Protein-based bacterial nanocompartments

Abstract

Provided herein are compositions, methods, systems and kits for generation of protein nanocompartment encapsulins for concurrent RNA and protein encapsulation within bacterial cells. In particular, provided herein are reagents and methodologies for generation and use of encapsulins providing simultaneous in vivo packaging of specific RNAs and specific proteins of interest.

Description

[0001] PROTEIN-BASED BACTERIAL NANOCOMPARTMENTS

[0002] STATEMENT REGARDING FEDERAL FUNDING

[0003] This invention was made with government support under GM133325 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] SEQUENCE LISTING

[0005] The text of the computer readable sequence listing filed herewith, titled “42908- 601_SEQUENCE_LISTING”, created February 27, 2025, having a file size of 11,194 bytes, is hereby incorporated by reference in its entirety.

[0006] FIELD

[0007] Provided herein are compositions, methods, systems and kits for generation of protein nanocompartment encapsulins for concurrent RNA and protein encapsulation within bacterial cells. In particular, provided herein are reagents and methodologies for generation and use of encapsulins providing simultaneous in vivo packaging of specific RNAs and specific proteins of interest.

[0008] BACKGROUND

[0009] Protein nanocages provide an engineering platform for diverse biotechnological and biomedical applications. Synthetic encapsulation shells that self-assemble in vivo (z.e., encapsulins) may comprise nucleic acid-binding peptides without disrupting native protein packaging mechanisms, and may be purified from bacterial cells. Protein nanocompartment encapsulins are capable of size selective in vivo RNA packaging, may simultaneously load multiple functional RNAs, and support concurrent in vivo packaging of RNAs and proteins of use, for example, in codelivery of therapeutic and other RNAs with proteins to elicit synergistic effects. (Kwon S, Giessen TW. Engineered Protein Nanocages for Concurrent RNA and Protein Packaging In Vivo. ACS Synth Biol. 2022 Oct 21; 11(10):3504-3515.) A limitation of conventional encapsulins is the inability to provide simultaneous in vivo packaging of specific RNAs of interest, in contrast to non-specific loading together with specific proteins of interest. SUMMARY

[0010] Provided herein are compositions, methods, systems and kits for generation of protein nanocompartment encapsulins for concurrent RNA and protein encapsulation within bacterial cells. In particular, provided herein are reagents and methodologies for generation and use of encapsulins providing simultaneous in vivo packaging of specific RNAs and specific proteins of interest. Exemplary, non-limiting compositions, systems, kits and methods are described below.

[0011] In some embodiments, the present invention provides a composition comprising a nanocompartment (e.g., an encapsulin) that contains a protein of interest and a specific RNA of interest (e.g., to the exclusion of other RNA molecules that are not of interest (e.g., with respect to RNA, consisting of or consisting essentially of the specific RNA of interest)). In some embodiments, the composition comprises an RNA binding protein fused to an encapsuling shell protein. In some embodiments, the composition comprises the specific RNA of interest fused to a target RNA sequence that binds to the RNA binding protein. In some embodiments, the nanocompartment composition is generated in vivo by expressing the two fusions so as to cause the specific RNA of interest to be selectively packaged into the nanocompartment. Also provided herein are methods for generating such composition.

[0012] For example, in some embodiments, the present invention provides a method of generating an encapsulin comprising a specific target RNA of interest, comprising: providing an encapsulin shell protein nucleic acid sequence; providing an RNA binding protein nucleic acid sequence; fusing the RNA binding protein nucleic acid sequence to the encapsulin shell protein nucleic acid sequence to generate an RNA binding protein encapsulin shell protein nucleic acid fusion sequence; inserting the RNA binding protein encapsulin shell protein nucleic acid fusion sequence into a first vector under control of a first promoter to generate a first plasmid; providing an RNA sequence that binds the RNA binding protein; providing a specific target RNA sequence of interest; fusing the specific target RNA sequence of interest to the RNA sequence that binds the RNA binding protein to generate a specific target RNA sequence of interest RNA sequence that binds the RNA binding protein fusion sequence; inserting the specific target RNA sequence of interest_RNA sequence that binds the RNA binding protein fusion sequence into a second vector under control of a second promoter to generate a second plasmid; co-transforming a bacterium with the first plasmid and the second plasmid; and auto-inducing the co-transformed bacterium to generate the encapsulin comprising the specific target RNA sequence of interest. In some embodiments, the method comprises an encapsulin shell protein nucleic acid sequence that is a Myxococcus xanthus (MxT3), a Thermotaga maritima (TmTl) or a Quasibacillus thermotolerans (QtT4) encapsulin shell protein nucleic acid sequence. In some embodiments, the RNA binding protein nucleic acid sequence is Escherichia A bacteriophage antiterminator protein N peptide (AN) nucleic acid sequence. In some embodiments, the first vector is a pETDuet vector. In some embodiments, the first promoter is an inducible T7 promoter. In some embodiments, the RNA binding sequence that binds the RNA binding protein is a BoxB RNA sequence. In some embodiments, the second vector is a pCDFDuet vector. In some embodiments, the second promoter is an inducible P70 promoter. In some embodiments, the bacterium is Escherichia coli BL21(DE3). In some embodiments, the cotransforming is electroporation co-transforming. In some embodiments, the method comprises isolating the encapsulin comprising said specific target RNA sequence of interest. In some embodiments, the method comprises purifying the isolated encapsulin comprising the specific target RNA sequence of interest. In some embodiments, the method comprises detecting and / or monitoring at least one property of the encapsulin comprising the specific target RNA sequence of interest wherein the at least one property comprises at least one of nucleic acid sequencing, RNA sequencing, tissue sectioning, immunohistochemistry, optical detection, light intensity detection optical imaging, microscopy, photography and videography.

[0013] In some embodiments, the present invention provides a composition comprising a cotransformed bacterium wherein said co-transformed bacterium is generated by a method comprising: providing an encapsulin shell protein nucleic acid sequence; providing an RNA binding protein nucleic acid sequence; fusing the RNA binding protein nucleic acid sequence to the encapsulin shell protein nucleic acid sequence to generate an RNA binding protein encapsulin shell protein nucleic acid fusion sequence; inserting the RNA binding protein encapsulin shell protein nucleic acid fusion sequence into a first vector under control of a first promoter to generate a first plasmid; providing an RNA sequence that binds the RNA binding protein; providing a specific target RNA sequence of interest; fusing the specific target RNA sequence of interest to the RNA sequence that binds the RNA binding protein to generate a specific target RNA sequence of interest RNA sequence that binds the RNA binding protein fusion sequence; inserting the specific target RNA sequence of interest RNA sequence that binds the RNA binding protein fusion sequence into a second vector under control of a second promoter to generate a second plasmid; co-transforming a bacterium with the first plasmid and thesecond plasmid; and auto-inducing the cotransformed bacterium to generate the encapsulin comprising the specific target RNA sequence of interest.

