Compositions comprising modified circular polyribonucleotides and uses thereof

Abstract

To provide pharmaceutical compositions and preparations of modified circular polyribonucleotides, and to provide uses thereof.SOLUTION: The present disclosure provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient and a circular polyribonucleotide comprising a first portion of contiguous unmodified nucleotides. In some embodiments, the circular polyribonucleotide comprises at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides. In some embodiments, the modified circular polyribonucleotide is delivered to a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application is a continuation of U.S. Provisional Patent Application No. 62 / 823,573, filed March 25, 2019. The entire contents of which are incorporated herein by reference. can be. [Background technology]

[0002] Certain cyclic polyribonucleotides are present in human tissues and cells, including those of healthy individuals. It is ubiquitous in Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides a method for preparing a compound comprising: a pharmaceutically acceptable carrier or excipient and a first part of contiguous unmodified nucleotides; In one embodiment, the present invention provides a pharmaceutical composition comprising a cyclic polyribonucleotide comprising the amino acid sequence of The cyclic polyribonucleotide comprises at least one modified polyribonucleotide and a contiguous non- In one embodiment, the modified circular polyribonucleic acid comprises a first portion of modified nucleotides. The oxide is delivered to the subject.

[0004] The present disclosure provides a method for reducing or decreasing the immunogenicity of cyclic polyribonucleotides in a subject. 1. A method for producing a hybrid modified cyclic polyribonucleotide comprising the steps of: The hybrid modified cyclic polyribonucleotide contains at least one modified nucleotide and 5% A first nucleotide sequence comprising at least about 5 to 1000 consecutive nucleotides having the following modified nucleotides: and administering to the subject a hybrid modified cyclic polyribonucleotide comprising the moiety: and comparing the modified cyclic polyribonucleotide to the corresponding unmodified cyclic polyribonucleotide in the subject's cells or tissue. to obtain reduced immunogenicity for the hybrid modified cyclic polyribonucleotide. In one embodiment, a method is provided comprising administering a cyclic polyribonucleotide to a subject. The method for reducing or decreasing the immunogenicity of a compound comprising the steps of: providing, wherein the hybrid modified cyclic polyribonucleotide comprises at least one and a first portion of contiguous unmodified nucleotides; administering a hybrid modified cyclic polyribonucleotide to a subject; and The hybrid modified cyclic polyribonucleotides are more potent than the corresponding unmodified cyclic polyribonucleotides in tissues. and obtaining a reduced or diminished immunogenicity against the polyribonucleotide. In embodiments, the first portion comprises an IRES. In one embodiment, the first nucleotide comprises at least about 5 to 1000 consecutive unmodified nucleotides. The part 1 is 5'-methylcytidine pseudouridine or N1-methyl-pseudouridine In one embodiment, 5% or less of the nucleotides in the first portion are In one embodiment, 5% or less of the IRES in the first portion are modified nucleotides. The nucleotide is a modified nucleotide.

[0005] The present disclosure provides a method for expressing one or more expression sequences in a subject, the method comprising: Providing a modified cyclic polyribonucleotide, The nucleotides contain at least one modified nucleotide and no more than 5% modified nucleotides. a first portion comprising at least about 5 to 1000 contiguous nucleotides having the sequence administering the hybrid-modified cyclic polyribonucleotide to a subject; and Expression and comparison of one or more expression sequences in fully modified circular polyribonucleotide equivalents in tissues and obtaining increased expression of one or more expression sequences in comparison to the control. In some embodiments, the method for expressing one or more expression sequences in a subject comprises at least one a modified polyribonucleotide of the present invention, a first portion of contiguous unmodified nucleotides, and one or more nucleotide sequences providing a hybrid modified circular polyribonucleotide comprising the desired sequence; administering a modified cyclic polyribonucleotide to a subject; and compared to the expression of one or more expression sequences in a fully modified circular polyribonucleotide equivalent, and obtaining increased expression of one or more expression sequences. In one embodiment, the first portion comprises at least about 5-10 In one embodiment, the first portion comprises 5'-methyl It lacks lucitidine, pseudouridine, or N1-methyl-pseudouridine. In some embodiments, no more than 5% of the nucleotides in the first portion are modified nucleotides. In one embodiment, 5% or less of the nucleotides in the IRES of the first portion are modified It is a nucleotide.

[0006] The present disclosure provides a method for increasing the stability of a cyclic polyribonucleotide in a subject, comprising: providing a hybrid-modified cyclic polyribonucleotide, The modified cyclic polyribonucleotide contains at least one modified nucleotide and 5% or less modified a first portion comprising at least about 5 to 1000 contiguous nucleotides, administering a hybrid modified cyclic polyribonucleotide to a subject; The hybridization of the ribonucleotides with the unmodified cyclic polyribonucleotides in the target cells or tissues is and obtaining increased stability for the lid-modified cyclic polyribonucleotide. In one embodiment, a method for increasing the stability of a cyclic polyribonucleotide in a subject is provided. The method of adding comprises the steps of providing a hybrid modified cyclic polyribonucleotide, The hybrid modified cyclic polyribonucleotide comprises at least one modified polyribonucleoside. a first portion of a hybrid modified circular polynucleotide and a second portion of a hybrid modified circular polynucleotide; administering to a subject a ribonucleotide containing a corresponding non-ribonucleotide in a cell or tissue of the subject; Hybrid modified cyclic polyribonucleotides compared to modified cyclic polyribonucleotides and obtaining increased stability against I. In one embodiment, the first portion is In one embodiment, the first portion comprises at least about 5 to 1000 contiguous non-linear cleavage regions. In one embodiment, the first portion comprises a 5'-methylcytidine. , pseudouridine, or N1-methyl-pseudouridine. In one embodiment, 5% or less of the nucleotides in the first portion are modified nucleotides. In embodiments, 5% or less of the nucleotides in the IRES of the first portion are modified nucleotides. It is Do.

[0007] In one embodiment, a method for reducing the immunogenicity of a cyclic polyribonucleotide in a subject The method includes providing a hybrid modified cyclic polyribonucleotide, The modified cyclic polyribonucleotide comprises at least one modified nucleotide and at least a first portion comprising about 5 to 1,000 consecutive unmodified nucleotides; and administering the modified cyclic polyribonucleotide to a subject; and detecting the presence of the modified cyclic polyribonucleotide in cells or tissues of the subject. The hybrid modified circular polyribonucleotides are more potent than the corresponding unmodified circular polyribonucleotides. and obtaining reduced immunogenicity to the nucleotide.

[0008] In one embodiment, a method for reducing the immunogenicity of a cyclic polyribonucleotide in a subject 1. A method for producing a hybrid modified cyclic polyribonucleotide comprising the steps of: A hybrid-modified cyclic polyribonucleotide is a polyribonucleotide that contains at least one modified nucleotide and at least one a first portion comprising at least about 5 to 1000 consecutive unmodified nucleotides; administering the lid-modified cyclic polyribonucleotide to a subject; and The hybrid modified cyclic polyribonucleotides are compared to the corresponding unmodified cyclic polyribonucleotides in and obtaining reduced immunogenicity to ribonucleotides.

