranded arm is individually 24 base pairs long. In some embodiments, each single-stranded arm is individually 25 base pairs long. In some embodiments, each single-stranded arm is individually 26 base pairs long. In some embodiments, each single-stranded arm is individually 27 base pairs long. In some embodiments, each single-stranded arm is individually 28 base pairs long. In some embodiments, each single-stranded arm is individually 29 base pairs long. In some embodiments, each single-stranded arm is individually 30 base pairs long. In some embodiments, each single-stranded arm is individually 31 base pairs long. In some embodiments, each single-stranded arm is individually 32 base pairs long. In some embodiments, each single-stranded arm is individually 33 base pairs long. In some embodiments, each single-stranded arm is 34 base pairs long. In some embodiments, each single-stranded arm is 35 base pairs long. In some embodiments, each single-stranded arm is 36 base pairs long. In some embodiments, each single-stranded arm is 37 base pairs long. In some embodiments, each single-stranded arm is 38 base pairs long. In some embodiments, each single-stranded arm is 39 base pairs long. In some embodiments, each single-stranded arm is 40 base pairs long. In some embodiments, each single-stranded arm is 41 base pairs long. In some embodiments, each single-stranded arm is 42 base pairs long. In some embodiments, each single-stranded arm is 43 base pairs long. In some embodiments, each single-stranded arm is 44 base pairs long. In some embodiments, each single-stranded arm is individually 45 base pairs long. In some embodiments, each single-stranded arm is individually 46 base pairs long. In some embodiments, each single-stranded arm is individually 47 base pairs long.In some embodiments, each single-stranded arm is individually 48 base pairs long. In some embodiments, each single-stranded arm is individually 49 base pairs long. In some embodiments, each single-stranded arm is individually 50 base pairs long. In some embodiments, each single-stranded arm is individually 55 base pairs long. In some embodiments, each single-stranded arm is individually 60 base pairs long. In some embodiments, each single-stranded arm is individually 65 base pairs long. In some embodiments, each single-stranded arm is individually 70 base pairs long. In some embodiments, each single-stranded arm is individually 75 base pairs long. In some embodiments, each single-stranded arm is individually 80 base pairs long. In some embodiments, each single-stranded arm is individually 85 base pairs long. In some embodiments, each single-stranded arm is individually 90 base pairs long. In some embodiments, each single-stranded arm is 95 base pairs long. In some embodiments, each single-stranded arm is 100 base pairs long. In some embodiments, each single-stranded arm is 125 base pairs long. In some embodiments, each single-stranded arm is 150 base pairs long. In some embodiments, each single-stranded arm is 175 base pairs long. In some embodiments, each single-stranded arm is 200 base pairs long. In some embodiments, each single-stranded arm is 200 base pairs long.

[0086] In some embodiments, an oligonucleotide may include one or two hinge segments that junction either of the single-stranded arms to the double-stranded region. Each hinge segment may be 1–4 nucleotides long, 2–4 nucleotides long, 1 nucleotide long, 2 nucleotides long, 3 nucleotides long, or 4 nucleotides long. In some embodiments, the nucleotide sequence of the hinge segment on the 5' side relative to the double-stranded region is CA, and the nucleotide sequence of the hinge segment on the 3' side relative to the double-stranded region is AC. The hinge segments should be understood as separate from the double-stranded portion and the single-stranded arms. For example, a first oligonucleotide may include a first single-stranded arm of 50 nucleotides, a complementary portion of 35 nucleotides, and a second single-stranded arm of 50 nucleotides, thus including a first oligonucleotide of 135 nucleotides. Alternatively, the first oligonucleotide may comprise a 50-nucleotide-length first single-stranded arm, a 4-nucleotide-length first hinge segment, a 35-nucleotide-length complementary portion, a 4-nucleotide-length second hinge segment, and a 50-nucleotide-length second single-stranded arm, thus comprising a 143-nucleotide-length first oligonucleotide. These examples are merely illustrative to illustrate how oligonucleotide lengths can be determined and should not be interpreted restrictively in any way.

[0087] Each single-stranded arm has a nucleic acid sequence that may be the same as or different from any other single-stranded arm. In some embodiments, the nucleotide sequences of each of the four single-stranded arms are identical. In some embodiments, three of the four single-stranded arms have nucleotide sequences that are identical to each other and different from the nucleotide sequence of the fourth single-stranded arm. In some embodiments, two of the four single-stranded arms have nucleotide sequences that are identical to each other and different from the nucleotide sequences of the other two single-stranded arms. In some embodiments, two of the four single-stranded arms have nucleotide sequences that are identical to each other, the other two of the four single-stranded arms have nucleotide sequences that are identical to each other, and the nucleotide sequences of the first two single-stranded arms are different from the nucleotide sequences of the second two single-stranded arms. In some embodiments, the nucleotide sequences of each of the four single-stranded arms are different. Therefore, the nucleotide sequence of any particular single-stranded arm provides flexibility in design selection and can be used to specifically bind to or be conjugated to a particular linked oligonucleotide (which may or may not be further conjugated to a targeting agent or cargo molecule as shown herein) or to another single-stranded arm of another monomeric nucleic acid carrier. In some embodiments, one or more single-stranded arms are complementary to the linked oligonucleotide conjugated to the targeting agent. In some embodiments, one or more single-stranded arms are complementary to a DNA dendrimer-binding sequence (DBS) as shown herein. In some embodiments, one or more single-stranded arms are complementary to the linked oligonucleotide conjugated to the targeting agent, and one or more single-stranded arms are complementary to a DNA dendrimer-binding sequence (DBS) as shown herein. Any two nucleic acid sequences with a degree of complementarity can be used to ligate the single-stranded arms of a DNA dendrimer to a linked oligonucleotide or DBS, where the two nucleic acid strands comprise the DBS and the corresponding single-stranded arms.Those skilled in the art can readily recognize complementary and non-complementary nucleic acid base pairs and thus conceive of two nucleic acid sequences having a desired degree of complementarity. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 30%-100% complementary, 35%-100% complementary, 40%-100% complementary, 45%-100% complementary, 50%-100% complementary, 55%-100% complementary, 60%-100% complementary, 65%-100% complementary, 70%-100% complementary, 75%-100% complementary, 80%-100% complementary, 85%-100% complementary, 90%-100% complementary, 95%-100% complementary, or any value or range in between. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and a linked oligonucleotide, or a single-stranded arm and a DBS) include sequences that are 30% to 95% complementary, 30% to 90% complementary, 30% to 85% complementary, 30% to 80% complementary, 30% to 75% complementary, 30% to 70% complementary, 30% to 65% complementary, 30% to 60% complementary, 30% to 55% complementary, 30% to 50% complementary, 30% to 45% complementary, 30% to 40% complementary, 30% to 35% complementary, or any value or range in between.

[0088] In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and a linked oligonucleotide, or a single-stranded arm and a DBS) include sequences that are less than 30% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and a linked oligonucleotide, or a single-stranded arm and a DBS) include sequences that are 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary, or any value or range between them. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 30% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 35% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 40% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 45% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 50% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 55% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and a linked oligonucleotide, or a single-stranded arm and a DBS) include sequences that are 60% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and a linked oligonucleotide, or a single-stranded arm and a DBS) include sequences that are 61% complementary.In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 62% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 63% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 64% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 65% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 66% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 67% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 68% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 69% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 70% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 71% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 72% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 73% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and a linked oligonucleotide, or a single-stranded arm and a DBS) include sequences that are 74% complementary.In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 75% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 76% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 77% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 78% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 79% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 80% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 81% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 82% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 83% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 84% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 85% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 86% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and a linked oligonucleotide, or a single-stranded arm and a DBS) include sequences that are 87% complementary.In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 88% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 89% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 90% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 91% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 92% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 93% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 94% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 95% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 96% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 97% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 98% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and linked oligonucleotide, or a single-stranded arm and DBS) include sequences that are 99% complementary. In some embodiments, the two nucleic acid sequences (e.g., a single-stranded arm and a linked oligonucleotide, or a single-stranded arm and a DBS) include sequences that are 100% complementary.

[0089] DNA dendrimers are commercially available. Furthermore, the structural design and construction of DNA dendrimers are generally known in the art. See, for example, U.S. Patents 5,175,270 and 6,274,723 (which are incorporated herein by reference in their entirety). The initiating monomer constitutes the approximate center of the dendrimer, depending on the type of branching formation. The three-dimensional construction of extension monomers around the initiating monomer constitutes the internal volume of the dendrimer. The outermost layer of extension monomers forms the surface of the dendrimer. Thus, the construction of a dendrimer results in a three-dimensional shape, usually a substantially spherical shape including layers of extension monomers (but not limited thereto). The outer layer contains numerous binding sites.