[0014] In some embodiments, the present invention provides a system, comprising a first plasmid comprising an RNA binding protein encapsulin shell protein nucleic acid fusion sequence in a first vector under control of a first promoter; a second plasmid comprising an RNA target sequence of interest_RNA binding sequence that binds an RNA binding protein fusion sequence in a second vector under control of a second promoter; and a bacterium comprising the first plasmid and the second plasmid.

[0015] In some embodiments, the present invention provides a kit, comprising: an encapsulin shell protein nucleic acid sequence; an RNA binding protein nucleic acid sequence; an RNA sequence that binds the RNA binding protein; a first plasmid comprising a first vector under control of a first promoter; a second plasmid comprising a second vector under control of a second promoter; and a bacterium.

[0016] In some embodiments, the present invention provides an encapsulin comprising a specific target RNA sequence of interest generated by a method of the present claims.

[0017] In some embodiments, the present invention provides use of an encapsulin comprising a specific target RNA sequence of interest generated by a method of the present claims.

[0018] In some embodiments, the present invention provides a cell comprising an encapsulin, the encapsulin comprising a protein / RNA complex that selectively encapsulates a specific RNA of interest into the encapsulin. In some embodiments, the protein / RNA complex comprises an Escherichia X bacteriophage antiterminator protein N peptide (XN) and a BoxB RNA sequence.

[0019] DESCRIPTION OF THE FIGURES

[0020] Figure 1 shows a schematic diagram of an engineered dual cargo-loaded protein nanocage comprising RNA and protein.

[0021] Figure 2A shows size exclusion chromatography of XN_MxT3_Brocolli (Superose 6).

[0022] Figure 2B shows size exclusion chromatography of XN_MxT3_BoxB-Brocolli (Superose 6).

[0023] Figure 2C shows SDS-PAGE analysis of purified ZN_MxT3_ BoxB Broccoli / Broccoli. Figure 3 A shows negative stain electron microscopy of XN_MxT3_BoxB-Brocolli encapsulins. Scale bar: 50 nm.

[0024] Figure 3B shows negative stain electron microscopy of XN_MxT3_Brocolli. Scale bar: 50 nm.

[0025] Figures 4A and 4B show Broccoli fluorescence of cell lysate (Fig. 4A) and purified encapsulin (Fig. 4B).

[0026] Figure 5 shows a TBE urea gel (6%) run with RNA extraction from purified XN_MxT3_ BoxB Broccoli / Broccoli. BoxB Broccoli: 123 bp (without T7 terminator: 76 bp) Broccoli: 104 bp (without T7 terminator: 57bp).

[0027] Figure 6 shows next generation sequencing of RNA extracted from purified XN_MxT3_ BoxB Broccoli / Broccoli

[0028] Figure 7 show next generation sequencing of RNA extracted from XN_MxT3_ Broccoli.

[0029] Figure 8 shows RNA-binding peptides / aptamer combinations and RNA -sequences.

[0030] DEFINITIONS

[0031] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0032] In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.”

[0033] The term “one or more,” as used herein, refers to a number higher than one. For example, the term “one or more” encompasses any of the following: two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, fifty or more, 100 or more, or an even greater number.

[0034] The term “one or more but less than a higher number,” “two or more but less than a higher number,” “three or more but less than a higher number,” “four or more but less than a higher number,” “five or more but less than a higher number,” “six or more but less than a higher number,” “seven or more but less than a higher number,” “eight or more but less than a higher number,” “nine or more but less than a higher number,” “ten or more but less than a higher number,” “eleven or more but less than a higher number,” “twelve or more but less than a higher number,” “thirteen or more but less than a higher number,” “fourteen or more but less than a higher number,” or “fifteen or more but less than a higher number” is not limited to a higher number. For example, the higher number can be 10,000, 1,000, 100, 50, etc. For example, the higher number can be approximately 50 (e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2).

[0035] As used herein, the term a “nucleic acid” or “nucleic acid molecule” generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA. “Nucleic acids” include, without limitation, single- and double-stranded nucleic acids. As used herein, the term “nucleic acid” also includes DNA as described above that contains one or more modified bases. Thus, DNA with a backbone modified for stability or for other reasons is a “nucleic acid.” The term “nucleic acid” as it is used herein embraces such chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA characteristic of viruses and cells, including for example, simple and complex cells.

[0036] The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or “nucleic acid” refer to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides for DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides for RNA are uracil, adenine, cytosine, and guanine.

[0037] The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or of a polypeptide or its precursor. A functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained. The term “portion” when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, “a nucleotide comprising at least a portion of a “gene” may comprise fragments of the gene or the entire gene.

[0038] The term “gene” also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends, e.g., for a distance of about 1 kb on either end, such that the gene corresponds to the length of the full- length mRNA (e.g., comprising coding, regulatory, structural and other sequences). The sequences that are located 5' of the coding region and that are present on the mRNA are referred to as 5' non-translated or untranslated sequences. The sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' nontranslated or 3' untranslated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0039] In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' ends of the sequences that are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, posttranscriptional cleavage, and polyadenylation.

[0040] The term “primer” refers to an oligonucleotide, whether occurring naturally as, e.g., a nucleic acid fragment from a restriction digest, or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid template strand is induced, (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer, and the use of the method.

[0041] The term “probe” refers to an oligonucleotide (e.g., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly, or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification, and isolation of particular nucleic acid sequences (e.g., a “capture probe”). It is contemplated that any probe used in the embodiments of the present disclosure may, in some embodiments, be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the various embodiments of the present disclosure be limited to any particular detection system or label.

[0042] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a sub-portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR’s) regulated under the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a sub-portion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0043] As used herein, the term “information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term “information related to a subject” refers to facts or data pertaining to a subject e.g., a human, plant, or animal). The term “genomic information” refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes, percentage methylation, allele frequencies, RNA expression levels, protein expression, phenotypes correlating to genotypes, etc.

[0044] DETAILED DESCRIPTION

[0045] Provided herein are compositions, methods, systems and kits for generation of protein nanocompartment encapsulins for concurrent RNA and protein encapsulation within bacterial cells. In particular, provided herein are reagents and methodologies for generation and use of encapsulins providing simultaneous in vivo packaging of specific RNAs and specific proteins of interest. Exemplary, non-limiting compositions, systems, kits and methods are described below.

