Photoreactive compositions and methods for producing circular polyribonucleotides

Abstract

The present disclosure relates, generally, to compositions and methods for producing, purifying, and using circular RNA.

Description

PHOTOREACTIVE COMPOSITIONS AND METHODS FOR PRODUCING CIRCULAR POLYRIBONUCLEOTIDESBackgroundThere is a need for methods of producing, purifying, and using circular polyribonucleotides.Summary of the InventionThe disclosure provides compositions and methods for producing, purifying, and using circular RNA.In one aspect, featured is a complex that includes (a) a linear polyribonucleotide including a first annealing region and a second annealing region; and (b) a bridging agent having a first photoreactive crosslinking agent and a second photoreactive crosslinking agent. The bridging agent is configured to bind to the first annealing region and the second annealing region on the linear polyribonucleotide. Each photoreactive crosslinking agent is configured to form a covalent adduct with the linear polyribonucleotide upon irradiation with light.In some embodiments, the bridging agent is or includes a polynucleotide. In some embodiments, the polynucleotide includes an aptamer.In some embodiments, the polynucleotide includes a third annealing region configured to hybridize to the first annealing region on the linear polyribonucleotide and a fourth annealing region configured to hybridize to the second annealing region on the linear polyribonucleotide.In some embodiments, the first annealing region and the second annealing region are each from 5 to 200 (e.g., 6 to 200, 7 to 200, 8 to 200, 9 to 200, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 30, 6 to 30, 7 to 30, 8 to 30, or 9 to 30, e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) ribonucleotides.In some embodiments, the first annealing region has at least 50% (e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%) complementarity to the third annealing region, and the second annealing region has at least 50% (e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%) complementarity to the fourth annealing region. In some embodiments, the first annealing region has at least 80% complementarity to the third annealing region, and the second annealing region has at least 80% complementarity to the fourth annealing region. In some embodiments, the first annealing region has at least 90% complementarity to the third annealing region, and the second annealing region has at least 90% complementarity to the fourth annealing region.In some embodiments, the first annealing region has zero or one mismatch with the third annealing region, and the second annealing region has zero or one mismatch with the fourth annealing region.In some embodiments, the third annealing region includes the first photoreactive crosslinking agent and the fourth annealing region includes the second photoreactive crosslinking agent.In some embodiments, the first photoreactive crosslinking agent and / or the second photoreactive crosslinking agent is a photoreactive nucleotide analog.In some embodiments, the photoreactive nucleotide analog crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the linear polyribonucleotide upon photoirradiation.In some embodiments, the photoreactive crosslinking agent includes 5-bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-Th iouridine, a diazirine, a phenylselenide, a furan, or an abasic site.In some embodiments, the carbazole is 3-cyanovinylcarbazole, 4-methylpyranocarbazole, or pyranocarbazole.In some embodiments, the coumarin is 7-hydroxycoumarin.In some embodiments, the first crosslinking agent includes a first photoreactive nucleotide analog and the second crosslinking agent includes a second photoreactive nucleotide analog.In some embodiments, the polynucleotide includes a plurality of photoreactive crosslinking agents.In some embodiments, the polynucleotide includes a plurality of photoreactive nucleotide analogs.In some embodiments, at least one of the plurality of photoreactive nucleotide analogs is attached to the 3’ end of the polynucleotide.In some embodiments, at least one of the plurality of photoreactive nucleotide analogs is attached to the 5’ end of the polynucleotide.In some embodiments, at least one of the plurality of photoreactive nucleotides analogs is located at an internal position within the polynucleotide.In some embodiments, the polynucleotide includes 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides between each of the photoreactive nucleotide analogs.In some embodiments, the 3’ end of the polynucleotide has at least 1 (e.g., at least (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide from the nearest photoreactive nucleotide analog.In some embodiments, the 3’ end of the polynucleotide has from 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the nearest photoreactive nucleotide analog.In some embodiments, the 5’ end of the polynucleotide has at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide from the nearest photoreactive nucleotide analog.In some embodiments, the 5’ end of the polynucleotide has from 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the nearest photoreactive nucleotide analog.In some embodiments, the polynucleotide has a structure A-P1 -B-P2-C, wherein each of A and B is optionally absent or polynucleotide of at least 1 nucleotide (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more), C is optionally absent or is a linker, and each of P1 and P2 are, independently, a photoreactive crosslinking agent (e.g., a photoreactive nucleotide analog). For example, in some embodiments, A is absent or has at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide. In some embodiments, B is absent or has at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide. Insome embodiments, C is a nucleotide. In some embodiments, C is a polynucleotide having at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide. In some embodiments, A includes the third annealing region. In some embodiments, C includes the fourth annealing region.In some embodiments, each of the photoreactive nucleotide analogs crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the linear polyribonucleotide upon photoirradiation.In some embodiments, the polynucleotide is single stranded.In some embodiments, the polynucleotide is double stranded and includes a first strand and a second strand. In some embodiments, the first strand of the double stranded polynucleotide includes the third annealing region and the fourth annealing region, and the second strand of the polynucleotide includes the first photoreactive crosslinking agent and the second photoreactive crosslinking agent.In some embodiments, the first strand of the polynucleotide is longer than the second strand of the polynucleotide.In some embodiments, the first strand of the polynucleotide includes a 5’ overhang and a 3’ overhang.In some embodiments, the first strand anneals to the linear polyribonucleotide and the second strand is configured to fill a gap of the linear polyribonucleotide formed by annealing of the first strand.In some embodiments, the linear polyribonucleotide includes two nicks in the phosphodiester backbone prior to photoirradiation.In some embodiments, the polynucleotide includes a 5’ terminal photoreactive crosslinking agent and a 3’ terminal crosslinking agent.In some embodiments, the linear polyribonucleotide includes a 5’ terminal uridine and a 3’ terminal uridine.In some embodiments, the first photoreactive crosslinking agent and the second photoreactive crosslinking agent is each, independently, a uridine or thymidine.In some embodiments, the first photoreactive crosslinking agent and the second photoreactive crosslinking agent each crosslinks to a ribonucleotide at an adjacent position in the linear polyribonucleotide upon photoirradiation.In some embodiments, the polynucleotide is linear.In some embodiments, the polynucleotide includes a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.In some embodiments, the polynucleotide is from 10 to 2,000 (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11 ,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000) nucleotides in length.In some embodiments, the bridging agent includes a structure PN1-L-PN2, wherein PN1 includes a first polynucleotide, PN2 includes a second polynucleotide, and L is a linker of one or more atoms.In some embodiments, the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.In some embodiments of any of the above embodiments, the polynucleotide has a structure A-P1 -B-P2-C, wherein each of A and B is optionally absent or polynucleotide of at least 1 nucleotide (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more), C is optionally absent or is a linker, and each of P1 and P2 are, independently, a photoreactive crosslinking agent (e.g., a photoreactive nucleotide analog). For example, in some embodiments, A is absent or has at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide. In some embodiments, B is absent or has at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide. In some embodiments, C is a nucleotide. In some embodiments, C is a nucleotide having at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide. In some embodiments, A includes the third annealing region. In some embodiments, C includes the fourth annealing region.In some embodiments, the bridging agent includes a polypeptide. In some embodiments, the polypeptide includes an antibody or antigen-binding fragment thereof, an enzyme, or a purification tag.In some embodiments, the first photoreactive crosslinking agent and / or second photoreactive crosslinking agent is a photoreactive amino acid analog.In some embodiments, the photoreactive amino acid analog crosslinks to a complementary ribonucleotide within the linear polyribonucleotide upon photoirradiation.In some embodiments, the photoreactive amino acid analog is an alkyl diazirene-based, arylazide-based, benzophenone-based unnatural amino acid, or A / -e-[2-(furan-2-yl)ethoxy]carbonyl- lysine.In some embodiments, the polypeptide includes a plurality of photoreactive crosslinking agents.In some embodiments, each of the plurality of photoreactive crosslinking agents is a photoreactive amino acid analog.In some embodiments, each of the photoreactive amino acids crosslinks to a complementary ribonucleotide within the linear polyribonucleotide upon photoirradiation.In some embodiments, the polypeptide includes an RNA recognition motif selected from a K homology domain, zinc finger motif, Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, THUMP domain, YT521 -B homology domain, double stranded RNA binding domain, helicase domain, cold shock domain, S1 domain, Sm domain, La motif, Piwi-Argonaute-Zwille domain, or intrinsically disordered region.In some embodiments, the bridging agent includes a structure PP1-L-PP2, wherein PP1 includes a first polypeptide, PP2 includes a second polypeptide, and L is a linker of one or more atoms.In some embodiments, the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.In some embodiments, the bridging agent includes a small molecule.In some embodiments, the small molecule includes an intercalator.In some embodiments, the linear polyribonucleotide includes a coding region.In some embodiments, the coding region encodes a polypeptide.In some embodiments, the linear polyribonucleotide includes an internal ribosomal entry site (IRES) operably linked to the coding region.In some embodiments, the linear polyribonucleotide is from 1 ,000 to 20,000 (e.g., 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11 ,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000) ribonucleotides in length.In some embodiments, the bridging agent includes a functional group. In some embodiments, the functional group includes a thiol group, an N-hydroxysuccinimide ester, a polypeptide, a polynucleotide, an azide, a dye, or a targeting moiety.In some embodiments, the bridging agent includes a tag (e.g., a purification tag). In some embodiments, the tag includes biotin.In some embodiments, the targeting moiety includes a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid, or a combination thereof. In some embodiments, the targeting moiety includes a small molecule.In some embodiments, the small molecule is selected from folic acid, urea, a-mannose, high mannose, ursodeoxycholic acid, an endosomal escape agent, or lithocholic acid.In some embodiments, the targeting moiety includes a polypeptide. In some embodiments, the polypeptide is a cell-penetrating peptide. In some embodiments, the polypeptide is selected from ASSLNIA (SEQ ID NO: 19), M12, RGD, melittin, LPS-binding protein (LBP) peptide, an adiposehoming peptide, or an endolytic peptide. In some embodiments, the polypeptide is an antibody or a target-binding fragment thereof. In some embodiments, the antibody or target-binding fragment thereof is selected from a monoclonal antibody or target-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, or a tandem scFv (taFv). In some embodiments, the antibody or target-binding fragment thereof is selected from an anti-FcRn antibody, an anti-MR antibody, an anti-CD205 antibody, an anti-CD169 antibody, an anti-CD14 antibody, an anti-CD36 antibody, an anti-CD5 antibody, an anti -CD71 antibody, an anti-CD38 antibody, or an anti- prohibin antibody.In some embodiments, the polypeptide is a nanobody. In some embodiments, the nanobody is selected from an anti-transferrin nanobody, an anti-HER2 nanobody, or an anti-EGFR nanobody. In some embodiments, the targeting moiety includes a carbohydrate. In some embodiments, the carbohydrate includes a saccharide, disaccharide, or polysaccharide. In some embodiments, the carbohydrate includes mannose, galactose, or glucose. In some embodiments, the carbohydrate includes GalNAc or mannose 6-phosphate. In some embodiments, the carbohydrate includes a mono-, di-, tri-, or tetra-GalNAc. In some embodiments, the carbohydrate is tri-GalNAc.In some embodiments, the targeting moiety includes a lipid. In some embodiments, the lipid includes a fatty acid. In some embodiments, the fatty acid is a saturated, monounsaturated, or polyunsaturated fatty acid. In some embodiments, the fatty acid is a branched or unbranched chain including from 4 to 40 (e.g., 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, or 40) main-chain carbon atoms. In some embodiments, the fatty acid includes squalene, stearic acid, oleic acid, palmitic acid, linoleic acid, stearic acid, lauric acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), eicosapentaenoic acid (EPA), octadecanoic acid, myristic acid, anadamide, a-tocopherol, a-tocopherol succinate, or a retinoic acid. In some embodiments, the fatty acid includes docosanoic acid. In some embodiments, the fatty acid includes docosahexanoic acid. In some embodiments, the fatty acid includes myristic acid. In some embodiments, the lipid includes a steroid or sterol selected from cholesterol, tocopherol, ursodeoxycholic acid, or lithocholic acid. In some embodiments, the steroid or sterol is cholesterol. In some embodiments, the steroid or sterol is tocopherol. In some embodiments, the lipid includes a fatsoluble vitamin selected from vitamin A, vitamin D, vitamin E, vitamin K, or an analog or metabolite thereof. In some embodiments, the lipid includes a phospholipid. In some embodiments, the phospholipid is selected from phosphocholine (PC), PC-docosahexaenoic acid (PC-DHA), PC- docosanoic acid (PC-DCA), PC-eicosapentaenoic acid (PC-EPA), PC-lithocholic acid (PC-LA), PC- retinoic acid (PC-RA), and PC-a-tocopherol succinate (PC-TS).In some embodiments, the targeting moiety includes an oligonucleotide. In some embodiments, the targeting moiety includes an aptamer.In another aspect, featured is a covalently closed polyribonucleotide that includes (a) a linear polyribonucleotide including a first annealing region and a second annealing region; and (b) a bridging agent. The bridging agent is bound to and covalently attached to the first annealing region of the linear polyribonucleotide with a first covalent attachment; and to the second annealing region of the linear polyribonucleotide with a second covalent attachment.In some embodiments, the bridging agent is or includes a polynucleotide. In some embodiments, the polynucleotide includes an aptamer.In some embodiments, the polynucleotide includes a third annealing region configured to hybridize to the first annealing region on the linear polyribonucleotide and a fourth annealing region configured to hybridize to the second annealing region on the linear polyribonucleotide.In some embodiments, the first annealing region and the second annealing region are each from 5 to 200 (e.g., 6 to 200, 7 to 200, 8 to 200, 9 to 200, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 30, 6 to 30, 7 to 30, 8 to 30, or 9 to 30, e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) ribonucleotides.In some embodiments, the first annealing region has at least 50% (e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%) complementarity to the third annealing region, and the second annealing region has at least 50% (e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%) complementarity to the fourth annealing region. In some embodiments, the first annealing region has at least 80% complementarity to the third annealing region, and the second annealingregion has at least 80% complementarity to the fourth annealing region. In some embodiments, the first annealing region has at least 90% complementarity to the third annealing region, and the second annealing region has at least 90% complementarity to the fourth annealing region.In some embodiments, the first annealing region has zero or one mismatch with the third annealing region, and the second annealing region has zero or one mismatch with the fourth annealing region.In some embodiments, the polynucleotide is single stranded.In some embodiments, the polynucleotide is double stranded and includes a first strand and a second strand.In some embodiments, the first strand of the polynucleotide is longer than the second strand of the polynucleotide.In some embodiments, the first strand of the polynucleotide includes a 5’ overhang and a 3’ overhang.In some embodiments, the first strand anneals to the linear polyribonucleotide and the second strand is configured to fill a gap of the linear polyribonucleotide formed by annealing of the first strand.In some embodiments, the linear polyribonucleotide includes two nicks in the phosphodiester backbone prior to photoirradiation.In some embodiments, the polynucleotide is linear.In some embodiments, the polynucleotide includes a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.In some embodiments, the polynucleotide is from 10 to 2,000 (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11 ,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000) nucleotides in length.In some embodiments, the bridging agent includes a structure PN1-L-PN2, wherein PN1 includes a first polynucleotide, PN2 includes a second polynucleotide, and L is a linker of one or more atoms.In some embodiments, the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.In some embodiments, the bridging agent includes a polypeptide. In some embodiments, the polypeptide includes an antibody or antigen-binding fragment thereof, an enzyme, or a purification tag.In some embodiments, the polypeptide includes an RNA recognition motif selected from a K homology domain, zinc finger motif, Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, THUMP domain, YT521 -B homology domain, double stranded RNA binding domain, helicase domain, cold shock domain, S1 domain, Sm domain, La motif, Piwi-Argonaute-Zwille domain, or intrinsically disordered region.In some embodiments, the bridging agent includes a structure PP1-L-PP2, wherein PP1 includes a first polypeptide, PP2 includes a second polypeptide, and L is a linker of one or more atoms.In some embodiments, the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.In some embodiments, the bridging agent includes a small molecule.In some embodiments, the small molecule includes an intercalator.In some embodiments, the linear polyribonucleotide includes a coding region.In some embodiments, the coding region encodes a polypeptide.In some embodiments, the linear polyribonucleotide includes an internal ribosomal entry site (IRES) operably linked to the coding region.In some embodiments, the linear polyribonucleotide is from 1 ,000 to 20,000 (e.g., 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11 ,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000) ribonucleotides in length.In some embodiments, the bridging agent includes a functional group. In some embodiments, the functional group includes a thiol group, an N-hydroxysuccinimide ester, a polypeptide, a polynucleotide, an azide, a dye, or a targeting moiety.In some embodiments, the bridging agent includes a tag (e.g., a purification tag). In some embodiments, the tag includes biotin. In another aspect, featured is a method of forming a covalently closed polyribonucleotide. The method includes irradiating the complex of any of the above embodiments with light.In another aspect, featured is a method of forming a covalently closed polyribonucleotide. The method includes the step of (a) forming a complex with (i) a linear polyribonucleotide that includes a first annealing region and a second annealing region; and (ii) a bridging agent that includes a first photoreactive crosslinking agent and a second photoreactive crosslinking agent. The bridging agent binds to the first annealing region and the second annealing region on the linear polyribonucleotide. The method further includes the step of (b) irradiating the complex with light, wherein each photoreactive crosslinking agent forms a covalent adduct with the linear polyribonucleotide.In some embodiments, a wavelength of the irradiated light is from 340-410 nm (e.g., from 350-370 nm, e.g., 365 nm or 366 nm). In some embodiments, the complex is irradiated for 1 to 120 (e.g., 5, 10, 20, 30, 60, 90, or 120) minutes.In some embodiments, the method further includes irradiating the complex with light at a second wavelength to release the bridging agent (e.g., polynucleotide) from the linear polyribonucleotide. The second wavelength of the irradiated light may be from 300-320 nm (e.g., 312 nm).In some embodiments, the method further includes contacting a cell with the complex to deliver the complex to the cell. The complex may be irradiated prior to contacting. The complex may be irradiated after contacting.In some embodiments, the cell is a eukaryotic cell (e.g., a mammalian cell, such as a human cell)In some embodiments, the complex is delivered to the cell ex vivo.In some embodiments, the complex is delivered to the cell in vivo.In some embodiments, the complex is administered to a subject.In some embodiments, the complex is irradiated prior to administration.In some embodiments, the complex is irradiated after administration.In some embodiments, administering the cell to the subject treats a disease or disorder in the subject.In some embodiments, the bridging agent includes a functional group. In some embodiments, the functional group includes a thiol group, an N-hydroxysuccinimide ester, a polypeptide, a polynucleotide, an azide, a dye, or a targeting moiety.In some embodiments, the bridging agent includes a tag.In some embodiments, the method further includes contacting the complex with a capture agent that binds the tag.In some embodiments, the tag includes biotin, and the capture agent includes streptavidin.In some embodiments, the method further includes separating the complex that includes the bridging agent that is bound to the capture agent.In some embodiments, the capture agent is conjugated to a column.In another aspect, featured is a method of separating a circular polyribonucleotide that includes a tag from a plurality of polyribonucleotides that includes a mixture of linear polyribonucleotides and circular polyribonucleotides. The method includes (a) providing a sample that includes (i) a plurality of linear polyribonucleotides, each having a first annealing region and a second annealing region; and (ii) a bridging agent that includes a first photoreactive crosslinking agent, a second photoreactive crosslinking agent, and a functional group that includes a tag. The bridging agent binds to the first annealing region and the second annealing region on one of the plurality of linear polyribonucleotides, wherein the bridging agent and the linear polyribonucleotide forms a complex. The method further includes (b) irradiating the complex with light, wherein each photoreactive crosslinking agent forms a covalent adduct with the linear polyribonucleotide, thereby producing a plurality of polyribonucleotides, wherein a subset of the plurality of polyribonucleotides includes the circular polyribonucleotide having the tag. The method further includes (b) contacting the sample with a capture agent that binds the tag; and (c) separating the circular polyribonucleotide having the tag that is bound to the capture agent from the plurality of polyribonucleotides in the sample.In some embodiments, the linear polyribonucleotides lack the tag.In some embodiments, step (d) includes immobilizing the capture agent.In some embodiments, the capture agent is conjugated to a column.In some embodiments, the tag includes biotin, and the capture agent includes streptavidin.In another aspect, featured is a covalently closed polyribonucleotide produced by any of the methods described herein.DefinitionsTo facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the disclosure. Terms such as "a", "an," and "the" are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The term "or" is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or”. The terminology herein is used to describe specific embodiments, but their usage is not to be taken as limiting, except as outlined in the claims.As used herein, any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.As used herein, the term “about” refers to a value that is within ± 10% of a recited value.As used herein, the term “bridging agent” refers to a molecule containing at least two photoreactive crosslinking agents configured to form covalent adducts with another molecule. The bridging agent may crosslink with a linear polyribonucleotide upon irradiation with light, thereby forming a circular polyribonucleotide.As used herein, the term “carrier” is a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., a circular polyribonucleotide) into a cell by a covalent modification of the circular polyribonucleotide, via a partially or completely encapsulating agent, or a combination thereof. Non-limiting examples of carriers include carbohydrate carriers (e.g., an anhydride-modified phytoglycogen or glycogen-type material), nanoparticles (e.g., a nanoparticle that encapsulates or is covalently linked binds to the circular polyribonucleotide), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., a protein covalently linked to the circular polyribonucleotide), or cationic carriers (e.g., a cationic lipopolymer or transfection reagent).As used herein, the terms “circular polyribonucleotide” and “circular RNA” are used interchangeably and mean a polyribonucleotide molecule that is continuous. In some embodiments, a circular polyribonucleotide has a structure having no free ends (i.e., no free 3’ or 5’ ends), for example a polyribonucleotide molecule that forms a circular or end-less structure through covalent or non- covalent bonds. The circular polyribonucleotide may be, e.g., a covalently closed polyribonucleotide.As used herein, the term “circularization efficiency” is a measurement of resultant circular polyribonucleotide versus its non-circular (e.g., linear) starting material.As used herein, the terms “disease,” “disorder,” and “condition” each refer to a state of sub- optimal health, for example, a state that is or would typically be diagnosed or treated by a medical professional.By “heterologous” is meant to occur in a context other than in the naturally occurring (native) context. A “heterologous” polynucleotide sequence indicates that the polynucleotide sequence is being used in a way other than what is found in that sequence’s native genome. For example, a “heterologous promoter” is used to drive transcription of a sequence that is not one that is natively transcribed by that promoter; thus, a “heterologous promoter” sequence is often included in anexpression construct by means of recombinant nucleic acid techniques. The term "heterologous" is also used to refer to a given sequence that is placed in a non-naturally occurring relationship to another sequence; for example, a heterologous coding or non-coding nucleotide sequence is commonly inserted into a genome by genomic transformation techniques, resulting in a genetically modified or recombinant genome.As used herein “increasing fitness” or “promoting fitness” of a subject refers to any favorable alteration in physiology, or of any activity carried out by a subject organism, as a consequence of administration of a peptide or polypeptide described herein, including, but not limited to, any one or more of the following desired effects: (1 ) increased tolerance of biotic or abiotic stress by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (2) increased yield or biomass by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (3) modified flowering time by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) increased resistance to pests or pathogens by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more, (4) increased resistance to herbicides by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5) increasing a population of a subject organism (e.g., an agriculturally important insect) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (6) increasing the reproductive rate of a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (7) increasing the mobility of a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (8) increasing the body weight of a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (9) increasing the metabolic rate or activity of a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (10) increasing pollination (e.g., number of plants pollinated in a given amount of time) by a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (1 1 ) increasing production of subject organism (e.g., insect, e.g., bee or silkworm) byproducts (e.g., honey from a honeybee or silk from a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (12) increasing nutrient content of the subject organism (e.g., insect) (e.g., protein, fatty acids, or amino acids) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; or (13) increasing a subject organism’s resistance to pesticides (e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion)) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more, (14) increasing health or reducing disease of a subject organism such as a human or non-human animal. An increase in host fitness can be determined in comparison to a subject organism to which the modulating agent has not been administered. Conversely, “decreasing fitness” of a subject refers to any unfavorable alteration in physiology, or of any activity carried out by a subject organism, as a consequence of administration of a peptide or polypeptide described herein, including, but not limited to, any one or more of the following intended effects: (1 ) decreased tolerance of biotic or abiotic stress by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, 100% or more; (2) decreased yield or biomass by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (3) modified flowering time by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) decreased resistance to pests or pathogens by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more, (4) decreased resistance to herbicides by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5) decreasing a population of a subject organism (e.g., an agriculturally important insect) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (6) decreasing the reproductive rate of a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (7) decreasing the mobility of a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (8) decreasing the body weight of a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (9) decreasing the metabolic rate or activity of a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (10) decreasing pollination (e.g., number of plants pollinated in a given amount of time) by a subject organism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (11 ) decreasing production of subject organism (e.g., insect, e.g., bee or silkworm) byproducts (e.g., honey from a honeybee or silk from a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (12) decreasing nutrient content of the subject organism (e.g., insect) (e.g., protein, fatty acids, or amino acids) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; or (13) decreasing a subject organism’s resistance to pesticides (e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion)) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more, (14) decreasing health or reducing disease of a subject organism such as a human or non-human animal. A decrease in host fitness can be determined in comparison to a subject organism to which the modulating agent has not been administered. It will be apparent to one of skill in the art that certain changes in the physiology, phenotype, or activity of a subject, e.g., modification of flowering time in a plant, can be considered to increase fitness of the subject or to decrease fitness of the subject, depending on the context (e.g., to adapt to a change in climate or other environmental conditions). For example, a delay in flowering time (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% fewer plants in a population flowering at a given calendar date) can be a beneficial adaptation to later or cooler springtimes and thus be considered to increase a plant’s fitness; conversely, the same delay in flowering time in the context of earlier or warmer springtimes can be considered to decrease a plant’s fitness.As used herein, the terms “linear RNA” or “linear polyribonucleotide” or “linear polyribonucleotide molecule” are used interchangeably and mean polyribonucleotide molecule having a 5’ and 3’ end. One or both of the 5’ and 3’ ends may be free ends or joined to another moiety. Linear RNA includes RNA that has not undergone circularization (e.g., is pre-circularized) and can be used as a starting material for circularization.As used herein, the term “modified ribonucleotide” means a nucleotide with at least one modification to the sugar, the nucleobase, or the internucleoside linkage.As used herein, the term “naked delivery” is a formulation for delivery to a cell without the aid of a carrier and without covalent modification to a moiety that aids in delivery to a cell. A naked delivery formulation is free from any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, naked delivery formulation of a circular polyribonucleotide is a formulation that includes a circular polyribonucleotide without covalent modification and is free from a carrier.The term “pharmaceutical composition” is intended to also disclose that the circular or linear polyribonucleotide included within a pharmaceutical composition can be used for the treatment of the human or animal body by therapy.The term “photoreactive crosslinking agent,” refers to any compound that is configured to form a covalent adduct with another molecule upon irradiation with light. For example, a photoreactive crosslinking agent may covalently attach to a ribonucleotide within a circular polyribonucleotide upon irradiation with light. In embodiments, a photoreactive crosslinking agent may be able to form a covalent attachment to a ribonucleotide within the circular polyribonucleotide that is reversible. In embodiments, the photoreactive crosslinking agent can be a photoreactive nucleotide analog or photoreactive amino acid analog.The term “photoreactive nucleotide analog” refers to any nucleic acid analog that is configured to form a covalent adduct with another molecule upon irradiation with light. For example, the photoreactive nucleotide analog may be present within a polynucleotide and be configured to covalently attach to a circular polyribonucleotide upon irradiation with light. In some embodiments, the photoreactive nucleotide analog can covalently attach to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of a complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. Examples of photoreactive nucleotide analogs include, but are not limited to, 4-thiouridene (4sU), 5-bromo-2’-deoxyuridine (BrdU), coumarin derivatives, 3-cyanovinylcarbazole D-threoninol (CNVD) derivatives, 3-cyanovinylcarbazole nucleoside (CNVK) derivatives, diazirene derivatives, phenylselenide derivatives, psoralen derivatives, or pyranocarbazole nucleoside (PCX) derivatives. For a review of certain photoreactive nucleotide analogs, see Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 and Tavakoli et al., RSC Adv. 12, 6484-6507, 2022. In embodiments, the covalent attachment of the photoreactive nucleotide analog to the ribonucleotide is reversible, e.g., it may be removed when irradiated with light, e.g., of a second different wavelength.The term “photoreactive amino acid analog” refers to any amino acid analog that is configured to form a covalent adduct with another molecule upon irradiation with light. For example, the photoreactive amino acid analog may be present within a polypeptide that covalently attaches to a circular polyribonucleotide upon irradiation with light. In some embodiments, the photoreactive amino acid analog can covalently attach to a nearby ribonucleotide within the circular polyribonucleotide upon photoirradiation. Examples of photoreactive amino acid analogs include but are not limited to diazirene-based, aryl azide-based, benzophenone-based unnatural amino acid, or A / -e-[2-(furan-2-yl)ethoxy]carbonyl-lysine . In embodiments, the covalent attachment of the photoreactive amino acid analog to the ribonucleotide is reversible, e.g., it may be removed when irradiated with light, e.g., a second different wavelength.The term “polynucleotide” as used herein means a molecule including one or more nucleic acid subunits, or nucleotides, and can be used interchangeably with “nucleic acid” or “oligonucleotide”. A polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. A nucleotide can include a nucleoside and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides are nucleotides in which the sugar is ribose. Polyribonucleotides or ribonucleic acids, or RNA, can refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose. As used herein, a polyribonucleotide sequence that recites thymine (T) is understood to represent uracil (U).As used herein, the term “polyribonucleotide cargo” herein includes any sequence including at least one polyribonucleotide. In embodiments, the polyribonucleotide cargo includes one or multiple expression sequences, wherein each expression sequence encodes a polypeptide. In embodiments, the polyribonucleotide cargo includes one or multiple noncoding sequences, such as a polyribonucleotide having regulatory or catalytic functions. In embodiments, the polyribonucleotide cargo includes a combination of expression and noncoding sequences. In embodiments, the