[0090] More specifically, DNA dendrimers can be prepared by a protocol having the following characteristics: (i) The starting material is a double-stranded region of DNA having 5' and 3' single-stranded overhangs or "binding arms" (e.g., four binding arms in total) attached to the double-stranded stem, referred to as the "start monomer," the name reflecting its role in the construction of the dendrimer. The 5' and 3' binding arms of each start monomer are annealed to complementary binding arms on an "extension monomer" having a similar composition and morphology. (ii) A subset of the four binding arms of each extension monomer is complementary to the binding arms of the start monomer. The non-complementary binding arms of the extension monomer are inert to annealing to the start monomer. Typically, four extension monomers can anneal to the start monomer to produce a monolayer or single-layer dendrimer in solution. (iii) To add another layer of extended monomer to the dendrimer, similar but distinguishable extended monomers are usually added, in which case each monomer has a subset of its four binding arms that are complementary to the binding arms on the dendrimer. Thus, a single-layer dendrimer can be progressively converted to a double-layer dendrimer, and so on, until a dendrimer of the desired size is obtained. The following table provides details of the structural elements of various dendrimer preparations. These are merely illustrative and not intended to be limiting in any sense. [Table 1]

[0091] After construction, DNA dendrimers can be crosslinked to maintain and stabilize their structure. Crosslinking of hybridized regions between monomers (i.e., intermonomer crosslinking), or crosslinking of hybridized regions between monomers and nucleic acids with detectable labels, and between stem units (intramonomer crosslinking), can stabilize the structure of polynucleotide dendrimers. Similarly, any hybridized region of any DNA-based carrier can be crosslinked to stabilize the carrier. Individual units of a carrier can also be crosslinked to a multimolecular carrier. Such crosslinking chemical reactions are well known in the art. See, for example, Cimino et al., Annu. Rev. Biochem. 54:1151-1193 (1985), Shi et al., Biochemistry 25:5895-5902 (1986), and Cimino et al., Biochemistry 25:3013-3020 (1986). Also see U.S. Patent No. 4,196,281. Non-limiting examples of suitable crosslinking agents include, in particular, psoralens (including, but not limited to, 8-methoxypsoralen and angelicin), mitomycin C, daunomycin, ethidium diazide, cisplatin, transplatin, carboplatin, 8-methoxypsoralen, mechloretamine, oxaliplatin, and carbodiimide compounds.

[0092] Polynucleotide chains used as monomers or other DNA-based carriers for dendrimers can be prepared by standard techniques of nucleic acid synthesis. These techniques may be biological or chemical. The techniques and procedures are typically carried out according to conventional methods in the relevant art and various general references (e.g., Sambrook et al., 2001 (cited above), Ausubel et al., eds., 2005 (cited above), and Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC).

[0093] In some embodiments, polynucleotides are chemically synthesized using methods known in the art. See, for example, Gait, 1985, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, England). In other embodiments, polynucleotides are enzymatically synthesized using polymerase chain reaction (PCR). One PCR method suitable for producing single-stranded polynucleotides is multicycle PCR using a single primer, which amplifies the single strand. As is well known in the art, nucleic acids can be purified by any suitable means before their use. For example, nucleic acids can be purified by reverse-phase or ion-exchange HPLC, size exclusion chromatography, or gel electrophoresis. Those skilled in the art will understand that the purification method depends in part on the size of the nucleic acid being purified.

[0094] The outer layer of a DNA dendrimer may have at least two types of binding arms. These binding arms can be used to bind one or more types of parts. In some embodiments, the DNA dendrimer of the composition can bind to or attach to a target part, cargo, support molecule, etc.

[0095] In some embodiments, DNA-based carriers may associate with secondary carriers, which may function as scaffolds for the DNA-based carriers. Examples of such secondary carriers include, but are not limited to, liposomes, non-DNA dendrimers, polymer carriers, microbubbles, paramagnetic and ferromagnetic particles, self-assembling polymers, polymerosomes, filomicelles, albumin particles, and lipoproteins. Self-assembling polymers are polymers formed by the self-assembly of single-molecule constituent units. These constituent units may be amphiphilic copolymers containing hydrophilic components (e.g., polyethyleneimine or polyethylene glycol) and hydrophobic components (e.g., aliphatic polyesters) in a core-shell structure. These structures, not maintained by direct conjugation or crosslinking of constituent units, are known as micelles, and their forms may vary, for example, from spherical micelles ("polymerosomes") to elongated or filamentous rods ("filomicelles"). The sizes of these structures can also vary, ranging from nanometers to micrometers. Once formed by self-assembly, these structures can, alternatively, be further chemically crosslinked to increase their stability. Hydrophobic cargoes can be embedded in the hydrophobic regions of the support, while hydrophilic cargoes can be incorporated into an internal aqueous core or an external hydrophilic corona.

[0096] In some embodiments, the DNA-based carrier is ligated to the surface of the secondary carrier. In some embodiments, the DNA-based carrier is not ligated to the secondary carrier but associates with it by being present in the same composition. In some embodiments, the cargo and targeting portions can be ligated to the DNA-based carrier or scaffold carrier by methods described herein and methods known in the art.

[0097] B. Targeting moiety In some embodiments, the DNA-based carrier is directed to specific cells by linking the targeting moiety to a carrier, secondary carrier, and / or cargo. In some embodiments, the targeting moiety is linked to the DNA-based carrier. In some embodiments, the targeting moiety is linked to a secondary carrier. The targeting moiety may be an antibody, a naturally occurring ligand or functional derivative thereof for a receptor, a vitamin, a hormone, a small molecular mimetic of a naturally occurring ligand, a peptide, a polypeptide, a peptide mimetic, a carbohydrate, a lipid, an FN3 domain, an aptamer, a nucleic acid, a toxin, a microbial component, or any other molecule that specifically binds to a cell surface molecule and induces endocytosis of the binding moiety.

[0098] While not bound by any particular theory, the targeting moiety can specifically bind to molecules on the cell surface of target cells. The targeting moiety can bind to cell surface molecules, thereby, in some embodiments, inducing endocytosis of DNA-based carriers. Non-limiting examples of cell surface molecules that can be targeted include cell surface proteins, carbohydrates, and lipids. Potentially targeted cell surface molecules include those associated with classical endocytosis and those associated with non-classical endocytosis.

[0099] In some embodiments, the target cell surface molecule is a cell adhesion molecule (CAM). Examples of cell adhesion molecules useful in the present invention include, but are not limited to, nerve-specific adhesion molecules (e.g., NCAM) and systemic cell adhesion molecules. Examples of systemic CAMs include cell adhesion molecules (e.g., ICAM-1, ICAM-2, ICAM-3), platelet / endothelial cell adhesion molecules (PECAM), activated leukocyte cell adhesion molecules (ALCAM), B lymphocyte cell adhesion molecules (BL-CAM), vascular cell adhesion molecules (VCAM), mucosal vascular adresin cell adhesion molecules (MAdCAM), CD44, LFA-2 (CD2), LFA-3 (CD58), and basidine (CD147). In some embodiments, the cell surface molecule is CD71.

[0100] In some embodiments, the targeting portion is an antibody that specifically binds to a target cell surface molecule. In some embodiments, the antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, humanized antibodies, synthetic antibodies, heavy chain antibodies, and biologically active fragments of antibodies, the biologically active fragments being Fab fragments, F(ab')2 fragments, and sc-Fv fragments.

[0101] In the compositions and methods of the present invention, when the antibody used as the targeting portion is a polyclonal antibody (IgG), the antibody is produced by inoculating a suitable animal with a target cell surface molecule. The antibody produced in the inoculated animal, which specifically binds to the cell surface molecule, is then isolated from the liquid obtained from the animal. Antibodies can be produced in this manner in several non-human mammals, including, but not limited to, goats, sheep, horses, camels, rabbits, and donkeys. Methods for producing polyclonal antibodies are well known in the art and are described, for example, in Harlow, et al. (1988, Antibodies, A Laboratory Manual, Cold Spring Harbor, NY).

[0102] Monoclonal antibodies directed against the full-length cell surface molecule or fragment thereof may be prepared using any well-known monoclonal antibody preparation procedure, such as those described in Harlow et al. (1988, Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and Tuszynski et al. (1988, Blood, 72:109-115). Human monoclonal antibodies may be prepared by the method described in U.S. Patent Application Publication No. 2003 / 0224490. Monoclonal antibodies directed against an antigen are generated from mice immunized with the antigen using the standard procedures cited herein. Nucleic acids encoding monoclonal antibodies obtained using the procedures described herein may be cloned and sequenced using techniques available in the art, such as those described in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and the references cited therein.

[0103] To prepare a targeted DNA-based carrier, such as a DNA dendrimer, the targeting moiety may be ligated to a DNA-based carrier, or to a composition containing a DNA-based carrier, or to a secondary carrier or cargo associated with it. A single targeting moiety may be ligated to a DNA-based carrier or secondary carrier. Alternatively, multiple (e.g., two or more) targeting moieties may be ligated to a DNA-based carrier, secondary carrier, or cargo. When multiple targeting moieties are ligated to a carrier or cargo, the moieties may target the same cell surface molecule or different cell surface molecules. If different cell surface molecules are targeted, these molecules may be associated with the same or different endocytosis pathways. The targeting moiety may also not be associated with an endocytosis pathway. If different cell surface molecules are targeted, these cell surface molecules may be present in the same cell type or in different cell types.

[0104] The linkage can be non-covalent or covalent. The targeted moiety can be directly linked to one or more polynucleotide chains constituting the DNA-based carrier. Alternatively, the targeted moiety can be linked to a linker molecule, which in turn is linked to the DNA-based carrier. In some embodiments where the DNA-based carrier is a DNA dendrimer, the linker molecule is an oligonucleotide containing a sequence substantially complementary to the sequence present on one of the binding arms on the surface of the DNA dendrimer. Thus, the targeted moiety is linked to the DNA dendrimer indirectly and non-covalently by hybridization of the oligonucleotide to the binding arm. This approach is also applicable to other DNA-based carriers. Optionally, the hybridized oligonucleotide is also crosslinked to the DNA dendrimer. The crosslinking chemistry is disclosed elsewhere herein. If crosslinking is not performed, the hybridization between the DNA dendrimer and the oligonucleotide linked to the targeted moiety must persist for a sufficiently long period under the conditions of use. The measurement of the binding free energy of nucleic acid molecules is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133, Freier et al., 1986, Proc. Nat. Acad. Sci. USA 83: 9373-9377, Turner et al., 1987, J. Am. Chem. Soc. 109: 3783-3785, and Chavali et al., 2005, Bioinformatics 21(20): 3918-3925).