[0046] Protein nanocages are biocompatible with a shell-like structure that supports generation of multifunctional and atomically defined nano-devices by modifying both their inner and outer surfaces through genetic manipulation followed by expression, purification and scale-up. Among naturally occurring protein nanocages, encapsulins provide an engineering platform for applications in medicine, catalysis, and nanotechnology. Encapsulins are self-assembling protein compartments composed of a type of shell protomer possessing the HK97 phage-like fold. They are able to assemble into Tl, T3 and T4 shells that are widely distributed throughout bacterial and archaeal domains. A key feature of encapsulins is the capacity to selectively encapsulate dedicated cargo proteins in vivo. Native cargo proteins may comprise N- or C-terminal domains or targeting peptides (TPs) for efficient cargo loading during shell self-assembly. This feature may be used to package nonnative cargo proteins into the encapsulin shell via simple genetic fusion of TPs to proteins of interest. Engineered encapsulins have utilites as nanoreactors, drug delivery systems, imaging agents and immunotherapies. Genetic and chemical shell modification supports small-molecule conjugation, peptide loop insertion, pore modification and fusion of protein domains to the N- and C-terminus of the encapsulin protomer. Encapsulins capable of triggered reversible disassembly for in vitro cargo loading and stimulus-responsive cargo release have been generated.

[0047] Encapsulation of nucleic acids in vivo provides technologies for RNA regulation and cytosolic sampling, and RNA- and DNA-based therapeutics. However, present technologies are limited by pharmacokinetic properties, difficulty in overcoming cell membranes, susceptibility to nucleases, inherent immunogenicity and rapid clearance from the body. Due to desirable properties and engineerability encapsulins provide an alternate strategy for nucleic acid packaging and delivery. As well, nucleic acid encapsulation in encapsulins supports concurrent sequestration and colocalization of specific proteins and specific nucleic acids for codelivery of distinct types of functional macromolecules acting in either an orthogonal or a synergistic manner.

[0048] In some embodiments, the present invention provides encapsulins as nanoencapsulation platforms for simultaneous specific RNA and specific protein packaging. In some embodiments, to support encapsulin loading with specific RNAs, the present invention provides an encapsulin shell protein MxT3 genetically fused to the kN peptide resulting in kN being displayed on the interior of the assembled protein shell. kN specifically binds a short RNA target sequence termed BoxB. BoxB may be genetically linked to a target RNA of interest to generate efficient self-assembly of target RNA-loaded MxT3 upon co-expression in A. coli.

[0049] In some embodiments, the present invention provides 2 types of specific therapeutic macromolecules ie., a specific RNA macromolecule and a specific protein macromolecule in a single nanocage. In some embodiments, codelivery of a specific RNA and aspecific protein may comprise any combination of therapeutic RNAs and proteins of interest to cells to target, for example, multiple intracellular target classes at the same time. In some embodiments, specific siRNA and antibodies are combined against the same target in a single nanocagebased delivery vehicle for improved suppression due to dual action at both the mRNA and protein level. In some embodiments, release of cargo from protein nanocages is promoted through inducing disassembly of the nanocage. In some embodiments, high cytosolic concentrations of specific RNA, for example specific tRNA, competes for Dps-N binding thereby liberating the delivered RNA cargo. In some embodiments, low pH in the late endosome promotes release of encapsulated nucleic acids. In some embodiments, copackaging of a specific RNA and a specific protein in a single nanocage provides codelivery to each target cell. In some embodiments, concurrent administration of the same therapeutics via separate delivery methods generates heterogeneous populations of singly and doubly targeted cells.

[0050] In some embodiments of the present invention, encapsulin-based protein cages (i.e., Dps Encs) concurrently package specific functional RNAs and specific proteins of interest in vivo. In some embodiments, Dps Encs protect encapsulated specific RNAs from nuclease digestion. In some embodiments, the present invention comprises different sizes of Dps Encs, for example, MxTl (18 nm, luminal volume: ~905 nm3), TmTl (24 nm, ~3054 nm3), MxT3 (32 nm, ~9203 nm3), and QtT4 (42 nm, ~24,429 nm3) spanning over an order of magnitude in correlation with the specific RNA loading capacity per shell. In some embodiments, Dps_MxT3 encapsulins are capable of colocalizing and protecting 2 specific functional RNAs, e.g., the split aptamer Split Broccoli (SB). In some embodiments, the SB binding partner DFHBI-1T may access the shell interior. In some embodiments, access is via the 5-, 3-, or 2-fold pores natively present in MxT3. In some embodiments, Dps_MxT3 specifically sequesters a TP -tagged co-expressed cargo protein while simultaneously packaging a specific RNA.

[0051] In some embodiments, the present invention provides encapsulin-based Dps Encs that co-package specific RNA and specific proteins in vivo in a single step. In some embodiments, in situ assembly of functional nanocages simplifies purification, and avoids nonphy si ologi cal in vitro conditions often required for disassembly and cargo loading of other protein nanocages. In some embodiments, the intrinsic specificity of encapsulins of the present invention provide methods for packaging coexpressed TP -tagged proteins that assemble into highly homogeneous cargo-loaded cages with minimal nonspecific loading. In some embodiments, after purification in vivo eGFP-loaded Dps_MxT3 of the present invention comprise minimal background of non-TP -tagged proteins. In some embodiments, encapsulin-based nanocage designs comprising Dps-N are replaced with RNA-binding peptides and / or domains that bind RNA in a sequence-specific manner. In some embodiments, functional specific RNAs are tagged with a packaging RNA sequence to generate sequence-selective in vivo specific RNA loading. In some embodiments of the present invention, protein nanocages loaded in vivo find use as living therapeutics. In living therapeutics, engineered bacteria are used as a drug delivery modality to reach a target site of interest. At the target, bioactive molecules are continuously produced locally by the bacteria to increase therapeutic effects with minimal systemic side effects. In some embodiments, nanocage systems that do not require in vitro assembly are locally assembled in vivo and released. In some embodiments, Dps Encs comprise cell targeting generated by genetic fusion of cell penetrating peptides or targeting systems to the encapsulin C-terminus exposed on the shell exterior. In some embodiments of the present invention, the Dps Encs provide encapsulins for codelivery of specific therapeutic RNAs and specific proteins that provide homogeneous synergistic effects at a single cell level. In some embodiments, the present invention provides co-delivery of proteinaceous bio-PROTAC effectors and siRNA that simultaneously target one or more specific diseasecausing genes at the mRNA and protein level. In some embodiments, the target comprises antiviral therapy, anticancer therapy, and / or treatment of metabolic diseases. In some embodiments, the present invention comprises co-delivery of Argonaute 2 (Ago2) and siRNA to promote siRNA efficiency. In some embodiments, the present invention comprises codelivery of self-replicating RNA encoding a non-native immunogenic epitope for use as a cancer immunotherapy. In some embodiments, the present invention is combined with, or provided as, living therapeutics to target tumor cells. In some embodiments, the present invention comprises co-delivery of mRNA-based vaccines and protein-based adjuvants.