[0009] In some embodiments, the first portion is at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive In one embodiment, the first portion comprises 5'-methylcytosine. In one embodiment, the first portion is devoid of uridine or pseudouridine. In some embodiments, the nucleotides in the circular polyribonucleic acid sequence are modified nucleotides. In one embodiment, the nucleic acid is a hybrid modified circular polyribonucleic acid. The nucleotides are a) at least about 0.7 times more nucleotide-rich than the corresponding unmodified cyclic polyribonucleotide ,0.8,0.9,1.0,1.1,1.2,1.3,1.5,1.6,1.8,2,2 b) have 0.2, 2.5, 2.8, or 3-fold higher expression of the corresponding unmodified circular polyribonucleotides; nucleotides, at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2 , 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4 0.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9 c) a corresponding unmodified circular polyribonucleotide having a half-life that is 0.0, 9.5, or 10.0 times longer; or d) have a half-life longer than that of nucleotides; or d) RIG-I, TLR-3, TLR-7, T Among LR-8, MDA-5, LGP-2, OAS, OASL, PKR, and IFN-β When assessed by expression or signal transduction or activation of at least one of at least about 1.1, 1.2, 1.3, 1.4, 1.6, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2. .5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3 .8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7 0.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times less immunogenic. In some embodiments, the at least one modified nucleotide is a) N(6) methyl adenosine 1'-nucleotide. a) 2'-O-methylcytidine; b) 5'-methylcytidine; and c) 2'-O-methylcytidine. , 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'- Deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP ), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylamino Aminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2 '-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), LNA, ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5' -Anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleoside nucleotides, thiophosphonate nucleotides, and 2'-fluoroN3-P5'-phosphoramidates or c) any modified nucleotide from Table 2. In an embodiment, at least about 10% of the hybrid modified cyclic polyribonucleotides 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% In one embodiment, % of the nucleotides are modified nucleotides. Modified cyclic polyribonucleotides are proteins, peptides, The biomolecule comprises a binding site configured to bind to a DNA, RNA, or cellular target. In one embodiment, the hybrid modified cyclic polyribonucleotide comprises one or more expression In one embodiment, the first portion comprises an IRE sequence consisting of unmodified nucleotides. In one embodiment, one or more of the hybrid modified cyclic polyribonucleotides The above expressed sequences have a) higher translation efficiency than their fully modified circular polyribonucleotide counterparts; b) at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2. 8-fold or 3-fold higher translation efficiency; c) higher than the corresponding unmodified cyclic polyribonucleotide or d) a translation efficiency of at least about 0.7 times that of the corresponding unmodified circular polyribonucleotide. ,0.8,0.9,1.0,1.1,1.2,1.3,1.5,1.6,1.8,2,2 0.2, 2.5, 2.8, or 3 times higher translation efficiency.

[0010] In some embodiments, the method of expressing one or more expression sequences in a subject comprises expressing one or more providing a hybrid modified circular polyribonucleotide comprising an expression sequence, Hybrid modified cyclic polyribonucleotides are those containing at least one modified nucleotide and a At least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, a first portion comprising 700, 800, 900, or 1000 consecutive unmodified nucleotides; administering a hybrid modified cyclic polyribonucleotide to a subject; the corresponding one of the fully modified cyclic polyribonucleotide equivalents in the cells or tissues of interest obtaining increased expression of one or more of the expression sequences compared to the expression of the expression sequences above. nothing.

[0011] In some embodiments, a method for increasing the stability of a cyclic polyribonucleotide in a subject providing a hybrid modified cyclic polyribonucleotide, The lid-modified cyclic polyribonucleotide comprises a modified cyclic polyribonucleotide and at least Approximately 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, a first portion comprising 800, 900, or 1000 consecutive unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to a subject; The hybrids are more readily modified than the corresponding unmodified cyclic polyribonucleotides in cells or tissues. and obtaining increased stability for the modified cyclic polyribonucleotide.

[0012] In certain embodiments, the first portion comprises an IRES.

[0013] In one aspect, the present disclosure provides a method for treating a pulmonary arthritis with a pharmaceutically acceptable carrier or excipient and a hybrid modification. A pharmaceutical composition comprising a cyclic polyribonucleotide, the pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide. The nucleotide comprises at least one modified nucleotide and a first portion, the first portion being , at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 60 Pharmaceutical compositions comprising 0, 700, 800, 900, or 1000 consecutive unmodified nucleotides In certain embodiments of this aspect, the first portion contains no more than 5% modified nucleotides. Contains octide.

[0014] In another aspect, the present disclosure provides a method for treating a pulmonary arthritis with a pharmaceutically acceptable carrier or excipient and a hybrid modified A pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, The nucleotide comprises at least one modified nucleotide and a first portion, is at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 6 00, 700, 800, 900, or 1000 contiguous nucleotides, and the first portion comprises The pharmaceutical composition lacks 5'-methylcytidine or pseudouridine. In certain embodiments of the aspect, the first portion comprises no more than 5% modified nucleotides.

[0015] In some embodiments, the pharmaceutical composition comprises a hybrid modified cyclic polyribonucleotide. , wherein the hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide. ocide; first binding of a first target, e.g., RNA, DNA, protein, or cellular target a first portion comprising a first binding site configured to bind to a moiety (wherein the first binding The portion comprises a first circular polyribonucleotide (circular RNA) linker consisting of unmodified nucleotides. wherein the first target and the hybrid modified circular polyribonucleic acid Reotide forms a complex.

[0016] In some embodiments, the pharmaceutical composition comprises a hybrid modified cyclic polyribonucleotide. , the hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide; Binding to a first binding moiety on a first target, e.g., RNA, DNA, protein, or cellular target. a first portion comprising a first binding site configured to bind to the The first cyclic polyribonucleotide (cyclic RNA) binding motif composed of unmodified nucleotides and a second binding moiety configured to bind to a second binding moiety of a second target. wherein the second binding moiety is a second circular RNA-binding motif, The binding moiety is different from the second binding moiety and is capable of binding to the first target, the second target, and the hybrid modification. The circular polyribonucleotide forms a complex, and the first target or the second target is Not RNA.

[0017] In one embodiment, a pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide. Thus, a hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide. a first binding site configured to bind to a first binding moiety of a first target; wherein the first binding moiety is a first circular polyribonucleotide (circular RNA) binding moiety. a second binding motif configured to bind to a second binding moiety of a second target; wherein the second binding moiety is a second circular RNA-binding motif. , the first binding moiety is different from the second binding moiety, and both the first target and the second target are In one embodiment, the hybrid modified circular polyribonucleoside is a microRNA. The cyclic polyribonucleotides have lower immunogenicity than the corresponding unmodified cyclic polyribonucleotides. In this form, the hybrid modified cyclic polyribonucleotide is a corresponding unmodified cyclic polyribonucleotide. from ribonucleotides, at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8 ,2,2.2,2.5,2.8,3,3.2,3.3,3.5,3.8,4.0,4.2 , 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 , 9.0, 9.5, or 10.0 times longer half-lives. Lid-modified cyclic polyribonucleotides are more soluble than the corresponding unmodified cyclic polyribonucleotides. In one embodiment, the hybrid modified circular polyribonucleic acid has a long half-life. RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, O Expression or signaling of at least one of AS, OASL, PKR, and IFN-β The corresponding unmodified cyclic polyribonucleotide, as assessed by cyclic transfer or activation, than at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2 0.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8 , 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0 times less immunogenic. In one embodiment, the hybrid modification Cyclic polyribonucleotides have a longer half-life than the corresponding unmodified cyclic polyribonucleotides. In one embodiment, at least one modified nucleotide has an N(6) methyl From the group consisting of adenosine (m6A), 5'-methylcytidine, and pseudouridine In one embodiment, at least one modified nucleic acid is selected from 2'-O-methyl, 2'-O-Methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-de Oxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP) , 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylamino 2'-O-DMAP, 2'-dimethylaminoethyloxyethyl -O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), LNA, ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'- Anhydrohexitol nucleic acid (HNA), morpholino, and methylphosphonate nucleotides , thiophosphonate nucleotides, and 2'-fluoro N3-P5'-phosphoramidiates In one embodiment, the hybrid modified circular polyribonucleic acid is selected from the group consisting of: At least about 10%, 20%, 30%, 40%, 50%, 60%, 70% of the nucleotides 80%, 90%, 95%, or 99% of the nucleotides are modified nucleotides. In embodiments, the modified cyclic polyribonucleotide is a polyribonucleotide consisting of unmodified nucleotides. configured to bind to peptides, proteins, biomolecules, DNA, RNA, or cellular targets In one embodiment, the first portion comprises a binding site. In this embodiment, the modified cyclic polyribonucleotide has an internal ribonucleotide consisting of unmodified nucleotides. In one embodiment, the first portion comprises an IRES. In certain embodiments, the IRES contains no more than 5% modified nucleotides.