polyribonucleotide cargo includes one or more polyribonucleotide sequence described herein, such as one or multiple regulatory elements, internal ribosomal entry site (IRES) elements, or spacer sequences.As used interchangeably herein, the terms “polyA,” “poly A sequence,” or “polyA tail” refer to an untranslated, contiguous region of a nucleic acid molecule of at least 5 nucleotides in length and consisting of adenosine residues. In some embodiments, a polyA tail is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides in length. In some embodiments, a polyA tail is located 3’ to (e.g., downstream of) an open reason frame (e.g., an open reading frame encoding a polypeptide), and the polyA tail is 3’ to a termination element (e.g., a Stop codon) such that the polyA is not translated. In some embodiments, a polyA tail is located 3’ to a termination element and a 3’ untranslated region.As used herein, the elements of a nucleic acid are “operably connected” if they are positioned on the vector such that they can be transcribed to form a linear RNA that can then be circularized into a circular RNA using the methods provided herein.Polydeoxyribonucleotides or deoxyribonucleic acids, or DNA, means macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds. A nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate. A nucleotide means a deoxyribonucleoside polyphosphate, such as, e.g., a deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate(dTTP) dNTPs, that include detectable tags, such as luminescent tags or markers (e.g., fluorophores). A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e., C, T or U, or variant thereof). In some examples, a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof. In some cases, a polynucleotide is a short interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. In some cases, a polynucleotide molecule is circular. A polynucleotide can have various lengths. A nucleic acid molecule can have a length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. A polynucleotide can be isolated from a cell or a tissue. Embodiments of polynucleotides include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.Embodiments of polynucleotides, e.g., polyribonucleotides or polydeoxyribonucleotides, include polynucleotides that contain one or more nucleotide variants, including nonstandard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s) or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6- adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6- diaminopurine and the like. In some cases, nucleotides include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Non-limiting examples of such modifications include phosphate chains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphates). In embodiments, nucleic acid molecules are modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone. In embodiments, nucleic acid molecules contain amine -modified groups, such as amino allyl 1 -dUTP (aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine reactive moieties, such as N-hydroxysuccinimide esters (NHS). Alternatives to standard DNA base pairs or RNA basepairs in the polynucleotides of the present disclosure can provide higher density in bits per cubic mm, higher safety (resistant to accidental or purposeful synthesis of natural toxins), easier discrimination in photo-programmed polymerases, or lower secondary structure. Such alternative base pairs compatible with natural and mutant polymerases for de novo or amplification synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul ;8(7) :612-4, which is herein incorporated by reference for all purposes.As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single molecule or a multi-molecular complex such as a dimer, trimer, or tetramer. They can also include single chain or multichain polypeptides such as antibodies or insulin and can be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.As used herein, the term “plant-modifying polypeptide” refers to a polypeptide that can alter the genetic properties (e.g., increase gene expression, decrease gene expression, or otherwise alter the nucleotide sequence of DNA or RNA), epigenetic properties, or biochemical or physiological properties of a plant in a manner that results in a change in the plant’s physiology or phenotype, e.g., an increase or a decrease in plant fitness.As used herein, the term “regulatory element” is a moiety, such as a nucleic acid sequence, that modifies expression of an expression sequence within the circular or linear polyribonucleotide.As used herein, a “spacer” refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions.As used herein, the term “sequence identity” is determined by alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm. Sequences are referred to as "substantially identical” or “essentially similar” when they share at least a certain minimal percentage of sequence identity when optimally aligned (e.g., when aligned by programs such as GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) I 8 (proteins) and gap extension penalty = 3 (nucleotides) 12 (proteins). For nucleotides the default scoring matrix used is nwsgapdna, and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity are determined, e.g., using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121 -3752 USA, or EmbossWin version 2.10.0 (using the program “needle”). Alternatively or additionally, percent identity is determined by searching against databases, e.g., using algorithmssuch as FASTA, BLAST, etc. Sequence identity refers to the sequence identity over the entire length of the sequence.As used herein, “structured” with regard to RNA refers to an RNA sequence that is predicted by the RNAFold software or similar predictive tools to form a structure (e.g., a hairpin loop) with itself or other sequences in the same RNA molecule.As used herein, the term "subject" refers to an organism, such as an animal, plant, or microbe. In embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In embodiments, the subject is a human. In embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, bison, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc. In embodiments, the subject is an invertebrate agricultural pest or an invertebrate that is parasitic on an invertebrate or vertebrate host. In embodiments, the subject is a plant, such as an angiosperm plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte. In embodiments, the subject is a eukaryotic alga (unicellular or multicellular). In embodiments, the subject is a plant of agricultural or horticultural importance, such as row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, groundcovers, and turf grasses.As used here, the term “targeting moiety” refers to a moiety that binds specifically to a cell or a portion thereof (e.g., an extracellular, intracellular, or membrane portion thereof) thereby promoting intracellular delivery of the moiety and any cargo bound or complexed thereto (e.g., a polyribonucleotide). A targeting moiety may include a lipid, a small molecule, a carbohydrate, a polypeptide, a nucleic acid (e.g., an aptamer), or a combination thereof. In some embodiments, a targeting moiety interacts with the cellular membrane thereby promoting intracellular delivery. In some embodiments, a targeting moiety promotes endosomal delivery and / or endosomal escape. In some embodiments, a targeting moiety promotes cellular delivery that is not specific to any cell type. In some embodiments, the targeting moiety binds preferentially to a particular cell type and therefore promotes cell-type specific delivery.As used herein, the term “treat,” or “treating,” refers to a prophylactic or therapeutic treatment of a disease or disorder (e.g., an infectious disease, a cancer, a toxicity, or an allergic reaction) in a subject. The effect of treatment can include reversing, alleviating, reducing severity of, curing, inhibiting the progression of, reducing the likelihood of recurrence of the disease or one or more symptoms or manifestations of the disease or disorder, stabilizing (i.e. , not worsening) the state of the disease or disorder, or preventing the spread of the disease or disorder as compared to the state or the condition of the disease or disorder in the absence of the therapeutic treatment. Embodimentsinclude treating plants to control a disease or adverse condition caused by or associated with an invertebrate pest or a microbial (e.g., bacterial, fungal, oomycete, or viral) pathogen. Embodiments include treating a plant to increase the plant’s innate defense or immune capability to tolerate pest or pathogen pressure.As used herein, the term “termination element” is a moiety, such as a nucleic acid sequence, that terminates translation of the expression sequence in the circular or linear polyribonucleotide.As used herein, the term “translation efficiency” is a rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, translation efficiency can be expressed as amount of protein or peptide produced per given amount of transcript that codes for the protein or peptide, e.g., in a given period of time, e.g., in a given translation system, e.g., an cell-free translation system like rabbit reticulocyte lysate.As used herein, the term “translation initiation sequence” is a nucleic acid sequence that initiates translation of an expression sequence in the circular or linear polyribonucleotide.As used herein, the term “therapeutic polypeptide” refers to a polypeptide that when administered to or expressed in a subject provides some therapeutic benefit. In embodiments, a therapeutic polypeptide is used to treat or prevent a disease, disorder, or condition in a subject by administration of the therapeutic peptide to a subject or by expression in a subject of the therapeutic polypeptide. In alternative embodiments, a therapeutic polypeptide is expressed in a cell and the cell is administered to a subject to provide a therapeutic benefit.As used herein, a "vector" means a piece of DNA, that is synthesized (e.g., using PCR), or that is taken from a virus, plasmid, or cell of a higher organism into which a foreign DNA fragment can be or has been inserted for cloning or expression purposes. In some embodiments, a vector can be stably maintained in an organism. A vector can include, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linearized plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like. In one embodiment, the vectors provided herein include a multiple cloning site (MCS). In another embodiment, the vectors provided herein do not include an MCS.Brief Description of the DrawingsFIGS. 1A and 1B are schematic drawings showing exemplary photocircularization methods. FIG. 1 A depicts a linear polyribonucleotide and a polynucleotide with two photoreactive crosslinking agents (shown as circles). The top panel of FIG. 1 A depicts a single stranded polynucleotide that anneals at each end to the linear polyribonucleotide. The two photoreactive crosslinking agents form covalent adducts with the polyribonucleotide upon irradiation (shown as an X). In the bottom panel, a double stranded polynucleotide is used with one strand annealing with the linear polyribonucleotide and the second strand containing photoreactive crosslinking agents at the ends of the strand. FIG. 1 B shows how a polynucleotide anneals to the linear polyribonucleotide to form a closed circular polyribonucleotide upon irradiation.FIGS. 2A and 2B are gels showing the efficiency of circularization of linear polyribonucleotides formed after irradiation with a linear polynucleotide containing two photoreactive crosslinking agents. Circular and linear polyribonucleotides were separated by polyacrylamide gel electrophoresis with 6% TBU (FIG. 2A) or 10% TBU (FIG. 2B). Cuvettes with small volumes of 140 pL maximum (S) or large volumes of 300 pL maximum and a greater surface area (L) were used to store samples during irradiation.FIGS. 3A and 3B are a gel and a graph showing the circularization efficiency via splint ligation using 2, 3, 4, 5, 6, 7, 8, or 10 mM of MgCl2. Circular and linear polyribonucleotides were separated by polyacrylamide gel electrophoresis.FIGS. 4A and 4B are a gel and a graph showing the photocircularization efficiency via irradiation. Circular and linear polyribonucleotides were separated by polyacrylamide gel electrophoresis.FIGS. 5A and 5B are graphs showing the amount of luminescence measured in HEK293 cells (FIG. 5A) and A549 cells (FIG. 5B) transfected with photocircularized polyribonucleotides encoding a luciferase polypeptide (eRNA). The photocircularized polyribonucleotides were photocircularized with an irradiation time of 0, 15, 30, or 45 minutes. The photocircularized polyribonucleotides, linear polyribonucleotide control, and self-circularized polyribonucleotide control (LAA3808) were delivered with lipofectamine MessengerMax (MM) into the cells.FIGS. 6A and 6B are graphs showing the concentration of cytokines (IFNB) measured in A549 cells transfected with photocircularized polyribonucleotides encoding a luciferase polypeptide (eRNA) and delivered with lipofectamine MessengerMAX (MM). The photocircularized polyribonucleotides were photocircularized with an irradiation time of 0, 15, 30, or 45 minutes. FIG. 6B shows the same data as FIG. 6A but excludes the self-circularized polyribonucleotide control to emphasize the results of the photocircularized polyribonuclotides against the linear polyribonucleotide control.FIGS. 7A and 7B are graphs showing the concentration of cytokines (IP10) measured in A549 cells transfected with photocircularized polyribonucleotides encoding a luciferase polypeptide (eRNA) and delivered with lipofectamine MessengerMAX (MM). The photocircularized polyribonucleotides were photocircularized with an irradiation time of 0, 15, 30, or 45 minutes. FIG. 7B shows the same data as FIG. 7A but excludes the self-circularized polyribonucleotide control to emphasize the results of the photocircularized polyribonuclotides against the linear polyribonucleotide control.FIG. 8 is a gel showing photocircularization efficiency. The annealing buffer was water, HEPES buffered saline 1x (HBS), HBS with 250 mM NaCI and 5 mM MgCI2(M), or Tris-HCI (pH = 7.0) with 2 mM MgCI2(Mg). The photocircularized polyribonucleotides were photocircularized with an irradiation time of 0 or 30 minutes. Linear and circular polyribonucleotides were separated via polyacrylamide gel electrophoresis.FIG. 9 is a gel showing reversibility of photocircularization. Linear polyribonucleotides were circularized via irradiation at 366 nm and reversibly linearized via irradiation at 312 nm.FIG. 10 is a schematic drawing showing exemplary photocircularization methods for modification of photocircularized polyribonucleotides. A bridging agent with two photoreactivecrosslinking agents and a functional group anneals at each end to the linear polyribonucleotide. The two photoreactive crosslinking agents form covalent adducts with the polyribonucleotide upon irradiation, thus producing a photocircularized polyribonucleotide with a functional group that can be used to further attach a chemical moiety.FIG. 11 is a schematic drawing showing exemplary photocircularization methods for modification and purification of photocircularized polyribonucleotides. A bridging agent with two photoreactive crosslinking agents and a tag anneals at each end to the linear polyribonucleotide. The two photoreactive crosslinking agents form covalent adducts with the polyribonucleotide upon irradiation. The photocircularized polyribonucleotide is incubated with a capture agent that binds to the tag, thereby separating the photocircularized polyribonucleotides from the uncircularized linear polyribonucleotides without the tagged bridging agent.Detailed DescriptionThe present invention features compositions and methods for producing a circular polyribonucleotide (circular RNA). Circular polyribonucleotides described herein are particularly useful for delivering a polynucleotide cargo (e.g., encoding a gene or protein) to a target cell.A circular polyribonucleotide may be produced from a linear polyribonucleotide in which a bridging agent containing photoreactive crosslinking agents circularizes the linear polyribonucleotide upon irradiation with light, thereby forming a circular polyribonucleotide. The compositions and methods described herein include a complex that includes a linear polyribonucleotide including a first annealing region and a second annealing region and a bridging agent having a first photoreactive crosslinking agent and a second photoreactive crosslinking agent. The bridging agent is configured to bind to the first annealing region and the second annealing region on the linear polyribonucleotide. Each photoreactive crosslinking agent is configured to form a covalent adduct with the linear polyribonucleotide upon irradiation with light. The bridging agent may be, for example, a polynucleotide, a polypeptide, or a small molecule. Also featured are covalently closed polyribonucleotides in which the bridging agent is bound to and covalently attached to the first annealing region and the second annealing region.By using photoreactive crosslinking agents, the linear molecule exhibits increased circularization efficiency as compared to other polyribonucleotide constructs that lack these features. Furthermore, by using photoreactive crosslinking agents, the linear molecule does not need to be treated with an exogenous enzyme, such as a ligase, to produce the circular polyribonucleotide. This is particularly advantageous for producing a circular product in a single pot reaction. The molecules, methods of producing, and uses thereof are described in more detail below.Bridging AgentsThe complexes described herein include a bridging agent that bridges the linear polyribonucleotide to form a circular polyribonucleotide. The bridging agent includes at least two photoreactive crosslinking agents that crosslink with the linear polyribonucleotide upon irradiation with light, thereby covalently closing the circle. The bridging agent may contain one or more bindingmoieties configured to interact with the linear polyribonucleotide, e.g., to bridge the ends of the polyribonucleotide before irradiation. The bridging agent may be or include, for example, a polynucleotide, a polypeptide, or a small molecule.Polynucleotide Bridging AgentsIn some embodiments, the bridging agent is or includes a polynucleotide. In some embodiments, the polynucleotide includes an aptamer. The polynucleotide may be, for example, a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid. The polynucleotide may be single stranded or double stranded (FIG. 1 A).In some embodiments, the polynucleotide includes a third annealing region configured to hybridize to the first annealing region on the linear polyribonucleotide and a fourth annealing region configured to hybridize to the second annealing region on the linear polyribonucleotide.In some embodiments, the first annealing region and the second annealing region are each from 5 to 200 (e.g., 6 to 200, 7 to 200, 8 to 200, 9 to 200, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 30, 6 to 30, 7 to 30, 8 to 30, or 9 to 30, e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) ribonucleotides.In some embodiments, the first annealing region has at least 50% (e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%) complementarity to the third annealing region, and the second annealing region has at least 50% (e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%) complementarity to the fourth annealing region. In some embodiments, the first annealing region has at least 80% complementarity to the third annealing region, and the second annealing region has at least 80% complementarity to the fourth annealing region. In some embodiments, the first annealing region has at least 90% complementarity to the third annealing region, and the second annealing region has at least 90% complementarity to the fourth annealing region.In some embodiments, the first annealing region has zero or one mismatch with the third annealing region, and the second annealing region has zero or one mismatch with the fourth annealing region.In some embodiments, the third annealing region includes the first photoreactive crosslinking agent and the fourth annealing region includes the second photoreactive crosslinking agent.In some embodiments, each of the photoreactive nucleotide analogs crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the linear polyribonucleotide upon photoirradiation.In some embodiments, the polynucleotide is single stranded.In some embodiments, the polynucleotide is double stranded and includes a first strand and a second strand. In some embodiments, the first strand of the double stranded polynucleotide includes the third annealing region and the fourth annealing region, and the second strand of the double stranded polynucleotide includes the first photoreactive crosslinking agent and the second photoreactive crosslinking agent. In some embodiments, the first strand of the double stranded polynucleotide is longer than the second strand of the double stranded polynucleotide. In someembodiments, the first strand of the double stranded polynucleotide includes a 5’ overhang and a 3’ overhang. In some embodiments, the first strand of the double stranded polynucleotide anneals to the linear polyribonucleotide and the second strand of the double stranded polynucleotide is configured to fill a gap of the linear polyribonucleotide formed by annealing of the first strand of the double stranded polynucleotide.In some embodiments, the linear polyribonucleotide includes two nicks in the phosphodiester backbone prior to photoirradiation.In some embodiments, the polynucleotide includes a 5’ terminal photoreactive crosslinking agent and a 3’ terminal crosslinking agent.In some embodiments, the linear polyribonucleotide includes a 5’ terminal uridine and a 3’ terminal uridine.In some embodiments, the first photoreactive crosslinking agent and the second photoreactive crosslinking agent is each, independently, a uridine or thymidine.In some embodiments, the first photoreactive crosslinking agent and the second photoreactive crosslinking agent each crosslinks to a ribonucleotide at an adjacent position in the linear polyribonucleotide upon photoirradiation.In some embodiments, the polynucleotide is linear.In some embodiments, the polynucleotide includes a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.In some embodiments, the polynucleotide is from 10 to 2,000 (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11 ,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000) nucleotides in length.In some embodiments, the bridging agent includes a structure PN1-L-PN2, wherein PN1 includes a first polynucleotide, PN2 includes a second polynucleotide, and L is a linker of one or more atoms. In some embodiments, the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.In some embodiments, the polynucleotide has a structure A-P1 -B-P2-C, wherein each of A and B is optionally absent or polynucleotide of at least 1 nucleotide (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more), C is optionally absent or is a linker, and each of P1 and P2 are, independently, a photoreactive crosslinking agent (e.g., a photoreactive nucleotide analog). For example, in some embodiments, A is absent or has at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide. In some embodiments, B is absent or has at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide. In some embodiments, C is a nucleotide. In some embodiments, C is a nucleotide having at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide. In some embodiments, A includes the third annealing region. In some embodiments, C includes the fourth annealing region.In some embodiments, the polynucleotide includes a plurality of photoreactive nucleotide analogs. In some embodiments, at least one of the plurality of photoreactive nucleotide analogs is attached to the 3’ end of the polynucleotide. In some embodiments, at least one of the plurality of photoreactive nucleotide analogs is attached to the 5’ end of the polynucleotide. In some embodiments, at least one of the plurality of photoreactive nucleotides analogs is located at an internal position within the polynucleotide.In some embodiments, the polynucleotide includes 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides between each of the photoreactive nucleotide analogs. In some embodiments, the 3’ end of the polynucleotide has at least 1 (e.g., at least (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide from the nearest photoreactive nucleotide analog. In some embodiments, the 3’ end of the polynucleotide has from 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 5’ end of the polynucleotide has at least 1 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more) nucleotide from the nearest photoreactive nucleotide analog. In some embodiments, the 5’ end of the polynucleotide has from 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the nearest photoreactive nucleotide analog.In some embodiments, the photoreactive crosslinking agent is attached to the 5’ end of the polynucleotide. In some embodiments, the photoreactive crosslinking agent is attached to the 3’ end of the polynucleotide. In some embodiments, the photoreactive crosslinking agent is a photoreactive nucleotide analog. In some embodiments, the photoreactive nucleotide analog replaces a single nucleotide within the polynucleotide. In some embodiments, the photoreactive nucleotide analog is located at an internal position within the polynucleotide. In some embodiments, the 3’ end of the polynucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog. In some embodiments, the 3’ end of the polynucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the photoreactive nucleotide analog. In some embodiments, the 5’ end of the polynucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog. In some embodiments, the 5’ end of the polynucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the photoreactive nucleotide analog.In some embodiments, the polynucleotide includes one, two, three, four, five, six, seven, eight, nine, ten or more photoreactive crosslinking agents. In some embodiments, each photoreactive crosslinking agent is a photoreactive nucleotide analog. In some embodiments, each of the photoreactive nucleotide analogs replaces a single nucleotide within the polynucleotide. In some embodiments, each photoreactive nucleotide analog is located at an internal position within the polynucleotide. In some embodiments, the 3’ end of the polynucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 3’ end of the polynucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35nucleotides) from the nearest photoreactive nucleotide analog. In some embodiments, the 5’ end of the polynucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 5’ end of the polynucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the nearest photoreactive nucleotide analog. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the 5’ end of the polynucleotide. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the 3’ end of the polynucleotide. In some embodiments, the polynucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) between each of the photoreactive nucleotide analogs. For example, the polynucleotide may have one photoreactive nucleotide analog attached to the 5’ end and a second photoreactive nucleotide analog 8 nucleotides away from the 5’ end. The polynucleotide may have one photoreactive nucleotide analog attached to the 5’ end, a second photoreactive nucleotide analog 10 nucleotides away from the 5’ end, and a third photoreactive nucleotide analog 15 nucleotides away from the 3’ end and 8 nucleotides away from the second photoreactive nucleotide analog.In some embodiments, the polynucleotide is from about 8-500 nucleotides in length (e.g., about 8-20, 8-30, 8-40, 8-50, 8-60, 8-70, 8-80, 8-90, 8-100, 8-200, 8-300, 8-400, 20-50, 20-100, 20- 200 20-300, 20-400, 30-50, 30-100, 30-200, 30-300, 30-400, 50-100, 50-200, 50-300, or 50-400 nucleotides in length). In some embodiments, the polynucleotide or a portion thereof has at least 20% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%) complementarity to a portion of the sequence of the linear polyribonucleotide. In some embodiments, the polynucleotide or a portion thereof has from 20% to 90% (e.g., from 20-90%, 20-90%, 20-90% 20-90%, 30%-90%, 40%-90%, or 50%-90%) complementarity to a portion of the sequence of the linear polyribonucleotide. The polynucleotide may be DNA, RNA, a synthetic nucleic acid, or a hybrid. The polynucleotide may include one or more nucleic acid modifications as described herein.In some embodiments, the polynucleotide is annealed (e.g., shares at least partial complementarity sufficient for binding with) and covalently bound to (e.g., photo-crosslinked) a sequence with a binding region of the linear polyribonucleotide. In some embodiments, the linear polyribonucleotide includes either a coding region (e.g., including an expression sequence), the binding region is sufficiently distant in sequence and space from the coding region or target binding region so that it does not interfere with or minimizes interference with translation efficiency. In some embodiments, the binding region on the polynucleotide includes from 1 to 100 ribonucleotides (e.g., from 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 ribonucleotides). For example, the binding region may include from 10 to 90 ribonucleotides (e.g., from 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 80, 30 to 80, 40 to 80, 50 to 80, 60 to 80, or 70 to 80 ribonucleotides). In some embodiments, the polynucleotide has from 50% to 100% (e.g., from 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%) complementarity to a portion of the binding region of the linear polyribonucleotide. Forexample, the polynucleotide may have from 80% to 100% (e.g., from 85% to 100%, 90% to 100%, 95% to 100%, 80% to 95%, 80% to 90%, or 80% to 85%) and complementarity to a portion of the binding region of the linear polyribonucleotide. In some embodiments, the binding region has zero or one mismatch with the polynucleotide.Polypeptide Bridging AgentsIn some embodiments, the bridging agent is or includes a polypeptide. In some embodiments, the polypeptide includes an antibody or antigen-biding fragment thereof, an enzyme, or a purification tag. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the N- terminus of the polypeptide. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the C-terminus of the polypeptide. In some embodiments, the photoreactive crosslinking agent is a photoreactive amino acid analog. In some embodiments, the photoreactive amino acid analog replaces a single amino acid within the polypeptide. In some embodiments, the photoreactive amino acid analog is located at an internal position within the polypeptide. In some embodiments, the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog. In some embodiments, the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the photoreactive amino acid analog.In some embodiments, the polypeptide includes one, two, three, four, five, six, seven, eight, nine, ten or more photoreactive crosslinking agents. In some embodiments, each photoreactive crosslinking agent is a photoreactive amino acid analog. In some embodiments, each of the photoreactive amino acid analogs replaces a single amino acid within the polypeptide. In some embodiments, each photoreactive amino acid analog is located at an internal position within the polypeptide. In some embodiments, the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog. In some embodiments, the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the nearest photoreactive amino acid analog. In some embodiments, the N- terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the nearest photoreactive amino acid analog. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the N-terminus of the polypeptide. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the C-terminus of the polypeptide. In someembodiments, the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) between each of the photoreactive amino acid analogs. For example, a polypeptide may have one photoreactive amino acid analog attached to the N-terminus and a second photoreactive amino acid analog 8 amino acids away from the N-terminus. The polypeptide may have one photoreactive amino acid analog attached to the N-terminus, a second photoreactive amino acid analog 10 amino acids away from the N-terminus, and a third photoreactive amino acid analog 15 amino acids away from the C-terminus and 8 amino acids away from the second photoreactive amino acid analog.The polypeptide may include an RNA binding protein domain. In some embodiments, the RNA binding protein domain (RBP) may a be an RNA recognition motif (RRM), K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521 -B homology domain, a double stranded RNA binding domain, a helicase domain, a cold shock domain, an S1 domain, an Sm domain, a La motif, a Piwi-Argonaute-Zwille domain, or an intrinsically disordered region.In some embodiments, the RNA binding protein domain may be an RNA recognition motif (RRM). RRMs are the most common and well-studied RNA-binding domain. The Protein Data Bank includes over 500 known structures which have an RRM. RRMs average 90 amino acids in size and adopt a p1a1 p2p3a2p4 topology forming two a helices against an antiparallel p sheet, which houses the conserved RNA-binding ribonucleoprotein 1 and ribonucleoprotein 2 motifs in the central p1 and p3 strands. In some embodiments, RRMs interact with from 2 to 8 nucleotides (e.g., 2, 3, 4, 5, 6, 7, and 8 nucleotides) in single-stranded RNA commonly. This interaction may occur through several sequential stacking interactions and hydrogen bonds with ribonucleoprotein motifs, often with nanomolar affinities. Each RRM may have its own sequence preferences, often for degenerate sequences such as GU-rich tracts. In some embodiments, the RBP includes multiple ribonucleoprotein motifs. The combination of consecutive ribonucleoprotein motifs in an RBP dramatically increases binding affinity and specificity.In some embodiments, the polypeptide has a K homology domain. The K homology (KH) domain was first discovered in heterogeneous nuclear ribonucleoprotein K (hnRNPK). The KH domain includes about 70 amino acid residues, and typically recognizes about 4 nucleotides in single stranded RNA. KH domains may adopt either a type I p1a1a2p2p'a' topology, in eukaryotes, or the reverse type II a'p'p1a1 a2p2 topology, in prokaryotes, with a conserved “GXXG” RNA-binding motif (SEQ ID NO: 1 ) located between the a1 and a2 helices. RNA binding may occur in a hydrophobic pocket of the KH domain and may include several hydrogen bonds coordinated by the “GXXG” motif (SEQ ID NO: 1 ). Proteins having a KH domain and RNA have few stacking interactions. Furthermore, proteins having a KH domain usually have weak micromolar RNA affinities. Multiple KH domains in an RBP can independently or synergistically increase binding specificity.In some embodiments, the polypeptide includes a zinc finger motif. Zinc finger motifs may be found in a large family of proteins that average 30 amino acids in size and form a simple ppa topology in which residues in the p hairpin turn and a helix are coordinated by a Zn2+ ion. Zinc finger motifpolypeptides may bind DNA but have been additionally shown to bind RNA. Zinc finger motifs include subtypes that interact with RNA and include amino acid sequences of CCHC (SEQ ID NO: 2), CCCH (SEQ ID NO: 3), CCCC (SEQ ID NO: 4), and CCHH (SEQ ID NO: 5) subtypes, where C and H refer to the interspersed cysteine and histidine residues that coordinate the zinc atom, respectively. The zinc finger motif subtypes display a range of sequence and structural specificities. The CCHC (SEQ ID NO: 2) subtype recognizes stem-loop elements in RNA through contacts with bases in the loop and the phosphate backbone of the stem. The CCCH (SEQ ID NO: 3) and CCCC (SEQ ID NO: 4) subtypes tend to recognize 3 nucleotide repeats through multiple such zinc finger motifs in one RBP. These contacts may be formed through hydrogen bonds with bases and the insertion of aromatic side chains that stack between bases. The versatile and abundant CCHH (SEQ ID NO: 5) subtype interacts with both single-stranded and double-stranded RNA. Thus, designer zinc finger motifs may be used for directed binding of RNA sequences.In some embodiments, the RNA binding protein includes a Pumilio homology domain. The Pumilio family of proteins occurs in most eukaryotes and is defined by the Pumilio homology domain. The Pumilio homology domain is very large, consisting of eight a-helical repeats of a highly conserved 36-amino acid sequence that forms a concave RNA-binding surface. Each repeat recognizes one unpaired RNA base through hydrogen bonds and a stabilizing stacking interaction, where the full domain recognizes up to 8 nucleotides in single-stranded RNA with low-nanomolar affinity. Wild-type Pumilio homology domain repeats do not specifically recognize cytosine; however, protein engineering has produced repeats that do. This protein engineering combined with the PUF domain’s predictable base recognition code allow modular design of Pumilio proteins that recognize from 8 nucleotides to 10-nucleotides sequences containing all RNA bases.In some embodiments, the polypeptide includes a pentatricopeptide repeat domain. Pentatricopeptide repeats include about 35 amino acid residues in length and form two antiparallel a helices. 