[0105] In some embodiments, the linker is a secondary IgG Fc-specific antibody linked to a DNA-based carrier. In some embodiments, the antibody specifically binds to the Fc portion of a primary IgG antibody, e.g., a targeting portion or an intervening antibody. The secondary antibody is preferably species-specific, being the source of the primary antibody. For example, if the targeting portion is a human IgG antibody, the secondary antibody is anti-human IgG. In another embodiment, the secondary antibody recognizes the epitope of the entire primary antibody, not just the Fc portion. Alternatively, the linker is an Fc-gamma receptor that specifically binds to the Fc portion of the IgG antibody. In any of these embodiments, the carrier can be readily linked to any targeting portion that is an IgG antibody.

[0106] Non-covalent couplings include, but are not limited to, affinity coupling pairs, such as biotin-streptavidin, and immunoaffinity pairs with sufficiently high affinity to maintain coupling during use, which are well known in the art. The art also contains a wealth of conjugation chemistry useful for covalently coupling target portions to DNA-based carriers, secondary carriers, or cargoes, either directly or via linkers. Covalent coupling techniques recognized in the art are disclosed, for example, in U.S. Patents 5,416,016, 6,335,435, 6,528,631, 6,861,514, and 6,919,439, which are incorporated herein by reference in their entirety. Other conjugation chemistry is disclosed in U.S. Patent Application Publication 20040249178, which is incorporated herein by reference in its entirety. Other conjugation chemistrys include: p-hydroxybenzoic acid linkers (Chang-Po et al., 2002, Bioconjugate Chem. 13(3): 525-529); native ligation (Stetsenko et al., 2000, J Org. Chem. 65: 4900-4908); disulfide-bridged conjugates (Oehlke et al., 2002, Eur J. Biochem. 269: 4025-4032 and Rogers et al., 2004, Nuc Acids Res. 32(22) 6595-6604); maleimide linkers (Zhu et al., 1993, Antisense Res Dev. 3: 265-275); thioester linkers (Ede et al., 1994, Bioconjugate Chem. 5:373-378); Diels-Alder cycloaddition reaction (Marchan et al., 2006. Nuc Acids Res. 34(3):e24, 2006 Feb. 14 Epub); U.S. Patent No. 6,656,730, etc.For an overview of peptide-oligonucleotide conjugation chemical reactions, see also Tung et al., 2000, Bioconjugate Chem. 11:605-618, Zatsepin et al., 2005, Curr Pharm Des. 11(28):3639-3654, and Juliano, 2005, Curr Opin Mol. Ther. 7(2):132-136.

[0107] C. Cargo The compositions described herein may also include cargoes. In some embodiments, the cargoes are directly or indirectly bound to DNA dendrimers. Cargoes bound to DNA dendrimers associate with the DNA dendrimers via linkers or other types of molecules (adapters), etc. One, two, or more different cargoes may be delivered by a targeted DNA-based carrier. Cargoes that may be delivered by the compositions disclosed herein include, but are not limited to, a variety of agents, such as, but are not limited to, therapeutic agents, contrast agents, monitoring agents, chemotherapeutic agents, anti-carcinogenic agents, anti-angiogenic agents, tumor suppressants, antibacterial agents, enzyme replacement agents, gene expression regulators, and expression constructs containing nucleic acids that encode therapeutic proteins or nucleic acids. In some embodiments, the cargoes may be exogenous or endogenous substances. Cargoes include any molecule that induces an effect in cells, including any protein, nucleic acid, small molecule, carbohydrate, or lipid. Cargoes may include peptides, proteins (including enzymes, antibodies, and peptide hormones), cytoskeletal ligands, nucleic acids, small molecules, non-peptide hormones, etc. The nucleic acids and cargo polynucleotides that can be delivered by the methods of the present invention include synthetic and native nucleic acid materials, including DNA, RNA, transposon DNA, antisense nucleic acids, dsRNA, siRNA, transcription RNA, messenger RNA, ribosomal RNA, nucleolar small RNA, microRNA, ribozymes, plasmids, and expression constructs. If the cargo is a nucleic acid or contains one, the nucleic acid may be a separate entity from the DNA-based carrier. In these embodiments, the DNA-based carrier is not the cargo itself.

[0108] In some embodiments, the cargo is a polynucleotide comprising at least one promoter and at least one coding sequence encoding at least one molecule of interest. For example, a cargo polynucleotide may comprise one promoter and two or more coding sequences encoding different molecules of interest, or one promoter for each coding sequence present. In some embodiments, the promoter or a group of promoters is a tissue-specific promoter, and the composition comprising the cargo polynucleotide transcribes or translates at least one molecule of interest only when present in target cells compatible with the tissue-specific promoter. Tissue-specific promoters are known in the art, and any such tissue-specific promoter is within the scope of this disclosure.

[0109] In some embodiments, the molecule of interest is a gene product, such as an enzyme, protein, or receptor. In some embodiments, the cargo polynucleotide encodes two or more molecules of interest. In some embodiments, at least one of the molecules of interest is a detectable molecule, such as a protein that is tagged and detectable by bioluminescence, fluorescence, radiation signaling, or some combination thereof. In some embodiments, such molecules of interest are useful for in vivo distribution studies and other quantitative and / or real-time assessments.

[0110] In some embodiments, cargo polynucleotides may have one or more structures or topologies. For example, a cargo polynucleotide may exist as a fully circular double-stranded DNA or RNA without free 5' or 3' ends. In some embodiments, a cargo polynucleotide is a plasmid. In some embodiments, a cargo polynucleotide may be a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, and a linear polynucleotide with one open end and one closed end. In some embodiments, a nicked circular polynucleotide is a fully circular double-stranded DNA or RNA having at least one break in one strand of DNA or RNA. As used herein, a nicked circular polynucleotide is understood to be a circular polynucleotide having a gap of 1 to 100 bases compared to an unnicked polynucleotide. In some embodiments, the nick produces a base gap having a value or range between 1-100 bases, 2-100 bases, 3-100 bases, 4-100 bases, 5-100 bases, 6-100 bases, 7-100 bases, 8-100 bases, 9-100 bases, 10-100 bases, 15-100 bases, 20-100 bases, 25-100 bases, 30-100 bases, 35-100 bases, 40-100 bases, 45-100 bases, 50-100 bases, 60-100 bases, 70-100 bases, 80-100 bases, 90-100 bases, or any value or range between these.In some embodiments, the nicks result in gaps of 1–90 bases, 1–80 bases, 1–70 bases, 1–60 bases, 1–50 bases, 1–45 bases, 1–40 bases, 1–35 bases, 1–30 bases, 1–25 bases, 1–20 bases, 1–15 bases, 1–10 bases, 1–9 bases, 1–8 bases, 1–7 bases, 1–6 bases, 1–5 bases, 1–4 bases, 1–3 bases, 1–2 bases, or any value or range between them. In some embodiments, the nicks may be due to the action of a nicking restriction enzyme. In some embodiments, the linear polynucleotide has defined 5' and 3' ends, either or both of which are optionally closed by covalent bonds.

[0111] Different topologies or structures may be used for different bindings to DNA dendrimers.

[0112] Described herein is a plasmid comprising a plasmid backbone containing at least two restriction sites, at least one promoter, and at least one coding sequence encoding at least one molecule of interest, the plasmid may form cargo polynucleotides having various topologies, such as those shown herein. For example, a gene cassette containing a promoter sequence and a coding sequence encoding at least one molecule of interest may be inserted into the plasmid backbone. The resulting plasmid may have a fully circular topology, and if such topology is desired, no further steps are required. However, if a different topology is desired, the plasmid may be contacted with one or more restriction enzymes to cleave one or both nucleotide strands of the plasmid and produce a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open end and one closed end. In some embodiments, the closed ends of the linear polynucleotide are formed by TelN protelomerase.

[0113] In some embodiments, cargo polynucleotides are transported to the nucleus of a cell. In some embodiments, cargo polynucleotides include one or more DNA targeting sequences (DTSs) or nuclear localization signals (NLSs). DTSs and NLSs are, independently, short amino acid sequences that target proteins and associated nucleic acids for translocation into the nucleus. In non-limiting examples, NLSs that can be encoded by cargo polynucleotides have amino acid sequences that have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to YPDEVKRKKKP (SEQ ID NO: 1), SLLESPFDKPDEVKRKKKPPTSHQSDATAEDDSSSKKK (SEQ ID NO: 2), PKKKRKV (SEQ ID NO: 15), or KKPNKKKRKE (SEQ ID NO: 46). These are non-limiting examples of NLS sequences, and any NLS may be used. In some embodiments, the NLS includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the NLS includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 2.In some embodiments, the NLS includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 46. In some embodiments, the NLS includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 46. In some embodiments, the NLS includes any one of the amino acid sequences of SEQ ID NO: 1, 2, 15, or 46. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, at least one DTS, at least one NLS, or both, may be located anywhere within or related to the cargo polynucleotide. For example, the DTS, NLS, or both may be located upstream or downstream of at least one promoter of the cargo polynucleotide. In such a molecule, the polypeptide is ligated to the cargo polynucleotide.