[0018] In one embodiment, the hybrid modified cyclic polyribonucleotide comprises one or more isomers of In some embodiments, the hybrid modified cyclic polyribonucleotide comprises the sequence A hybrid modified circular polyribonucleotide comprising one or more expression sequences and an IRES is , 5'-methylcytidine, pseudouridine, or a combination thereof outside the IRES In one embodiment, one or more of the hybrid modified cyclic polyribonucleotides The expressed sequences have higher translation efficiency than the corresponding fully modified circular polyribonucleotides. In some embodiments, one or more expression sequences of a hybrid modified circular polyribonucleotide are is at least about 0.7, 0.8, 0.9, 10.1, 11.2, 12.3, 13.4, 14.5, 15.6, 16.7, 17.8, 18.9, 20.9, 22.1, 23.2, 24.3, 25.4, 26.5, 27.6, 28.7, 29.8, 9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, In one embodiment, the fully modified circular polyribonucleotide has a translation efficiency that is 2.8 or 3 times higher. The nucleotide equivalents comprise at least one modified nucleotide outside the first portion and the first In one embodiment, the hybrid modification comprises more than 5% modified nucleotides in the portion of One or more of the expression sequences of the circular polyribonucleotide may be a corresponding unmodified circular polyribonucleotide. In one embodiment, the hybrid modified cyclic polynucleotide has a higher translation efficiency than the oxidase. One or more expressed sequences of polyribonucleotides are , at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1 0.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher translation efficiency. In the embodiment, one or more expression sequences of the hybrid modified circular polyribonucleotide are It has a higher translation efficiency than its fully modified cyclic polyribonucleotide equivalent. Therefore, one or more of the expressed sequences of the hybrid modified circular polyribonucleotides have more than 10% modification. a fully modified circular polyribonucleotide having a first portion containing a modified nucleotide, In one embodiment, the hybrid modified cyclic polyribonucleotide One or more of the expression sequences must be 100% modified with pseudouridine or 5' methylcytosine. The modified circular polyribonucleotide has a higher translation efficiency than a fully modified circular polyribonucleotide having a first portion comprising the In one embodiment, one or more expression sequences of the hybrid modified circular polyribonucleotide are The sequence comprises a fully modified circular polyribonucleotide sequence having a first portion comprising a modified nucleotide. Equal to at least about 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5 ,2.8,3,3.2,3.3,3.5,3.8,4.0,4.2,4.5,4.8,5 .0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or In one embodiment, the translation efficiency of the hybrid Contains modified cyclic polyribonucleotides or fully modified cyclic polyribonucleotide equivalents Measured either in cells or in an in vitro translation system (e.g., rabbit reticulocyte lysate). In some embodiments, the hybrid modified cyclic polyribonucleotide is It has the ability to do ring-circle translation.

[0019] In one embodiment, each of the one or more expression sequences is a hybrid modified cyclic polynucleotide. separated from the subsequent expressed sequence by a staggered element of ribonucleotides, and one or more Rolling circle translation of the expressed sequence produces at least two polypeptide molecules. In certain embodiments, the pharmaceutically acceptable carrier or excipient is ethanol. In one embodiment, the staggered elements are derived from (a) two translations of a single expressed sequence; or (b) preventing the production of a single polypeptide from more than one translation of two or more expressed sequences; In one embodiment, the staggered element is a sequence separated from one or more expressed sequences. In one embodiment, the stagger element comprises a portion of one or more expression sequences. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises a laurin The hybrid modified circular polyribonucleotides are capable of hybrid circle translation. All polyribonucleotides produced during rolling circle translation of lid-modified circular polyribonucleotides At least 50%, at least 60%, at least 70%, or at least at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% distinct polypeptides. each of the distinct polypeptides is a single translation of one or more expressed sequences. In one embodiment, the hybrid modified cyclic polynucleotide is produced from one or more translation rounds. Ribonucleotides are hybrid modified circular polyribonucleotides rolling circle At least 50% of all polypeptides (mol / mol) produced during translation, at least 6 0%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% % are distinct polypeptides, and the amount of distinct products across all polypeptides The ratio is tested in an in vitro translation system. In one embodiment, the in vitro translation system comprises rabbit reticulocyte lysate. In certain embodiments, the staggered element comprises one or more and the stagger element is at the 3' end of at least one of the expression sequences of the hybrid modified circular Structured to stall ribosomes during rolling-circle translation of polyribonucleotides In one embodiment, the staggered element is selected from the group consisting of 2A sequences and 2A-like sequences. In one embodiment, the staggered element encodes a peptide sequence selected from the group consisting of G In one embodiment, the stagger element encodes a sequence having a C-terminal sequence that is P. , a C-terminal conjugate of D(V / I)ExNPGP (where x = any amino acid) In one embodiment, the staggered element encodes a sequence having a GDV sequence. ESNPGP, GDIEENPGP, VEPNPGP, IETNPGP, GDIESNP GP, GDVELNPGP, GDIETNPGP, GDVEPGP, GDVEENPG P,GDVEQNPGP,IESNPGP,GDIELNPGP,HDIETNPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEFNPGP, encoding a sequence selected from the group consisting of In one embodiment, a stagger element is located at the 3' end of each of the one or more expression sequences. In one embodiment, the first nucleotide in the hybrid modified cyclic polyribonucleotide is The staggered elements of the expression sequence are the first expression sequence in the hybrid modified circular polyribonucleotide. It is located upstream (5' end) of the first translation initiation sequence of the expression sequence following the sequence, and contains the stagger element and the The distance between the translation initiation sequences of the first and subsequent expressed sequences is In some embodiments, the first amino acid in the hybrid modified cyclic polyribonucleotide is The staggered element of the expression sequence of 1 is the first in the hybrid modified circular polyribonucleotide It is located upstream (5' end) of the first translation initiation sequence of the expression sequence following expression, and is a hybrid modification. The modified circular polyribonucleotides are subsequently translated to form the corresponding hybrid modified circular polyribonucleotides. a second staggered sequence upstream of the second translation initiation sequence of the second expressed sequence in ribonucleotides; The corresponding hybrid modified circular polyribonucleotide containing the element is subsequently translated. and the second staggered element in the corresponding hybrid modified circular polyribonucleotide is , a second translation initiation sequence, a staggered element in a hybrid modified circular polyribonucleotide, and For example, at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold greater than the distance between the first translation start site and the , 8, 9, 10, or more times greater. The distance between the element and the first translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt , 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt , 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt , 75 nt, or more. In some embodiments, the second staggered element and the second The distance between the stagger element and the first translation start is at least 2 times greater than the distance between the stagger element and the first translation start. nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt , 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt , 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt , 60 nt, 65 nt, 70 nt, 75 nt, or more. and an expression sequence following the first expression sequence in the hybrid modified circular polyribonucleotide. is an expression sequence other than the first expression sequence. In one embodiment, the hybrid modified circle The subsequent expression sequence of the first expression sequence in the polyribonucleotide is be.