[0052] EXPERIMENTAL EXAMPLES

[0053] Experimental Methods

[0054] XN_MxT3 sequence protein sequence

[0055] MDAQTRRRERRAEKQAQWKAANGG5GG5PDFLGHAENPLREEEWARLNETVIQV ARRSLVGRRILDIYGPLGAGVQTVPYDEFQGVSPGAVDIVGEQETAMVFTDARKFKT IPIIYKDFLLHWRDIEAARTHNMPLDVSAAAGAAALCAQQEDELIFYGDARLGYEGL MTANGRLTVPLGDWTSPGGGFQAIVEATRKLNEQGHFGPYAVVLSPRLYSQLHRIY EKTGVLEIETIRQLASDGVYQSNRLRGESGVVVSTGRENMDLAVSMDMVAAYLGAS RMNHPFRVLEALLLRIKHPDAICTLEGAGATERR (SEQ ID No : 1) Bold: XN peptide, Italic: linker

[0056] RNA sequences

[0057] Broccoli is an RNA aptamer able to specifically bind the fluorogenic small molecule DFHBI-1T used as a read-out for specific RNA-loading.

[0058] BoxB Broccoli

[0059] GGGCCCGGAAAAAGGGCCCGCGGAGACGGUCGGGUCCAGA UA UUCGUA UCUG UCGAGUAGAGUGUGGGCUCCGCAAGAGCAGAACCCCGGGGGGCCGCGAAACGG GUCUUGAGGGGUUUUUUG (SEQ ID No. : 2)

[0060] Bold: BoxB, Italic: Broccoli, Underlined: T7 terminator GCGGAGACGGUCGGGUCCAGA UA UUCGUA UCUGUCGAGUAGAGUGUGGGCUCCG G4HUAGCAUAACCCCUUGGGGCCUCUAAACGGGUCUUGAGGGGUUUUUUG (SEQ ID NO.: 3)

[0061] Italic: Broccoli, Underlined: T7 terminator

[0062] Cloning, co-expression and purification of LN_MxT3 with BoxB Broccoli / Broccoli

[0063] For plasmid assembly, the XN_MxT3 gene was inserted into the pETDuet vector under the control of an IPTG inducible T7 promoter. For BoxB Broccoli / Broccoli transcription, either BoxB Broccoli or Broccoli was inserted into the pCDFDuet vector under the control of the constitutive P70 promoter. The resulting plasmids were used to cotransformed A. coll BL21(DE3).

[0064] For expression, auto-induction was used in order for the target RNAs (BoxB Broccoli / Broccoli) to be constitutively transcribed first followed by XN_MxT3 expression. 250 mL of fresh ZYM-5052 auto-inducing medium containing appropriate antibiotics was inoculated 1 : 1000 using a 5 mL overnight culture and grown at 30°C for 24 hours. Cells were harvested via centrifugation (5,000 g, 12 min, 4°C).

[0065] For purification, cell pellets were suspended in 5 mL / g (wet cell mass) of Tris buffer (20 mM Tris, 150 mM NaCl, pH 7.5). Lysis components [lysozyme (0.5 mg / mL), Benzonase nuclease (25 units / mL), MgCh (1.5 mM), and SIGMAFAST EDTA-free protease inhibitor cocktail (one tablet per 100 mL)] were added, and cells were incubated on ice for 15 min. Samples were then sonicated at 60% amplitude and a pulse time of 10 s on and 20 s off for 5 min total (Model 120 Sonic Dismembrator, Fisher Scientific). After sonication, samples were clarified by centrifugation (10,000 g, 15 min, 4 °C). To the supernatant, NaCl and PEG-8000 were added to a final concentration of 0.5 M and 10%, respectively, and incubated on ice for 50 min, followed by centrifugation (8,000 g, 10 min, 4 °C). The supernatant was removed, and the pellet was resuspended in 3 mL of Tris buffer (pH 7.5) and filtered using a 0.2 pm syringe filter. The filtered sample was subjected to SEC using a Sephacryl S-500 16 / 60 column and Tris buffer (pH 7.5) at a flow rate of 1 mL / min. Fractions were evaluated using SDS-PAGE and encapsulin-containing fractions were combined, concentrated, and dialyzed using Amicon filter units (100 kDa MWCO) and Tris buffer without NaCl (20 mM Tris, pH 7.5). The low salt sample was then loaded on a HiPrep DEAE FF 16 / 10 Ion Exchange column at a flow rate of 3 mL / min to remove nucleic acid contamination. Encapsulin- containing fractions were concentrated, centrifuged (10,000 g, 10 min, 4 °C), and then subjected to SEC using a Superose 6 10 / 300 GL column and Tris buffer (pH 7.5) at a flow rate of 0.5 mL / min. Purified proteins were stored in Tris buffer (pH 7.5) at 4 °C until further use.

[0066] Broccoli fluorescence experiments Lysate-based experiments

[0067] 50 mL of fresh ZYM-5052 auto-inducing medium containing appropriate antibiotics was inoculated 1 : 1000 using a 5 mL overnight culture of either XN_MxT3_BoxB_Broccoli- or XN_MxT3_Broccoli-expressing cells. Cultures were grown at 30 °C for 24 hours. As a control, 50 mL of E. coli BL21 (DE3) without transformed plasmids was similarly grown in ZYM-5052 auto-inducing medium at 30 °C for 24 h. Harvested cells were resuspended in 5 mL of Tris buffer (pH 7.5) and sonicated at 60% amplitude and a pulse time of 10s on and 20s off for 3 min 30s total. Lysates were clarified by centrifugation (10,000 g, 15 min, 4 °C), and supernatants were filtered using 0.2 pm syringe filters. For each sample, two 100 pL aliquots were prepared. To one of the two aliquots of each sample, 14.7 pL of DFHBI-1T (final concentration: 1 mM) was added and incubated at 37 °C for 40 min, followed by fluorescence measurements using a Synergy Hl plate reader configured with filter sets for green fluorescence (A-x = 472 nm, Xem= 507 nm). For the second aliquot, 1.5 pL of MgCh (final concentration: 1.5 mM) and 1 pL of Benzonase (250 units) were added and incubated overnight at room temperature. The following day, 15 pL of DFHBI-1T (final concentration: 1 mM) was added and incubated at 37 °C for 40 min, followed by fluorescence analysis. For fluorescence measurements, 25 pL of each sample was loaded per well in triplicate into a black flat bottom 384-well plate. Background fluorescence from the control E. coli BL21(DE3) sample without plasmid was subtracted from all samples, yielding final fluorescence intensities.

[0068] Purified encapsulin-based experiments

[0069] Broccoli fluorescence was measured using purified ZN_MxT3_BoxB_Broccoli and XN_MxT3_Broccoli. To 77 pL of each sample containing 3 pmol of RNA-loaded protein shell, 3 pL of DFHBI-1T (final concentration: 300 pM) was added and incubated at 37 °C for 40 min, followed by fluorescence analysis as described above. As background, 75 pL of Tris buffer (pH 7.5) was used and subtracted from the fluorescence signal of each sample. RNA extraction from XN_MxT3_ BoxB Broccoli / Broccoli

[0070] RNA was extracted from purified XN_MxT3_BoxB_Broccoli and XN_MxT3_Broccoli samples via phenol-chloroform extraction, followed by ethanol precipitation. Phenol:chloroform:isoamyl alcohol (25:24: 1, pH 8) was used for phenol-chloroform extraction, and after ethanol precipitation, the desalted nucleic acid extracts were dissolved in TEN buffer (Tris 10 mM, EDTA 1 mM, pH 8) and stored at -80 °C. Quantification of RNA was carried out using a Nanodrop Spectrophotometer from ThermoFisher Scientific, Inc. (USA).