2-30 repeats form a solenoid-shaped scaffold that binds specific single-stranded RNA sequences with nanomolar affinity. Two residues in each repeat determine base-specific binding through hydrogen bonds, enabling the development of pentatricopeptide repeat domains designed to bind specified single-stranded RNA sequences.In some embodiments, the polypeptide includes a pseudouridine synthase and archaeosine transglycosylase (PUA) domain. PUA domains include a range from 67 to 94 amino acids residues (e.g., 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, and 94 amino acid residues), with a p1 a1 p2p3p4p5a2p6 architecture that forms a pseudobarrel encased by two a helices. PUA domains may contact double-stranded RNA, and its adjacent loops or overhangs through extensive hydrogen bonds may contact all parts of the RNA. These contacts are typically formed by a glycine-rich loop between a1 and p2 or a2 and p6. Unlike many other domains, PUA domains are not found as tandem repeats.In some embodiments, the polypeptide includes a THUMP domain. Named for thiouridine synthase, methyltransferase, and pseudouridine synthase, the THUMP domain is found in numerous tRNA-modifying enzymes. THUMP domains are always found in proximity to RNA-modifying domains and often in proximity to an N-terminal ferredoxin-like domain. THUMP domains include about 100amino acids residues and display a a1a2p1a3p2p2 topology that forms parallel a helices flanking a p sheet.In some embodiments, the polypeptide includes a YT521 -B homology (YTH) domain. The YTH domain is found in the YTH family of proteins that identify N6-methyladenosine (m6A) marks in RNA. The YTH domain ranges from 100 to 150 amino acids residues in length and forms a six- stranded p barrel surrounded by four or five a helices. Three residues in the hydrophobic core of the p barrel trap the methyl group of m6A in an aromatic cage consisting of hydrogen bonds with the adenosine and IT interactions between tryptophan rings and the methyl group. The YTH domain specifically binds m6A over unmodified adenosines.In some embodiments, the polypeptide includes a double-stranded RNA binding domain or motif. The double-stranded RNA binding domain or motif may include from 65 to 70 amino acids residues. Double-stranded RNA binding domains specifically recognize and bind double-stranded RNA and are found in proteins with roles in viral protection, RNAi, and cellular transport. Doublestranded RNA binding domains often appear as tandem repeats or in combination with other functional RNA-binding domains, such as RNA-editing or helicase domains. The double-stranded RNA binding domain is made up of an a1 p1 p2p3a2 fold that forms an antiparallel p sheet flanked by a helices on one face. Double-stranded RNA binding domains specifically recognize the structure of an A-form RNA helix, spanning up to 16 base pairs with hydrogen-bond contacts to the phosphodiester backbone and 2' OH. In some cases, double-stranded RNA binding domains have demonstrated base-specific contacts, such as to bases in adjacent loops.In some embodiments, the polypeptide includes a helicase domain. Helicase domains are found in all forms of life in helicase proteins, which unwind both DNA and double-stranded RNA. Helicases include six superfamilies, of which superfamily 1 and superfamily 2 contain all the eukaryotic RNA helicases. RNA-binding helicases include the Upf1 -like family in superfamily 1 and the DEAD-box, DEAH, RIG-l-like, Ski2-like, and NS3 families in superfamily 2. The remaining superfamilies, 3-6, contain bacterial and viral helicases that form multimeric rings. Helicase domains are very large, containing 350-400 amino acid residues. In superfamily 1 and superfamily 2, the helicase domain is composed of two “recombinase A (recA)-like” subdomains, each of which contains an ATP-catalytic core, a nucleic-acid-binding region, and subdomains that coordinate the two. Within families of helicases these subdomains are quite conserved. Helicase monomers in the ring-forming superfamilies of helicases are similarly quite large and composed of multiple subdomains. Bound RNA is surrounded by recA-like domains or, in the case of multimeric helicases, RNA is pulled through the center of the ring. Contacts with RNA are dominated by hydrogen bonds to phosphate and sugar moieties, but contacts with bases have only occasionally been observed. Affinities to RNA for proteins having a helicase domain are often in the nanomolar range, although they vary greatly by helicase and are modulated by other subdomains of the helicase. ATP binding generally promotes higher affinity to RNA by causing the helicase RNA-binding regions to clamp.In some embodiments, the polypeptide may include a cold shock domain. The cold shock domain is found in a large family of proteins associated with cold adaptation found in all domains of life. Cold shock domains are composed of about 70 amino acids residues or more and five antiparallelp strands that form a common p barrel structure known as an oligosaccharide / oligonucleotide-binding fold. Cold shock domains contain the conserved ribonucleoprotein 1 and ribonucleoprotein 2 motifs common to RNA recognition motifs, which bind single-stranded RNA. Cold shock domains contact 3 or 4 nucleotides through sequential stacking interactions and hydrogen bonds with bases, achieving nanomolar affinities. Cold shock domain containing proteins vary greatly in the types of sequences they recognize.In some embodiments, the polypeptide includes an S1 RNA-binding domain. The S1 domain includes about 70 amino acid residues, which forms a 5-stranded antiparallel p barrel in the same oligonucleotide binding fold family as the cold shock domain. S1 domains are additionally found in several exoribonucleases and eukaryotic translation initiation factors and in combination with other RNA-binding domains such as the KH domain or cold shock domains. Despite their abundance, very little structural information is available for S1 domains in complex with RNA. S1 domains interact with both single-stranded RNA and double-stranded RNA in the context of the RNA-binding channel of exoribonucleases.In some embodiments, the polypeptide includes a Sm-RNA binding motif. The Sm RNA- binding motif is found in Sm and like-Sm proteins in eukaryotes and archaea and in Hfq protein in prokaryotes. The Sm motif consists of about 70 residues with an a1 p1 p2p3p4p5 topology that forms a curved antiparallel p sheet. Sm-containing proteins readily multimerize through interactions between strands p4 and p5 in two Sm motifs. For example, Sm-Sm interactions link the seven human Sm proteins that make up the protein core of small nuclear ribonucleoproteins. The Sm multimers bind RNA with nanomolar affinity. Two Sm motifs form a 6-nucleotide binding surface that binds specific bases, often uridines, through hydrogen bonds and stacking interactions.In some embodiments, the polypeptide includes a La motif. La motif includes about 90 amino acid residues and is found in eukaryotic La and La-related proteins (LARPs). The La motif consists of five a helices and three p strands that form a small antiparallel p sheet against a modified winged- helix fold. The winged-helix structure itself is common to several other RNA — binding proteins. La motifs are always found adjacent to at least one RNA recognition motif, where the combination of these two domains likely evolved as a unit. In La proteins, the dual La motif and RNA recognition motif region tightly binds the UUU-OH elements at the 3' ends of polymerase-l Il-transcribed small RNAs. Binding occurs in a cleft between the La motif and the RNA recognition motif rather than the traditional RNA-binding surfaces of either the RNA recognition motif or the La motif winged-helix fold. Several uracil bases stack with highly conserved aromatic residues in the La motif, and hydrogen bonds from both the La motif and RNA-recognition motif coordinate bases, phosphates, and the terminating 2' OH. These contacts result in low-nanomolar affinities of the La motif for 3'-terminal UUU-OH elements.In some embodiments, the polypeptide includes a Piwi-Argonaute-Zwille or PIWI RNA-binding domains. These RNA binding domains define the Argonaute family of proteins found in eukaryotes. These domains are found on opposite sides of the Argonaute protein, both domains facilitate binding of small interfering RNA and microRNA guides to mRNA targets. The Piwi-Argonaute-Zwille domain occurs in Dicer proteins in addition to Argonaute proteins. Crystal structures of the Piwi-Argonaute-Zwille domain display a six-stranded p barrel topped with two a helices and flanked on the opposite side by a special appendage containing a p hairpin and short a helix. A binding pocket formed between this appendage and the p barrel binds the 2 nucleotide 3' overhang in guide RNAs with low- micromolar affinity. Binding is coordinated mostly by conserved tyrosine residues that form hydrogen bonds with the phosphate backbone and sugar hydroxyls of the two terminal nucleotides. The PIWI RNA-binding domain tertiary structure forms an Rnase H-like fold consisting of a five-stranded p sheet flanked by a helices on both faces. The PIWI domain has endonucleolytic activity in some cases, but primarily stabilizes the gRNA-mRNA duplex seed region through hydrogen bonds with the gRNA backbone of nucleotides 3-5 and the 5' overhang base.In some embodiments, the polypeptide includes an intrinsically disordered region. Intrinsically disordered regions are unstructured and often consist of repeats of arginine / serine residues, arginine / glycine, arginine- or lysine-rich patches, or short linear motifs of amino acids. Despite their lack of structure, intrinsically disordered regions have been found to dominate the composition in over 20% of RNA binding proteins. Intrinsically disordered regions may be the sole RNA-binding domain in an RNA binding protein and may actually drive the majority of protein-RNA interactions in the cell. Like globular RNA-binding domains, intrinsically disordered regions are conserved, often occur multiple times in one RNA binding protein, and can coordinate RNA binding in concert with other domains. Intrinsically disordered regions have been shown to drive higher affinity to RNA in RNA binding proteins that contain ordered RNA-binding domains and can themselves transition to an ordered state once bound to RNA. Intrinsically disordered regions show little RNA sequence dependence, however, suggesting that these regions’ high affinity for RNA is predominantly driven by electrostatic attraction to the phosphodiester backbone.In some embodiments, the bridging agent includes a structure PP1-L-PP2, wherein PP1 includes a first polypeptide, PP2 includes a second polypeptide, and L is a linker of one or more atoms.In some embodiments, the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.Small Molecule Bridging AgentsIn some embodiments, the bridging agent includes a small molecule. In some embodiments, the small molecule includes an intercalator. In some embodiments, the small molecule may bind to a complementary group of the linear polyribonucleotide by way of click chemistry. For a review, see, e.g., De Fazio et al., Chem. Soc. Rev. 50, 13410, 2021 .Photoreactive Crosslinking AgentsThe disclosure provides a complex that includes a linear polyribonucleotide and a bridging agent (e.g., a polynucleotide, polypeptide, or small molecule) containing at least two photoreactive crosslinking agents. The photoreactive crosslinking agents provide at least two covalent attachments between the bridging agent and the linear polyribonucleotide upon irradiation with light, e.g., at a first wavelength. In some embodiments, the photoreactive crosslinking agent is reversibly attached. Forexample, one or both of the covalent attachments may be removed upon irradiation with light, e.g., at a second wavelength that is different from the first wavelength. In some embodiments, the photoreactive crosslinking agents are attached to the end of the bridging agent. In some embodiments, the photoreactive crosslinking agents are located at an internal position within the bridging agent.The bridging agent may contain a plurality of photoreactive crosslinking agents. In some embodiments, each of the photoreactive crosslinking agents are the same. In some embodiments, the photoreactive crosslinking agents are different from one another. In some embodiments, each of the photoreactive crosslinking agents is located at an internal position within the bridging agent. In some embodiments, at least one of the photoreactive crosslinking agents is attached to an end of the bridging agent. In some embodiments, at least one of the photoreactive crosslinking agents is attached to a second end of the bridging agent. The photoreactive crosslinking agent may be, for example, a photoreactive nucleotide analog or a photoreactive amino acid analog.Photoreactive Nucleotide AnalogsIn some embodiments, the bridging agent includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) photoreactive nucleotide analogs. In some embodiments, each of the one or more photoreactive nucleotide analogs replaces a single nucleotide within the polynucleotide. Each of the one or more photoreactive nucleotide analogs may include a nucleotide or nucleoside modified to contain a photoreactive group. In some embodiments, the photoreactive nucleotide analog crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the opposing ribonucleotide within the linear polyribonucleotide upon photoirradiation. The disclosure provides photoreactive nucleotide analogs included within the polynucleotide. Photoreactive nucleotide analogs include, for example, 4-thiouridene (4sU), 5-bromo-2’-deoxyuridine (BrdU), coumarin derivatives, 3-cyanovinylcarbazole D-threoninol (CNVD) derivatives, 3- cyanovinylcarbazole nucleoside (CNVK) derivatives, diazirene derivatives, phenylselenide derivatives, psoralen derivatives, or pyranocarbazole nucleoside (PCX) derivatives.In some embodiments, the photoreactive crosslinking agent includes 5-bromo-2’-deoxyuridine (BrdU), a carbazole (e.g., a vinylcarbazole), a psoralen, a coumarin, 4’-thiouridine, a diazirine, a phenylselenide, a furan, or an abasic site.In some embodiments, the carbazole is 3-cyanovinylcarbazole, 4-methylpyranocarbazole, or pyranocarbazole.In some embodiments, the coumarin is 7-hydroxycoumarin.In some embodiments, the photoreactive nucleotide analog is one disclosed in U.S. Pat. No. 8,481 ,714, the disclosure of which is hereby incorporated by reference in its entirety.In some embodiments, the photoreactive crosslinking agent includes a compound of Formula (I):wherein, in the formula (I), Rarepresents a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen, wherein, in the formula (I), Ri and R2 each independently represent a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen, and wherein, in the formula (I), Rb represents a sugar, a polysaccharide, a polyether, a polyol, a polypeptide chain, or a water-soluble synthetic polymer.In some embodiments, the photoreactive crosslinking agent is the compound according to Formula (I), wherein Rb is represented by any one of Formulae (II) - (V):wherein, in Formula (IV), n is represented as any number 2-5 (e.g., 2, 3, 4, or 5).In some embodiments, the photoreactive crosslinking agent is the compound of any one of Formulae (VI) - (X):wherein, in Formula (IX), n is represented as any number 2 to 5 (e.g., 2, 3, 4, or 5).In some embodiments, the photoreactive crosslinking agent includes a compound of Formularepresents a hydrogen or methoxy group, wherein in Formula (XII), R3 represents a hydrogen, a methyl group, or a methoxy group, wherein in Formula (XII), FU represents an aminomethyl group, an azidomethyl group, or a hydrogen group, wherein in Formula (XII), Rs represents a hydrogen or methyl group.In some embodiments, the photoreactive crosslinking agent is the compound of any one of Formulae (XIII) - (XVII):In some embodiments, the photoreactive nucleotide analog may be 4sU. The structure of 4sU mimics the structure of uridine analogs but gains photoreactive properties by replacing the carbonyl oxygen at position 4 of the ring with a sulfur atom. Upon irradiation with light having a wavelength of 356 nm, photoreactive 4sU within a polynucleotide covalently attaches to a nearby cytosine nucleobase through a [2+2] cycloaddition reaction.In some embodiments, the photoreactive nucleotide analog may be BrdU. Upon irradiation with light having wavelength of 308 nm, BrdU generates 2’-deoxyuridin-5-yl radicals.In some embodiments, the photoreactive nucleotide analog may be a coumarin analog or a derivative thereof. Coumarin and derivatives thereof located within the polynucleotide covalently attaches to a nearby thymine nucleobase through a [2+2] cycloaddition reaction upon irradiation with light having a wavelength of 250 nm, resulting in the formation of a syn-cycloaddition photo-adduct. Irradiating the syn-cycloaddition photo-adduct with light having a wavelength of 254 nm reverses the reaction. An example of a coumarin derivative that is a photoreactive nucleotide analog is 7- hydroxycoumarin (see, e.g., Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 , which is hereby incorporated by reference in its entirety).In some embodiments, the photoreactive nucleotide analog may beCNVK or derivatives thereof.CNVK and derivatives thereof within a polynucleotide have a vinyl group that is aligned with the C5-C6 double bond of a pyrimidine nucleobase located 1 base downstream of an opposing nucleotide within a nearby polynucleotide. Upon irradiation with light having a wavelength of 366 nm,CNVK or a derivative thereof covalently attaches with a nearby pyrimidine-containing nucleobase through a [2+2] cycloaddition, resulting in the formation of the photo-adduct. Pyrimidine-containing nucleobases include cytosine, thymine, and uracil. Irradiating the photo-adduct with light having a wavelength of 312 nm reverses the reaction. Irradiation with longer wavelength light, as compared to the 254 nm wavelength light used for reversing photo-adduct formation, circumvents the photo-induced DNA damage at shorter wavelengths. The trans isomer compared to the cis isomer ofCNVK is the reactive species for photoreactive crosslinking (see, e.g., Fujimoto et al. J. Am. Chem. Soc.135, 16161 -16167, 2013 which is hereby incorporated by reference in its entirety). Another example of aCNVK derivative is n-CNVK which includes a linker of variable lengths (n = 2-5). Upon irradiation, n-CNVK can form a covalent bond with a nearby pyrimidine nucleobase at locations other than the 1 base downstream position of an opposing nucleotide depending on the linker length (see, e.g., Fujimoto et al. Photochem. Photobiol. Sci. 19, 776-782, 2020, which is hereby incorporated by reference in its entirety).In some embodiments, the photoreactive nucleotide analog may beCNVD, a derivative ofCNVK.CNVD and derivatives thereof within a polynucleotide have a vinyl group that is aligned with the C5-C6 double bond of a pyrimidine nucleobase located 1 base downstream of an opposing nucleotide within a nearby polynucleotide.CNVD is a derivative ofCNVK that has the same chemical structure ofCNVK except for a D-threoninol linker in the place of the deoxyribose sugar which acts to increase the flexibility as compared toCNVK, increasing the photo-induced crosslinking reaction rate due to minimization of entropic losses during hybridization (see, e.g., Sakamoto et al. Org. Let. 17, 936-939, 2015, which is hereby incorporated by reference in its entirety). Upon irradiation with light having a wavelength of 365 nm,CNVD or a derivative thereof covalently attaches with a nearby pyrimidine- containing nucleobase through a [2+2] cycloaddition, resulting in the formation of the photo-adduct. Pyrimidine-containing nucleobases include cytosine, thymine, and uracil. Irradiating the photo-adduct with light having a wavelength of 312 nm reverses the reaction. Irradiation with longer wavelength light, as compared to the 254 nm wavelength light used for reversing photo-adduct formation, circumvents photo-induced DNA damage at shorter wavelengths.In some embodiments, the photoreactive nucleotide analog may be a diazirene derivative. Upon irradiation with light having a wavelength of 365 nm, a diazirene derivative becomes a reactive carbene intermediate. The reactive carbene intermediates rapidly form a covalent bond with a nearby nucleobase through C-C, C-H, O-H, or X-H (X = heteroatom) insertions. The reactive carbene intermediate can react with all nucleobases, including adenine, cytosine, guanine, thymine, and uracil, forming a covalent attachment with a nearby nucleobase. An example of a diazirene derivative is 3- phenyl-3-trifluoromethyl-3- / 7-diazirine which is attached to the sugar ring through an acetal linkage (see, e.g., Nakamoto et al. J. Org. Chem. 79, 2463-2472, 2014, which is hereby incorporated by reference in its entirety).In some embodiments, the photoreactive nucleotide analog may be a phenyl selenide analog. Upon irradiation with light having a wavelength of 350 nm, phenyl selenide becomes a radical intermediate which has the capability of alkylating the N1 or N6 position of nearby purine nucleobases, including adenine. An example of a phenyl selenide analog is phenyl selenide-modified 2’-deoxythymidine (see, e.g., Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 , which is hereby incorporated by reference in its entirety).In some embodiments, the photoreactive nucleotide analog may be a psoralen analog or a derivative thereof. Psoralen and derivatives thereof are tricyclic compounds that may intercalate in any AT or AU region of hybridized DNA and RNA sequences, respectively. Psoralen and derivatives thereof react through their furan or pyrone photoreactive site to covalently attach the C5-C6 double bond of pyrimidine residues. This forms a covalent attachment between two nearby pyrimidine nucleobases through a [2+2] cycloaddition reaction upon irradiation with light having a wavelength of 365 nm, resulting in the formation of a cyclobutene photo-adduct. The resulting cyclobutene photoadduct may be in the syn- or cis- configuration. Pyrimidine residues are present among cytosine, thymine, and uracil bases. Irradiating the cyclobutene photo-adduct with light having a wavelength of 254 nm reverses the reaction. An example of a psoralen derivative is 4’-aminomethyltrioxsalen (see, e.g., Velema et al. JACS Au 3, 316-332, 2023, which is hereby incorporated by reference in its entirety).In some embodiments, the photoreactive nucleotide analog may be pyranocarbazolenucleoside (PCX) and derivatives thereof. The photoreactivePCX and derivatives thereof within a polynucleotide include a vinyl group that is aligned with the C5-C6 double bond of a pyrimidine nucleobase located 1 base downstream of an opposing nucleotide within a nearby a polynucleotide. Upon irradiation with light having a wavelength of 400 nm,PCX covalently attaches with a nearby, pyrimidine-containing nucleobase through a [2+2] cycloaddition, resulting in the formation of the photo-adduct. Pyrimidine-containing nucleobases include cytosine, thymine, and uracil. Irradiating the photo-adduct with light having a wavelength of 312 nm reverses the reaction. Irradiation with longer wavelength light, as compared to the 254 nm wavelength light used for reversing photo-adduct formation, circumvents the photo-induced DNA damage at shorter wavelengths. An example of a pyranocarbozole derivative isPCX with a D-threoninol linker in the place of the deoxyribose sugar (PCXD) which acts to increase the flexibility as compared toPCX, increasing the photo-induced crosslinking reaction rate due to minimization of entropic losses during hybridization (see, e.g., Fujimoto et al. RSC Adv. 9, 30693-30697, 2019, which is hereby incorporated by reference in its entirety). Another example of a pyranocarbozole derivative isPCX that includes a methyl group at the 4-position ofPCX. 4-methylpyranocarbozole nucleoside (MEPK) is able to differentiate between thymine and cytosine, enabling selectivity toward thymine (see, e.g., Mihara et al. Org. Biomol. Chem. 19, 9860-9866, 2021 , which is hereby incorporated by reference in its entirety).In some embodiments, the polynucleotide may include another photoreactive nucleotide analog which allows for the polynucleotide bridging agent to covalently attach to the polyribonucleotide described herein. For a review of certain photoreactive nucleotide analogs, see,e.g., Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 ; Tavakoli et al., RSC Adv. 12, 6484-6507, 2022; and Velema et al. JACS Au 3, 316-332, 2023.Photoreactive Amino Acid AnalogsIn some embodiments, the bridging agent includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) photoreactive amino acid analogs. In some embodiments, the photoreactive amino acid analog replaces an amino acid within a polypeptide bridging agent. The one or more photoreactive amino acid analogs may include an amino acid modified to contain a photoreactive group. Photoreactive nucleotide analogs include, for example, aryl azide-based, benzophenone-based, or diazirene-based unnatural amino acids or A / -e-[2-(furan-2-yl)ethoxy]carbonyl-lysine.In some embodiments, the photoreactive amino acid analog may be an aryl azide-based unnatural amino acid. Upon irradiation with light having a wavelength of 250-350 nm, aryl azide-based unnatural amino acids generate a reactive nitrene intermediate. The reactive nitrene intermediate can covalently bond with a nearby nucleobase through C-H and X-H insertions. An example of an arylazide-based unnatural amino acid is tetrafluorophenylazide (see, e.g., Smith et al Future Med. Chem. 7, 159-183, 2015, which is hereby incorporated by reference in its entirety).In some embodiments, the photoreactive amino acid analog may be a benzophenone-based unnatural amino acid. Benzophenone-based unnatural amino acids can be easily prepared and are further beneficial due to their inertness to solvents. Upon irradiation with light having a wavelength of 350-365 nm, benzophenone-based unnatural amino acids generate a reactive diradical intermediate, containing reactive triplet carbonyl states. The reactive diradical intermediate can covalently attach to a nearby nucleobase through C-H insertion. Benzophenone-based unnatural amino acids are activated with light of a longer wavelength, reducing the risk of damage to biomolecules; however, irradiation times are longer, risking the potential for non-specific interactions.In some embodiments, the photoreactive amino acid analog may be a diazirene-based unnatural amino acid. Upon irradiation with light having a wavelength of 350-380 nm, diazirine-based unnatural amino acids generate a reactive carbene intermediate. The reactive carbene intermediates rapidly form a covalent bond with a nearby nucleobase through C-C, C-H, O-H, or X-H (X = heteroatom) insertions. The reactive carbene intermediate can react with all nucleobases, including adenine, cytosine, guanine, thymine, and uracil, forming a covalent attachment with a nearby nucleobase. One example of an alkyl diazirene-based amino acid is a pyrrolysine analog that bears an alkyl diazirene reactive group attached by a short, flexible C2 linker (see, e.g., Dziuba et al. ChemBioChem 2020, 21, 88-93, which is hereby incorporated by reference in its entirety).In some embodiments, the photoreactive amino acid analog may be A / -e-[2-(furan-2- yl)ethoxy]carbonyl-lysine. A / -e-[2-(furan-2-yl)ethoxy]carbonyl-lysine is genetically encodable and can be irradiated with red light to covalently attach the amino acid to a nearby nucleobase (see, e.g., Moritz et al. Angew. Chem. 2013, 52, 4690-4693, , which is hereby incorporated by reference in its entirety).Functional Groups, Tags, and Targeting MoietiesIn some embodiments, the bridging agent includes a functional group. In some embodiments, the functional group includes a thiol group, an N-hydroxysuccinimide ester, a polypeptide, a polynucleotide, an azide, a dye, or a targeting moiety. A targeting moiety binds specifically to a cell or a portion thereof (e.g., an extracellular, intracellular, or membrane portion thereof), thereby promoting intracellular delivery of the moiety and any cargo bound or complexed thereto. Upon incorporation of the bridging agent into the linear polyribonucleotide, the functional group or tag is covalently attached upon photocirculizarization.In some embodiments, the bridging agent includes a tag (e.g., a purification tag). In some embodiments, the tag includes biotin.In some embodiments, the targeting moiety includes a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid, or a combination thereof. In some embodiments, the targeting moiety includes a small molecule.In some embodiments, the small molecule is selected from folic acid, urea, a-mannose, high mannose, ursodeoxycholic acid, an endosomal escape agent, or lithocholic acid.In some embodiments, the targeting moiety includes a polypeptide. In some embodiments, the polypeptide is a cell-penetrating peptide. In some embodiments, the polypeptide is selected from ASSLNIA (SEQ ID NO: 19), M12, RGD, melittin, LPS-binding protein (LBP) peptide, an adiposehoming peptide, or an endolytic peptide. In some embodiments, the polypeptide is an antibody or a target-binding fragment thereof. In some embodiments, the antibody or target-binding fragment thereof is selected from a monoclonal antibody or target-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, or a tandem scFv (taFv). In some embodiments, the antibody or target-binding fragment thereof is selected from an anti-FcRn antibody, an anti-MR antibody, an anti-CD205 antibody, an anti-CD169 antibody, an anti-CD14 antibody, an anti-CD36 antibody, an anti-CD5 antibody, an anti -CD71 antibody, an anti-CD38 antibody, or an anti- prohibin antibody.In some embodiments, the polypeptide is a nanobody. In some embodiments, the nanobody is selected from an anti-transferrin nanobody, an anti-HER2 nanobody, or an anti-EGFR nanobody. In some embodiments, the targeting moiety includes a carbohydrate. In some embodiments, the carbohydrate includes a saccharide, disaccharide, or polysaccharide. In some embodiments, the carbohydrate includes mannose, galactose, or glucose. In some embodiments, the carbohydrate includes GalNAc or mannose 6-phosphate. In some embodiments, the carbohydrate includes a mono-, di-, tri-, or tetra-GalNAc. In some embodiments, the carbohydrate is tri-GalNAc.In some embodiments, the targeting moiety includes a lipid. In some embodiments, the lipid includes a fatty acid. In some embodiments, the fatty acid is a saturated, monounsaturated, or polyunsaturated fatty acid. In some embodiments, the fatty acid is a branched or unbranched chain including from 4 to 40 (e.g., 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, or 40) main-chain carbon atoms. In some embodiments, the fatty acid includes squalene, stearic acid, oleic acid, palmitic acid, linoleic acid,stearic acid, lauric acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), eicosapentaenoic acid (EPA), octadecanoic acid, myristic acid, anadamide, a-tocopherol, a-tocopherol succinate, or a retinoic acid. In some embodiments, the fatty acid includes docosanoic acid. In some embodiments, the fatty acid includes docosahexanoic acid. In some embodiments, the fatty acid includes myristic acid. In some embodiments, the lipid includes a steroid or sterol selected from cholesterol, tocopherol, ursodeoxycholic acid, or lithocholic acid. In some embodiments, the steroid or sterol is cholesterol. In some embodiments, the steroid or sterol is tocopherol. In some embodiments, the lipid includes a fatsoluble vitamin selected from vitamin A, vitamin D, vitamin E, vitamin K, or an analog or metabolite thereof. In some embodiments, the lipid includes a phospholipid. In some embodiments, the phospholipid is selected from phosphocholine (PC), PC-docosahexaenoic acid (PC-DHA), PC- docosanoic acid (PC-DCA), PC-eicosapentaenoic acid (PC-EPA), PC-lithocholic acid (PC-LA), PC- retinoic acid (PC-RA), and PC-a-tocopherol succinate (PC-TS).In some embodiments, the targeting moiety includes an oligonucleotide. In some embodiments, the targeting moiety includes an aptamer.PolyribonucleotidesThe disclosure provides polyribonucleotides (e.g., linear precursors) that are complexed with a bridging agent or circular polyribonucleotides produced by a method as described herein, e.g., using irradiation. The elements described in the following sections may be present in a linear polyribonucleotide precursor or a circular polyribonucleotide upon photocircularization.Expression SequencesIn some embodiments, the polyribonucleotide includes at least one expression sequence (e.g., coding region) that encodes a polypeptide. In some embodiments, the polypeptide is functional when expressed in a cell. In some embodiments, the polyribonucleotide includes or more expression sequences.The encoded polypeptide may have a length from about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1 ,000 to about 2,500 amino acids, or any range therebetween. In some embodiments, the polypeptide has a length of less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1 ,500 amino acids, less than about 1 ,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less may be useful.Some examples of a polypeptides include a fluorescent tag or marker, an antigen, a peptide therapeutic, a synthetic or analog peptide from a naturally-bioactive peptide, an agonist or antagonist peptide, an anti-microbial peptide, a pore-forming peptide, a bicyclic peptide, a targeting or cytotoxic peptide, a degradation or self-destruction peptide, and degradation or self-destruction peptides. Peptides useful in the invention described herein also include antigen-binding peptides, e.g., antigen binding antibody or antibody-like fragments, such as single chain antibodies, nanobodies (see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21 (7): 1076-113). Such antigen binding peptides may bind a cytosolic antigen, a nuclear antigen, an intra-organellar antigen.The polypeptide may be produced in substantial amounts. As such, the polypeptide may be any proteinaceous molecule that can be produced. A polypeptide can be a polypeptide that can be secreted from a cell, or localized to the cytoplasm, nucleus, or membrane compartment of a cell. Some polypeptides include, but are not limited to, at least a portion of a viral envelope protein, metabolic regulatory enzymes (e.g., that regulate lipid or steroid production), an antigen, a toleragen, a cytokine, a toxin, enzymes whose absence is associated with a disease, and polypeptides that are not active in an animal until cleaved (e.g., in the gut of an animal), and a hormone.In some embodiments, the polyribonucleotide includes an expression sequence encoding a protein e.g., a therapeutic protein. In some embodiments, therapeutic proteins that can be expressed from the circular polyribonucleotide disclosed herein have antioxidant activity, binding, cargo receptor activity, catalytic activity, molecular carrier activity, molecular function regulator, molecular transducer activity, nutrient reservoir activity, protein tag, structural molecule activity, toxin activity, transcription regulator activity, translation regulator activity, or transporter activity. Some examples of therapeutic proteins may include, but are not limited to, an enzyme replacement protein, a protein for supplementation, a protein vaccination, antigens (e.g. tumor antigens, viral, bacterial), hormones, cytokines, antibodies, immunotherapy (e.g. cancer), cellular reprogramming / transdifferentiation factor, transcription factors, chimeric antigen receptor, transposase or nuclease, immune effector (e.g., influences susceptibility to an immune response / signal), a regulated death effector protein (e.g., an inducer of apoptosis or necrosis), a non-lytic inhibitor of a tumor (e.g., an inhibitor of an oncoprotein), an epigenetic modifying agent, epigenetic enzyme, a transcription factor, a DNA or protein modification enzyme, a DNA-intercalating agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a competitive inhibitor for an enzyme, a protein synthesis effector or inhibitor, a nuclease, a protein fragment or domain, a ligand or a receptor, and a CRISPR system or component thereof.In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include human proteins, for instance, receptor binding protein, hormone, growth factor, growth factor receptor modulator, and regenerative protein (e.g., proteins implicated in proliferation and differentiation, e.g., therapeutic protein, for wound healing). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotidedisclosed herein include EGF (epithelial growth factor). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include enzymes, for instance, oxidoreductase enzymes, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzyme, and desaturases. In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include an intracellular protein or cytosolic protein. In some embodiments, the circular polyribonucleotide expresses a NanoLuc® luciferase (nLuc). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include a secretary protein, for instance, a secretary enzyme. In some cases, the circular polyribonucleotide expresses a secretary protein that can have a short half-life therapeutic in the blood or can be a protein with a subcellular localization signal, or protein with secretory signal peptide. In some embodiments, the circular polyribonucleotide expresses a Gaussia Luciferase (gLuc). In some cases, the circular polyribonucleotide expresses a non-human protein, for instance, a fluorescent protein, an energytransfer acceptor, or a protein-tag like Flag, Myc, or His. In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide include a GFP. In some embodiments, the circular polyribonucleotide expresses tagged proteins, .e.g., fusion proteins or engineered proteins containing a protein tag, e.g., chitin binding protein (CBP), maltose binding protein (MBP), Fc tag, glutathione-S-transferase (GST), AviTag, Calmodulin-tag, polyglutamate tag; E-tag, FLAG-tag), HA- tag, His-tag, Myc-tag, NE-tag, S-tag, SBP-tag, Softag 1 , Softag 3, Spot-tag, Strep-tag; TC tag, Ty tag, V5 tag ; VSV-tag; or Xpress tag.In some embodiments, the circular polyribonucleotide encodes the expression of an antibody, e.g., an antibody fragment, or a portion thereof. In some embodiments, the antibody expressed by the circular polyribonucleotide can be of any isotype, such as IgA, IgD, IgE, IgG, or IgM. In some embodiments, the circular polyribonucleotide expresses a portion of an antibody, such as a light chain, a heavy chain, a Fc fragment, a CDR (complementary determining region), a Fv fragment, or a Fab fragment, a further portion thereof. In some embodiments, the circular polyribonucleotide expresses one or more portions of an antibody. For instance, the circular polyribonucleotide can include more than one expression sequence, each of which expresses a portion of an antibody, and the sum of which can constitute the antibody. In some cases, the circular polyribonucleotide includes one expression sequence coding for the heavy chain of an antibody, and another expression sequence coding for the light chain of the antibody. In some cases, when the circular polyribonucleotide is expressed in a cell or a cell-free environment, the light chain and heavy chain can be subject to appropriate modification, folding, or other post-translation modification to form a functional antibody.Regulatory ElementsIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes one or more regulatory elements. In some embodiments, the polyribonucleotide includes a regulatory element, e.g., a sequence that modifies expression of an expression sequence within the polyribonucleotide.A regulatory element may include a sequence that is located adjacent to an expression sequence that encodes an expression product. A regulatory element may be linked operatively to the adjacent sequence. A regulatory element may increase an amount of product expressed as compared to an amount of the expressed product when no regulatory element exists. In addition, one regulatory element can increase amounts of products expressed for multiple expression sequences attached in tandem. Hence, one regulatory element can enhance the expression of one or more expression sequences. Multiple regulatory elements are well-known to persons of ordinary skill in the art.In some embodiments, the regulatory element is a translation modulator. A translation modulator can modulate translation of the expression sequence in the polyribonucleotide. A translation modulator can be a translation enhancer or suppressor. In some embodiments, the polyribonucleotide includes at least one translation modulator adjacent to at least one expression sequence. In some embodiments, the polyribonucleotide includes a translation modulator adjacent each expression sequence. In some embodiments, the translation modulator is present on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide (s).In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site.Further examples of regulatory elements are described, e.g., in paragraphs