[0020] In one embodiment, the hybrid modified cyclic polyribonucleotide comprises a) an encoding a) putogens; b) staggered elements; c) regulatory elements; d) replication elements; and f) pseudo-double-stranded secondary structures. In one embodiment, the hybrid structure comprises at least one structural element selected from the group consisting of: Doped circular polyribonucleotides have a) higher translation efficiency than their linear counterparts; b) multiple translation c) stoichiometric translation efficiency of the product; c) lower immunogenicity than the equivalent lacking the encryptogen; d) a) increased half-life over linear equivalents; and b) persistence during cell division. In one embodiment, the hybrid modified circular polyribonucleic acid The octides are at least 5%, at least 10%, at least 15%, less than their linear counterparts. at least 20%, at least 30%, at least 40%, at least 50%, at least 6 0%, at least 70%, at least 80%, at least 90%, at least 100%, At least 150%, at least 2x, at least 3x, at least 4x, at least 5x fold, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 1 0-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher translation efficiency. In some embodiments, the hybrid modified cyclic polyribonucleotide has a linear equivalent. In one embodiment, the hybrid modification has a translation efficiency at least 5 times higher than that of the hybrid modification. Circular polyribonucleotides are composed of a) 5'-UTR; b) 3'-UTR; c) poly-A sequence d) a 5'-cap; e) a termination element; f) susceptibility to exonuclease degradation; and g) lacking at least one of binding to a cap-binding protein. In one embodiment, one or more of the expression sequences includes a Kozak initiation sequence. The helical structure comprises at least one double-stranded RNA having at least one non-double-stranded segment. In one embodiment, the pseudohelical structure comprises a repeating sequence, e.g., an A segment. In one embodiment, the nucleic acid sequence comprises a first sequence and a second sequence linked by an encoding sequence. In one embodiment, the encryptogen comprises a splicing element. , a protein binding site, e.g., a ribonucleotide binding protein. In this context, the encryptogen may, for example, induce immunosuppression to evade an immune response, such as a CTL response. In one embodiment, the hybrid modified circular polyribonucleic acid comprises an immunoprotein binding site. Nucleotides include, for example, RIG-I, TLR-3, TLR-7, TLR-8, and MDA-5. , LGP-2, OAS, OASL, PKR, and IFN-β. lacking cryptogens as assessed by expression, signaling, or activation In one embodiment, the hybrid In some embodiments, the modified cyclic polyribonucleotide further comprises a riboswitch. In some embodiments, the hybrid modified cyclic polyribonucleotide further comprises an aptazyme. In the form, the hybrid modified circular polyribonucleotide comprises a non-canonical translation initiation sequence, For example, GUG and CUG start codons, translation initiation codons that initiate expression under stress conditions, e.g. In one embodiment, the one or more expressed sequences encode a peptide. In some embodiments, the hybrid modified cyclic polyribonucleotide is a regulatory nucleic acid, e.g. In one embodiment, the hybrid modified circular polyribonucleotides The nucleotides range in size from about 20 bases to about 20 kb. Therefore, the hybrid modified cyclic polyribonucleotide is a cyclic polyribonucleotide that is a cyclic polyribonucleotide. In one embodiment, the hybrid modified circular polyribonucleic acid is synthesized by A gene is a gene that contains multiple expression vectors having either the same or different nucleotide sequences. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises a sequence The nucleic acid is substantially resistant to solutions, such as exonucleases.

[0021] In one embodiment, the hybrid modified cyclic polyribonucleotide is a first target, For example, the first binding moiety may be RNA, DNA, a protein, or a cell membrane. a first binding site formed (wherein the first binding moiety is a first cyclic polyribonucleotide (circular RNA) binding motif); and a second binding moiety of a second target. a second binding site composed of a second circular RNA-binding motif; a modified cyclic polyribonucleotide comprising a first linking moiety and a second linking moiety; The first target, the second target, and the hybrid modified circular polyribonucleic acid are distinct from the fragment. The target forms a complex, and the first target or the second target is not a microRNA.

[0022] In one embodiment, the hybrid modified cyclic polyribonucleotide is a first target a first binding site configured to bind to a first binding moiety, wherein the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding motif; and a second target a second binding site configured to bind to a second binding moiety of interest (wherein the second binding moiety The second circular RNA-binding motif is a hybrid modified circular polyribonucleic acid (HPC) the first binding moiety is different from the second binding moiety and binds to the first target and the second target. Both targets are microRNAs.

[0023] In some embodiments, the first and second targets interact with each other. In one embodiment, the complex regulates a cellular process. are the same, and the first and second binding sites bind to different moieties. In one embodiment, the first and second targets are different. The ribonucleotide may comprise one or more nucleotides configured to bind to a third or more binding moieties. In one embodiment, the one or more targets are the same. In some embodiments, one or more binding sites are configured to bind to different moieties. In one embodiment, the hybrid modified ring When contacted with the first and second targets, the polyribonucleotides react with the first or second targets. In one embodiment, the first and second targets regulate a cellular process associated with a complex. In certain embodiments, the cellular processes are involved in the development of a disease or condition. In certain embodiments, the cellular process involves the synthesis of hybrid modified circular polyribonucleic acid. In certain embodiments, the cellular process is associated with the development of a disease or condition. In one embodiment, the first target comprises a deoxyribonucleic acid (DNA) molecule and the second target comprises a In one embodiment, the complex is capable of directing transcription of a DNA molecule, Regulating the epigenetic remodeling or degradation of DNA molecules. In an embodiment, the first target comprises a first protein and the second target comprises a second protein. In one embodiment, the complex comprises a first protein, a second protein, a third protein, a fourth protein, a fifth protein, a sixth protein, a sixth protein, a sixth protein, a seventh ... modulating protein translocation or signal transduction, or modulating natural protein function, or Inhibiting the formation of a complex formed by direct interaction between the first and second proteins. In one embodiment, the first target comprises a first ribonucleic acid (RNA) molecule and a second In one embodiment, the target comprises a second RNA molecule. In one embodiment, the first target comprises a protein and the second target comprises a The target comprises an RNA molecule. In one embodiment, the complex regulates the translocation of a protein. or the formation of complexes formed by direct interactions between proteins and RNA molecules In one embodiment, the first binding moiety comprises a receptor and the second binding moiety comprises a substrate for the receptor. In certain embodiments, the complex inhibits activation of the receptor. In some embodiments, the modified cyclic polyribonucleotide binds to a binding moiety of a target. wherein the binding moiety comprises a ribonucleic acid (RNA) binding motif. and the hybrid modified circular polyribonucleotide is translationally deficient or In one embodiment, the target is a non-microRNA. The red-modified cyclic polyribonucleotide is configured to bind to a binding moiety of a target. The hybridization site comprises a ribonucleic acid (RNA) binding motif. The modified cyclic polyribonucleotide is translationally deficient or translationally defective, The target is a microRNA. In one embodiment, the target comprises a DNA molecule. In some embodiments, the attachment of the linking moiety to the hybrid modified cyclic polyribonucleotide is In one embodiment, the target comprises a protein. In some embodiments, the binding moiety to the hybrid modified circular polyribonucleotide is The binding inhibits the protein's interaction with other molecules. The protein is a receptor, and binding of the first binding moiety to the modified cyclic polyribonucleotide results in the receptor In one embodiment, the protein is a first enzyme, wherein The hybrid-modified cyclic polyribonucleotide is coupled to a second enzyme configured to bind to the second enzyme. and further comprising a binding site for the first and second hybrid modified circular polyribonucleotides. The binding of the target enzyme modulates the enzymatic activity of the first and second enzymes. The target comprises a messenger RNA (mRNA) molecule. The binding of the binding moiety to the red-modified circular polyribonucleotide interferes with the translation of the mRNA molecule. In some embodiments, the target comprises a ribosome. The attachment of a binding moiety to a hybrid modified circular polyribonucleotide prevents interference with the translation process. In some embodiments, the target comprises a circular RNA molecule. , the attachment of the binding moiety to the hybrid modified circular polyribonucleotide forms a circular RNA molecule. In one embodiment, binding to a hybrid modified circular polyribonucleotide The binding of the moieties sequesters the microRNA molecule. The decorated circular polyribonucleotide is configured to bind to a binding moiety on the membrane of a cellular target. The binding moiety is a ribonucleic acid (RNA) binding motif. In embodiments, the hybrid modified cyclic polyribonucleotide is a nucleotide sequence that is linked to a second cellular target. and a second binding site configured to bind to a second binding moiety, wherein The second binding moiety is a second RNA binding motif. The doped modified cyclic polyribonucleotide is configured to bind to both targets. In the embodiment, the hybrid modified cyclic polyribonucleotide is linked to a second linking moiety. a second binding site configured to bind to the hybrid modified circular polyribonucleic acid; Binding of both targets to the oxidase induces a conformational change in the first target, thereby induces downstream signaling of the target.

[0024] In certain embodiments, the present disclosure is formulated in a carrier, such as a membrane or lipid bilayer. Compositions as described herein are provided.

[0025] In one aspect, the present disclosure provides a method for administering to a subject having a disease or condition a pharmaceutical composition as described herein. A method of treatment is provided comprising administering the composition.

[0026] In one aspect, the disclosure provides a method of making a pharmaceutical composition, comprising: The present invention provides a method for producing a hybrid modified circular polyribonucleotide comprising:

[0027] In one aspect, the present disclosure provides a method for producing a hybrid modified circular polyribonucleic acid (HMA) polymer as described herein. A method for making a nucleic acid fragment, comprising: The method includes the step of circularizing the polyribonucleotide to form a bridge-modified circular polyribonucleotide.