[0114] In some embodiments, one or more DTS or NLS sequences may be bound to or ligated to a cargo polynucleotide instead of being located within the cargo polynucleotide. In some embodiments, one or more DTS or NLS sequences may be contained in any adapter molecule described herein, which is ligated to a cargo polynucleotide. In some embodiments, one or more DTS or NLS sequences may be ligated directly to a cargo polynucleotide without associating with a corresponding adapter molecule. In some embodiments, a DTS or NLS sequence may be bound to a cargo polynucleotide via a modified nucleotide. For example, a DTS or NLS sequence may be bound to a nicked circular cargo polynucleotide at a nick site, optionally, in this case, the modified nucleotide is first inserted at the nick site.

[0115] In some embodiments, a cargo comprising the cargo polynucleotide described herein may be directly linked to a DNA dendrimer by any covalent or noncovalent interaction or linkage described herein, such as a hydrogen bond. Thus, in some embodiments, the cargo polynucleotide includes a DNA dendrimer-binding sequence (DBS) that links the cargo polynucleotide to the DNA dendrimer. In some embodiments, the DBS includes a nucleic acid sequence complementary to the nucleic acid sequence of the DNA dendrimer. In non-limiting examples, the DBS includes a nucleic acid sequence complementary to at least one binding arm of the DNA dendrimer. In some embodiments, the DBS is linked to the DNA dendrimer by a hydrogen bond.

[0116] In some embodiments, the DBS includes a nucleic acid sequence selected from one of the following: TAGAGGTAACAACTAGCGTACAA (SEQ ID NO: 3), TAGAGGTAACAACTAGCGTACAATTTTTTTTTTT (SEQ ID NO: 4), TCAGCTTGTACTCTAGTTGTTACCTCTAATGCTGGACC (SEQ ID NO: 10), GCACCCTACAGAGTAACCTAGATTGATCAAACACCTCA (SEQ ID NO: 11), CCTCAGCTTGTACTCTAGTTGTTACCTCTAATGCTGGACCTCAGC (SEQ ID NO: 22), or CCTCAGCACCCTACAGAGTAACCTAGATTGATCAAACACCTCAGC (SEQ ID NO: 23). In some embodiments, the DBS includes nucleic acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to any one of sequence numbers 3, 4, 22, or 23. In some embodiments, the DBS includes nucleic acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 3. In some embodiments, the DBS includes a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 4.In some embodiments, the DBS includes a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 10. In some embodiments, the DBS includes a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 11. In some embodiments, the DBS includes a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 22. In some embodiments, the DBS includes a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 23. In some embodiments, the DBS includes one of the nucleic acid sequences of SEQ ID NOs: 3, 4, 22, or 23. In some embodiments, the DBS includes the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DBS includes the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the DBS includes the nucleic acid sequence of SEQ ID NO: 10.In some embodiments, the DBS includes the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the DBS includes the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the DBS includes the nucleic acid sequence of SEQ ID NO: 23.

[0117] In some embodiments, the DBS includes a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10, provided that the nucleic acid sequence includes at least 20 consecutive nucleic acids of the sequence of SEQ ID NO: 10. The at least 20 consecutive nucleic acids of SEQ ID NO: 10 may be any 20 consecutive nucleic acids. Therefore, at least 20 consecutive nucleic acids may include nucleic acids 1-20 of SEQ ID NO: 10, nucleic acids 2-21 of SEQ ID NO: 10, nucleic acids 3-22 of SEQ ID NO: 10, nucleic acids 4-23 of SEQ ID NO: 10, nucleic acids 5-24 of SEQ ID NO: 10, nucleic acids 6-25 of SEQ ID NO: 10, nucleic acids 7-26 of SEQ ID NO: 10, nucleic acids 8-27 of SEQ ID NO: 10, nucleic acids 9-28 of SEQ ID NO: 10, nucleic acids 10-29 of SEQ ID NO: 10, nucleic acids 11-30 of SEQ ID NO: 10, or nucleic acids 12-31 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 1-20 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 2-21 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 3-22 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 4-23 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 5-24 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 6-25 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 7-26 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 8-27 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 9-28 of SEQ ID NO: 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 10-29 of SEQ ID NO: 10.In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 11-30 of sequence number 10. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 12-31 of sequence number 10.

[0118] In some non-limiting embodiments, a DBS includes a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10, wherein the DBS includes at least 20 consecutive nucleic acids of the sequence of SEQ ID NO: 10, and the DBS includes 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 20 consecutive nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 21 consecutive nucleic acids of the sequence of SEQ ID NO: 10.In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 22 consecutive nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 23 consecutive nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 24 consecutive nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 25 consecutive nucleic acids of the sequence of SEQ ID NO: 10.In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 26 consecutive nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 27 consecutive nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 28 consecutive nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 29 consecutive nucleic acids of the sequence of SEQ ID NO: 10.In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 30 consecutive nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 10 contains at least 31 consecutive nucleic acids of the sequence of SEQ ID NO: 10.

[0119] In some embodiments, the DBS includes a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11, provided that the nucleic acid sequence includes at least 20 consecutive nucleic acids of the sequence of SEQ ID NO: 11. The at least 20 consecutive nucleic acids of SEQ ID NO: 11 may be any 20 consecutive nucleic acids. Therefore, at least 20 consecutive nucleic acids may include nucleic acids 1-20 of SEQ ID NO: 11, nucleic acids 2-21 of SEQ ID NO: 11, nucleic acids 3-22 of SEQ ID NO: 11, nucleic acids 4-23 of SEQ ID NO: 11, nucleic acids 5-24 of SEQ ID NO: 11, nucleic acids 6-25 of SEQ ID NO: 11, nucleic acids 7-26 of SEQ ID NO: 11, nucleic acids 8-27 of SEQ ID NO: 11, nucleic acids 9-28 of SEQ ID NO: 11, nucleic acids 10-29 of SEQ ID NO: 11, nucleic acids 11-30 of SEQ ID NO: 11, or nucleic acids 12-31 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 1-20 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 2-21 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 3-22 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 4-23 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 5-24 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 6-25 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 7-26 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 8-27 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 9-28 of SEQ ID NO: 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 10-29 of SEQ ID NO: 11.In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 11-30 of sequence number 11. In some embodiments, at least 20 consecutive nucleic acids include nucleic acids 12-31 of sequence number 11.

[0120] In some non-limiting embodiments, a DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11, wherein the DBS comprises at least 20 consecutive nucleic acids of the sequence of SEQ ID NO: 11, and comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 20 consecutive nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 21 consecutive nucleic acids of the sequence of SEQ ID NO: 11.In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 22 consecutive nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 23 consecutive nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 24 consecutive nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 25 consecutive nucleic acids of the sequence of SEQ ID NO: 11.In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 26 consecutive nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 27 consecutive nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 28 consecutive nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 29 consecutive nucleic acids of the sequence of SEQ ID NO: 11.In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 30 consecutive nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, a DBS containing a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 11 contains at least 31 consecutive nucleic acids of the sequence of SEQ ID NO: 11.

[0121] In some embodiments, the DBS sequence further comprises a polythymine sequence at either its 5' or 3' end. These are non-limiting examples of DBS molecules, and any DBS sequence complementary to the dendrimer arm may be used. In some embodiments, the DBS and dendrimer arm have a complementarity percentage as shown herein.

[0122] D. Adapter molecule Cargo can also be linked to a DNA dendrimer by an adapter molecule as shown herein. In some embodiments, the adapter molecule includes a DBS sequence that links the adapter molecule to a DNA dendrimer and a cargo-binding region that links to a cargo (e.g., a cargo polynucleotide as shown herein).

[0123] In some embodiments, the DBS includes a nucleic acid sequence complementary to the nucleic acid sequence of the DNA dendrimer. For example, the DBS includes a nucleic acid sequence complementary to at least one binding arm of the DNA dendrimer. In some embodiments, the DBS binds to the DNA dendrimer by hydrogen bonds. In some embodiments, the DBS includes any DBS sequence disclosed herein. In some embodiments, any DBS sequence complementary to the dendrimer arm may be used.

[0124] In some embodiments, the cargo-binding region may be linked to the cargo polynucleotide by various linkers known in the art. For example, the cargo-binding region may include a nucleic acid sequence that is complementary to at least a portion of the cargo polynucleotide and / or links to the cargo by DNA ligation. In another example, the cargo-binding region may be linked to the cargo polynucleotide by chemical coupling, for example, click chemistry or EDC crosslinking, but not limited to these. In some embodiments, the cargo-binding region includes at least one nucleotide having an amine, azide, or other reactive group. In some embodiments, the cargo-binding region includes at least one cysteine ​​residue.

[0125] The adapter molecule may also contain other regions or features. In some embodiments, such regions or features may be located between the DBS and the cargo binding region, but may also be located in any order within the adapter molecule.

[0126] In some embodiments, the adapter molecule further comprises at least one purification region. In some embodiments, the purification region comprises at least one purification tag or affinity tag. The purification tag or affinity tag may be any such chemical tag or amino acid tag known in the art, for example, a polyhistidine tag, for example, His6, His12, etc.

[0127] In some embodiments, the adapter molecule further comprises a DTS, an NLS, or both, as shown herein. In some embodiments, the DTS and NLS regions are located between the DBS and the cargo binding region. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 1, 2, 15, or 46. In some embodiments, the NLS includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the NLS includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 15.In some embodiments, the NLS includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 46. In some embodiments, the NLS includes the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments, the NLS includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the NLS has the amino acid sequence of SEQ ID NO: 2. In some embodiments, the NLS has the amino acid sequence of SEQ ID NO: 15. In some embodiments, the NLS has the amino acid sequence of SEQ ID NO: 46.