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[0161] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.Internal Ribosomal Entry SitesIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences). In embodiments, the IRES is located between a heterologous promoter and the 5’ end of a coding sequence.A suitable IRES element to include in a polyribonucleotide includes an RNA sequence capable of engaging a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.In some embodiments, the IRES element is derived from the DNA of an organism including, but not limited to, a virus, a mammal, or a Drosophila. Such viral DNA may be derived from, but is not limited to, picomavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, Drosophila DNA from which an IRES element is derived includes, but is not limited to, an Antennapedia gene from Drosophila melanogaster.In some embodiments, if present, the IRES sequence is an IRES sequence of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis invictavirus 1 , Rhopalosiphum padi virus, Reticuloendotheliosis virus, fuman poliovirus 1 , Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1 , Human Immunodeficiency Virus type 1 , Homalodisca coagulata virus- 1 , Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 , Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1 , Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1 , Human AML1 / RUNX1 , Drosophila antennapedia, Human AQP4, Human AT1 R, Human BAG-I, Human BCL2, Human BiP, Human c-IAPI , Human c-myc, Human elF4G, Mouse NDST4L, Human LEF1 , Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-I, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Salivirus, Cosavirus, Parechovirus, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1 , Human c-src, Human FGF-I, Simian picomavirus, Turnip crinkle virus, Aichivirus, Crohivirus, Echovirus 11 , an aptamer to elF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 / 2). In yet another embodiment, the IRES is an IRES sequence of Coxsackievirus B3 (CVB3). In a further embodiment, the IRES is an IRES sequence of Encephalomyocarditis virus. In a further embodiment, the IRES is an IRES sequence of Theiler's encephalomyelitis virus. In some embodiments, the IRES sequence has more than 90% sequence identify with one of the foregoing IRES sequences.In some embodiments, the polyribonucleotide includes at least one IRES flanking at least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, the IRES flanks both sides of at least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, the polyribonucleotide includes one or more IRES sequences on one or both sides of each expression sequence, leading to separation of the resulting peptide(s) and or polypeptide(s).In some embodiments, a polyribonucleotide described herein includes an IRES (e.g., an IRES operably linked to a coding region). For example, the polyribonucleotide may include any IRES as described in Fan et al. Nature Communications 13(1 ):3751 -3765, 2022 doi: 10.1038 / s41467-022- 31327-y; Chen et al. Nature Biotechnology 41 :262-272, 2023; Chen et al. Mol. Ce / / 81 (20):4300-4318, 2021 ; Jopling et al. Oncogene 20:2664-2670, 2001 ; Baranick et al. PNAS 105(12):4733-4738, 2008; Lang et al. Molecular Biology of the Cell 13(5) :1792-1801 , 2002; Dorokhov et al. PNAS 99(8):5301 - 5306, 2002; Wang et al. Nucleic Acids Research 33(7) :2248-2258, 2005; Petz et a. Nucleic Acids Research 35(8):2473-2482, 2007; Chen et al. Science 268:415-417, 1995; and International Publication No. WQ2020 / 198403; International Patent Publication No. WO 2021 / 263124 A2; and International Publication No. WO2022 / 271965, each of which is hereby incorporated by reference in their entirety.Signal SequencesIn some embodiments, a polypeptide expressed from a circular polyribonucleotide disclosed herein includes a secreted protein, for example, a protein that naturally includes a signal sequence, orone that does not usually encode a signal sequence but is modified to contain one. In some embodiments, the polypeptide encoded by the circular polyribonucleotide includes a secretion signal. For example, the secretion signal may be the naturally encoded secretion signal for a secreted protein. In another example, the secretion signal may be a modified secretion signal for a secreted protein. In other embodiments, the polypeptide encoded by the circular polyribonucleotide does not include a secretion signal.In some embodiments, a circular polyribonucleotide encodes multiple copies of the same polypeptide (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more). In some embodiments, at least one copy of the polypeptide includes a signal sequence and at least one copy of the polypeptide does not include a signal sequence. In some embodiments, a circular polyribonucleotide encodes plurality of polypeptides (e.g., a plurality of different polypeptides or a plurality of polypeptides having less than 100% sequence identity), where at least one of the plurality of polypeptides includes a signal sequence and at least one copy of the plurality of polypeptides does not include a signal sequence.In some embodiments, the signal sequence is a wild-type signal sequence that is present on the N-terminus of the corresponding wild-type polypeptide, e.g., when expressed endogenously. In some embodiments, the signal sequence is heterologous to the polypeptide, e.g., is not present when the wild-type polypeptide is expressed endogenously. A polyribonucleotide sequence encoding a polypeptide may be modified to remove the nucleotide sequence encoding a wild-type signal sequence and / or add a sequence encoding a heterologous signal sequence.A polypeptide encoded by a polyribonucleotide may include a signal sequence that directs the polypeptide to the secretory pathway. In some embodiments, the signal sequence may direct the polypeptide to reside in certain organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes). In some embodiments, the signal sequence directs the polypeptide to be secreted from the cell. For secreted proteins, the signal sequence may be cleaved after secretion, resulting in a mature protein. In other embodiments, the signal sequence may become embedded in the membrane of the cell or certain organelles, creating a transmembrane segment that anchors the protein to the membrane of the cell, endoplasmic reticulum, or Golgi apparatus. In certain embodiments, the signal sequence of a transmembrane protein is a short sequence at the N-terminal of the polypeptide. In other embodiments, the first transmembrane domain acts as the first signal sequence, which targets the protein to the membrane.In some embodiments, a polypeptide encoded by a polyribonucleotide includes either a secretion signal sequence, a transmembrane insertion signal sequence, or does not include a signal sequence.Cleavage DomainsA circular polyribonucleotide of the disclosure can include a cleavage domain (e.g., a stagger element or a cleavage sequence).The term “stagger element” refers to a moiety, such as a nucleotide sequence, that induces ribosomal pausing during translation. In some embodiments, the stagger element is a non-conservedsequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence - D(V / l)ExNPGP, where x= any amino acid (SEQ ID NO: 6). In some embodiments, the stagger element may include a chemical moiety, such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereof.In some embodiments, the circular polyribonucleotide includes at least one stagger element adjacent to an expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element adjacent to each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide(s). In some embodiments, the stagger element is a portion of the one or more expression sequences. In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and each of the one or more expression sequences is separated from a succeeding expression sequence by a stagger element on the circular polyribonucleotide. In some embodiments, the stagger element prevents generation of a single polypeptide (a) from two rounds of translation of a single expression sequence or (b) from one or more rounds of translation of two or more expression sequences. In some embodiments, the stagger element is a sequence separate from the one or more expression sequences. In some embodiments, the stagger element includes a portion of an expression sequence of the one or more expression sequences.In some embodiments, the circular polyribonucleotide includes a stagger element. To avoid production of a continuous expression product, e.g., peptide or polypeptide, while maintaining rolling circle translation, a stagger element may be included to induce ribosomal pausing during translation. In some embodiments, the stagger element is at 3’ end of at least one of the one or more expression sequences. The stagger element can be configured to stall a ribosome during rolling circle translation of the circular polyribonucleotide. The stagger element may include, but is not limited to a 2A-like, or CHYSEL (SEQ ID NO: 7) (cis-acting hydrolase element) sequence. In some embodiments, the stagger element encodes a sequence with a C-terminal consensus sequence that is X1X2X3EX5NPGP (SEQ ID NO: 8), where Xi is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid. In some embodiments, this sequence includes a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence - D(V / I)EXNPGP (SEQ ID NO: 9), where x= any amino acid. Some nonlimiting examples of stagger elements includes GDVESNPGP (SEQ ID NO: 10), GDIEENPGP (SEQ ID NO: 1 1 ), VEPNPGP (SEQ ID NO: 12), IETNPGP (SEQ ID NO: 13), GDIESNPGP (SEQ ID NO: 14), GDVELNPGP (SEQ ID NO: 15), GDIETNPGP (SEQ ID NO: 16), GDVENPGP (SEQ ID NO: 17), GDVEENPGP (SEQ ID NO: 18), GDVEQNPGP (SEQ ID NO: 19), IESNPGP (SEQ ID NO: 20), GDIELNPGP (SEQ ID NO: 21 ), HDIETNPGP (SEQ ID NO: 22), HDVETNPGP (SEQ ID NO: 23), HDVEMNPGP (SEQ ID NO: 24), GDMESNPGP (SEQ ID NO: 25), GDVETNPGP (SEQ ID NO: 26), GDIEQNPGP (SEQ ID NO: 27), and DSEFNPGP (SEQ ID NO: 28).In some embodiments, the stagger element described herein cleaves an expression product, such as between G and P of the consensus sequence described herein. As one non-limiting example, the circular polyribonucleotide includes at least one stagger element to cleave the expression product.In some embodiments, the circular polyribonucleotide includes a stagger element adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element after each expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element is present on one or both sides of each expression sequence, leading to translation of individual peptide(s) and or polypeptide(s) from each expression sequence.In some embodiments, a stagger element includes one or more modified nucleotides or unnatural nucleotides that induce ribosomal pausing during translation. Unnatural nucleotides may include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Examples such as these are distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule. Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage I to the phosphodiester backbone), and any combination thereof that can induce ribosomal pausing during translation. Some of the exemplary modifications provided herein are described elsewhere herein.In some embodiments, the stagger element is present in the circular polyribonucleotide in other forms. For example, in some exemplary circular polyribonucleotides, a stagger element includes a termination element of a first expression sequence in the circular polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a first translation initiation sequence of an expression succeeding the first expression sequence. In some examples, the first stagger element of the first expression sequence is upstream of (5’ to) a first translation initiation sequence of the expression succeeding the first expression sequence in the circular polyribonucleotide. In some cases, the first expression sequence and the expression sequence succeeding the first expression sequence are two separate expression sequences in the circular polyribonucleotide. The distance between the first stagger element and the first translation initiation sequence can enable continuous translation of the first expression sequence and its succeeding expression sequence.In some embodiments, the first stagger element includes a termination element and separates an expression product of the first expression sequence from an expression product of its succeeding expression sequences, thereby creating discrete expression products. In some cases, the circular polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the succeeding sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide including a stagger element of a second expression sequence that is upstream of a second translation initiation sequence of an expression sequence succeeding the second expression sequence is not continuously translated. In some cases, there is only one expression sequence in the circular polyribonucleotide, and the first expression sequence and its succeeding expression sequence are the same expression sequence. In some exemplary circular polyribonucleotides, a stagger element includes a first termination element of a first expression sequence in the circular polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a downstream translation initiation sequence. In some such examples, the first stagger element is upstream of (5’ to) a first translation initiation sequence of thefirst expression sequence in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence enables continuous translation of the first expression sequence and any succeeding expression sequences.In some embodiments, the first stagger element separates one round expression product of the first expression sequence from the next round expression product of the first expression sequences, thereby creating discrete expression products. In some cases, the circular polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the first expression sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide including a stagger element upstream of a second translation initiation sequence of a second expression sequence in the corresponding circular polyribonucleotide is not continuously translated. In some cases, the distance between the second stagger element and the second translation initiation sequence is at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x greater in the corresponding circular polyribonucleotide than a distance between the first stagger element and the first translation initiation in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt,30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater. In some embodiments, the distance between the second stagger element and the second translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater than the distance between the first stagger element and the first translation initiation. In some embodiments, the circular polyribonucleotide includes more than one expression sequence.Examples of stagger elements are described in paragraphs