[0071] Constructs and transformation

[0072] Constructs except Dps-N-fused encapsulins (Dps Encs) were acquired from Integrated DNA Technologies (IDT) as E. coli codon-optimized gBlocks. Genes for Dps Encs were obtained through overhang PCR using the native encapsulin genes as templates, adding Dps-N in the process. Genes except Split Broccoli (SB) were cloned into the pETDuet-1 vector, while SB was inserted into pCDFDuet-1 using Gibson Assembly. Top and Bottom of SB separated with a 270 bp spacer were inserted into a single pCDFDuet-1 vector. E. coli BL21 (DE3) cells were transformed with the assembled plasmids via electroporation and were confirmed through Sanger sequencing (Eurofins Scientific).

[0073] Protein Expression and Purification

[0074] Expression experiments were conducted using lysogeny broth (LB) medium supplemented with the appropriate selection marker [100 mg / mL ampicillin (pETDuet-1), 50 mg / mL spectinomycin (pCDFDuet-1), or both], 500 mL of fresh LB medium was inoculated 1 : 100 using a 5 mL overnight culture, grown at 37 °C to OD600 of 0.4-0.5, and then induced with 0.1 mM IPTG. After induction, cultures were grown at 30 °C overnight for ca. 18 h and harvested via centrifugation (8000 g, 10 min, 4 °C). The resulting cell pellets were frozen and stored at -20 °C until further use.

[0075] Frozen cell pellets were resuspended in 5 mL / g (wet cell mass) of Tris buffer (20 mM Tris, 150 mM NaCl, pH 7.5). Lysis components [lysozyme (0.5 mg / mL), Benzonase nuclease (25 units / mL), MgCh (1.5 mM), and SIGMAFAST EDTA-free protease inhibitor cocktail (one tablet per 100 mL)] were added, and cells were incubated on ice for 15 min. Samples were then sonicated at 55% amplitude and a pulse time of 10 s on and 20 s off for 5 min total (Model 120 Sonic Dismembrator, Fisher Scientific). After sonication, samples were clarified by centrifugation (10,000 g, 15 min, 4 °C). NaCl and PEG-8000 were added to the supernatant to a final concentration of 0.5 M and 10%, respectively, and incubated on ice for 40 min, followed by centrifugation (8000 g, 10 min, 4 °C). The supernatant was removed, and the pellet was resuspended in 3 mL of Tris buffer (pH 7.5) and filtered using a 0.2 pm syringe filter.

[0076] The filtered sample was subjected to SEC using a Sephacryl S-500 16 / 60 column and Tris buffer (pH 7.5) at a flow rate of 1 mL / min. Fractions were evaluated using SDS-PAGE and encapsulin-containing fractions were combined, concentrated, and dialyzed using Amicon filter units (100 kDa MWCO) and Tris buffer without NaCl (20 mM Tris, pH 7.5). The low salt sample was then loaded on a HiPrep DEAE FF 16 / 10 Ion Exchange column at a flow rate of 3 mL / min to remove nucleic acid contamination. Encapsulin-containing fractions were concentrated, centrifuged (10,000 g, 10 min, 4 °C), and then subjected to SEC using a Superose 6 10 / 300 GL column and Tris buffer (pH 7.5) at a flow rate of 0.5 mL / min. Purified proteins were stored in Tris buffer (pH 7.5) at 4 °C until further use.

[0077] Transmission Electron Microscopy

[0078] Encapsulin samples for negative-stain TEM were diluted to 0.15 mg / mL in Tris buffer (pH 7.5). Gold grids (200-mesh coated with a Formvar-carbon film, EMS) were made hydrophilic by glow discharge at 5 mA for 60 s (easiGlow, PELCO). 4 pL of sample was added to the grid and incubated for 1 min, wicked with filter paper, and washed with 0.75% uranyl formate before staining with 0.75% uranyl formate for 1 min. Stain was removed using filter paper, and the grid was dried for at least 20 min before imaging. TEM micrographs were captured using a Morgagni transmission electron microscope at 100 keV at the University of Michigan Life Sciences Institute.

[0079] DLS Analysis

[0080] Sizing and poly dispersity measurements were condcuted on an Uncle instrument (Unchained Labs) at 15 °C in triplicate. Encapsulin samples were adjusted to 0.5 mg / mL of monomer using Tris buffer (pH 7.5), centrifuged (10,000 g, 10 min, 4 °C), and then immediately analyzed via DLS.

[0081] RNA Extraction

[0082] RNA was extracted from purified Dps Enc samples via phenol-chloroform extraction, followed by ethanol precipitation. Phenol: chloroform: isoamyl alcohol (25:24: 1, pH 8) was used for phenol-chloroform extraction. After ethanol precipitation, the desalted nucleic acid extracts were dissolved in TEN buffer (Tris 10 mM, EDTA 1 mM, pH 8) and stored at -80 °C. E. coli total RNA was purchased from ThermoFisher Scientific (AM7940). Quantification of RNA was conducted using a Nanodrop Spectrophotometer from ThermoFisher Scientific, Inc. (USA).

[0083] Nuclease Challenge of Extracted RNA and RNA-Loaded Dps Encs

[0084] DNase (ThermoFisher Scientific, EN0521), RNase (ThermoFisher Scientific, EN0531), and Benzonase (Sigma Aldrich, E8263) were used for nuclease challenge experiments of extracted RNA and RNA-loaded Dps Encs. For nuclease incubation experiments, 1 pL (1.5 pL) of DNase, RNase, and Benzonase was added to 9 pL (13.5 pL) of extracted RNA (RNA-loaded Dps_Encs) samples (final concentration: 10, 5, and 25 U / mL, respectively), followed by 30 min incubation at 37 °C.

[0085] Native Gel Electrophoresis

[0086] Native Agarose Gel Electrophoresis

[0087] 3% native agarose gels were used to determine the nucleic acid encapsulation capacity of Dps Encs and to demonstrate the nuclease resistance of Dps Encs shell. U TAE buffer was used to make agarose gels. The amount of Dps Enc loaded per lane was adjusted for each Dps Enc encapsulin so as to visualize nucleic acid signal after GelRed staining, while corresponding Nat Encs were loaded at equal amounts for direct comparison. Per lane, 15 pL of sample was loaded with an additional 2 pL of 70% (v / v) aqueous glycerol. Gel electrophoresis was carried out using 1 x TAE buffer at a constant voltage of 90 V for 35 min. Gels were first stained with GelRed to visualize nucleic acids and then stained with Coomassie blue to visualize proteins. Nucleic acid encapsulation capacity of Nat Encs and Dps Encs was compared via gel densitometry, first, normalizing the intensity of nucleic acid bands by their corresponding protein band (N / P). Then, N / P values of Dps Encs were normalized by N / P values of the corresponding Nat Encs for comparison. Band intensities of nucleic acid and protein were measured using Fiji / ImageJ v2.1.0 / 1.53c.