[0128] In some embodiments, the DTS includes any sequence that promotes interaction with DNA. For example, the DTS sequence may include transcription factor binding sequences, etc. Such sequences are known in the art and are within the scope of this disclosure. Alternatively, those skilled in the art may utilize DTS synthetically derived from known sequences that promote interaction with DNA. Such DTS are within the scope of this disclosure. Furthermore, those skilled in the art can screen putative DTS sequences to provide suitable functional DTS sequences, i.e., sequences that promote interaction with DNA. Such DTS are within the scope of this disclosure.

[0129] In some embodiments, the DTS is SV40-DTS. In some embodiments, the DTS comprises a nucleic acid sequence that behaves substantially the same as SV40-DTS. In some embodiments, the SV40-DTS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCA (Sequence ID 8). In some embodiments, the SV40-DTS comprises the nucleic acid sequence of Sequence ID 8.

[0130] In some embodiments, the DTS or NLS region further includes at least one spacer located either before or after the DTS or NLS region. If two spacers are present, they are located immediately before and after the DTS, NLS, or both. The spacers are generally known in the art and include a variety of flexible or semi-flexible amino acids or molecules. In some embodiments, one or more spacers optionally include polyglycine with alanine and / or serine residues. In some embodiments, the spacer includes an amino acid sequence selected from either GGGG (SEQ ID NO: 5) or GGGGGG (SEQ ID NO: 29). In some embodiments, the spacer includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer includes the amino acid sequence of SEQ ID NO: 29. In some embodiments, one or more spacers include polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PGLA), or any combination thereof. In some embodiments, one or more spacers may optionally be substituted with saturated or unsaturated hydrocarbon chains containing 3 to 6 carbon atoms.

[0131] In some embodiments, the adapter molecule further comprises a cell-permeable peptide sequence (CPP). CPPs are a group of short-chain peptides that have the ability to increase membrane entry and can be used to assist in the transport of molecules across the cell membrane (see Xu et al., 2019, J Control Release, 309:106-124 for a review). Various CPPs may be included in the adapter molecule to assist in the entry of DNA dendrimer-based compositions into target cells or target nuclei. For example, in some embodiments, the CPP has the amino acid sequence QPRRRPRRKKRG (SEQ ID NO: 6). In some embodiments, the CPP is located between the DBS and the cargo-binding region.

[0132] In some embodiments, the cargo is separable from the DNA dendrimer. Therefore, in some embodiments, the adapter molecule further includes one or more cleavage sites. The cleavage sites can be located anywhere between the DBS and the cargo-binding region. In some embodiments, the cleavage sites are located adjacent to or nearly adjacent to the DBS. For example, in some embodiments, the cleavage site is a valine-citrulline / p-aminobenzylcarbamate (val-cit) cleavable linker. Val-cit linkers are known in the art and are used to release cargo from a targeted site in drug conjugation. With respect to the exemplary sequences shown herein, the val-cit linker may be represented by the sequence VCit, where Cit is citrulline. Other cleavable linkers known in the art, such as self-cleaving ribozyme cleavage sites or adjustable pH-sensitive linkers, may also be included (see, for example, Choy et al., Bioconjugate Chem. (2016) 27(3):824-830).

[0133] In some embodiments, the adapter molecule further comprises at least one flexible linker. In some embodiments, the flexible linker is located between the DNS and the cargo binding region. In some embodiments, the at least one flexible linker is polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PGLA), (GG)n (SEQ ID NO: 31), (GGGGS)n (SEQ ID NO: 7), (GGGGA)n (SEQ ID NO: 27), or any combination thereof, where each n is independently 1 to 5.

[0134] In some embodiments, the adapter molecule includes a purification region. In some embodiments, the purification region includes the amino acid sequence HHHHHHC (SEQ ID NO: 28).

[0135] The adapter molecules described herein may have none of the elements disclosed herein, some of them, or all of them. In non-limiting examples, an adapter molecule may include, in addition to the DBS and cargo binding region, one or more purification regions, DTS, NLS, CPP, spacers, cleavage sites, flexible linkers, or any combination thereof. In some embodiments, an adapter molecule includes, in any order, one or any combination of the DBS, flexible linker, CPP, NLS, purification regions, spacers, and cargo binding regions, e.g., those shown herein. Non-limiting examples of adapter molecules are listed below, including the following elements in the 5' to 3' direction: [Table 2]

[0136] It should be understood that the adapter molecules listed in the previous table are contiguous sequences containing various specific sequence identifiers. Therefore, the adapter molecule represented by the sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTTT (SEQ ID NO: 4)-HHHHHHC (SEQ ID NO: 28) is a contiguous sequence where the thymine at the 3' end of SEQ ID NO: 4 is conjugated to the histidine at the N end of SEQ ID NO: 28, and the hyphens (-) separating the different parts of the contiguous sequence indicate conjugation.

[0137] The adapter molecule represented by the sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTTT (SEQ ID NO: 4)-GGGGGG (SEQ ID NO: 29)-YPDEVKRKKKP (SEQ ID NO: 1) is a continuous sequence in which the thymine at the 3' end of SEQ ID NO: 4 is conjugated to the glycine at the N-terminus of SEQ ID NO: 29, and the glycine at the C-terminus of SEQ ID NO: 29 is conjugated to the tyrosine at the N-terminus of SEQ ID NO: 1. The hyphens (-) separating the various parts of the continuous sequence indicate conjugation.

[0138] The adapter molecule represented by the sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTTT (SEQ ID NO: 4)-GGGGGG (SEQ ID NO: 29)-(VCit)-GGGG (SEQ ID NO: 5)-YPDEVKRKKKP (SEQ ID NO: 1)-GGGG (SEQ ID NO: 5)-HHHHHHC (SEQ ID NO: 28) is a continuous sequence, where the thymine at the 3' end of SEQ ID NO: 4 is conjugated to the glycine at the N-terminus of SEQ ID NO: 29, and the glycine at the C-terminus of SEQ ID NO: 29 is a val-cit linker. It is conjugated to VCit, which is further conjugated to the N-terminal glycine of SEQ ID NO: 5, the C-terminal glycine of SEQ ID NO: 5 is conjugated to the N-terminal tyrosine of SEQ ID NO: 1, the C-terminal proline of SEQ ID NO: 1 is conjugated to the N-terminal glycine of SEQ ID NO: 5, and the C-terminal glycine of SEQ ID NO: 5 is conjugated to the N-terminal histidine of SEQ ID NO: 28, where hyphens (-) separating different parts of a continuous sequence indicate conjugation.

[0139] The adapter molecule represented by the sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTTT (SEQ ID NO: 4)-GGGGGG (SEQ ID NO: 29)-(VCit)-QPRRRPRRKKRG (SEQ ID NO: 6)-SLLESPFDKPDEVKRKKKPPTSHQSDATAEDDSSSKKK (SEQ ID NO: 2)-GGGG (SEQ ID NO: 5)-HHHHHHC (SEQ ID NO: 28) is a continuous sequence, where the thymine at the 3' end of SEQ ID NO: 4 is conjugated to the glycine at the N-terminus of SEQ ID NO: 29, and the C-terminus of SEQ ID NO: 29 The glycine in this sequence is conjugated to the val-cit linker (VCit), which is further conjugated to the N-terminal glutamine in sequence number 6; the C-terminal glycine in sequence number 6 is conjugated to the N-terminal serine in sequence number 2; the C-terminal lysine in sequence number 2 is conjugated to the N-terminal glycine in sequence number 5; and the C-terminal glycine in sequence number 5 is conjugated to the N-terminal histidine in sequence number 28. Here, hyphens (-) separating different parts of a continuous sequence indicate conjugation.

[0140] As shown herein, protein-oligonucleotide conjugations, i.e., amino acid-nucleic acid conjugations, are known in the art, and any such conjugation techniques can be utilized to construct the adapter molecules described above. Thus, conjugations between various parts of a contiguous sequence indicated by the hyphen (-) above are known in the art and may be any suitable conjugations as shown herein.

[0141] In some embodiments, the adapter molecule includes the sequences shown in the table below. In some embodiments, the adapter molecule includes the sequences shown in the table below from the 5' to the 3' end of the nucleotide sequence and from the N-terminus to the C-terminus of the amino acid sequence. [Table 3]

[0142] In some embodiments, the adapter molecule includes the sequences of SEQ ID NO: 4 and SEQ ID NO: 28 from the 5' end to the 3' end. In some embodiments, the adapter molecule includes the sequences of SEQ ID NO: 4, SEQ ID NO: 29, and SEQ ID NO: 1 from the 5' end to the 3' end. In some embodiments, the adapter molecule includes the sequences of SEQ ID NO: 4, SEQ ID NO: 29, VCit, SEQ ID NO: 5, SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 28 from the 5' end to the 3' end. In some embodiments, the adapter molecule includes the sequences of SEQ ID NO: 4, SEQ ID NO: 29, VCit, SEQ ID NO: 6, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 28 from the 5' end to the 3' end.

[0143] In some embodiments, the adapter molecule includes sequences of SEQ ID NO: 4 and SEQ ID NO: 28 from the 5' end to the 3' end, where the sequences are conjugated to each other. In some embodiments, the adapter molecule includes sequences of SEQ ID NO: 4, SEQ ID NO: 29, and SEQ ID NO: 1 from the 5' end to the 3' end, where the sequences are conjugated to each other. In some embodiments, the adapter molecule includes sequences of SEQ ID NO: 4, SEQ ID NO: 29, VCit, SEQ ID NO: 5, SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 28 from the 5' end to the 3' end, where the sequences are conjugated to each other. In some embodiments, the adapter molecule includes sequences of SEQ ID NO: 4, SEQ ID NO: 29, VCit, SEQ ID NO: 6, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 28 from the 5' end to the 3' end, where the sequences are conjugated to each other.