[0028] In one aspect, the present disclosure provides engineered cells comprising the compositions described herein. do.

[0029] definition While the present invention will be described with respect to particular embodiments and with reference to certain drawings, the present invention The present invention is not limited thereto, but is limited only by the claims. Terms generally should be understood in their ordinary sense unless otherwise indicated.

[0030] The term "pharmaceutical composition" refers to the therapeutic effect of a cyclic polyribonucleotide contained within a pharmaceutical composition. It is also intended to disclose that the present invention may be used for the treatment of the human or animal body by therapy. It is therefore equivalent to "cyclic polyribonucleotide for therapeutic use." Something is intended to be.

[0031] As described herein, cyclic polyribonucleotides, such cyclic polyribonucleosides Compositions containing such cyclic polyribonucleotides, methods of using such cyclic polyribonucleotides, etc. Elements such as replication elements, expression sequences, stagger elements, and encryptogens (see, e.g., Examples 10) or for example, expression sequences, stagger elements and regulatory elements (see, e.g., Examples 32 and 40) are used to produce different Technical effects (e.g., increased translation efficiency over linear counterparts in Examples 10 and 40 and How is it used to achieve increased half-life over the linear equivalent in Example 40? The following description is based in part on examples that illustrate how the present invention can be implemented. It is to be understood that various variations of the specific discoveries and combinations that have been made are contemplated, particularly based on these examples. It is based on

[0032] As used herein, "circular RNA (circRNA)" or "circular polyribonucleotides" refers to RNA that is The terms "nucleotidyl polynucleotide" and "circular RNA" are synonyms. and structures that have no free ends (i.e., no free 3' and / or 5' ends). Polyribonucleotide molecules having a structure, e.g., cyclic or polyribonucleotides, which are linked together via covalent or non-covalent bonds. means a polyribonucleotide that forms an endless structure.

[0033] As used herein, "modified cyclic polyribonucleotide" or "modified cyclic R NA (modified circular RNA)" or "modified circular RNA (mod The terms "identified circRNA" and "identified circRNA" are used synonymously and are used with at least one modification. Modified circular RNA refers to a cyclic polyribonucleotide containing modified nucleotides. It may or may not be uniformly modified along its entire length.

[0034] As used herein, "hybrid modified cyclic polyribonucleotide equivalents" refers to " or "hybrid modified cyclic polyribonucleotide" or "hybrid modified cyclic RN A (hybrid modified circRNA)" or "hybrid modified circ The term "hybrid modified circular RNA" are used interchangeably and have the same nucleotide sequence as the reference modified circular polyribonucleotide and the first of consecutive nucleotides containing 5% or less modified nucleotides described herein In certain embodiments, the term "cyclic polyribonucleotide" refers to a modified cyclic polyribonucleotide having a contiguous The first portion of the nucleotides comprises unmodified nucleotides (i.e., modified nucleotides (e.g., containing no contiguous unmodified nucleotides or only unmodified nucleotides). The first portion contains an IRES. The hybrid modified circular RNA is modified along its entire length. For example, in certain embodiments, a hybrid modified ring RNA is composed of [(all cytosines = methylcytosines) + (all uridines = pseudouridines) In another example, the modified hybrid is (all adenosines = m6a) + unmodified IRES].

[0035] As used herein, "fully modified cyclic poly(amic acid)" refers to a poly(amic acid) "ribonucleotide equivalent" or "completely modified" "fied) cyclic polyribonucleotide equivalent" or "full-length modified circular polyribonucle The terms "modified circular RNA" and "fully modified circular RNA" are used interchangeably and are not intended to be construed as limiting the scope of the invention. a control hybrid circular polyribonucleotide having the same nucleotide sequence as the modified circular polyribonucleotide; a first polyribonucleotide containing 5% or less modified nucleotides corresponding to a first portion of the polyribonucleotide; For example, the first portion may be 5% or more of a modified cyclic polyribonucleotide having a The modified IRES includes an IRES with the following modified nucleotides (i.e., a modified IRES). The RNA may or may not be uniformly modified along the entire length of the molecule. The fully modified cyclic polyribonucleotide contains an IRES with more than 5% modified nucleotides. a first portion, wherein 50% of the nucleotides outside the first portion are modified nucleotides; (e.g., 50% of the external uridines in the first portion are pseudouridines). In one embodiment, the fully modified cyclic polyribonucleotide is Another example is the modified IRES. In the fully modified cyclic polyribonucleotide, [(all adenosines = m6a) + modified Decorative IRES.

[0036] As used herein, the term "modified ribonucleotide" refers to any of the nucleotides listed in Table 1 below. Natural unmodified nucleotides adenosine (A) and uridine (U) are shown by the chemical formulas in unmodified natural ribonucleic acids such as guanine (G), cytidine (C), and monophosphates Any ribonucleotide analogue having one or more chemical modifications relative to the chemical composition of the ribonucleotide. In one embodiment, the chemical modification of the modified ribonucleotide is Any one or more of the ribonucleotides, such as the sugar, nucleobase, or internucleoside linkage for functional groups (e.g., phosphate / phosphodiester linkages / phosphodiesters) It is a modification to the ether skeleton.

[0037] As used herein, the term "linear equivalent" refers to a circular polyribonucleic acid. The nucleotide sequence is the same as or similar to the nucleotide sequence of ... , 80%, 75%, or any percentage of sequence similarity therebetween), and Polyribonucleotide molecules (and fragments thereof) with two free ends (i.e. , the non-cyclized form of a circularized polyribonucleotide (and fragments thereof). In embodiments, the linear equivalent (e.g., pre-circularized form) is a cyclic polyribonucleotide. A nucleotide sequence that is the same or similar to (e.g., 100%, 95%, 90%, 85%, 8 0%, 75%, or any percentage sequence similarity therebetween) and the same or similar A polyribonucleotide molecule (and its fragments) having the nucleic acid modification and having two free ends. fragments) (i.e., the non-cyclized forms of the circularized polyribonucleotides (and their fragments) In some embodiments, the linear equivalent is a cyclic polyribonucleotide. The same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%) %, 75%, or any percentage of sequence similarity therebetween) and different nucleic acid modifications or a polyribonucleotide molecule having no nucleic acid modification and having two free ends. Polyribonucleotides (and fragments thereof) (i.e., non-cyclized forms of cyclized polyribonucleotides ( and fragments thereof). In some embodiments, the linear equivalent is a polyribonucleic acid. A fragment of a nucleotide molecule is a linear equivalent that is shorter than the linear equivalent polyribonucleotide molecule. A linear equivalent is any portion of a polyribonucleotide molecule. In certain embodiments, a linear equivalent In one embodiment, the linear equivalent further comprises a 5' cap. In one embodiment, the linear equivalent further comprises a 3'UTR. In some embodiments, the linear equivalent further comprises a 5'UTR.

[0038] As used herein, the term "fragment" refers to a fragment of a nucleotide molecule. "Nucleotide" refers to any portion of a nucleotide molecule that is at least one nucleotide shorter than the first nucleotide. For example, the nucleotide molecule may be a circular polyribonucleotide molecule, the fragments of which may be A polyribonucleotide or any number of polyribonucleotides that are part of a circular polyribonucleotide molecule. As another example, the nucleotide molecule can be a linear polyribonucleotide. The fragment may be a ribonucleotide molecule, the fragment being a linear polyribonucleotide molecule. can be a monoribonucleotide or any number of contiguous polyribonucleotides, .

[0039] As used herein, the term "encryptogen" refers to the ability of immune cells to Reduce, avoid and / or prevent detection and / or Nucleic acid sequences of circular polyribonucleotides that are useful for reducing the induction of an immune response against It is a structure.

[0040] As used herein, the term "expressed sequence" refers to the expression of a product, e.g., a peptide or or a nucleic acid sequence encoding a polypeptide or a regulatory nucleic acid. An exemplary expressed sequence encoding a gene can include multiple nucleotide triplets, which Each of these can code for an amino acid and are called "codons."