[0088] 2% native agarose gels were used for nucleic acid extracted from purified Dps Encs. The extracted nucleic acid was incubated with nucleases and loaded on the gels along with undigested nucleic acid for comparison. Per lane, 10 pL of sample was loaded with an additional 10 pL of 2 RNA loading buffer. Gel electrophoresis was carried out in 1 x TAE buffer at a constant voltage of 125 V for 25-30 min. The gel was stained with GelRed to visualize nucleic acids. Native Polyacrylamide Gel Electrophoresis

[0089] Native polyacrylamide gel electrophoresis analyses were conducted in an Invitrogen XCell SureLock using NativePAGE 3 to 12% bis-tris mini protein gels with 1 x NativePAGE Anode Buffer and l x NativePAGE Cathode Buffer. 850 fmol of encapsulin shells was loaded per lane to maintain equivalent amounts of shells across all lanes for comparative analysis. The number of shells was calculated as follows: # of shells = [protein concentration (mg / mL)] / [protomer v (g / mol) x # of protomer per shell]. Protein concentration was measured by A280 using Nanodrop, and absorption coefficient was calculated for each Dps Enc based on the protomer sequence. Native PAGE gels were run at a constant voltage of 150 V for 1 h, followed by an additional 1 h run at 250 V at 4 °C. Gels were then stained, first with GelRed for nucleic acid visualization and then with Coomassie blue for protein detection. For eGFP_MxTP_Dps_MxT3, the gel was first exposed to UV light for eGFP visualization before staining with GelRed and Coomassie blue.

[0090] To quantify and compare the amount of RNA loaded in each Dps Enc encapsulin, gel densitometry of GelRed-stained gels was carried out using Fiji / ImageJ v2.1.0 / 1 ,53c. Pixel intensities of bands were background subtracted, yielding final overall intensities per band for comparisons.

[0091] Split Broccoli (SB) Fluorescence Experiments

[0092] 50 mL of fresh LB medium containing appropriate antibiotic(s) was inoculated using an overnight 1 mL culture of either SB-, SB + Nat_MxT3-, or SB + Dps_MxT3 -expressing cells. Cultures were grown at 37 °C to an OD600 of 0.4-0.5, then induced with 0.2 mM IPTG, and further grown for 5 h at 30 °C. As a control, 50 mL of E.coli BL21 (DE3) without transformed plasmids was similarly grown in LB at 30 °C for 5 h. Harvested cells were resuspended in 5 mL of Tris buffer (pH 7.5) and sonicated at 55% amplitude and a pulse time of 10 s on and 20 s off for 3 min 30 s total. Lysates were clarified by centrifugation (10,000 g, 15 min, 4 °C), and supernatants were filtered using 0.2 pm syringe filters. For each sample, two 100 pL aliquots were prepared. 14.7 pL of DFHBI-1T (final concentration: 1 mM) was added to one of the 2 aliquots of each sample, and incubated at 37 °C for 40 min, followed by fluorescence measurements using a Synergy Hl plate reader configured with filter sets for green fluorescence (L-x = 472 nm, L-m = 507 nm). For the other aliquot, 1 pL of MgCh (final concentration: 1.5 mM) and 1 pL of Benzonase (250 units) were added and incubated overnight at room temperature. The following day, 15 pL of DFHBI-1T (final concentration: 1 mM) was added and incubated at 37 °C for 40 min, followed by fluorescence analysis. For fluorescence measurements, 25 pL of each sample was loaded per well in triplicate into a black-flat bottom 384-well plate. Background fluorescence from the control E.coli BL21(DE3) sample without plasmid was subtracted from all samples, yielding final fluorescence intensities.

[0093] SB fluorescence was also measured using purified Nat_MxT3, Dps_MxT3, SB + Nat_MxT3, and SB + Dps_MxT3 samples. 2 pL of DFHBI-1T (final concentration: 200 pM) was added to 75 pL of each sample containing 5 pmol of capsid, and incubated at 37 °C for 40 min, followed by fluorescence analysis as above. As background, 75 pL of Tris buffer (pH 7.5) was used and subtracted from the fluorescence signal of each sample.

[0094] Protein Identification

[0095] In-gel digestion with trypsin was performed using a robot (ProGest, DigiLab) with the following protocol: (a) washed with 25 mM ammonium bicarbonate, followed by acetonitrile, (b) reduced with 10 mM dithiothreitol at 60 °C, (c) alkylated with 50 mM iodoacetamide at RT, (d) digested with sequencing grade trypsin (Promega) at 37 °C for 4 h, and (e) quenched with formic acid, and the supernatant was analyzed directly without further processing.

[0096] Half of each digested sample was analyzed by nano LC-MS / MS with a Waters M- Class HPLC system interfaced to a ThermoFisher Fusion Lumos mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75 pm analytical column at 350 nL / min; both columns were packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in a data-dependent mode, with the Orbitrap operating at 60,000 FWHM and 15,000 FWHM for MS and MS / MS, respectively. The instrument was run with a 3 s cycle for MS and MS / MS.

[0097] Encapsulin-Based In Vivo RNA Encapsulation System

[0098] Three naturally occurring assembly states of encapsuling (Tl, T3, and T4) provided a range of RNA packaging nanocages with different dimensions, luminal volume and charge. The three established encapsulin systems from Thermotoga maritima (TmTl), Myxococcus xanthus (MxT3), and Quasibacillus thermotolerans (QtT4) were used as engineering scaffolds

[0099] To provide encapsulins with the ability to bind and encapsulate nucleic acid, genetically fused the Escherichia coli Dps-N peptide (MSTAKLVKSKATN) originating from the DNA-binding protein from starved cells (Dps) was fused to the N-terminus of the encapsulin protomer via a flexible six-residue linker (GGSGGS), yielding the Dps Enc fusion constructs. In some embodiments, Dps-N consists of the 13 N-terminal residues of Dps and includes 3 positively charged lysines and is able to bind to both DNA and RNA. In some embodiments, binding arise from the electrostatic interaction of the positively charged lysine residues with the negatively charged DNA / RNA phosphate backbone. Dps-N fusion constructs provide broad specificity in use as a nucleic acid-binding peptide. In assembled Tm, Mx, and Qt encapsulins, the N-termini of all protomers are pointed toward the shell interior. In engineered Dps Encs, 3 additional positive charges per protomer were introduced to the encapsulin lumen resulting in overall charge increases of +180 (Tl), +540 (T3), and +720 (T4) fusion constructs. This increased positive charge of the shell interior serves to five the encapsulation of RNA during shell self-assembly. In some embodiments, Dps Encs provide in vivo packaging of native or overexpressed RNAs while minimizing concurrent DNA packaging. Due to the relatively small size of encapsulins and that few if any DNA molecules small enough to be encapsulated inside encapsulin shells are present inside cells.