[0144] E. Supporting molecules The compositions herein may also include one or more support molecules. Support molecules as shown herein may be used for a wide range of tasks, including, but not limited to, protecting DNA dendrimers and cargo polynucleotides from nuclease degradation, enhancing transfection efficiency, assisting nuclear delivery, and condensing the size of DNA dendrimers, cargo polynucleotides, or both. Support molecules may be covalently or noncovalently linked to DNA dendrimers in the same or similar manner as the targeting moiety and cargo. However, in some embodiments, one or more support molecules may not be linked to DNA dendrimers and may associate with the composition by other means.

[0145] While we do not wish to be constrained by theory, since many polynucleotide structures have a negatively altered amine skeleton, a support molecule with sufficient positive charge can associate with the composition via charge attraction. Thus, in some embodiments, one or more support molecules associate with the composition via charge attraction. In some embodiments, one or more support molecules have a net positive charge high enough to allow them to interact with a negatively charged DNA dendrimer and optionally compress it. In some embodiments, one or more support molecules have a net positive charge low enough to avoid aggregation or other cytotoxic side effects.

[0146] Furthermore, such support molecules can also associate with cargo polynucleotides and DNA dendrimers simultaneously. Therefore, in some embodiments, the compositions described herein comprise a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting site, where the cargo polynucleotide is not covalently linked to the DNA dendrimer. The support molecule can thus associate the cargo polynucleotide with the DNA dendrimer regardless of the topology of the cargo polynucleotide. However, if the cargo polynucleotide has a perfectly circular topology with no nicks, sites, or regions for direct covalent linkage to a DNA dendrimer or adapter molecule, one or more support molecules can associate the cargo polynucleotide with the DNA dendrimer to form a composition.

[0147] The support molecules described herein may be used individually, as repetitions, or in combination within a single molecular entity or in a complex mixture of individual molecular entities. In some embodiments, the support molecules may be added to the composition before the association of the cargo, DNA dendrimer, and optionally the adapter molecule. In some embodiments, the support molecules may be added to the composition after the association of the cargo, DNA dendrimer, and optionally the adapter molecule. In some embodiments, but not limited to, the support molecules described herein are stabilizers.

[0148] While we do not wish to be bound by any particular theory, pore sizes differ from cell type to cell type, allowing only the uptake of materials of the same size. To enable delivery to cells with smaller pore sizes, particles must be reduced in size or compressed. In addition to size, particle size and particle protection can improve the durability of scaffolds and payloads. To date, various stabilizer components have been developed and tested for the purpose of both compressing nanoparticles and protecting them from degrading enzymes. A net positive charge allows the stabilizer to interact with negatively charged nucleic acids via electrostatic interactions, thereby compressing them. This compression allows for protection from nucleases because the stabilizer is present on the surface of the nanostructure interacting with the in vivo environment. This charge needs to be sufficient to complex the nucleic acids, but should not be high enough to cause aggregation or other cytotoxic side effects due to the positive charge. Thus, stabilizers can consist of biocompatible peptides and / or polymers. The term “biocompatible,” as used herein, refers to a substance that has minimal toxicity in vitro / in vivo, is excretable, and / or can be broken down into units that can be processed by the body. Therefore, in some embodiments, the stabilizer includes a peptide moiety that enhances cellular uptake and nuclear localization. The CPP and NLS sequences are elements added to the stabilizer sequence if the initial basic stabilizer (positively charged compression moiety only) successfully compresses the nucleic acid and protects it from nucleases.

[0149] In some embodiments, the stabilizer is a supporting molecule. In some embodiments, the supporting molecule comprises a biocompatible peptide, polymer, or both. Thus, in some embodiments, the supporting molecule is a peptide-based supporting molecule or a polymer-based supporting molecule. In some embodiments, the supporting molecule comprises a CPP sequence, an NLS sequence, or both. In some embodiments, the CPP sequence is any CPP sequence disclosed herein. In some embodiments, the NLS sequence is any NLS sequence disclosed herein.

[0150] In some embodiments, the stabilizer is a peptide-based stabilizer. In some embodiments, the supporting molecule is a peptide-based supporting molecule. In some embodiments, the supporting molecule is ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK(histone H1 sequence, SEQ ID NO: 12);WRRRGFGRRR(SEQ ID NO: 13);GRKKRRQRRRPQ(SEQ ID NO: 14);PKKKRKV(SV40 NLS sequence, SEQ ID NO: 15); GLFHAIAHFIHGGWHGLIHGWYG (SEQ ID NO: 16); WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 17); HHHHHHHHHH (SEQ ID NO: 18); (KK)n (SEQ ID NO: 32) (where n = 2 to 15); GLFHAIAHFIHGGWHGLIHGWYGP (H5WYG sequence, SEQ ID NO: 9); WSQ (Spacer sequence); AAEAEAKAKAEAEAKAK (SEQ ID NO: 33), AAEAEAEAKAKAKAKAK (SEQ ID NO: 34); GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 35), ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKATPKKST The amino acid sequence includes KKTPKKAKK (SEQ ID NO: 36), THRPPMWSPVWP-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSKKPNKKKRKE (SEQ ID NO: 37), THRPPMWSPVWP-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSPKKKRKVC (SEQ ID NO: 38), THRPPMWSPVWP-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKC (SEQ ID NO: 39), ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSPKKKRKV (SEQ ID NO: 40), ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSKKPNKKKRKE (SEQ ID NO: 41), THRPPMWSPVWP (SEQ ID NO: 42), or any combination thereof.In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42, or any combination thereof. In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 9. In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 12. In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 13.In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 14. In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 15. In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 16. In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 17. In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 18. In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 32. In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 33.In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 34. In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 35. In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 36. In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 37.In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 38. In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 39. In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40. In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41. In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 42. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 12. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 13. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 14. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 15. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 16. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 32. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 33. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 34.In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 35. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 36. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 37. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 38. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 39. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 40. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 41. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 42. In some embodiments, the supporting molecule includes the amino acid sequence of WSQ.

[0151] In some embodiments, the supporting molecule comprises an amino acid sequence selected from one of the following: GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 19); HHHHHHHHHHATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 20); or GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVGRKKRRQRRRPQWRRRGFGRRR (SEQ ID NO: 21). In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs. In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 20.In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 21. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 20. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 21.

[0152] In some embodiments, the stabilizer is a polymer-based stabilizer. In some embodiments, the support molecule is a polymer-based support molecule. In some embodiments, the support molecule is a REG-based support molecule. In some embodiments, the support molecule is a REG polymer support molecule. In some embodiments, the support molecule contains an amino acid sequence selected from one of the following: KKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 24); KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 26). In some embodiments, the supporting molecule includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs.In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with SEQ ID NO: 25. In some embodiments, the supporting molecule contains an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with SEQ ID NO: 26. In some embodiments, the supporting molecule contains the amino acid sequence of SEQ ID NO: 24. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 25. In some embodiments, the supporting molecule includes the amino acid sequence of SEQ ID NO: 26.

[0153] In some embodiments, any of the supporting molecules shown herein may further include additional portions, for example, a visualization portion, or a portion for supporting further chemical modification. In some non-limiting embodiments, the visualization portion is Cy3, or any other low molecular weight fluorophore known in the art. In some non-limiting embodiments, the portion for supporting further chemical modification is dibenzylcyclooctin (DBCO), etc. The visualization portion may be conjugated to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the supporting molecule. Similarly, the chemical modification portion may be conjugated to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the supporting molecule. The visualization portion and / or chemical modification portion may be conjugated directly to the supporting molecule or via a cleavable linker, such as a val-cit linker, as shown herein.

[0154] In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 37 further includes a visualization portion at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the N-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising a sequence and a visualization portion having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 37 is given by the sequence:THRPPMWSPVWP-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSKKPNKKKRKE-Cy3 (SEQ ID NO: 44).

[0155] In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 38 further includes a visualization portion at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the N-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising a sequence and a visualization portion having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 38 is given by the sequence:THRPPMWSPVWP-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSPKKKRKVC-Cy3 (SEQ ID NO: 45).

[0156] In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 39 further includes a visualization portion at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the N-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising a sequence and a visualization portion having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 39 is given by the sequence:THRPPMWSPVWP-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKC-Cy3 (SEQ ID NO: 46).

[0157] In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 40 further comprises a visualization moiety, a chemically modified moiety, or a combination thereof. In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40 further includes a visualization portion at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the N-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40 further includes a chemically modified moiety at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the chemically modified moiety is at the N-terminus of the amino acid sequence. In some embodiments, the chemically modified moiety is at the C-terminus of the amino acid sequence. In some embodiments, the chemically modified moiety is at both the N-terminus and C-terminus of the amino acid sequence.In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40 further comprises both a visible portion and a chemically modified portion. In some embodiments, the visible portion is at the N-terminus of the amino acid sequence, and the chemically modified portion is at the C-terminus of the amino acid sequence. In some embodiments, the visible portion is at the C-terminus of the amino acid sequence, and the chemically modified portion is at the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40, as well as both a visible portion and a chemically modified portion, is given by the sequence:DBCO-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSPKKKRKV-Cy3 (SEQ ID NO: 47).