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[0175] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.In some embodiments, a plurality of polypeptides encoded by a circular ribonucleotide may be separated by an IRES between each polypeptide (e.g., each polypeptide is operably linked to a separate IRES). For example, a circular polyribonucleotide may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence. The IRES may be the same IRES between all polypeptides. The IRES may be different between different polypeptides.In some embodiments, the plurality of polypeptides may be separated by a 2A self-cleaving peptide. For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first polypeptide, a 2A, and a second polypeptide.In some embodiments, the plurality of polypeptides may be separated by a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first polypeptide, a protease cleavage site (e.g., a furin cleavage site), and a second polypeptide.In some embodiments, the plurality of polypeptides may be separated by a 2A self-cleaving peptide and a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a firstpolypeptide, a 2A, a protease cleavage site (e.g., a furin cleavage site), and a second polypeptide. A circular polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first polypeptide, a protease cleavage site (e.g., a furin cleavage site), a 2A, and a second polypeptide. A tandem 2A and furin cleavage site may be referred to as a furin-2A (which includes furin-2A or 2A-furin, arranged in either orientation).Furthermore, the plurality of polypeptides encoded by the circular ribonucleotide may be separated by both IRES and 2A sequences. For example, an IRES may be between one polypeptide and a second polypeptide while a 2A peptide may be between the second polypeptide and the third polypeptide. The selection of a particular IRES or 2A self-cleaving peptide may be used to control the expression level of a polypeptide under control of the IRES or 2A sequence. For example, depending on the IRES and or 2A peptide selected, expression on the polypeptide may be higher or lower.In some embodiments, a circular polyribonucleotide includes at least one cleavage sequence. In some embodiments, the cleavage sequence is adjacent to an expression sequence. In some embodiments, the cleavage sequence is between two expression sequences. In some embodiments, cleavage sequence is included in an expression sequence. In some embodiments, the circular polyribonucleotide includes from 2 to 10 cleavage sequences. In some embodiments, the circular polyribonucleotide includes from 2 to 5 cleavage sequences. In some embodiments, the multiple cleavage sequences are between multiple expression sequences; for example, a circular polyribonucleotide may include three expression sequences two cleavage sequences such that there is a cleavage sequence in between each expression sequence. In some embodiments, the circular polyribonucleotide includes a cleavage sequence, such as in an immolating circRNA or cleavable circRNA or self-cleaving circRNA. In some embodiments, the circular polyribonucleotide includes two or more cleavage sequences, leading to separation of the circular polyribonucleotide into multiple products, e.g., miRNAs, linear RNAs, smaller circular polyribonucleotide, etc.In some embodiments, a cleavage sequence includes a ribozyme RNA sequence. A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNA, but they have also been found to catalyze the aminotransferase activity of the ribosome. Catalytic RNA can be “evolved” by in vitro methods. Similar to riboswitch activity discussed above, ribozymes and their reaction products can regulate gene expression. In some embodiments, a catalytic RNA or ribozyme can be placed within a larger non-coding RNA such that the ribozyme is present at many copies within the cell for the purposes of chemical transformation of a molecule from a bulk volume. In some embodiments, aptamers and ribozymes can both be encoded in the same non-coding RNA.In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, a polyribonucleotide may encode two or more polypeptides, e.g., where the two or more polypeptides are encoded by a single open-reading frame (ORF). For example, two or more polypeptides may be encoded by a single open-reading frame, the expression of which is controlled by an IRES. In some embodiments, the ORF further encodes a polypeptide linker, e.g., such that the expression product of the ORF encodes two or more polypeptides each separated by a sequenceencoding a polypeptide linker (e.g., a linker of 5-200, 5 to 100, 5 to 50, 5 to 20, 50 to 100, or 50 to 200 amino acids). The polypeptide linker may include a cleavage site, for example, a cleavage site recognized and cleaved by a protease (e.g., an endogenous protease in a subject following administration of the polyribonucleotide to that subject). In such embodiments, a single expression product including the amino acid sequence of two or more polypeptides is cleaved upon expression, such that the two or more polypeptides are separated following expression. Exemplary protease cleavage sites are known to those of skill in the art, for example, amino acid sequences that act as protease cleavage sites recognized by a metalloproteinase (e.g., a matrix metalloproteinase (MMP), such as any one or more of MMPs 1 -28), a disintegrin and metalloproteinase (ADAM, such as any one or more of ADAMs 2, 7-12, 15, 17-23, 28-30 and 33), a serine protease (e.g., furin), urokinasetype plasminogen activator, matriptase, a cysteine protease, an aspartic protease, or a cathepsin protease. In some embodiments, the protease is MMP9 or MMP2. In some embodiments, the protease is matriptase.In some embodiments, a circular polyribonucleotide described herein is an immolating circular polyribonucleotide, a cleavable circular polyribonucleotide, or a self-cleaving circular polyribonucleotide. A circular polyribonucleotide can deliver cellular components including, for example, RNA, long non-coding RNA (IncRNA), long intergenic non-coding RNA (lincRNA), microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), small interfering RNA (siRNA), or small hairpin RNA (shRNA). In some embodiments, a circular polyribonucleotide includes miRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites; (iii) degradable linkers; (iv) chemical linkers; and / or (v) spacer sequences. In some embodiments, circRNA includes siRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites (e.g., ADAR); (iii) degradable linkers (e.g., glycerol); (iv) chemical linkers; and / or (v) spacer sequences. Non-limiting examples of self-cleavable elements include hammerhead, splicing element, hairpin, hepatitis delta virus (HDV), Varkud Satellite (VS), and glmS ribozymes.Translation Initiation SequencesIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes at least one translation initiation sequence. In some embodiments, the polyribonucleotide includes a translation initiation sequence operably linked to an expression sequence.In some embodiments, the polyribonucleotide encodes a polypeptide and may include a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotide includes the translation initiation sequence, e.g., Kozak sequence, adjacent to an expression sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, the translation initiation sequence, e.g., Kozak sequence, is present on one or both sides of each expression sequence, leading to separation of the expression products. In some embodiments, the polyribonucleotide includes at least one translation initiation sequenceadjacent to an expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single stranded region of the polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs

[0163] -

[0165] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.The polyribonucleotide may include more than 1 start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 start codons. Translation may initiate on the first start codon or may initiate downstream of the first start codon.In some embodiments, the polyribonucleotide may initiate at a codon which is not the first start codon, e.g., AUG. Translation of the polyribonucleotide may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG. In some embodiments, translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress induced conditions. As a non-limiting example, the translation of the polyribonucleotide may begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the polyribonucleotide translation may begin at alternative translation initiation sequence, CTG / CUG. As another non-limiting example, the polyribonucleotide translation may begin at alternative translation initiation sequence, GTG / GUG. As another non-limiting example, the polyribonucleotide may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g., CGG, GGGGCC (SEQ DI NO: 93), CAG, CTG.Termination ElementsIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes at least one termination element. In some embodiments, the polyribonucleotide includes a termination element operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination element.In some embodiments, the polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element. In some embodiments, the polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the polyribonucleotide is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product.In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element. In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the circular polyribonucleotide iscontinuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product, e.g., peptides or polypeptides, due to lack of ribosome stalling or fall-off. In such an embodiment, rolling circle translation expresses a continuous expression product through each expression sequence. In some other embodiments, a termination element of an expression sequence can be part of a stagger element. In some embodiments, one or more expression sequences in the circular polyribonucleotide includes a termination element. However, rolling circle translation or expression of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide is performed. In such instances, the expression product may fall off the ribosome when the ribosome encounters the termination element, e.g., a stop codon, and terminates translation. In some embodiments, translation is terminated while the ribosome, e.g., at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.In some embodiments, the circular polyribonucleotide includes a termination element at the end of one or more expression sequences. In some embodiments, one or more expression sequences include two or more termination elements in succession. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome completely disengages with the circular polyribonucleotide. In some such embodiments, production of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide may require the ribosome to reengage with the circular polyribonucleotide prior to initiation of translation. Generally, termination elements include an in-frame nucleotide triplet that signals termination of translation, e.g., UAA, UGA, UAG. In some embodiments, one or more termination elements in the circular polyribonucleotide are frame-shifted termination elements, such as but not limited to, off-frame or -1 and + 1 shifted reading frames (e.g., hidden stop) that may terminate translation. Frame-shifted termination elements include nucleotide triples, TAA, TAG, and TGA that appear in the second and third reading frames of an expression sequence. Frame-shifted termination elements may be important in preventing misreads of mRNA, which is often detrimental to the cell. In some embodiments, the termination element is a stop codon.Further examples of termination elements are described in paragraphs

[0169] -

[0170] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.Untranslated RegionsIn some embodiments, a circular polyribonucleotide includes untranslated regions (UTRs). UTRs of a genomic region including a gene may be transcribed but not translated. In some embodiments, a UTR may be included upstream of the translation initiation sequence of an expression sequence described herein. In some embodiments, a UTR may be included downstream of an expression sequence described herein. In some instances, one UTR for first expression sequence is the same as or continuous with or overlapping with another UTR for a second expression sequence.Exemplary untranslated regions are described in paragraphs

[0197] -

[0201] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.In some embodiments, a circular polyribonucleotide includes a polyA tail. Exemplary polyA tails are described in paragraphs