[0041] As used herein, the term "immunity protein binding site" refers to an immune protein. In one embodiment, the immunoglobulin-binding protein is a nucleotide sequence that binds to the immunoglobulin. The site serves to mask the cyclic polyribonucleotide as being exogenous, e.g. For example, the immune protein binding site is a site where the cyclic polyribonucleotide is bound by the immune protein. can be bound by a protein (e.g., a competitive inhibitor) that prevents it from being recognized and bound, This reduces or avoids the immune response to cyclic polyribonucleotides. As used herein, the term "immunity protein" refers to, for example, an immunogen, e.g., Any protein or peptide associated with an immune response to, for example, cyclic polyribonucleotides. Non-limiting examples of immune proteins include T cell receptors (TCRs), antibodies ( immunoglobulins), major histocompatibility complex (MHC) proteins, complement proteins and R NA-binding proteins are examples.

[0042] As used herein, the term "quasi-helical structure" refers to a circular polyribonucleic acid The higher-order structure of a cyclic polyribonucleotide, wherein at least a portion of the cyclic polyribonucleotide is a helical structure. It folds into a structure.

[0043] As used herein, the phrase "quasi-double-stranded secondary structure" refers to a circular polyribonucleotide. A higher-order structure of nucleotides, wherein at least a portion of the cyclic polyribonucleotide is Forms an internal duplex.

[0044] As used herein, the term "regulatory element" refers to a circular polyribonucleic acid These are portions such as nucleic acid sequences that regulate the expression of an expression sequence within the gene.

[0045] As used herein, the term "repetitive nucleotide sequence" refers to a sequence of DNA or or repetitive nucleic acid sequences within stretches of RNA or throughout the genome. In some embodiments, the repetitive nucleotide sequence comprises a poly-CA or poly-TG (UG) sequence. In the form, the repeated nucleotide sequence is repeated in the Alu family of introns. Contains arrays.

[0046] As used herein, the term "replication element" refers to a molecule that is useful for replication or is a sequence and / or motif that initiates transcription of a circular polyribonucleotide.

[0047] As used herein, the term "stagger element" refers to a staggered element that is present on the ribosome during translation. In one embodiment, the staggered portion is a nucleotide sequence that induces pausing. The element consists of a non-conserved sequence of amino acids with a strong α-helical propensity, followed by the consensus sequence -D (V / I)ExNPG P, where x=any amino acid. In the method, the stagger element may be glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid or the like. The compound may contain chemical moieties such as any combination of these.

[0048] As used herein, the term "substantially resistant" refers to a condition in which the At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, It means having 95%, 96%, 97%, 98% or 99% resistance.

[0049] As used herein, the term "stoichiometric translation" refers to the synthesis of a circular polyribonucleic acid. The production of substantially equal expression products translated from the nucleic acid sequences is achieved by, for example, For a cyclic polyribonucleotide with , the expression products of the two expression sequences may have substantially equal amounts, e.g., The difference in amount (e.g., molar difference) between the , 8%, 9%, 10%, 15%, or 20%, or any percentage therebetween This may mean that it can be a page.

[0050] As used herein, the term "translation initiation sequence" refers to the sequence of a circular polyribonucleic acid sequence. It is a nucleic acid sequence that initiates translation of an expression sequence in a nucleotide.

[0051] As used herein, the term "termination element" refers to a terminal element of a circular polyribonucleic acid. A portion of a gene, such as a nucleic acid sequence, that stops the translation of an expression sequence in a gene.

[0052] As used herein, the term "translation efficiency" refers to the rate at which ribonucleotide transcript production In one embodiment, the expression level refers to the rate or amount of protein or peptide production from a given organism. Translation efficiency can be measured, for example, in a given translation system, e.g., in vitro, such as rabbit reticulocyte lysate. in a translation system or in an in vivo translation system such as a eukaryotic or prokaryotic cell, e.g. A given amount of protein or peptide-encoding transcript produced in a given period of time The amount of protein or peptide obtained may be expressed as the amount of protein or peptide obtained.

[0053] As used herein, the term "circularization efficiency" refers to the efficiency of the resulting circular polyribonucleotides. It refers to the measurement of a nucleotide relative to its starting material.

[0054] As used herein, the term "immunogenic" refers to the ability to induce an immune response to a substance. In some embodiments, the immune system of an organism or certain immune cells may be induced to When exposed to an immunogenic agent, an immune response can be induced. In certain embodiments, the absence or lack of an immune response above a detectable threshold to the disease. When the immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic substance, an immune response is initiated. In certain embodiments, the non-immunogenic cyclic polynucleotides provided herein are not detectable. Ribonucleotides are those that produce an immunogenicity above a predetermined threshold as measured by an immunogenicity assay. does not induce a response to cyclic polyribonucleotides (e.g., using an immunogenicity assay) When measuring the innate immune response to a disease (e.g., measuring an inflammatory marker), The non-immunogenic polyribonucleotides provided herein inhibit the natural immune response at levels below a predetermined threshold. The predetermined threshold may result in the production of a response, for example, a response due to an innate immune response relative to a control. resulting in 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold increases in the levels of the marker It may be 9-fold or 10-fold or less.

[0055] As used herein, the term "pharmaceutically acceptable" means biologically or refers to a component that is not otherwise objectionable, e.g., a component that does not have significant undesirable biological effects without causing or adversely interacting with any of the other ingredients of the formulation in which it is contained. Without further elaboration, they can be incorporated into the pharmaceutical formulations of the present invention and administered to the subjects described herein. In certain embodiments, the term "pharmaceutically acceptable" is used to refer to an excipient. When a product is approved, it must meet the required standards of toxicity and manufacturing testing; It is the US Food and Drug Administration Inactive ingredients are included in the inactive ingredients guide prepared by the Inactive Ingredients Agency (INRA).

[0056] As used herein, the term "carrier" refers to a cyclic polyribonucleotide. by covalent modification, partially or completely by encapsulating agents, or a combination thereof facilitates transport or delivery of compositions (e.g., cyclic polyribonucleotides) into cells Carriers refer to compounds, compositions, reagents, or molecules. Non-limiting examples of carriers include carbohydrate carriers. bodies (e.g., anhydride-modified phytoglycogen or glycogen-type materials), nanoparticles ( For example, nanoparticles encapsulating or covalently attached to cyclic polyribonucleotides. ), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, proteins Protein carriers (e.g., proteins covalently bound to cyclic polyribonucleotides), or cationic Examples include ionic carriers (e.g., cationic lipopolymers or transfection reagents). It can be obtained.

[0057] As used herein, "naked delivery" refers to The term "carrier-free" refers to a compound that is carrier-free and has covalent modifications to moieties that aid in delivery to cells. Naked delivery formulations refer to formulations for delivery to cells that do not contain any transfection agent. an injection reagent, a cationic carrier, a carbohydrate carrier, a nanoparticle carrier, or a protein carrier For example, naked delivery formulations of cyclic polyribonucleotides do not contain covalent modifications. The formulation contains a cyclic polyribonucleotide that does not contain a carrier.

[0058] The term "diluent" refers to a composition described herein (e.g., a circular polyribonucleic acid The term "dilution" refers to a vehicle containing an inert solvent in which a composition containing an oxalate can be diluted or dissolved. The diluent can be an RNA solubilizing agent, a buffer, an isotonic agent, or a mixture thereof. The diluent may be a liquid or a solid. Non-limiting examples of liquid diluents include water or other Solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, carbonate Chill, Ethyl Acetate, Benzyl Alcohol, Benzyl Benzoate, Propylene Glycol, 1 ,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, Sorghum oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran Fatty acid esters of hydroxyfurfuryl alcohol, polyethylene glycol and sorbitan, and and 1,3-butanediol. Non-limiting examples of solid diluents include calcium carbonate. Sodium, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, Calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline Cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, Examples include dry starch, corn starch, or powdered sugar.

[0059] Incorporation by Reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. The patent or patent application is specifically and individually indicated to be incorporated by reference. and the same is incorporated herein by reference to the same extent.