[0158] In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 41 further includes a visualization moiety, a chemical modification moiety, or a combination thereof. In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further includes a visualization portion at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the N-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at the C-terminus of the amino acid sequence. In some embodiments, the visualization portion is located at both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further includes a chemically modified moiety at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the chemically modified moiety is at the N-terminus of the amino acid sequence. In some embodiments, the chemically modified moiety is at the C-terminus of the amino acid sequence. In some embodiments, the chemically modified moiety is at both the N-terminus and C-terminus of the amino acid sequence.In some embodiments, a supporting molecule containing a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further comprises both a visualization portion and a chemical modification portion. In some embodiments, the visualization portion is at the N-terminus of the amino acid sequence, and the chemical modification portion is at the C-terminus of the amino acid sequence. In some embodiments, the visualization portion is at the C-terminus of the amino acid sequence, and the chemical modification portion is at the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41, as well as both a visible portion and a chemically modified portion, is given by the sequence:DBCO-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSKKPNKKKRKE-Cy3 (SEQ ID NO: 48).

[0159] In some embodiments, polyethylene glycol (PEG) can be linked to or conjugated to any supporting molecule disclosed herein. The PEG molecule may be any PEG known in the art, including a variety of molecular weights. In some embodiments, PEG is PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000. In some embodiments, PEG is PEG400. In some embodiments, PEG is PEG500. In some embodiments, PEG is PEG1000. In some embodiments, PEG is PEG2000. In some embodiments, PEG is PEG3000. In some embodiments, PEG is PEG4000. In some embodiments, PEG is PEG-5000. In some embodiments, the combination of PEG molecules and supporting molecules increases the circulation time of the supporting molecule, shields positive charges, improves the stability of the composition, or results in any combination thereof. In some embodiments, the PEG molecule is bonded to the N-terminus of the supporting molecule. In some embodiments, the PEG molecule is bound to the C-terminus of the supporting molecule.

[0160] In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 9 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 12 further comprises a PEG molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 13 further comprises a PEG molecule.In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 14 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 15 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 16 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 17 further comprises a PEG molecule.In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with SEQ ID NO: 18 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with SEQ ID NO: 19 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 20 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 21 further comprises a PEG molecule.In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 24 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 25 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 26 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 32 further comprises a PEG molecule.In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 33 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 34 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 35 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 36 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 37 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 38 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 39 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 40 further comprises a PEG molecule.In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 42 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 43 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 44 further comprises a PEG molecule.In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 45 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 46 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 47 further comprises a PEG molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 48 further comprises a PEG molecule. In some embodiments, the supporting molecule comprises the amino acid sequence of WSQ and further comprises a PEG molecule.In some embodiments, a supporting molecule comprising an amino acid sequence and a PEG molecule having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, further comprises one or more spacer molecules. In some embodiments, the spacer molecules are as shown herein. In some embodiments, the spacer molecule comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule contains the amino acid sequence of WSQ.

[0161] In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, and A supporting molecule containing an amino acid sequence having sequence identity of 85%, 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%, or at least 99% further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 9 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 12 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 13 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 14 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 15 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 16 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 17 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 18 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 19 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 20 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 21 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 24 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 25 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 26 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 32 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 33 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 34 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 35 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 36 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 37 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 38 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 39 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 42 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 43 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 44 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 45 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 46 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 47 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, and less A supporting molecule comprising an amino acid sequence having 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%, or at least 99% sequence identity, and a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule, further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as shown herein. In some embodiments, the spacer molecule comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises the amino acid sequence of WSQ.

[0162] In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 9 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 12 further comprises a PEG2000 molecule.In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 13 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 14 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 15 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 16 further comprises a PEG2000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 17 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 18 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 19 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 20 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 21 further comprises a PEG2000 molecule.In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 24 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 25 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 26 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 32 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 33 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 34 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 35 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 36 further comprises a PEG2000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 37 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 38 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 39 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further comprises a PEG2000 molecule.In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 42 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 43 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 44 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 45 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to sequence number 46 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 47 further comprises a PEG2000 molecule. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 48 further comprises a PEG2000 molecule.In some embodiments, a supporting molecule comprising an amino acid sequence and a PEG2000 molecule having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises one or more spacer molecules. In some embodiments, the spacer molecules are as shown herein. In some embodiments, the spacer molecule contains the amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule contains the amino acid sequence of WSQ.

[0163] In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs. 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG molecule ligated or conjugated to the N-terminus or C-terminus of the amino acid sequence. In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and less A supporting molecule comprising an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus or C-terminus of the amino acid sequence.In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% A supporting molecule comprising an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% A supporting molecule comprising an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, A supporting molecule comprising an amino acid sequence having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus or C-terminus of the amino acid sequence, further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as shown herein. In some embodiments, the spacer molecule comprises the amino acid sequence of Sequence ID No. 5. In some embodiments, the spacer molecule comprises the amino acid sequence of WSQ.

[0164] In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% A supporting molecule comprising an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 9 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 12 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 13 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 14 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 15 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 16 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 17 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 18 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 19 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 20 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 21 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 24 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a support comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 25 further comprises PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecules linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 26 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 32 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 33 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 34 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 35 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 36 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 37 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 38 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 39 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 42 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 43 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 44 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 45 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 46 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 47 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence. In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 9 A supporting molecule comprising an amino acid sequence having 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the N-terminus of the amino acid sequence, further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as shown herein. In some embodiments, the spacer molecule comprises the amino acid sequence of Sequence ID No. 5. In some embodiments, the spacer molecule comprises the amino acid sequence of WSQ.

[0165] In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs. 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 9 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 12 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 13 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 14 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 15 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 16 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 17 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 18 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a support comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 19 further comprises PEG2000 molecules ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 20 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 21 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 24 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 25 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 26 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 32 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 33 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 34 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 35 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 36 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 37 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 38 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 39 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 42 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 43 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 44 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 45 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 46 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 47 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 48 further comprises a PEG2000 molecule ligated or conjugated to the N-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs. 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, and a PEG2000 molecule linked or conjugated to the N-terminus of the amino acid sequence, further comprises one or more spacer molecules. In some embodiments, the spacer molecules are as shown herein. In some embodiments, the spacer molecule contains the amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule contains the amino acid sequence of WSQ.

[0166] In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% A supporting molecule comprising an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 9 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 12 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 13 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 14 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 15 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 16 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 17 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 18 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 19 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 20 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 21 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 24 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 25 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 26 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 32 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, for A / B 33, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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% A supporting molecule containing an amino acid sequence having at least 99% sequence identity further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule containing an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 34 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 35 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 36 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 37 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 38 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 39 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 42 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 43 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 44 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 45 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 46 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 47 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, for any one of sequence numbers 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 9 A supporting molecule comprising an amino acid sequence having 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated to the C-terminus of the amino acid sequence, further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as shown herein. In some embodiments, the spacer molecule comprises the amino acid sequence of Sequence ID No. 5. In some embodiments, the spacer molecule comprises the amino acid sequence of WSQ.

[0167] In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs. 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 9 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 12 further comprises a PEG2000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 13 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 14 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 15 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 16 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 17 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 18 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 19 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 20 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 21 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 24 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 25 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 26 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 32 further comprises a PEG2000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 33 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 34 further comprises a PEG2000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 35 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 36 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 37 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 38 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 39 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 40 further comprises a PEG2000 molecule linked or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 41 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 42 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 43 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 44 further comprises a PEG2000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 45 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 46 further comprises a PEG2000 molecule ligated or conjugated to the C-terminus of the amino acid sequence.In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 47 further comprises a PEG2000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to SEQ ID NO: 48 further comprises a PEG2000 molecule linked or conjugated to the C-terminus of the amino acid sequence. In some embodiments, a supporting molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs. 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, and a PEG2000 molecule linked or conjugated to the C-terminus of the amino acid sequence, further comprises one or more spacer molecules. In some embodiments, the spacer molecules are as shown herein. In some embodiments, the spacer molecule contains the amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule contains the amino acid sequence of WSQ.

[0168] In some embodiments, a supporting molecule containing any one of the amino acid sequences of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG molecule. In some embodiments, a supporting molecule containing the amino acid sequence of SEQ ID NOs: 9 further comprises a PEG molecule. In some embodiments, a supporting molecule containing the amino acid sequence of SEQ ID NOs: 12 further comprises a PEG molecule. In some embodiments, a supporting molecule containing the amino acid sequence of SEQ ID NOs: 13 further comprises a PEG molecule. In some embodiments, a supporting molecule containing the amino acid sequence of SEQ ID NOs: 14 further comprises a PEG molecule. In some embodiments, a supporting molecule containing the amino acid sequence of SEQ ID NOs: 15 further comprises a PEG molecule. In some embodiments, a supporting molecule containing the amino acid sequence of SEQ ID NOs: 16 further comprises a PEG molecule. In some embodiments, a supporting molecule containing the amino acid sequence of SEQ ID NOs: 17 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 18 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 19 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 20 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 21 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 24 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 25 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 26 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 32 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 33 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 34 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 35 further comprises a PEG molecule.In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 36 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 37 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 38 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 39 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 40 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 41 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 42 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 43 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 44 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 45 further comprises a PEG molecule. In some embodiments, the supporting molecule containing the amino acid sequence of SEQ ID NO: 4...

Claims

1. DNA dendrimers, Targeted portion, and It contains an adapter molecule-cargo polynucleotide complex, A composition in which the DNA dendrimer is linked, conjugated, or associated with the targeting portion and the adapter molecule-cargo polynucleotide complex.

2. The composition according to claim 1, wherein the adapter molecule-cargo polynucleotide complex comprises an adapter molecule and a cargo polynucleotide.