[0202] -

[0205] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety. In some embodiments, a circular polyribonucleotide lacks a polyA tail.In some embodiments, a circular polyribonucleotide includes a UTR with one or more stretches of Adenosines and Uridines embedded within. These AU rich signatures may increase turnover rates of the expression product.Introduction, removal, or modification of UTR AU rich elements (AREs) may be useful to modulate the stability, or immunogenicity (e.g., the level of one or more marker of an immune or inflammatory response) of the circular polyribonucleotide. When engineering specific circular polyribonucleotides, one or more copies of an ARE may be introduced to the circular polyribonucleotide and the copies of an ARE may modulate translation and / or production of an expression product. Likewise, AREs may be identified and removed or engineered into the circular polyribonucleotide to modulate the intracellular stability and thus affect translation and production of the resultant protein.It should be understood that any UTR from any gene may be incorporated into the respective flanking regions of the circular polyribonucleotide.In some embodiments, a circular polyribonucleotide lacks a 5’-UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 3’-UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a polyA tail and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a termination element and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks an internal ribosomal entry site and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a cap and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 5’-UTR, a 3’-UTR, and an IRES, and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide includes one or more of the following sequences: a sequence that encodes one or more miRNAs, a sequence that encodes one or more replication proteins, a sequence that encodes an exogenous gene, a sequence that encodes a therapeutic, a regulatory element (e.g., translation modulator, e.g., translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids that targets endogenous genes (e.g., siRNA, IncRNAs, shRNA), and a sequence that encodes a therapeutic mRNA or protein.In some embodiments, a circular polyribonucleotide lacks a 5’-UTR. In some embodiments, the circular polyribonucleotide lacks a 3’-UTR. In some embodiments, the circular polyribonucleotide lacks a polyA tail. In some embodiments, the circular polyribonucleotide lacks a termination element.In some embodiments, the circular polyribonucleotide lacks an internal ribosomal entry site. In some embodiments, the circular polyribonucleotide lacks degradation susceptibility by exonucleases. In some embodiments, the fact that the circular polyribonucleotide lacks degradation susceptibility can mean that the circular polyribonucleotide is not degraded by an exonuclease, or only degraded in the presence of an exonuclease to a limited extent, e.g., that is comparable to or similar to in the absence of exonuclease. In some embodiments, the circular polyribonucleotide is not degraded by exonucleases. In some embodiments, the circular polyribonucleotide has reduced degradation when exposed to exonuclease. In some embodiments, the circular polyribonucleotide lacks binding to a cap-binding protein. In some embodiments, the circular polyribonucleotide lacks a 5’ cap.Spacer SequencesIn some embodiments, the polyribonucleotides described herein include one or more spacer sequences. A spacer refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacers may be present in between any of the nucleic acid elements described herein. Spacer may also be present within a nucleic acid element described herein.A spacer sequence may be used to separate an IRES from adjacent structural elements to martini the structure and function of the IRES or the adjacent element. A spacer can be specifically engineered depending on the IRES. In some embodiments, an RNA folding computer software, such as RNAFold, can be utilized to guide designs of the various elements of the vector, including the spacers.The spacer may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, each spacer region is at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. Each spacer region may be, e.g., from 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a polyA tail (or, e.g., polyA sequence). The first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a polyA-C tail (or, e.g., polyA-C sequence). In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a polyA-G tail (or, e.g., polyA-G sequence). In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a polyA-U tail (or, e.g., polyA-U sequence). In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a random sequence.In some embodiments, the spacer sequence can be, for example, at least 10 nucleotides in length, at least 15 nucleotides in length, or at least 30 nucleotides in length. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the spacer sequence is from 20 to 50 nucleotides in length. In certain embodiments, the spacer sequence is 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 or 50 nucleotides in length.The spacer sequences can be polyA tails, polyA-C tails, polyC tails, or poly-U tails.In some embodiments, the spacer sequences can be polyA-T, polyA-C, polyA-G, or a random sequence.Exemplary spacer sequences are described in paragraphs

[0293] -

[0302] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.ModificationsA polyribonucleotide (e.g., a linear or circular polyribonucleotide) as described herein may include one or more substitutions, insertions and / or additions, deletions, and covalent modifications with respect to reference sequences, in particular, the parent polyribonucleotide, are included within the scope of this disclosure.In some embodiments, a circular polyribonucleotide includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, polyA tail, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). In some embodiments, the first isolated nucleic acid includes messenger RNA (mRNA). In some embodiments, the polyribonucleotide includes at least one nucleoside selected from the group such as those described in