[0060] The following detailed description of the embodiments of the present invention is better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the embodiments illustrated herein are shown in the drawings. However, the present invention does not depend on the precise arrangements and instrumentalities of the embodiments shown in the drawings. It should be understood that this is not limiting. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for reducing the immunogenicity of a cyclic polyribonucleotide in a subject, comprising: providing a hybrid-modified cyclic polyribonucleotide, the hybrid-modified cyclic polyribonucleotide comprising a first portion comprising at least one modified nucleotide and at least about 5-1000 contiguous unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; obtaining reduced immunogenicity for said hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of said subject. (Item 2) 2. The method of claim 1, wherein the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive unmodified nucleotides. (Item 3) Item 1, wherein the first portion lacks 5'-methylcytidine or pseudouridine. Or the method according to 2. (Item 4) 4. The method according to any one of items 1 to 3, wherein the cyclic polyribonucleotide has translation ability. (Item 5) The hybrid modified cyclic polyribonucleotide is (a) has at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3 times higher expression than the corresponding unmodified cyclic polyribonucleotide; (b) has a half-life that is at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times longer than a corresponding unmodified cyclic polyribonucleotide; (c) has a longer half-life than the corresponding unmodified cyclic polyribonucleotide; or (d) the method of any one of paragraphs 1 to 4, having at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times less immunogenicity than a corresponding unmodified cyclic polyribonucleotide, as assessed by expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, and IFN-β. (Item 6) The at least one modified nucleotide is (a) N(6)methyladenosine (m6A), 5'-methylcytidine, and pseudouridine; (b) 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, and 2'-fluoro N3-P5'-phosphoramidite; or (c) The method according to any one of items 1 to 5, wherein the modified nucleotide is selected from the group consisting of any of the modified nucleotides in Table 2. (Item 7) 7. The method of any one of items 1 to 6, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the nucleotides of the hybrid modified cyclic polyribonucleotide are modified nucleotides. (Item 8) 8. The method of any one of items 1 to 7, wherein the hybrid modified cyclic polyribonucleotide comprises a binding site consisting of unmodified nucleotides and configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target. (Item 9) 8. The method according to any one of items 1 to 7, wherein the hybrid modified cyclic polyribonucleotide comprises one or more expression sequences. (Item 10) 10. The method of any one of items 1 to 7 or 9, wherein the first part comprises an IRES consisting of unmodified nucleotides. (Item 11) The one or more expression sequences of the hybrid modified circular polyribonucleotide are (a) Higher translation efficiency than fully modified circular polyribonucleotide equivalents; (b) a translation efficiency that is at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than a fully modified circular polyribonucleotide equivalent; (c) a higher translation efficiency than the corresponding unmodified circular polyribonucleotide; or (d) a translation efficiency that is at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than the corresponding unmodified cyclic polyribonucleotide 11. The method according to item 9 or 10, comprising: (Item 12) 1. A method for expressing one or more expression sequences in a subject, comprising: providing a hybrid modified cyclic polyribonucleotide comprising one or more expression sequences, wherein the hybrid modified cyclic polyribonucleotide comprises a first portion comprising at least one modified nucleotide and at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining increased expression of said one or more expressed sequences compared to the expression of the corresponding one or more expressed sequences in a fully modified circular polyribonucleotide equivalent in said subject's cells or tissues. (Item 13) 1. A method for increasing cyclic polyribonucleotide stability in a subject, comprising: providing a hybrid modified cyclic polyribonucleotide, wherein the hybrid modified cyclic polyribonucleotide comprises a first portion comprising a modified cyclic polyribonucleotide and at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; obtaining increased stability for said hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of said subject. (Item 14) 14. The method of claim 12 or 13, wherein the first portion comprises an IRES. (Item 15) 1. A pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising: at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding moiety of a first target, e.g., an RNA, DNA, protein, or cellular target, wherein the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding motif composed of unmodified nucleotides; wherein said first target and said hybrid modified cyclic polyribonucleotide form a complex. (Item 16) 1. A pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising: at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding moiety of a first target, e.g., an RNA, DNA, protein, or cellular target, wherein the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding motif composed of unmodified nucleotides; and a second binding site configured to bind to a second binding moiety of a second target, wherein said second binding moiety is a second circular RNA-binding motif; the first binding moiety is different from the second binding moiety; the first target, the second target, and the hybrid modified cyclic polyribonucleotide form a complex; The pharmaceutical composition, wherein the first target or the second target is not a microRNA. (Item 17) 1. A pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising: at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding moiety of a first target, wherein the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding motif; and a second binding site configured to bind to a second binding moiety of a second target, wherein said second binding moiety is a second circular RNA-binding motif; the first binding moiety is different from the second binding moiety; The pharmaceutical composition, wherein both the first target and the second target are microRNAs. [Brief explanation of the drawings]