[0100] Dps Encs and unmodified native Tm, Mx, and Qt controls (Nat Encs) were produced in E. coli and purified through a combination of polyethylene glycol (PEG) precipitation, ion exchange chromatography (IEC), and size-exclusion chromatography (SEC). SDS-PAGE analysis of purified Dps Encs and Nat Encs was used to confirm sample homogeneity. Further analyses using negative-stain transmission electron microscopy (TEM), dynamic light scattering (DLS), and analytical SEC indicated that all Dps Encs formed stable shells with similar size and appearance compared to the corresponding Nat Encs.

[0101] In Vivo Nucleic Acid Encapsulation and Resistance toward Nuclease Digestion

[0102] Native agarose gel electrophoresis with both protein and nucleic acid staining shows that all purified Dps Encs contained significantly more nucleic acid than the respective Nat Enc controls. To exclude nonspecific nucleic acid binding to the outside of encapsulin shells, Benzonase treatments and IEC are used during purifications. Dps MxTl and Dps_MxT3 have the highest relative nucleic acid packaging capacity with 20- and 11 -fold increases in signal when compared to their native forms. Dps TmTl and Dps_QtT4 yield moderate signal increases of 2.4- and 4-fold, respectively.

[0103] Purified Dps Encs were treated with DNase, RNase, or Benzonase to protect encapsulated nucleic acid from nuclease digestion due to the physical sequestration of nucleic acid inside a protein barrier. Encapsulin shells possess small pores at the 5-, 3-, and 2-fold symmetry axes with diameters ranging from 2 to 7 A that is too small to allow nuclease access to the shell interior. Once formed encapsulin shells are stable may only be disassembled under harsh nonphy si ologi cal conditions, thus making them suitablecontainers for protecting labile nucleic acids.

[0104] Analysis of Encapsulated Nucleic Acid Content and Size-Selective RNA Packaging

[0105] To identify the type and size distribution of encapsulated nucleic acid, total nucleic acid contents from purified Dps Encs were subjected to differential nuclease treatment using DNase, Exposure to DNase has no effect on the extracted nucleic acid samples, while RNase and Benzonase treatment results in complete digestion indicating that nucleic acid encapsulated in Dps Encs is RNA. In some embodiments, Dps Encs encapsulate specific RNAs in a size- selective manner within a relevant size range for, for example, delivery of specific siRNAs between 20 and 25 nt in length. Specific RNA packaging capacity per shell increases with shell diameter. Larger Dps Encs provide larger volumes for RNA packaging and contain more Dps-N-fused protomers that result in an increased number of positive luminal charges.

[0106] Simultaneous In Vivo Packaging of 2 Functional RNAs

[0107] In some embodiments, multiple nonendogenous functional RNAs are copackaged at the same time and protected from nucleases. The split fluorogenic aptamer Split Broccoli (SB) was used to coexpress its two RNAs— Top (97 nt) and Bottom (153 nt)— together with Dps_MxT3 Dps_MxT3 was used due to its a high upper size limit for RNA, low background, and high loading capacity. Benzonase was added to cleared cell lysates from cells expressing SB alone, SB + Nat_MxT3, or SB + Dps_MxT3 to remove free SB, to test the protective role of encapsulin shells, and to allow the detection of encapsulated SB via addition of the smallmolecule SB binding partner DFHBI-1T, thereby yielding a fluorescence readout. The highest SB fluorescence signal was observed for SB + Dps_MxT3, indicating that Dps_MxT3 packages both SB RNAs, protected them from nuclease digestion, and allows access of the small-molecule DFHBI-1T to the shell interior. Nat_MxT3 and Dps_MxT3 were purified alone or from cells coexpressing SB, followed by incubation with DFHBI-1T to generate higher SB fluorescence signals for Dps_MxT3, confirming our initial results.

[0108] Concurrent RNA and Protein Packaging In Vivo

[0109] The ability of Dps Encs to encapsulate RNA was combined with encapsulins’ native capacity for specific protein encapsulation. The Mx targeting peptide (MxTP, PEKRLTVGSLRR) with a flexible six-residue linker (GGSGGS) was genetically fused to the C -terminus of eGFP and cloned immediately upstream of the Dps_MxT3 gene for coexpression SDS-PAGE analysis of purified Dps_MxT3 confirmed the successful in vivo loading and copurification of MxTP -tagged eGFP. Protein cargo loading for Dps_MxT3 was comparable with Nat_MxT3. Negative-stain TEM analysis confirmed that eGFP-loaded Dps_MxT3 particles form homogeneous shells similar in size and appearance to Nat_MxT3. Concurrent loading of both eGFP and RNA with native PAGE analysis on purified eGFP- loaded Dps_MxT3 shells confirms that Nat_MxT3, eGFP-loaded Dps_MxT3 exhibit higher RNA signal intensity and eGFP fluorescence in the high-molecular-weight encapsulin band. Coelution of RNA and eGFP signals confirmsl copackaging of RNA and a specific heterologously expressed protein in vivo.

[0110] EXAMPLE 1

[0111] Encapsulin-containing fractions of the present invention were concentrated, centrifuged (10,000 g, 10 min, 4 °C), and then subjected to size exclusion chromatography (SEC) using a Superose 6 10 / 300 GL column and Tris buffer (pH 7.5) at a flow rate of 0.5 mL / min. As shown in Figure 2A, XN_MxT3_Brocolli (Superose 6) (2A) showed a single main peak with high 260 / 280 nm ratio at an elution volume (8.5 mL) corresponding to a T=3 encapsulin shell. As shown in SEC of XN_MxT3_BoxB-Brocolli (Superose 6) (2B) showed a single main peak with high 260 / 280 nm ratio at an elution volume (8.5 mL) corresponding to a T=3 encapsulin shell. Figure 2C shows SDS-PAGE analysis of purified XN_MxT3_ BoxB Broccoli / Broccoli (2C). These data indicate that Broccoli loading via BoxB / XN interaction does not compromise T=3 shell formation and yields clean and assembled encapsulin shells.

[0112] EXAMPLE 2

[0113] Figure 3 shows negative stain electron microscopy of XN_MxT3_BoxB-Brocolli encapsulins (3 A) and XN_MxT3_Brocolli (3B). Encapsulin samples for negative-stain TEM were diluted to 0.15 mg / mL in Tris buffer (pH 7.5). Gold grids (200-mesh coated with a Formvar-carbon film, EMS) were made hydrophilic by glow discharge at 5 mA for 60 s. 4 pL of sample was added to the grid and incubated for 1 min, wicked with filter paper, and washed with 0.75% uranyl formate before staining with 0.75% uranyl formate for 1 min. Stain was removed using filter paper, and the grid was dried for at least 20 min before imaging. TEM micrographs were captured using a Morgagni transmission electron microscope at 100 keV. Figure 3 A) shows BoxB Broccoli-loaded XN_MxT3 shells highlighting their homogeneity and structural integrity. Figure 3B) shows XN_MxT3 shells without loaded Broccoli.