[0311] of International Patent Publication No. WO2019 / 118919, which is incorporated herein by reference in its entirety.A polyribonucleotide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage I to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.In some embodiments, a polyribonucleotide includes at least one N(6)methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, the m6A modification can reduce immunogenicity (e.g., reduce the level of one or more marker of an immune or inflammatory response) of the circular polyribonucleotide.In some embodiments, a modification may include a chemical or cellular induced modification. For example, some non-limiting examples of intracellular RNA modifications are described by Lewisand Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.In some embodiments, chemical modifications to the ribonucleotides of a circular polyribonucleotide may enhance immune evasion. The circular polyribonucleotide may be synthesized and / or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5’ end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.), 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), base modifications (e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners), removal of bases (abasic nucleotides), or conjugated bases. The modified ribonucleotide bases may also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications may modulate expression, immune response, stability, subcellular localization, to name a few functional effects, of the circular polyribonucleotide. In some embodiments, the modification includes a bi-orthogonal nucleotide, e.g., an unnatural base. See for example, Kimoto et al, Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039 / c7cc06661 a, which is hereby incorporated by reference.In some embodiments, sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar one or more ribonucleotides of the circular polyribonucleotide may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of circular polyribonucleotide include, but are not limited to, circular polyribonucleotide including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Circular polyribonucleotides having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the circular polyribonucleotide will include ribonucleotides with a phosphorus atom in its internucleoside backbone.Modified polyribonucleotide backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Various salts, mixed salts and free acid forms are also included. In some embodiments, the circular polyribonucleotide may be negatively or positively charged.The modified nucleotides, which may be incorporated into the polyribonucleotide, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of thepolynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylenephosphonates).The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked to the circular polyribonucleotide is expected to reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5’-0- (1 -thiophosphate)-adenosine, 5’-0-(1 -thiophosphate)-cytidine (a-thio-cytidine), 5’-0-(1 -thiophosphate)- guanosine, 5’-0-(1 -thiophosphate)-uridine, or 5’-0-(1 -thiophosphate)-pseudouridine).Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.In some embodiments, a circular polyribonucleotide may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into circular polyribonucleotide, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4’-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1 -(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1 -(tetrahydrofuran-2-yl)pyrimidine-2,4(1 H,3H)-dione), troxacitabine, tezacitabine, 2’-deoxy-2’-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1 -beta-D-arabinofuranosylcytosine, N4-octadecyl-1 -beta- D-arabinofuranosylcytosine, N4- palmitoy 1-1 -(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5’-elaidic acid ester).A polyribonucleotide may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the circular polyribonucleotide, or in a given predetermined sequence region thereof. In some embodiments, the circular polyribonucleotide includes a pseudouridine. In some embodiments, the circular polyribonucleotide includes an inosine, which may aid in the immune system characterizing the circular polyribonucleotide as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability / reduced degradation. See for example, Yu, Z. et al.(2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.In some embodiments, all nucleotides in a polyribonucleotide (or in a given sequence region thereof) are modified. In some embodiments, the modification may include an m6A, which may augment expression; an inosine, which may attenuate an immune response; pseudouridine, which may increase RNA stability, or translational readthrough (stagger element), an m5C, which may increase stability; and a 2,2,7-trimethylguanosine, which aids subcellular translocation (e.g., nuclear localization).Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) may exist at various positions in a circular polyribonucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the circular polyribonucleotide, such that the function of the circular polyribonucleotide is not substantially decreased. A modification may also be a non-coding region modification. The circular polyribonucleotide may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1 % to 90%, from 1 % to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).Methods of UseIn some embodiments, a circular polyribonucleotide produced according to the methods described herein is used for the treatment or prevention of a disease or condition in a subject.For example, a circular polyribonucleotide as described herein may be administered to a subject (e.g., in a pharmaceutical composition). In some embodiments, the complex is irradiated prior to administration to a subject. In some embodiments the complex is irradiated after administration to a subject. In some embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). Inembodiments, the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusk.In some embodiments, the disclosure provides a method of modifying a subject by providing to the subject a composition or formulation described herein. In some embodiments, the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or formulation is or includes or a eukaryotic or prokaryotic cell including a nucleic acid described herein.In some embodiments, the disclosure provides a method of treating a disease or condition in a subject in need thereof by providing to the subject a composition or formulation described herein. In some embodiments, the composition or formulation is or includes a polynucleotide provided to a eukaryotic subject. In some embodiments, the composition or formulation is or includes a eukaryotic or prokaryotic cell including a polyribonucleotide described herein. In some embodiments, the polyribonucleotide is provided in an amount and for a duration sufficient to treat a disease or condition in a subject, e.g., in need thereof.Methods of MakingThe present disclosure includes a method of forming a circular polyribonucleotide, e.g., by forming covalent attachments between one or more photoreactive crosslinking agents and the linear polyribonucleotide upon irradiation with light. Such a method produces a closed circular polyribonucleotide in which the bridging agent is conjugated (e.g., directly, chemically-covalently conjugated, etc.) to the linear polyribonucleotide.In some embodiments, the method includes the step of forming a complex with a linear polyribonucleotide that includes a first annealing region and a second annealing region; and a bridging agent that includes a first photoreactive crosslinking agent and a second photoreactive crosslinking agent. The bridging agent binds to the first annealing region and the second annealing region on the linear polyribonucleotide. The method further includes the step of irradiating the complex with light, wherein each photoreactive crosslinking agent forms a covalent adduct with the linear polyribonucleotide.In some embodiments, a wavelength of the irradiated light is from 340-410 nm (e.g., from 350-370 nm, e.g., 365 nm or 366 nm).In some embodiments, a wavelength of the irradiated light is from 350-370 nm (e.g., 350, 351 , 352, 353. 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, or 370) nm. In some embodiments, the complex is irradiated with light at the wavelength for 1 to 120 (e.g., 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes.In some embodiments, the method further includes removing the covalent attachments by irradiating the complex with light at a second wavelength. In some embodiments, the second wavelength is 300-320 nm (e.g., 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311 , 312, 313, 314, 315, 316, 317, 318, 319, or 320) nm. In some embodiments, the complex is irradiated withlight at the second wavelength for 1 to 120 (e.g., 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes.In some embodiments, the complex is irradiated prior to administration to a cell or tissue. In some embodiments, the complex is irradiated after administration to a cell or tissue. In some embodiments, the complex is irradiated prior to administration to a subject. In some embodiments, the complex is irradiated after administration to a subject.Methods of Functionalization, Purification, and Targeted DeliveryIn some embodiments, the bridging agent includes a functional group or a tag (e.g., a purification tag). The functional group may be used for purification or targeted delivery or may be attached (e.g., conjugated) to an additional moiety, such as a purification tag or a targeting moiety for further use. Upon incorporation of the bridging agent into the linear polyribonucleotide, the functional group or tag is covalently attached upon photocirculizarization.In some embodiments, the functional group includes a thiol group, an N-hydroxysuccinimide ester, a polypeptide, a polynucleotide, an azide, a dye, or a targeting moiety.In some embodiments, the bridging agent includes a tag (e.g., a purification tag). In some embodiments, the tag includes biotin.In some embodiments, the targeting moiety includes a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid, or a combination thereof. In some embodiments, the targeting moiety includes a small molecule. In some embodiments, the bridging agent includes a tag. In some embodiments, following photocircularization, the method further includes contacting the complex with a capture agent that binds the tag. In some embodiments, the tag includes biotin, and the capture agent includes streptavidin.In some embodiments, the method further includes separating the complex that includes the bridging agent that is bound to the capture agent.In some embodiments, the capture agent is conjugated to a column.Also featured herein is a method of separating a circular polyribonucleotide that includes a tag from a plurality of polyribonucleotides that include a mixture of linear polyribonucleotides and circular polyribonucleotides. The method includes (a) providing a sample that includes (i) a plurality of linear polyribonucleotides, each having a first annealing region and a second annealing region; and (ii) a bridging agent that includes a first photoreactive crosslinking agent, a second photoreactive crosslinking agent, and a functional group that includes a tag. The bridging agent binds to the first annealing region and the second annealing region on one of the plurality of linear polyribonucleotides, wherein the bridging agent and the linear polyribonucleotide form a complex. The method further includes (b) irradiating the complex with light, wherein each photoreactive crosslinking agent forms a covalent adduct with the linear polyribonucleotide, thereby producing a plurality of polyribonucleotides, wherein a subset of the plurality of polyribonucleotides includes the circular polyribonucleotide having the tag. The method further includes (b) contacting the sample with a capture agent that binds the tag; and (c) separating the circular polyribonucleotide having the tag that is bound to the capture agent from the plurality of polyribonucleotides in the sample.In some embodiments, the linear polyribonucleotides lack the tag. In some embodiments, step (d) includes immobilizing the capture agent. In some embodiments, the capture agent is conjugated to a column. In some embodiments, the tag includes biotin, and the capture agent includes streptavidin.FormulationsIn some embodiments of the present disclosure a circular polyribonucleotide described herein may be formulated in composition, e.g., a composition for delivery to a cell, a plant, an invertebrate animal, a non-human vertebrate animal, or a human subject, e.g., an agricultural, veterinary, or pharmaceutical composition. In some embodiments, the circular polyribonucleotide is formulated in a pharmaceutical composition. In some embodiments, a composition includes a circular polyribonucleotide and a diluent, a carrier, an adjuvant, or a combination thereof. In a particular embodiment, a composition includes a circular polyribonucleotide described herein and a carrier or a diluent free of any carrier. In some embodiments, a composition including a circular polyribonucleotide with a diluent free of any carrier is used for naked delivery of the circular polyribonucleotide to a subject.SaltsIn some cases, a composition or pharmaceutical composition provided herein comprises one or more salts. For controlling the tonicity, a physiological salt such as sodium salt can be included a composition provided herein. Other salts can comprise potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride, or the like. In some cases, the composition is formulated with one or more pharmaceutically acceptable salts. The one or more pharmaceutically acceptable salts can comprise those of the inorganic ions, such as, for example, sodium, potassium, calcium, magnesium ions, and the like. Such salts can comprise salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid, or maleic acid. The polyribonucleotide can be present in either linear or circular form.Buffers / pHA composition or pharmaceutical composition provided herein can comprise one or more buffers, such as a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (e.g., with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers, in some cases, are included in the 5-20 mM range.A composition or pharmaceutical composition provided herein can have a pH between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8. The composition or pharmaceutical composition can have a pH of about 7. The polyribonucleotide can be present in either linear or circular form.Detergents / surfactantsA composition or pharmaceutical composition provided herein can comprise one or more detergents and / or surfactants, depending on the intended administration route, e.g., polyoxyethylene sorbitan esters surfactants (commonly referred to as “Tweens”), e.g., polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO / PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, e.g., octoxynol-9 (Triton X-100, or t- octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as “SPANs”), such as sorbitan trioleate (Span 85) and sorbitan monolaurate, an octoxynol (such as octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide (“CTAB”), or sodium deoxycholate. The one or more detergents and / or surfactants can be present only at trace amounts. In some cases, the composition can include less than 1 mg / ml of each of octoxynol-10 and polysorbate 80. Non-ionic surfactants can be used herein. Surfactants can be classified by their “HLB” (hydrophile / lipophile balance). In some cases, surfactants have a HLB of at least 10, at least 15, and / or at least 16. The polyribonucleotide can be present in either linear or circular form.DiluentsIn some embodiments, a composition of the disclosure includes a circular polyribonucleotide and a diluent. In some embodiments, a composition of the disclosure includes a linear polyribonucleotide and a diluent.A diluent can be a non-carrier excipient. A non-carrier excipient serves as a vehicle or medium for a composition, such as a circular polyribonucleotide as described herein. A non-carrier excipient serves as a vehicle or medium for a composition, such as a linear polyribonucleotide as described herein. Non-limiting examples of a non-carrier excipient include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, polymers, peptides, proteins, cells, hyaluronidases, dispersing agents, granulating agents, disintegrating agents, binding agents, buffering agents (e.g., phosphate buffered saline (PBS)), lubricating agents, oils, and mixtures thereof. A non-carrier excipient can be any one of the inactive ingredients approved by the United States Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database that does not exhibit a cell-penetrating effect. A non-carrier excipient can be any inactive ingredient suitable for administration to a non-human animal, for example, suitable for veterinary use. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation.In some embodiments, the circular polyribonucleotide may be delivered as a naked delivery formulation, such as including a diluent. A naked delivery formulation delivers a circular polyribonucleotide, to a cell without the aid of a carrier and without modification or partial or complete encapsulation of the circular polyribonucleotide, capped polyribonucleotide, or complex thereof.A naked delivery formulation is a formulation that is free from a carrier and wherein the circular polyribonucleotide is without a covalent modification that binds a moiety that aids in delivery to a cell or without partial or complete encapsulation of the circular polyribonucleotide. In some embodiments, a circular polyribonucleotide without a covalent modification that binds a moiety that aids in delivery to a cell is a polyribonucleotide that is not covalently bound to a protein, small molecule, a particle, a polymer, or a biopolymer. A circular polyribonucleotide without covalent modification that binds a moiety that aids in delivery to a cell does not contain a modified phosphate group. For example, a circular polyribonucleotide without a covalent modification that binds a moiety that aids in delivery to a cell does not contain phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.In some embodiments, a naked delivery formulation is free of any or all of: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. In some embodiments, a naked delivery formulation is free from phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2- dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1 ,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP), N-[1 -(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 -[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N- dimethyl-1 -propanaminium trifluoroacetate (DOSPA), 3B-[N — (N\N'-Dimethylaminoethane)- carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HC1 ), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1 ,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density lipoprotein (LDL), high- density lipoprotein (HDL), or globulin.In certain embodiments, a naked delivery formulation includes a non-carrier excipient. In some embodiments, a non-carrier excipient includes an inactive ingredient that does not exhibit a cellpenetrating effect. In some embodiments, a non-carrier excipient includes a buffer, for example PBS. In some embodiments, a non-carrier excipient is a solvent, a non-aqueous solvent, a diluent, a suspension aid, a surface-active agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersing agent, a granulating agent, a disintegrating agent, a binding agent, a buffering agent, a lubricating agent, or an oil.In some embodiments, a naked delivery formulation includes a diluent. A diluent may be a liquid diluent or a solid diluent. In some embodiments, a diluent is an RNA solubilizing agent, a buffer,or an isotonic agent. Examples of an RNA solubilizing agent include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of a buffer include 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2- Acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N- morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine, or phosphate. Examples of an isotonic agent include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, and sucrose.CarriersIn some embodiments, a composition of the disclosure includes a circular polyribonucleotide and a carrier. In some embodiments, a composition of the disclosure includes a linear polyribonucleotide and a carrier.In certain embodiments, a composition includes a circular polyribonucleotide as described herein in a vesicle or other membrane-based carrier. In certain embodiments, a composition includes a linear polyribonucleotide as described herein in a vesicle or other membrane-based carrier.In other embodiments, a composition includes the circular polyribonucleotide in or via a cell, vesicle or other membrane-based carrier. In other embodiments, a composition includes the linear polyribonucleotide in or via a cell, vesicle or other membrane-based carrier. In one embodiment, a composition includes the circular polyribonucleotide in liposomes or other similar vesicles. In one embodiment, a composition includes the linear polyribonucleotide in liposomes or other similar vesicles. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral, or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011 , Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 for review).Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011 , Article ID 469679, 12 pages, 2011. doi :10.1155 / 2011 / 469679 for review). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647- 652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.In certain embodiments, a composition of the disclosure includes a circular polyribonucleotide and lipid nanoparticles, for example lipid nanoparticles described herein. In certain embodiments, a composition of the disclosure includes a linear polyribonucleotide and lipid nanoparticles. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a circular polyribonucleotide molecule as described herein. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a linear polyribonucleotide molecule as described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of the SLN, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, may also be employed. These nanoparticles possess the complementary advantages of PNPs and liposomes. A PLN is composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell offers good biocompatibility. As such, the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390 / nano7060122.Additional non-limiting examples of carriers include carbohydrate carriers (e.g., an anhydride- modified phytoglycogen or glycogen-type material), protein carriers (e.g., a protein covalently linked to the circular polyribonucleotide or a protein covalently linked to the linear polyribonucleotide), or cationic carriers (e.g., a cationic lipopolymer or transfection reagent). Non-limiting examples of carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, and anhydride-modified phytoglycogen beta-dextrin. Non-limiting examples of cationic carriers include lipofectamine, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2- dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1 ,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP), N-[1 -(2,3- dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), 1 -[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N- dimethyl-1 -propanaminium trifluoroacetate (DOSPA), 3B-[N — (N\N'-Dimethylaminoethane)- carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HC1 ), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N- dimethylammonium bromide (DDAB), N-(1 ,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N- hydroxyethyl ammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC). Non-limiting examples of protein carriers include human serum albumin (HSA), low-density lipoprotein (LDL), high- density lipoprotein (HDL), or globulin.Exosomes can also be used as drug delivery vehicles for a circular RNA composition or preparation described herein. Exosomes can be used as drug delivery vehicles for a linear polyribonucleotide composition or preparation described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; https: / / doi.Org / 10.1016 / j.apsb.2O16.02.001 .Ex vivo differentiated red blood cells can also be used as a carrier for a circular RNA composition or preparation described herein. Ex vivo differentiated red blood cells can also be used as a carrier for a linear polyribonucleotide composition or preparation described herein. See, e.g., International Patent Publication Nos. WO2015 / 073587; WO2017 / 123646; WO2017 / 123644; WO2018 / 102740; WO2016 / 183482; WO2015 / 153102; WO2018 / 151829; WO2018 / 009838; Shi et al. 2014. Proc Natl Acad Sci USA. 111 (28): 10131-10136; US Patent 9,644,180; Huang et al. 2017. Nature Communications 8: 423; Shi et al. 2014. Proc Natl Acad Sci USA. 111 (28): 10131-10136.Fusosome compositions, e.g., as described in International Patent Publication No. WO2018 / 208728, can also be used as carriers to deliver a circular polyribonucleotide molecule described herein. Fusosome compositions, e.g., as described in WO2018 / 208728, can also be used as carriers to deliver a linear polyribonucleotide molecule described herein.Virosomes and virus-like particles (VLPs) can also be used as carriers to deliver a circular polyribonucleotide molecule described herein to targeted cells. Virosomes and virus-like particles (VLPs) can also be used as carriers to deliver a linear polyribonucleotide molecule described herein to targeted cells.Plant nanovesicles and plant messenger packs (PMPs), e.g., as described in International Patent Publication Nos. WO2011 / 097480, WO2013 / 070324, WO2017 / 004526, or W02020 / 041784 can also be used as carriers to deliver the circular RNA composition or preparation described herein. Plant nanovesicles and plant messenger packs (PMPs) can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein. Lipid reconstructed plant messenger packs (LPMPs), e.g., as described in International Patent Publication Nos. W02021 / 041301 , WO2023 / 069498, or WO2023 / 122080 can also be used as carriers to deliver the circular RNA composition or preparation described herein. Lipid reconstructed plant messenger packs (LPMPs) can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein. Lipid reconstructed natural messenger packs (LNMPs), e.g., as described in International Patent Publication Nos. WO2024 / 102434 can also be used as carriers to deliver the circular RNA composition or preparation described herein. Lipid reconstructed natural messenger packs (LNMPs) can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein. Bacteria-derived lipid compositions, e.g., as described in International Patent Publication Nos. WO2023 / 096858 can also be used as carriers to deliver the circular RNA composition or preparation described herein. Bacteria-derived lipid compositions can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein.Microbubbles can also be used as carriers to deliver a circular polyribonucleotide molecule described herein. Microbubbles can also be used as carriers to deliver a linear polyribonucleotide molecule described herein. See, e.g., US7115583; Beeri, R. et al., Circulation. 2002 Oct 1 ;106(14):1756-1759; Bez, M. et al., Nat Protoc. 2019 Apr; 14(4): 1015-1026; Hernot, S. et al., Adv Drug Deliv Rev. 2008 Jun 30; 60(10): 1153-1166; Rychak, J.J. et al., Adv Drug Deliv Rev. 2014 Jun; 72: 82-93. In some embodiments, microbubbles are albumin-coated perfluorocarbon microbubbles.The carrier including the circular polyribonucleotides described herein may include a plurality of particles. The particles may have median article size of 30 to 700 nanometers (e.g., 30 to 50, 50 to100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 100 to 500, 50 to 500, or 200 to 700 nanometers). The size of the particle may be optimized to favor deposition of the payload, including the circular polyribonucleotide into a cell. Deposition of the circular polyribonucleotide into certain cell types may favor different particle sizes. For example, the particle size may be optimized for deposition of the circular polyribonucleotide into antigen presenting cells. The particle size may be optimized for deposition of the circular polyribonucleotide into dendritic cells. Additionally, the particle size may be optimized for depositions of the circular polyribonucleotide into draining lymph node cells.Lipid NanoparticlesThe compositions, methods, and delivery systems provided by the present disclosure may employ any suitable carrier or delivery modality described herein, including, in certain embodiments, lipid nanoparticles (LNPs). Lipid nanoparticles, in some embodiments, comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO2019217941 ; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol).Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example those described in Table 4 of WO2019217941 , which is incorporated by reference — e.g., a lipid-containing nanoparticle can comprise one or more of the lipids in Table 4 of WO2019217941 . Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of WO2019217941 , incorporated by reference.In some embodiments, conjugated lipids, when present, can include one or more of PEG- diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG- DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'- di(tetradecanoyloxy)propyl-1 -0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypoly ethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing.In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009 / 127060 or US2010 / 0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.In some embodiments, the lipid particle comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticle comprises an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1 .In some embodiments, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1 :1 to about 25:1 , from about 10:1 to about 14:1 , from about 3:1 to about 15:1 , from about 4:1 to about 10:1 , from about 5:1 to about 9:1 , or about 6:1 to about 9:1 . The amounts of lipids and nucleic acid can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg / ml to about 30 mg / mL.Some non-limiting example of lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein includes,In some embodiments an LNP comprising Formula (i) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.In some embodiments an LNP comprising Formula (ii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.In some embodiments an LNP comprising Formula (Hi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.In some embodiments an LNP comprising Formula (v) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.In some embodiments an LNP comprising Formula (vi) is used to deliver a polyribonucleotide(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.In some embodiments an LNP comprising Formula (viii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.In some embodiments an LNP comprising Formula (ix) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.whereinX1is O, NR1, or a direct bond, X2is C2-5 alkylene, X3is C(=O) or a direct bond, R1is H or Me, R3is C1 -3 alkyl, R2is C1 -3 alkyl, or R2taken together with the nitrogen atom to which it is attached and 1 -3 carbon atoms of X2form a 4-, 5-, or 6-membered ring, or X1is NR1, R1and R2taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R2taken together with R3and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1is C2-12 alkylene, Y2is selected from(in either orientation), (in either orientation), (in either orientation), n is 0 to 3, R4is C1 -15 alkyl, Z1is C1 -6 alkylene or a direct bond,(in either orientation) or absent, provided that if Z1is a direct bond, Z2is absent;R5is C5-9 alkyl or C6-10 alkoxy, R6is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7is H or Me, or a salt thereof, provided that if R3and R2are C2 alkyls, X1is O, X2is linear C3 alkylene, X3is C(=0), Y1is linear Ce alkylene, (Y2)n-R4is, R4is linear C5 alkyl, Z1is C2 alkylene, Z2is absent, W is methylene, and R7is H, then R5and R6are not Cx alkoxy.In some embodiments an LNP comprising Formula (xii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.(xi)In some embodiments an LNP comprising Formula (xi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.In some embodiments an LNP comprises a compound of Formula (xiii) and a compound of Formula (xiv).In some embodiments an LNP comprising Formula (xv) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.In some embodiments an LNP comprising a formulation of Formula (xvi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein is made by one of the following reactions:In some embodiments an LNP comprising Formula (xxi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxi) is an LNP described by WO2021113777 (e.g., a lipid of Formula (1 ) such as a lipid of Table 1 of WO2021113777).wherein each n is independently an integer from 2-15; Li and L3 are each independently -OC(O)-* or - C(O)O-*, wherein indicates the attachment point to R1 or R3;R1 and R3 are each independently a linear or branched C9-C20 alkyl or C9-C20 alkenyl, optionally substituted by one or more substituents selected from a group consisting of oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkyl sulfonyl, and alkyl sulfonealkyl; andR2 is selected from a group consisting of:In some embodiments an LNP comprising Formula (xxii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxii) is an LNP described by WO2021 1 13777 (e.g., a lipid of Formula (2) such as a lipid of Table 2 of WO2021 1 13777).wherein each n is independently an integer from 1 -15; Ri and R2 are each independently selected from a group consisting of:Rs is selected from a group consisting of:In some embodiments an LNP comprising Formula (xxiii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxiii) is an LNP described by WO2021113777).whereinX is selected from -O-, -S-, or -OC(O)-*, wherein * indicates the attachment point to Ri;Ri is selected from a group consisting of:and R2 is selected from a group consisting of:In some embodiments, a composition described herein (e.g., a nucleic acid (e.g., a circular polyribonucleotide, a linear polyribonucleotide) or a protein) is provided in an LNP that comprises an ionizable lipid. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo- 6-(undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of US9,867,888 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 9Z.12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca- 9,12-dienoate (LP01 ), e.g., as synthesized in Example 13 of WO2015 / 095340 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Di((Z)-non-2-en-1-yl) 9- ((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., as synthesized in Example 7, 8, or 9 of US2012 / 0027803 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 1 ,1'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl) amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO2010 / 053572 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13- dimethyl-17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17-tetradecahydro- IH- cyclopenta[a]phenanthren-3-yl 3-(1 H-imidazol-4-yl)propanoate, e.g., Structure (I) from W02020 / 106946 (incorporated by reference herein in its entirety).In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. In some embodiments, the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids includingpolyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, a lipid nanoparticle may comprise a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA (e.g., a circular polyribonucleotide, a linear polyribonucleotide)) described herein, encapsulated within or associated with the lipid nanoparticle. In some embodiments, the nucleic acid is co-formulated with the cationic lipid. The nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the nucleic acid may be encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipid described herein, e.g., Formula (i), (ii), (ii), (vii) and / or (ix) encapsulates at least 1 %, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of an RNA molecule.Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016 / 031 1759; I of US201503761 15 or in US2016 / 0376224; I, II or III of US20160151284; I, IA, II, or 11 A of US20170210967; l-c of US20150140070; A of US2013 / 0178541 ; I of US2013 / 0303587 or US2013 / 0123338; I of US2015 / 0141678; II, III, IV, or V of US2015 / 0239926; I of US2017 / 01 19904; I or II of WO2017 / 1 17528; A of US2012 / 0149894; A of US2015 / 0057373; A of WO2013 / 1 16126; A of US2013 / 0090372; A of US2013 / 0274523; A of US2013 / 0274504; A of US2013 / 0053572; A of W02013 / 016058; A of W02012 / 162210; I of US2008 / 042973; I, II, III, or IV of US2012 / 01287670; I or II of US2014 / 0200257; I, II, or III of US2015 / 0203446; I or III of US2015 / 0005363; I, IA, IB, IC, ID, II, HA, IIB, IIC, HD, or lll-XXIV of US2014 / 0308304; of US2013 / 0338210; I, II, III, or IV of W02009 / 132131 ; A of US2012 / 0101 1478; I or XXXV of US2012 / 0027796; XIV or XVII of US2012 / 0058144; of US2013 / 0323269; I of US201 1 / 01 17125; I, II, or III of US201 1 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012 / 0202871 ; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US201 1 / 0076335; I or II of US2006 / 008378; I of US2013 / 0123338; I or X-A-Y-Z of US2015 / 0064242; XVI, XVII, or XVIII of US2013 / 0022649; I, II, or III of US2013 / 01 16307; I, II, or III of US2013 / 01 16307; I or II of US2010 / 0062967; l-X of US2013 / 0189351 ; I of US2014 / 0039032; V of US2018 / 0028664; I of US2016 / 0317458; I of US2013 / 0195920; 5, 6, or 10 of US10,221 ,127; HI-3 of WO2018 / 081480; I-5 or I-8 of W02020 / 081938; 18 or 25 of US9,867,888; A of US2019 / 0136231 ; II of W02020 / 219876; 1 of US2012 / 0027803; OF-02 of US2019 / 0240349; 23 of US10,086,013; cKK- E12 / A6 of Miao et al (2020); C12-200 of WO2010 / 053572; 7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al; TS-P4C2 of US9,708,628; I of W02020 / 106946; I of W02020 / 106946;and (1 ), (2), (3), or (4) of WO2021 / 113777. Exemplary lipids further include a lipid of any one of Tables 1 -16 of WO2021 / 113777.In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 IZ)-heptatriaconta- 6,9,28,3 I- tetraen-l9-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (I3Z,I6Z)- A,A-dimethyl-3- nonyldocosa-13, 16-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety).Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-0- monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-0-dimethyl PE), 18-l-trans PE, I- stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS).Other examples of non-cationic lipids suitable for use in the lipid nanoparticles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationiclipids are described in WO2017 / 099823 or US patent publication US2018 / 0028664, the contents of which is incorporated herein by reference in their entirety.In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018 / 0028664, incorporated herein by reference in its entirety. The non-cationic lipid can comprise, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , or 8:1 ).In some embodiments, the lipid nanoparticles do not comprise any phospholipids.In some aspects, the lipid nanoparticle can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2 -hydroxy)-ethyl ether, cholesteryl- (4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4 '-hydroxy)- butyl ether. Exemplary cholesterol derivatives are described in PCT publication W02009 / 127060 and US patent publication US2010 / 0130588, each of which is incorporated herein by reference in its entirety.In some embodiments, the component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.In some embodiments, the lipid nanoparticle can comprise a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG- lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)- conjugated lipid.Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG- dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'- di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US5, 885,613, US6,287,59I, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, US2017 / 0119904, US2018 / 0028664, and WO2017 / 099823, the contents of all of which areincorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formula III, lll-a-l, lll-a-2, lll-b-1 , lll-b-2, or V of US2018 / 0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, a PEG-lipid is of Formula II of US20150376115 or US2016 / 0376224, the content of both of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG- dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylg lycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG- disterylglycamide, PEG-cholesterol (l-[8'-(Cholest-5-en-3[beta]- oxy)carboxamido-3',6'-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG- DMB (3,4-Ditetradecoxylbenzyl- [omega]- methyl-poly(ethylene glycol) ether), and 1 ,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1 ,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from:In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.Exemplary conjugated lipids, i.e. , PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289A9, the contents of all of which are incorporated herein by reference in their entirety.In some embodiments, the PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. Molar ratios of the ionizablelipid, non-cationic-lipid, sterol, and PEG / conjugated lipid can be varied as needed. For example, the lipid particle can comprise 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non-cationic-lipid by mole or by total weight of the composition and 1 -10% conjugated lipid by mole or by total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10- 20% non- cationic-lipid by mole or by total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1 -10% conjugated lipid by mole or by total weight of the composition. The composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non- cationic-lipid by mole or by total weight of the composition. The composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition. The formulation may also be a lipid nanoparticle formulation, for example comprising 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1 -20% cholesterol by mole or by total weight of the composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50: 10:38.5: 1 .5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5: 1.5.In some embodiments, the lipid particle comprises ionizable lipid, non-cationic lipid (e.g., phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of noncationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5.In some embodiments, the lipid particle comprises ionizable lipid I non-cationic- lipid I sterol I conjugated lipid at a molar ratio of 50:10:38.5: 1 .5.In an aspect, the disclosure provides a lipid nanoparticle formulation comprising phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.In some embodiments, one or more additional compounds can also be included. Those compounds can be administered separately, or the additional compounds can be included in the lipid nanoparticles of the invention. In other words, the lipid nanoparticles can contain other compounds in addition to the nucleic acid or at least a second nucleic acid, different than the first. Without limitations, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.In some embodiments, the LNPs comprise biodegradable, ionizable lipids. In some embodiments, the LNPs comprise (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate, also called 3- ((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,I2Z)- octadeca-9,l2-dienoate) or another ionizable lipid. See, e.g., lipids of WO2019 / 067992, WO / 2017 / 173054, WO2015 / 095340, and WO2014 / 136086, as well as references provided therein. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.In some embodiments, the average LNP diameter of the LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about I mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.An LNP may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of an LNP, e.g., the particle size distribution of the lipidnanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. An LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of an LNP may be from about 0.10 to about 0.20.The zeta potential of an LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.The efficiency of encapsulation of a protein and / or nucleic acid, describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with an LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a protein and / or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.An LNP may optionally comprise one or more coatings. In some embodiments, an LNP may be formulated in a capsule, film, or table having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness or density.Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by W02020 / 061457 and WO2021 / 113777, each of which is incorporated herein by reference in its entirety. Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021 ). doi.org / 10.1038 / s41578-021 - 00358-0, which is incorporated herein by reference in its entirety (see, for example, exemplary lipids and lipid derivatives of Figure 2 of Hou et al.).In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certainembodiments, LNPs are formulated using the GenVoyJLM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-di linoleyl-4- dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51 (34):8529-8533 (2012), incorporated herein by reference in its entirety.LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910, both incorporated by reference, and are useful for delivery of circular polyribonucleotides and linear polyribonucleotides described herein.Additional specific LNP formulations useful for delivery of nucleic acids (e.g., circular polyribonucleotides, linear polyribonucleotides) are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO.In embodiments, a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) encoding at least a portion (e.g., an antigenic portion) of a protein or polypeptide described herein is formulated in an LNP, wherein: (a) the LNPs comprise a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, (b) the LNPs have a mean particle size of between 80 nm and 160 nm, and (c) the polyribonucleotide. In embodiments, the polyribonucelotide (e.g., circular polyribonucleotide, linear polyribonucleotide) formulated in an LNP is a vaccine.Exemplary dosing of polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) LNP may include about 0.1 , 0.25, 0.3, 0.5, 1 , 2, 3, 4, 5, 6, 8, 10, or 100 mg / kg (RNA). In some embodiments, a dose of a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) antigenic composition described herein is between 30-200 mcg, e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg.ExamplesThe following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their invention.Example 1. Photocircularization of a linear polyribonucleotide via a photoreactive polynucleotideThis example demonstrates photocircularization of a linear polyribonucleotide, wherein photocircularization includes annealing a polynucleotide with photoreactive crosslinking agents to the linear polyribonucleotide and irradiating the complex to form covalent attachments between the polynucleotide and resulting circular polyribonucleotide.The photoreactive polynucleotide containing two 3-carbovinylcarbazole nucleoside (CNVK) photoreactive crosslinking agents (SEQ ID NO: 29) was annealed to the linear polyribonucleotide that is -1500 nucleotides in length named LAA2367 (SEQ ID NO: 30) in a thermocycler by heating at 75°C for 30 seconds, heating at 75°C for 1 minute and 30 seconds, and cooling to 25°C for 2 minutes andrepeated one time. The annealing buffer was HEPES buffered saline 1x (HBS) containing 250 mM NaCI and 5 mM MgCl2 (M). Polyribonucleotides includingCNVK were resuspended in water. The resulting circular polyribonucleotide was stored at room temperature. Rectangular cuvettes with equal sides having small volumes of 140 pL maximum (S) or flat rectangular cuvettes with a larger surface area (i.e. , greater exposure to light) having large volumes of 300 pL maximum (L) were used for storing the samples during irradiation. The circular polyribonucleotide was irradiated with 366 nm light for various lengths of time to form covalent attachment. This resulted in the circular polyribonucleotide bound to the photoreactive polynucleotide via annealing and irradiation for:(a) 0 minutes;(b) 30 minutes with S;(c) 45 minutes with S;(d) 15 minutes with L;(e) 30 minutes with L; or(f) 45 minutes with L.Polyacrylamide gel electrophoresis was used to separate the contents of each sample. The sample and corresponding lane are shown in the table in FIGS. 2A and 2B. Each lane contained a 5 pL fraction of the sample and 5 pL of urea with glycerol. The polyacrylamide gel included either 6% or 10% TBU, and electrophoresis was run with a voltage of 250 V for 1 hour and 40 minutes. The gel was stained with SYBR safe dye and scanned using iBright. The results were compared to an RNA ladder and the linear polyribonucleotide that was not annealed nor irradiated. Results of this experiment are shown in FIGS. 2A and 2B. This experiment resulted in two distinct bands for lanes containing linear or circular polyribonucleotides. The lower band contains linear polyribonucleotides. The higher band contains circular polyribonucleotides. The band for circularized polyribonucleotides has a higher intensity compared to the band for linear polyribonucleotides for irradiated samples (Lanes 4-8 in FIGS. 2A and 2B).This experiment demonstrates the success of photocircularization to circularize a linear polyribonucleotide. This experiment also demonstrates that linear polyribonucleotides could be circularized via irradiation for 15-45 minutes.Example 2. Efficiency of circularizing a linear polyribonucleotide with a polynucleotide via photocircularization in comparison to splint ligationThis example demonstrates the efficiency of circularizing a linear polyribonucleotide with a polynucleotide via photocircularization in comparison to splint ligation.A linear polyribonucleotide that is -1200 nucleotides in length (SEQ ID NO: 31 ) named VLD53 was hybridized with a splint polynucleotide (SEQ ID NO: 32) and circularized via splint ligation. Splint ligation was performed with RNA ligase 1 and 2, 3, 4, 5, 6, 7, 8, or 10 mM of MgCl2 in RNA ligase reaction buffer. Polyacrylamide gel electrophoresis was used to separate the contents of each sample with a gel including 6% TBU, as described in Example 1 . Gel electrophoresis was run at 250 V for 2 hours, and the gel was stained with RNA loading dye. The results were compared to an RNA ladder and the results of the photocircularization experiment utilizing the polyacrylamide gel including6% TBU in Example 1 . The sample and corresponding lane for photocircularization are listed in the table in FIG. 4A. Results of this experiment are shown in FIGS. 3A, 3B, 4A, and 4B. This experiment resulted in two distinct bands for lanes containing splint ligated and photocircularized polyribonucleotides. The lower band contains linear polyribonucleotide. The higher band contains circular polyribonucleotides. The circularization efficiency for each lane was determined by calculating the percentage of circular polyribonucleotides in the sample after separation.This experiment demonstrates the comparability of photocircularization and splint ligation as methods for circularization of a linear polyribonucleotide with a polynucleotide. This experiment also demonstrates a higher average circularization efficiency with photocircularization (-54.8%) as compared to splint ligation (-40.3%).Example 3. Effect of photocircularization of a linear polyribonucleotide on the expression of a polypeptide encoded by a photocircularized polyribonucleotide in vitro across two different cell linesThis experiment demonstrates the effect of photocircularization of a linear polyribonucleotide on the expression of the polypeptide encoded by the resulting circular polyribonucleotide in vitro across two different cell lines.A linear polyribonucleotide encoding a Gaussia luciferase polypeptide (LAA2367) was photocircularized with the same protocol and photoreactive polyribonucleotide as described in Example 1 . The confluent human embryonic kidney cells (HEK293) were plated in a 96 well plate overnight. The following day, the cells were transfected with 1 pmol of the resulting photocircularized polyribonucleotides and a lipofectamine MessengerMax (MM) delivery agent in Opti-MEM media and incubated for 24 hours at 37 °C under 5% CO2. A different polyribonucleotide encoding a Gaussia luciferase polypeptide named LAA3808 (SEQ ID NO: 33) that is -87% circular RNA and ~6% linear RNA was self-circularized using a self-splicing ribozyme enzyme. LAA3808 includes the same internal ribosomal entry site (IRES) and spacer sequence as LAA 2367. After incubation for 24 hours, a luciferase assay was performed to assess polypeptide expression in the cells in comparison to samples containing cells transfected with no polyribonucleotide, the self-circularized LAA3808 with and without MM, and the linear LAA2367 that was not annealed nor irradiated and with MM. Results of this experiment are shown in FIG. 5A.This experiment demonstrates that expression of the encoded polypeptide was comparable between the photocircularized polyribonucleotide and the self-circularized polyribonucleotides. This experiment also demonstrates that expression of the polypeptide encoded by the polyribonucleotide was not hindered by photocircularization. This experiment also demonstrates enhanced expression of the polypeptide for photocircularized polyribonucleotides in comparison to linear polyribonucleotides.In another experiment, LAA2367 was photocircularized with the same protocol and photoreactive polyribonucleotide as described in Example 1 . The annealed but not irradiated sample is termed TO in this experiment. The confluent lung carcinoma epithelial cells (A549) were plated in a 96 well plate overnight. The following day, the cells were transfected with 1 pmol of the resulting photocircularized polyribonucleotides and a lipofectamine MessengerMax (MM) delivery agent in Opti-MEM media and incubated for 24 hours at 37 °C under 5% CO2. After incubation for 24 hours, a luciferase assay was performed to assess polypeptide expression in the cells in comparison to samples containing cells transfected with MM only, TO with MM, the linear LAA2367 that was not annealed nor irradiated and with MM, and the results of the prior experiment in Example 3. Results of this experiment are shown in FIG. 5B.This experiment demonstrates that the expression of the polypeptide encoded by the circular polyribonucleotide circularized via photocircularization was reproducible across two different cell lines: A549 and HEK293. This experiment also demonstrates that the expression of the polypeptide encoded by the polyribonucleotide is enhanced when circularized via photocircularization in comparison to annealing without subsequent irradiation and to the linear polyribonucleotide itself.Example 4. Effect of photocircularized polyribonucleotides on cytokine secretion in vitroThis experiment demonstrates the effect of photocircularization on the excretion of cytokines in vitro.A linear polyribonucleotide encoding a Gaussia luciferase polypeptide (LAA2367) was photocircularized with the same protocol and photoreactive polyribonucleotide as described in Example 1 . The annealed but not irradiated complex is termed TO in this experiment. The confluent lung carcinoma epithelial cells (A549) were plated in a 96 well plate overnight. The following day, the cells were transfected with 1 pmol of the resulting photocircularized polyribonucleotides and a lipofectamine MessengerMax (MM) delivery agent in Opti-MEM media and incubated for 24 hours. The transfected cells were incubated at 37 °C under 5% CO2. A different polyribonucleotide encoding a firefly luciferase polypeptide named Flue eRNA (SEQ ID NO: 34) was self-circularized using a selfsplicing ribozyme enzyme. After incubation for 24 hours, an enzyme-linked lectin assay was performed to measure the concentration of cytokines, IFN3 and IP10, secreted in the cells in comparison to samples containing cells transfected with no polyribonucleotide, the photoreactive polyribonucleotide without MM (pcirc eRNA only), the linear LAA2367 that was not annealed nor irradiated, and with MM, MM only, TO with MM, and the self-circularized Flue eRNA with MM. Results of this experiment are shown in FIGS. 6A, 6B, 7A, and 7B.This experiment demonstrates that the photocircularized polyribonuclotides exhibited lower levels of IFN3 and IP10 in comparison to the linear polyribonucleotide, the annealed but not irradiated linear polyribonucleotide and photoreactive polyribonucleotide complex, and to self-circularized polyribonucleotides when transfected into A549 cells with MM.Example 5. Effect of solvent on the photocircularization of linear polyribonucleotidesThis experiment demonstrates the effect of solvent on the photocircularization of linear polyribonucleotides.A linear polyribonucleotide encoding a Gaussia luciferase polypeptide (LAA2367) was photocircularized with the same annealing protocol and photoreactive polyribonucleotide as described in Example 1 . The resulting circular polyribonucleotides were irradiated with light having a wavelength of 366 nm for 0 or 30 minutes in:(a) water;(b) HEPES buffered saline 1x (HBS);(c) HBS including 250 mM NaCI and 5 mM MgCl2 (M); or(d) 50 mM Tris-HCI (pH = 7.0) including 2 mM MgCl2 (Mg).Polyacrylamide gel electrophoresis was used to separate the contents of each sample. The sample and corresponding lane are listed in the table in FIG. 8. Each lane contained a 5 pL fraction of the sample, 6 pL of 8 M urea with 10% glycerol, and 4 pL of RNA loading dye. The polyacrylamide gel included 6% TBU, and electrophoresis was run with a voltage of 250 V for 1 hour and 55 minutes. The gel was stained with SYBR safe dye and scanned using iBright. The results were compared to an RNA ladder and the linear polyribonucleotide that was not annealed nor irradiated. Results of this experiment are shown in FIG. 8. This experiment resulted in two distinct bands for lanes containing annealed then irradiated polyribonucleotides. The lower band contains linear polyribonucleotides. The higher band contains circular polyribonucleotides.This experiment demonstrates the circularization efficiency of photocircularization of linear polyribonucleotides is dependent on the choice of solvent. This experiment also demonstrates that linear polyribonucleotides photocircularized in HBS had a higher circularization efficiency in comparison to photocircularization in water, HBS including M, and Tris-HCI including MgCl2. This experiment also demonstrates the loss or degradation of photocircularized polyribonucleotides in HBS including M and Tris-HCI including MgChExample 6. Reversal of photocircularization via irradiating a photocircularized polyribonucleotide with light having a different wavelength than that used for forming the photocircularized polyribonucleotideThis experiment demonstrates the reversibility of photocircularization of a photocircularized polyribonucleotide by irradiating the photocircularized polyribonucleotide with a different wavelength of light than that used for photocircularization.A linear polyribonucleotide was bound to a photoreactive polynucleotide via:(a) annealing in water;(b) photocircularization in water;(c) annealing in HBS; or(d) photocircularization in HBS.The protocol for annealing was previously described in Example 1 . The same linear polyribonucleotide encoding a Gaussia luciferase polypeptide (LAA2367) and photoreactive polyribonucleotide described in Example 1 are used in this experiment. The samples were irradiated for 30 minutes at 366 nm. The photocircularization was then reversed by irradiating with light having a wavelength of 312 nm for 30 minutes. Polyacrylamide gel electrophoresis was used to separate the contents of each sample. Results of this experiment are shown in FIG. 9. This experiment resulted in two distinct bands for lanes containing annealed and photocircularized polyribonucleotides. The lower band contains linear polyribonucleotides. The higher band contains circular polyribonucleotides. A distinct band for circular polyribonucleotides in addition to a distinct band for linear polyribonucleotidesis only observed with the polyribonucleotide circularized via photocircularization in HBS 1x (Lane 8 of FIG. 9). After irradiation with light having a wavelength of 312 nm for 30 minutes, the band for circular polyribonucleotides is no longer visible (Lane 9 of FIG. 9), indicating reversibility of photocircularization.This experiment demonstrates the reversibility of photocircularization of a linear polyribonucleotide by irradiating with light having a different wavelength than used for photocircularization.Example 7. Functionalization of circularized polyribonucleotides via photocircularizationThis example demonstrates the use of photocircularization of a linear polyribonucleotide to incorporate a functional group by incorporating the functional group into the bridging agent prior to photocircularization.As shown in FIG. 10, a bridging agent with two photoreactive crosslinking agents and a functional group can anneal at each end to the linear polyribonucleotide. The two photoreactive crosslinking agents form covalent adducts with the polyribonucleotide upon irradiation, thus producing a photocircularized polyribonucleotide with a functional group that can be used to further attach a chemical moiety.The bridging agent may be designed in such a way that there is terminal end with the functional group, e.g., for further attachment of a targeting molecule.5'- AGACGCUACG[cnvK]AUUUUCCCA[cnvK]GUCCGUAGCGUCUUUA [Spacer][Functional group]- 3' (SEQ ID NO: 35)Adding a chemical functional group onto the circular polyribonucleotide is challenging considering the unmodified and covalently closed loop structure of circular RNA. However, by using photocircularization, any kind of chemical functional group can be attached to the RNA in a sitespecific manner. The bridging agent can include the functianol group, e.g., at the terminus of an oligonucleotide bridging agent, and the functional group is incorporated into the circular polyribonucleotide upon photocircularization.Example 8. Purification of circular polyribonucleotidesThis example demonstrates the use of photocircularization of a linear polyribonucleotide to purify circular polyribonucleotides by incorporating a purification tag into the bridging agent prior to photocircularization.As shown in FIG. 11 , a bridging agent with two photoreactive crosslinking agents and a purification tag can anneal at each end to the linear polyribonucleotide. The two photoreactive crosslinking agents form covalent adducts with the polyribonucleotide upon irradiation, thus producing a photocircularized polyribonucleotide with a purification tag can be used to separate circularized RNA from linear RNA.The bridging agent may be designed in such a way that there is terminal end with the purification tag, such as biotin.5'- AGACGCUACG[cnvK]AUUUUCCCA[cnvK]GUCCGUAGCGUCUUUA [Spacer][Biotin]-3' (SEQ ID NO: 36)The method can include contacting the sample with a capture agent, such as streptavidin, that binds the tag and separating the circular polyribonucleotide having the tag that is bound to the capture agent from the linear polyribonucleotides lacking the tag that are not bound to the capture agent. Upon immobilization of the capture agent, the photocircularized RNA can be separated from the linear RNA.Other Embodiments While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