3. The composition according to claim 2, wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding at least one molecule of interest, and the cargo polynucleotide is a nicked cyclic polynucleotide, a linear polynucleotide having closed 5' and 3' ends, a linear polynucleotide having open 5' and 3' ends, or a linear polynucleotide having one open end and one closed end, wherein the one open end and one closed end may be located at either the 5' or 3' end of the polynucleotide.

4. The composition according to claim 2, wherein the cargo polynucleotide further comprises at least one DNA targeting sequence (DTS).

5. The composition according to claim 4, wherein the cargo polynucleotide comprises two or more DTSs.

6. The composition according to claim 2, wherein the cargo polynucleotide further comprises at least one nuclear localization signal sequence (NLS).

7. The composition according to claim 4, wherein the cargo polynucleotide comprises at least one DTS and at least one NLS.

8. The composition according to claim 2, wherein the adapter molecule comprises a DNA dendrimer binding sequence (DBS) and a cargo binding region (CBR).

9. The composition according to claim 8, wherein the adapter is linked to the DNA dendrimer via the DBS, and the DBS includes a nucleic acid sequence complementary to the nucleic acid sequence of the DNA dendrimer.

10. The composition according to claim 8, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to any one of sequence numbers 3, 4, 10, 11, 22, or 23.

11. The composition according to claim 8, wherein the adapter molecule further comprises a tag, such as a tag that can be used as an affinity tag for isolating / purifying the composition complex.

12. The composition according to claim 8, wherein the adapter molecule further comprises at least one NLS, the at least one NLS being located between the DBS and the cargo binding region, or overlapping with the DBS and the cargo binding region.

13. The composition according to claim 12, wherein the at least one NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to any one of SEQ ID NOs: 1, 2, 15, or 43.

14. The composition according to claim 12, wherein the at least one NLS further comprises at least one spacer located before or after the at least one NLS, the at least one spacer comprising polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PGLA), glycine / alanine spacer, glycine / serine spacer, or any combination thereof.

15. The composition according to claim 14, wherein the at least one spacer comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to either SEQ ID NO: 5 or 29.

16. The composition according to claim 8, wherein the adapter molecule further comprises at least one DTS, the at least one DTS being located between the DBS and the CBR, or overlapping with the DBS and the CBR.

17. The composition according to claim 8, wherein the adapter molecule further comprises a cell-permeable peptide sequence (CPP), the CPP being located between the DBS and the CBR, or overlapping with the DBS and the CBR.

18. The composition according to claim 17, wherein the CPP comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to Sequence ID No.

6.

19. The composition according to claim 8, wherein the adapter molecule further comprises at least one cleavage site.

20. The composition according to claim 19, wherein the cutting portion is a val-cit linker.

21. The composition according to claim 8, wherein the adapter molecule further comprises at least one flexible linker selected from the group consisting of polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PGLA), (GG)n, (GGGGGS)n, or (GGGGGA)n, where each n is independently 1 to 5.

22. The composition according to claim 1, further comprising a support molecule.

23. The composition according to claim 22, wherein the supporting molecule is linked to or conjugated to the DNA dendrimer, or is not linked to or conjugated to the DNA dendrimer.

24. The aforementioned support molecule i) Condensing the size of the cargo polynucleotide, the DNA dendrimer, or both, ii) Molecules that assist nuclear delivery, iii) Protect the cargo polynucleotide, the DNA dendrimer, or both from nuclease degradation, and / or iv) The composition according to claim 22, which enhances the transfection efficiency of the composition.

25. The composition according to claim 22, wherein the supporting molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

26. The composition according to claim 22, further comprising a PEG molecule linked or conjugated to the N-terminus or C-terminus of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

27. The composition according to claim 26, wherein the PEG molecule is selected from one of PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

28. The composition according to any one of claims 1 to 27, wherein the targeted portion is selected from the group consisting of an antibody, a naturally occurring ligand or functional derivative thereof for a receptor, a vitamin, a hormone, a low molecular weight mimetic of a naturally occurring ligand, a peptide, a polypeptide, a peptide mimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, any other molecule that specifically binds to a cell surface molecule and induces endocytosis of the binding portion, or any combination thereof.

29. A composition comprising a cargo polynucleotide, a supporting molecule, and a DNA dendrimer linked to a targeting site.

30. The composition according to claim 29, wherein the targeted portion is selected from the group consisting of an antibody, a naturally occurring ligand or functional derivative thereof for a receptor, a vitamin, a hormone, a low molecular weight mimetic of a naturally occurring ligand, a peptide, a polypeptide, a peptide mimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, any other molecule that specifically binds to a cell surface molecule and induces endocytosis of the binding portion, or any combination thereof.

31. The cargo polynucleotide comprises at least one promoter, at least one coding sequence encoding at least one molecule of interest, and optionally a DBS. The cargo polynucleotide is a complete circular polynucleotide (e.g., a plasmid), a nicked circular polynucleotide, a linear polynucleotide having closed 5' and 3' ends, a linear polynucleotide having open 5' and 3' ends, or a linear polynucleotide having one open end and one closed end. The composition according to claim 29, wherein the one open end and the one closed end may be located at either the 5' or 3' of the cargo polynucleotide.

32. The composition according to claim 31, wherein the cargo polynucleotide is a closed cyclic polynucleotide or a cyclic polynucleotide containing a nic.

33. The composition according to claim 29, wherein the cargo polynucleotide further comprises at least one DTS.

34. The composition according to claim 33, wherein the cargo polynucleotide is a cyclic polynucleotide containing a nick, and the DTS is linked to the cargo polynucleotide at a single-strand DNA cleavage site (i.e., at the location of the nick).

35. The composition according to claim 29, wherein the cargo polynucleotide further comprises at least one NLS.

36. The composition according to claim 29, wherein the cargo polynucleotide comprises at least one DTS and at least one NLS.

37. The composition according to claim 31, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with respect to any one of sequence numbers 3, 4, 10, 11, 22, or 23.

38. The composition according to claim 29, wherein the supporting molecule is linked to or conjugated to the DNA dendrimer, or is not linked to or conjugated to the DNA dendrimer.

39. The aforementioned support molecule i) Condensing the size of the cargo polynucleotide, the DNA dendrimer, or both, ii) To assist in nuclear delivery, iii) Protect the cargo polynucleotide, the DNA dendrimer, or both from nuclease degradation, and / or iv) The composition according to claim 29, which enhances the transfection efficiency of the composition.

40. The composition according to claim 29, wherein the presence of the supporting molecule promotes the association of the cargo polynucleotide and the DNA dendrimer with each other.

41. The composition according to claim 29, wherein the supporting molecule has a positive charge.

42. The composition according to claim 29, wherein the supporting molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

43. The composition according to claim 29, further comprising a PEG molecule linked or conjugated to the N-terminus or C-terminus of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% sequence identity with any one of SEQ ID NOs. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

44. The composition according to claim 43, wherein the PEG molecule is selected from one of PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

45. The composition according to any one of claims 1 to 44, wherein the coding sequence of the cargo polynucleotide encodes an antibody, enzyme, protein, miRNA, siRNA, antisense RNA, etc.

46. The composition according to claim 22 or 29, wherein the nitrogen / phosphate (N / P) ratio of the supporting molecule is 0.5 to 10.

47. A pharmaceutical composition comprising the composition according to any one of claims 1 to 46 and a pharmaceutically acceptable carrier.

48. A method for delivering a molecule of interest to the nucleus of a target cell, comprising contacting the target cell with the composition according to any one of claims 1 to 46 or the pharmaceutical composition according to claim 47, wherein the targeted portion binds to the target cell.

49. A method for treating a disease, comprising administering a composition according to any one of claims 1 to 46 or a pharmaceutical composition according to claim 47 to a target to treat the disease, wherein the targeted portion binds to the target cells.

50. A plasmid comprising at least two restriction enzyme recognition sites, at least one promoter, at least one coding sequence encoding at least one molecule of interest, and optionally a plasmid backbone containing a DBS, wherein the plasmid can form cargo polynucleotides having various structures depending on whether the plasmid has one or more 5' ends and / or 3' ends, or neither.

51. A method for producing the composition according to any one of claims 1 to 28, To form a nicked cyclic polynucleotide, a linear polynucleotide having closed 5' and 3' ends, a linear polynucleotide having open 5' and 3' ends, or a linear polynucleotide having one open end and one closed end, the plasmid according to claim 50 is brought into contact with one or more restriction enzymes that cleave at the restriction enzyme recognition site. To form an adapter molecule-cargo polynucleotide complex, the cargo polynucleotide is linked to the adapter molecule, and The method comprising linking the adapter molecule-cargo polynucleotide complex and the targeting moiety to a DNA dendrimer in order to form the composition.

52. A method for producing the composition according to any one of claims 29 to 46, Contacting a plasmid according to claim 50, which includes a DNA dendrimer-binding sequence (DBS) to form a nicked cyclic polynucleotide, a linear polynucleotide having closed 5' and 3' ends, a linear polynucleotide having open 5' and 3' ends, or a linear polynucleotide having one open end and one closed end, with one or more restriction enzymes that cleave at the restriction enzyme recognition site, and The method comprising linking the cargo polynucleotide and the targeting moiety to a DNA dendrimer in order to form the composition.

53. A method for producing the composition according to any one of claims 29 to 46, The method comprising contacting an uncut circular plasmid according to claim 50, a DNA dendrimer linked to a targeting portion, and a support molecule capable of condensing the size of the cargo polynucleotide and the DNA dendrimer, such that the cargo polynucleotide and the DNA dendrimer associate with each other to form the composition.

54. A kit comprising the compositions described in claims 1 to 46, the pharmaceutical composition described in claim 47, the plasmid described in claim 50, or several combinations thereof.

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