[0114] EXAMPLE 3

[0115] Figure 4A shows Broccoli fluorescence of cell lysate. More fluorescence based on DFHBI-IT-Broccoli binding is observed for XN_MxT3_BoxB_Broccoli-expressing cells after nuclease treatment. These results indicate that Broccoli is more protected from digestion in this sample due to more efficient loading arising from the presence of BoxB. Figure 4B shows Broccoli fluorescence of purified encapsuling. Greater fluorescence based on DFHBI- IT-Broccoli binding is observed for the XN_MxT3_BoxB_Broccoli sample. These results indicate that more Broccoli co-purifies with the sample due to the improved specific loading of ZN_MxT3 with the co-transcribed BoxB Broccoli RNA.

[0116] EXAMPLE 4

[0117] Figure 5 shows a TBE urea gel (6%) with RNA extraction from purified ZN_MxT3_ BoxB Broccoli / Broccoli. RNA extracted from purified ZN_MxT3_BoxB_Broccoli encapsulins contains more target RNA compared to the negative control with BoxB fused to the target RNA.

[0118] EXAMPLE 5

[0119] Figure 6 shows next generation sequencing of RNA extracted from purified XN_MxT3_ BoxB Broccoli / Broccoli. These results indicate that target RNA (BoxB Broccoli) accounts for over 98% of total encapsulated RNA (1320 / (1320+16.9+4.99)* 100 = 98.4%).

[0120] EXAMPLE 6

[0121] Figure 7 shows next generation sequencing of RNA extracted from +N_MxT3_ Broccoli. No target RNA was detected in the negative control lacking BoxB.

[0122] EXAMPLE 7

[0123] Figure 8 shows alternate RNA-binding peptides / aptamer combinations and RNA- sequences in some embodiments of the present invention. References:

[0124] [1] Austin, R. J. et al. Designed arginine-rich RNA-binding peptides with picomolar affinity. J Am Chem Soc 124, 10966-10967 (2002);

[0125] [2], J Karn, C. et al. RNA binding by the tat and rev proteins of HIV-I. Biochimie. 73(1), 9- 16 (1991);

[0126] [3] Tok, J.D. et al. Binding of a cyclic BIV P-tat peptide with its TAR RNA construct.

[0127] Bioorganic & Medicinal Chemistry Letters. 11(1), 43-46 (2001);

[0128] [4], Hyun, S. et al. An RNA aptamer that selectively recognizes symmetric dimethylation of arginine 8 in the histone H3N-terminal peptide. Nucleic Acid Therapeutics vol. 21 157-163 (2011).

[0129] INCORPORATION BY REFERENCE

[0130] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

Claims

CLAIMSWe claim:

1. A method of generating a nanocompartment comprising a specific target RNA of interest, comprising, expressing in a host cell: i) an RNA binding protein encapsulin shell protein nucleic acid fusion sequence; and ii) a specific target RNA sequence of interest_RNA sequence that binds said RNA binding protein fusion sequence; under conditions that generate a nanocompartment comprising the specific target RNA sequence of interest.

2. The method of claim 1, wherein said RNA binding protein encapsulin shell protein nucleic acid fusion sequence is expressed in a first vector and said specific target RNA sequence of interest RNA sequence that binds said RNA binding protein fusion sequence is expressed in a second vector.

3. The method of claim 1, wherein said encapsulin shell protein nucleic acid sequence is a Myxococcus xanthus (MxT3), a Thermotaga maritima (TmTl) or a Quasibacillus thermotolerans (QtT4) encapsulin shell protein nucleic acid sequence.

4. The method of claim 1, wherein said RNA binding protein nucleic acid sequence is Escherichia bacteriophage antiterminator protein N peptide ( N) nucleic acid sequence.

5. The method of claim 2, wherein said first vector is a pETDuet vector.

6. The method of claim 2, wherein said first vector comprises an inducible T7 promoter.

7. The method of claim 1, wherein said RNA binding sequence that binds said RNA binding protein is a BoxB RNA sequence.

8. The method of claim 2, wherein said second vector is a pCDFDuet vector.

9. The method of claim 2, wherein said second vector comprises an inducible P70 promoter.

10. The method of claim 1, wherein said host cell is Escherichia coli BL21(DE3).

11. The method of claim 2, wherein said expressing comprises co-transforming said first vector and said second vector into said host cell.

12. The method of claim 11, wherein said co-transforming comprises electroporation.

13. The method of claim 1, further comprising the step of isolating said nanocompartment comprising said specific target RNA sequence of interest.

14. The method of claim 13, further comprising detecting and / or monitoring at least one property of said nanocompartment comprising said specific target RNA sequence of interest wherein said at least one property comprises at least one of nucleic acid sequencing, RNA sequencing, tissue sectioning, immunohistochemistry, optical detection, light intensity detection optical imaging, microscopy, photography, and videography.

15. A composition comprising a host cell generated by a method comprising: coexpressing in the host cell: i) an RNA binding protein encapsulin shell protein nucleic acid fusion sequence; and ii) a specific target RNA sequence of interest RNA sequence that binds said RNA binding protein fusion sequence; under conditions that generate a nanocompartment comprising the specific target RNA sequence of interest.

16. A system, comprising: a) a first plasmid comprising an RNA binding protein encapsulin shell protein nucleic acid fusion sequence in a first vector under control of a first promoter; b) a second plasmid comprising an RNA target sequence of interest RNA binding sequence that binds an RNA binding protein fusion sequence in a second vector under control of a second promoter; and g) a host cell comprising said first plasmid and said second plasmid.

17. A kit, comprising: a) an encapsulin shell protein nucleic acid sequence; b) an RNA binding protein nucleic acid sequence; c) an RNA sequence that binds said RNA binding protein; d) a first plasmid comprising a first vector under control of a first promoter; e) a second plasmid comprising a second vector under control of a second promoter; and f) optionally, a host cell.

18. A nanocompartment comprising a specific target RNA sequence of interest generated by a method of any of claims of 1-14.

19. Use of nanocompartment comprising a specific target RNA sequence of interest generated by a method of any of claims 1-14.

20. Use of a composition, system, or kit of any of claims 15-17.

21. A cell comprising an encapsulin, said encapsulin comprising a protein / RNA complex that selectively encapsulates a specific RNA of interest into said encapsulin.

22. The cell of claim 21, wherein said protein / RNA complex comprises an Escherichia A bacteriophage antiterminator protein N peptide (AN) and a BoxB RNA sequence.

Images