Claims

CLAIMS1 . A complex comprising:(a) a linear polyribonucleotide comprising a first annealing region and a second annealing region; and(b) a bridging agent comprising a first photoreactive crosslinking agent and a second photoreactive crosslinking agent wherein the bridging agent is configured to bind to the first annealing region and the second annealing region on the linear polyribonucleotide; and wherein each photoreactive crosslinking agent is configured to form a covalent adduct with the linear polyribonucleotide upon irradiation with light.

2. The complex of claim 1 , wherein the bridging agent comprises a polynucleotide.

3. The complex of claim 2, wherein the polynucleotide comprises an aptamer.

4. The complex of claim 2 or 3, wherein the polynucleotide comprises a third annealing region configured to hybridize to the first annealing region on the linear polyribonucleotide and a fourth annealing region configured to hybridize to the second annealing region on the linear polyribonucleotide.

5. The complex of claim 4, wherein the first annealing region and the second annealing region are each from 8 to 200 ribonucleotides.

6. The complex of claims 4 or 5, wherein the first annealing region has at least 80% complementarity to the third annealing region, and the second annealing region has at least 80% complementarity to the fourth annealing region.

7. The complex of any one of claims 4-6, wherein the first annealing region has zero or one mismatch with the third annealing region, and the second annealing region has zero or one mismatch with the fourth annealing region.

8. The complex of any one of claims 4-7, wherein the third annealing region comprises the first photoreactive crosslinking agent and the fourth annealing region comprises the second photoreactive crosslinking agent.

9. The complex of any one of claims 2-8, wherein the photoreactive crosslinking agent comprises 5- bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-thiouridine, a diazirine, a phenylselenide, a furan, or an abasic site.

10. The complex of claim 9, wherein the carbazole is 3-cyanovinylcarbazole, 4- methylpyranocarbazole, or pyranocarbazole.11 . The complex of claim 9, wherein the coumarin is 7-hydroxycoumarin.

12. The complex of any one of claims 2-8, wherein the first photoreactive crosslinking agent and / or the second photoreactive crosslinking agent is a photoreactive nucleotide analog.

13. The complex of claim 12, wherein the photoreactive nucleotide analog crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the linear polyribonucleotide upon photoirradiation.

14. The complex of any one of claims 2-8, wherein the first crosslinking agent comprises a first photoreactive nucleotide analog and the second crosslinking agent comprises a second photoreactive nucleotide analog.

15. The complex of claim 14, wherein the polynucleotide comprises a plurality of photoreactive nucleotide analogs.

16. The complex of claim 14 or 15, wherein at least one of the plurality of photoreactive nucleotide analogs is attached to the 3’ end of the polynucleotide.

17. The complex of any one of claims 14-16, wherein at least one of the plurality of photoreactive nucleotide analogs is attached to the 5’ end of the polynucleotide.

18. The complex of any one of claims 14-17, wherein at least one of the plurality of photoreactive nucleotides analogs is located at an internal position within the polynucleotide.

19. The complex of any one of claims 14-18, wherein the polynucleotide comprises 1 to 10 nucleotides between each of the photoreactive nucleotide analogs.

20. The complex of any one of claims 14-19, wherein the 3’ end of the polynucleotide has at least 1 nucleotide from the nearest photoreactive nucleotide analog.21 . The complex of claim 20, wherein the 3’ end of the polynucleotide has from 1 to 10 nucleotides from the nearest photoreactive nucleotide analog.

22. The complex of any one of claims 14-21 , wherein the 5’ end of the polynucleotide has at least 1 nucleotide from the nearest photoreactive nucleotide analog.

23. The complex of claim 22, wherein the 5’ end of the polynucleotide has from 1 to 10 nucleotides from the nearest photoreactive nucleotide analog.

24. The complex of any one of claims 14-23, wherein each of the photoreactive nucleotide analogs crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the linear polyribonucleotide upon photoirradiation.

25. The complex of any one of claims 2-24, wherein the polynucleotide is single stranded.

26. The complex of any one of claims 2-7, wherein the polynucleotide is double stranded and comprises a first strand and a second strand.

27. The complex of claim 26, wherein the first strand of the double stranded polynucleotide comprises the third annealing region and the fourth annealing region and the second strand of the double stranded polynucleotide comprises the first photoreactive crosslinking agent and the second photoreactive crosslinking agent.

28. The complex of claim 26 or 27, wherein the first strand of the double stranded polynucleotide is longer than the second strand of the double stranded polynucleotide.

29. The complex of claim 28, wherein the first strand of the double stranded polynucleotide comprises a 5’ overhang and a 3’ overhang.

30. The complex of any one of claims 26-29, wherein the first strand of the double stranded polynucleotide anneals to the linear polyribonucleotide and the second strand of the double stranded polynucleotide is configured to fill a gap of the linear polyribonucleotide formed by annealing of the first strand of the double stranded polynucleotide.31 . The complex of claim 30, wherein the linear polyribonucleotide comprises two nicks in the phosphodiester backbone prior to photoirradiation.

32. The complex of any one of claims 26-31 , wherein the double stranded polynucleotide comprises a 5’ terminal photoreactive crosslinking agent and a 3’ terminal crosslinking agent.

33. The complex of claim 32, wherein the linear polyribonucleotide comprises a 5’ terminal uridine and a 3’ terminal uridine.

34. The complex of claim 32 or 33, wherein the first photoreactive crosslinking agent and the second photoreactive crosslinking agent is each, independently, a uridine or thymidine.

35. The complex of any one of claims 26-34, wherein the first photoreactive crosslinking agent and the second photoreactive crosslinking agent each crosslinks to a ribonucleotide at an adjacent position in the linear polyribonucleotide upon photoirradiation.

36. The complex of any one of claims 2-35, wherein the polynucleotide is linear.

37. The complex of any one of claims 2-36, wherein the polynucleotide comprises a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.

38. The complex of any one of claims 2-37, wherein the polynucleotide is from 20 to 2,000 nucleotides in length.

39. The complex of any one of claims 2-38, wherein the bridging agent comprises a structure PNi-L- PN2, wherein PN1 comprises a first polynucleotide, PN2 comprises a second polynucleotide, and L is a linker of one or more atoms.

40. The complex of claim 39, wherein the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.41 . The complex of claim 1 , wherein the bridging agent comprises a polypeptide.

42. The complex of claim 41 , wherein the polypeptide comprises an antibody or antigen-biding fragment thereof, an enzyme, or a purification tag.

43. The complex of claim 41 or 42, wherein the first photoreactive crosslinking agent and / or second photoreactive crosslinking agent is a photoreactive amino acid analog.

44. The complex of any one of claims 41 -43, wherein the photoreactive amino acid analog crosslinks to a complementary ribonucleotide within the linear polyribonucleotide upon photoirradiation.

45. The complex of any one of claim 41 -44, wherein the photoreactive amino acid analog is an alkyl diazirene-based, arylazide-based, benzophenone-based unnatural amino acid, or A / -e-[2-(furan-2- yl)ethoxy]carbonyl-lysine.

46. The complex of any one of claims 41 -45, wherein the polypeptide comprises a plurality of photoreactive crosslinking agents.

47. The complex of claim 46, wherein each of the plurality of photoreactive crosslinking agents is a photoreactive amino acid analog.

48. The complex of claim 47, wherein each of the photoreactive amino acids crosslinks to a complementary ribonucleotide within the linear polyribonucleotide upon photoirradiation.

49. The complex of any one of claims 41 -48, wherein the polypeptide comprises an RNA recognition motif selected from a K homology domain, zinc finger motif, Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, THUMP domain, YT521 -B homology domain, double stranded RNA binding domain, helicase domain, cold shock domain, S1 domain, Sm domain, La motif, Piwi-Argonaute-Zwille domain, or intrinsically disordered region.

50. The complex of any one of claims 41 -49, wherein the bridging agent comprises a structure PPi-L- PP2, wherein PP1 comprises a first polypeptide, PP2 comprises a second polypeptide, and L is a linker of one or more atoms.51 . The complex of claim 50, wherein the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.

52. The complex claim 1 , wherein the bridging agent comprises a small molecule.

53. The complex of claim 52, wherein the small molecule comprises an intercalator.

54. The complex of any one of claims 1 -53, wherein the linear polyribonucleotide comprises a coding region.

55. The complex of claim 54, wherein the coding region encodes a polypeptide.

56. The complex of claim 54 or 55, wherein the linear polyribonucleotide comprises an internal ribosomal entry site (IRES) operably linked to the coding region.

57. The complex of any one of claims 1 -56, wherein the linear polyribonucleotide is from 1 ,000 to 20,000 ribonucleotides in length.

58. The complex of any one of claims 1 -57, wherein the bridging agent comprises a functional group.

59. The complex of claim 58, wherein the functional group comprises a thiol group, an N- hydroxysuccinimide ester, a polypeptide, a polynucleotide, an azide, a dye, or a targeting moiety.

60. The complex of any one of claims 1 -59, wherein the bridging agent comprises a tag.61 . The complex of claim 60, wherein the tag comprises biotin.

62. A covalently closed polyribonucleotide comprising:(a) a linear polyribonucleotide comprising a first annealing region and a second annealing region; and(b) a bridging agent; wherein the bridging agent is bound to and covalently attached to the first annealing region of the linear polyribonucleotide with a first covalent attachment, and to the second annealing region of the linear polyribonucleotide with a second covalent attachment.

63. The complex of claim 62, wherein the bridging agent comprises a polynucleotide.

64. The complex of claim 63, wherein the polynucleotide comprises an aptamer.

65. The complex of claim 63 or 64, wherein the polynucleotide comprises a third annealing region configured to hybridize to the first annealing region on the linear polyribonucleotide and a fourth annealing region configured to hybridize to the second annealing region on the linear polyribonucleotide.

66. The complex of claim 65, wherein the first annealing region and the second annealing region are each from 8 to 200 ribonucleotides.

67. The complex of claims 65 or 66, wherein the first annealing region has at least 80% complementarity to the third annealing region, and the second annealing region has at least 80% complementarity to the fourth annealing region.

68. The complex of any one of claims 65-67, wherein the first annealing region has zero or one mismatch with the third annealing region, and the second annealing region has zero or one mismatch with the fourth annealing region.

69. The complex of any one of claims 62-68, wherein the polynucleotide is single stranded.

70. The complex of any one of claims 62-68, wherein the polynucleotide is double stranded and comprises a first strand and a second strand.71 . The complex of claim 70, wherein the first strand of the double stranded polynucleotide is longer than the second strand of the double stranded polynucleotide.

72. The complex of claim 71 , wherein the first strand of the double stranded polynucleotide comprises a 5’ overhang and a 3’ overhang.

73. The complex of any one of claims 70-72, wherein the first strand of the double stranded polyribonucleotide anneals to the linear polyribonucleotide and the second strand of the double stranded polyribonucleotide is configured to fill a gap of the linear polyribonucleotide formed by annealing of the first strand of the double stranded polyribonucleotide.

74. The complex of any one of claims 63-72, wherein the polynucleotide is linear.

75. The complex of any one of claims 63-74, wherein the polynucleotide comprises a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.

76. The complex of any one of claims 63-75, wherein the polynucleotide is from 20 to 2,000 nucleotides in length.

77. The complex of any one of claims 64-76, wherein the bridging agent comprises a structure PNi-L- PN2, wherein PN1 comprises a first polynucleotide, PN2 comprises a second polynucleotide, and L is a linker of one or more atoms.

78. The complex of claim 77, wherein the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.

79. The complex of claim 62, wherein the bridging agent comprises a polypeptide.

80. The complex of claim 79, wherein the polypeptide comprises an antibody or antigen-biding fragment thereof, an enzyme, or a purification tag.81 . The complex of claim 79 or 80, wherein the polypeptide comprises an RNA recognition motif selected from a K homology domain, zinc finger motif, Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, THUMP domain, YT521 -B homology domain, double stranded RNA binding domain, helicase domain, cold shock domain, S1 domain, Sm domain, La motif, Piwi-Argonaute-Zwille domain, or intrinsically disordered region.

82. The complex of any one of claims 79-81 , wherein the bridging agent comprises a structure PP1-L- PP2, wherein PP1 comprises a first polypeptide, PP2 comprises a second polypeptide, and L is a linker of one or more atoms.

83. The complex of claim 82, wherein the linker is an amino acid linker, a nucleic acid linker, a small molecule linker, or a combination thereof.

84. The complex claim 62, wherein the bridging agent comprises a small molecule.

85. The complex of claim 84, wherein the small molecule comprises an intercalator.

86. The complex of any one of claims 62-85, wherein the linear polyribonucleotide comprises a coding region.

87. The complex of claim 86, wherein the coding region encodes a polypeptide.

88. The complex of claim 86 or 87, wherein the linear polyribonucleotide comprises an internal ribosomal entry site (IRES) operably linked to the coding region.

89. The complex of any one of claims 62-88, wherein the linear polyribonucleotide is from 1 ,000 to 20,000 ribonucleotides in length.

90. The complex of any one of claims 62-89, wherein the bridging agent comprises a functional group.91 . The complex of claim 90, wherein the functional group comprises a thiol group, an N- hydroxysuccinimide ester, a polypeptide, a polynucleotide, an azide, a dye, or a targeting moiety.

92. The complex of any one of claims 62-91 , wherein the bridging agent comprises a tag.

93. The complex of claim 92, wherein the tag comprises biotin.

94. A method of forming a covalently closed polyribonucleotide comprising irradiating the complex of any one of claims 1 -93 with light.

95. A method of forming a covalently closed polyribonucleotide comprising:(a) forming a complex with(i) a linear polyribonucleotide comprising a first annealing region and a second annealing region; and(ii) a bridging agent comprising a first photoreactive crosslinking agent and a second photoreactive crosslinking agent; wherein the bridging agent binds to the first annealing region and the second annealing region on the linear polyribonucleotide; and(b) irradiating the complex with light, wherein each photoreactive crosslinking agent forms a covalent adduct with the linear polyribonucleotide.

96. The method of claim 94 or 95, wherein a wavelength of the irradiated light is from 340-410 nm.

97. The method of claim 96, wherein the wavelength of the irradiated light is from 350-370 nm.

98. The method of claim 97, wherein the wavelength of the irradiated light is 365 nm or 366 nm.

99. The method of any one of claims 94-98, wherein the complex is irradiated for 1 to 120 minutes.

100. The method of any one of claims 94-99, further comprising irradiating the complex with light at a second wavelength to release each photoreactive crosslinking agent from the linear polyribonucleotide.101 . The method of claim 100, wherein the second wavelength of the irradiated light is from 300-320 nm.

102. The method of claim 101 , wherein the second wavelength of the irradiated light is 312 nm.

103. The method of any one of claims 92-102, further comprising contacting a cell with the complex to deliver the complex to the cell.

104. The method of claim 103, wherein the complex is irradiated prior to contacting.

105. The method of claim 103, wherein the complex is irradiated after contacting.

106. The method of any one of claims 103-105, wherein the cell is a eukaryotic cell.

107. The method of claim 106, wherein the eukaryotic cell is a mammalian cell.

108. The method of claim 107, wherein the mammalian cell is a human cell.

109. The method of any one of claims 103-108, wherein the complex is delivered to the cell ex vivo.

110. The method of any one of claims 103-108, wherein the complex is delivered to the cell in vivo.

111. The method of any one of claims 109-110, wherein the complex is administered to a subject.

112. The method of claim 111 , wherein the complex is irradiated prior to administration.

113. The method of claim 111 , wherein the complex is irradiated after administration.

114. The method of any one of claims 103-113, wherein administering the cell to the subject treats a disease or disorder in the subject.

115. The method of any one of claims 95-114, wherein the bridging agent comprises a functional group.

116. The method of claim 115, wherein the functional group comprises a thiol group, an N- hydroxysuccinimide ester, a polypeptide, a polynucleotide, an azide, a dye, or a targeting moiety.

117. The method of any one of claims 95-116, wherein the bridging agent comprises a tag.

118. The method of claim 117, further comprising contacting the complex with a capture agent that binds the tag.

119. The method of claim 118, wherein the tag comprises biotin, and the capture agent comprises streptavidin.

120. The method of claim 118 or 119, further comprising separating the complex comprising the bridging agent that is bound to the capture agent.121 . The method of any one of claims 118-120, wherein the capture agent is conjugated to a column.

122. A covalently closed polyribonucleotide formed by the method of any one of claims 95-121 .

123. A method of separating a circular polyribonucleotide comprising a tag from a plurality of polyribonucleotides comprising a mixture of linear polyribonucleotides and circular polyribonucleotides, the method comprising:(a) providing a sample comprising(i) a plurality of linear polyribonucleotides, each comprising a first annealing region and a second annealing region; and(ii) a bridging agent comprising a first photoreactive crosslinking agent, a second photoreactive crosslinking agent, and a functional group comprising a tag; wherein the bridging agent binds to the first annealing region and the second annealing region on one of the plurality of linear polyribonucleotides, wherein the bridging agent and the linear polyribonucleotide forms a complex;(b) irradiating the complex with light, wherein each photoreactive crosslinking agent forms a covalent adduct with the linear polyribonucleotide, thereby producing a plurality of polyribonucleotides, wherein a subset of the plurality of polyribonucleotides comprises the circular polyribonucleotide comprising the tag;(b) contacting the sample with a capture agent that binds the tag; and(c) separating the circular polyribonucleotide comprising the tag that is bound to the capture agent from the plurality of polyribonucleotides in the sample.

124. The method of claim 123, wherein the linear polyribonucleotides lack the tag.

125. The method of claim 123 or 124, wherein step (d) comprises immobilizing the capture agent.

126. The method of any one of claims 123-125, wherein the capture agent is conjugated to a column.

127. The method of any one of claims 123-126, wherein the tag comprises biotin, and the capture agent comprises streptavidin.

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