[0061] [Figure 1-1] 1A, 1B, and 1C show that the modified circular RNA was translated intracellularly. [Figure 1-2] 1A, 1B, and 1C show that the modified circular RNA was translated intracellularly. [Figure 1-3] 1A, 1B, and 1C show that the modified circular RNA was translated intracellularly. [Figure 2-1] Figures 2A, 2B, and 2C show that the modified circular RNA has reduced immunogenicity compared to unmodified circular RNA to cells, as assessed by MDA5, OAS, and IFN-β expression in transfected cells. [Figure 2-2] Figures 2A, 2B, and 2C show that the modified circular RNA has reduced immunogenicity compared to unmodified circular RNA to cells, as assessed by MDA5, OAS, and IFN-β expression in transfected cells. [Figure 2-3]Figures 2A, 2B, and 2C show that the modified circular RNA has reduced immunogenicity compared to unmodified circular RNA to cells, as assessed by MDA5, OAS, and IFN-β expression in transfected cells. [Figure 3] 1 shows that hybrid modified circular RNAs have reduced immunogenicity compared to unmodified circular RNAs as assessed by intracellular RIG-I, MDA5, IFN-β, and OAS expression. [Figure 4] FIG. 1 shows a schematic diagram of an exemplary in vitro generation process of circular RNA containing a start codon, an ORF (open reading frame) encoding GFP, a stagger element (2A), an encryptogen, and an IRES (internal ribosome entry site). [Figure 5] FIG. 1 shows a schematic diagram of an exemplary in vivo generation process of circular RNA. [Figure 6] An exemplary circular RNA design is shown, including a start codon, an ORF encoding GFP, a stagger element (2A), and an encryptogen. [Figure 7] FIG. 1 is a schematic diagram showing the in vivo stoichiometric protein expression of two different circular RNAs. [Figure 8] FIG. 1 is a schematic diagram showing in vivo protein expression in a mouse model from exemplary circular RNAs. [Figure 9] FIG. 1 shows a schematic diagram of an exemplary circular RNA with one double-stranded RNA segment that can be subjected to dot blot analysis for its structural information. [Figure 10] FIG. 1 shows a schematic diagram of an exemplary circular RNA with a pseudohelical structure (HDVmin) that can be subjected to SHAPE analysis for its structural information. [Figure 11] FIG. 1 shows a schematic diagram of an exemplary circular RNA with a functional pseudohelical structure (HDVmin) that exhibits HDAg binding activity. [Figure 12] FIG. 1 is a schematic diagram showing transcription, self-cleavage, and ligation of an exemplary self-replicating circular RNA. [Figure 13] 1 is a denaturing PAGE gel image showing the in vitro generation of various exemplary circular RNAs. [Figure 14] 1 is a graph summarizing the circularization efficiency of various exemplary circular RNAs. [Figure 15] 1 is a denaturing PAGE gel image showing the reduced degradation susceptibility of an exemplary circular RNA compared to its linear counterpart. [Figure 16] 1 is a denaturing PAGE gel image showing an exemplary circular RNA after an exemplary purification process. [Figure 17] 1 is a Western blot image showing expression of Flag protein (approximately 15 kDa) by an exemplary circular RNA lacking an IRES, cap, 5' and 3' UTRs. [Figure 18] 1 is a Western blot image showing rolling circle translation of an exemplary circular RNA. [Figure 19] 1 shows Western blot images demonstrating the production of distinct proteins or continuous long peptides from various exemplary circular RNAs with and without exemplary stagger elements. [Figure 20A] 1 is a Western blot image showing a comparison of protein expression between various exemplary circular RNAs with exemplary stagger or termination elements (stop codons). [Figure 20B] 1 is a graph summarizing signal intensities from Western blot analysis of protein products translated from two exemplary circular RNAs. [Figure 21] 1 is a graph summarizing the luciferase activity of the translation products of exemplary circular RNAs and their linear equivalents compared to vehicle control RNA. [Figure 22] 1 is a graph summarizing the amount of RNA at different collection time points in a time course experiment examining the half-life of an exemplary circular RNA. [Figure 23A] FIG. 10 is a graph showing qRT-PCR analysis of linear and circular RNA levels 24 hours after delivery into cells using primers that captured both linear and circular RNA. [Figure 23B] FIG. 1 is a graph showing qRT-PCR analysis of linear and circular RNA levels using primers specific for circular RNA. [Figure 24] 1 is an image showing blots of cell lysates from circular and linear RNA probed for EGF protein and β-tubulin loading control. [Figure 25] 1 is a graph showing qRT-PCR analysis of immune-related genes from 293T cells transfected with circular or linear RNA. [Figure 26] 1 is a graph showing the luciferase activity of proteins expressed from circular RNAs by rolling circle translation. [Figure 27] 1 is a graph showing the luciferase activity of proteins expressed from circular or linear RNA. [Figure 28] 1 is a graph showing the luciferase activity of proteins expressed from linear or circular RNA by rolling circle translation. [Figure 29] 1 is a graph showing the luciferase activity of proteins expressed from circular RNAs via IRES translation initiation. [Figure 30] 1 is a graph showing luciferase activity of proteins expressed from circular RNAs by IRES-initiated and rolling circle translation. [Figure 31] 1 is an image showing a protein blot of expression products from circular or linear RNA. [Figure 32] 1 is an image showing a protein blot of expression products from circular or linear RNA. [Figure 33] 1 shows the predicted structure of an exemplary circular RNA with a pseudo-double-stranded structure. [Figure 34] 1 shows the predicted structure of an exemplary circular RNA with pseudohelical structure. [Figure 35] 1 shows the predicted structure of an exemplary circular RNA with a pseudohelical structure linked to repeat sequences. [Figure 36] 1 presents experimental data showing that degradation of an exemplary circular RNA by RNAse H produced nucleic acid degradation products consistent with circular RNA but not concatemeric RNA. [Figure 37]Electrophoretic images of DNA of various lengths generated for the formation of various RNA lengths are shown. [Figure 38] Experimental data are shown confirming RNA circularization using RNAse R treatment and qPCR analysis for circular linkages of various lengths. [Figure 39] 1 shows the generation of an exemplary circular RNA with a protein binding site. [Figure 40] 1 shows the generation of an exemplary circular RNA with a miRNA binding site. [Figure 41] 1 shows the generation of an exemplary circular RNA by self-splicing. [Figure 42] Figure 1 shows experimental data demonstrating the higher stability of circular RNA in dividing cells compared to linear controls. [Figure 43] 1 shows experimental data demonstrating protein expression from an exemplary circular RNA with multiple expression sequences and rolling circle translation of an exemplary circular RNA with multiple expression sequences. [Figure 44] After injection into mice, circular RNA was detected at higher levels than linear RNA in the liver of mice at 3, 4 and 7 days after injection. [Figure 45A] Figure 1 shows that after injection of circular or linear RNA expressing Gaussia luciferase into mice, Gaussia luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23 days after administration of circular RNA, whereas the activity was detected in plasma only at 1 and 2 days after administration of modified linear RNA. [Figure 45B] Figure 1 shows that after injection of circular or linear RNA expressing Gaussia luciferase into mice, Gaussia luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23 days after administration of circular RNA, whereas the activity was detected in plasma only at 1 and 2 days after administration of modified linear RNA. [Figure 46] 1 shows that after injection of RNA, circular RNA, but not linear RNA, was detected in the liver and spleen at 16 days after administration of RNA. [Figure 47]Figure 1 shows that after RNA injection, linear RNA, but not circular RNA, was immunogenic as assessed by RIG-I, MDA-5, IFN-B, and OAS. [Figure 48] Various exemplary cyclization methods are shown. [Figure 49] We show that circular RNAs containing modified nucleotides have reduced immunogenicity in vivo compared to modified mRNA and compared to circular RNAs produced with only unmodified nucleotides. [Figure 50] We show that circular RNAs containing modified nucleotides have increased stability in vivo over their fully unmodified counterparts and over modified mRNAs. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention relates generally to pharmaceutical compositions and formulations of cyclic polyribonucleotides and their uses. In one embodiment, the cyclic polyribonucleotide comprises at least one modified polyribonucleotide. In some embodiments, the first portion comprises a ribonucleotide and a contiguous unmodified nucleotide. In one embodiment, the modified cyclic polyribonucleotide is delivered to a subject.

[0063] The present disclosure provides a method for reducing or decreasing the immunogenicity of cyclic polyribonucleotides in a subject. 1. A method for producing a hybrid modified cyclic polyribonucleotide comprising the steps of: The hybrid-modified cyclic polyribonucleotide comprises at least one modified polyribonucleotide. and a first portion of contiguous nucleotides having 5% or less modified nucleotides. administering a hybrid modified cyclic polyribonucleotide to a subject; and hybrid-modified cyclic polyribonucleotides compared to the corresponding unmodified cyclic polyribonucleotides in tissues. and obtaining reduced immunogenicity against cyclic polyribonucleotides. In some embodiments, the immunogenicity of a cyclic polyribonucleotide in a subject is reduced or The method of reducing comprises providing a hybrid modified cyclic polyribonucleotide, The hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide and and at least about 5-1000 consecutive nucleotides with 5% or less modified nucleotides. and administering the hybrid modified circular polyribonucleotide to a subject. and the corresponding unmodified cyclic polyribonucleotide in the cells or tissue of the subject. Compared to hybrid modified cyclic polyribonucleotides, reduced immunogenicity is obtained. In one embodiment, the method comprises administering a cyclic polyribonucleotide immunogen to a subject. The method for reducing or decreasing the affinity of a compound to a cyclic polyribonucleotide provides a hybrid modified cyclic polyribonucleotide. wherein the hybrid modified cyclic polyribonucleotide comprises at least one modified polyribonucleotide. a step of hybridizing the first portion of the hybridized nucleotides to a first portion of unmodified ribonucleotides and consecutive unmodified nucleotides; administering a modified cyclic polyribonucleotide to a subject; and The hybrid modified cyclic polyribonucleotides are compared to the corresponding unmodified cyclic polyribonucleotides in and obtaining reduced immunogenicity to the polynucleotide. The first portion comprises an IRES. In one embodiment, the first portion comprises at least about 5 In one embodiment, the first portion comprises 5' to 1000 contiguous unmodified nucleotides. -methylcytidine or pseudouridine is absent. No more than 5% of the nucleotides in the molecule are modified nucleotides.

[0064] The present disclosure provides a method for expressing one or more expression sequences in a subject, the method comprising: one modified polyribonucleotide, consecutive nucleotides with no more than 5% modified nucleotides and a hybrid modified circular polyribonucleic acid comprising one or more expression sequences. providing a hybrid modified cyclic polyribonucleotide to a subject; and administering the hybrid modified cyclic polyribonucleotide to a subject. and a corresponding one in a fully modified cyclic polyribonucleotide in a cell or tissue of interest. obtaining increased expression of one or more of the expression sequences compared to the expression of the expression sequences above. In one embodiment, the method comprises expressing one or more expression sequences in a subject. The method includes the steps of providing a hybrid modified cyclic polyribonucleotide, The lid-modified cyclic polyribonucleotide comprises at least one modified nucleotide and 5% or more a first nucleic acid sequence comprising at least about 5 to 1000 consecutive nucleotides having the following modified nucleotides: and administering the hybrid modified cyclic polyribonucleotide to a subject. and the production of one or more fully modified circular polyribonucleotide equivalents in the cells or tissues of the subject. obtaining increased expression of one or more of the expression sequences compared to the expression of the expression sequences above. In one embodiment, the method of expressing one or more expression sequences in a subject comprises at least a first portion of contiguous unmodified nucleotides; and a second portion of contiguous unmodified nucleotides. providing a hybrid modified cyclic polyribonucleotide comprising the above expression sequence; administering the hybrid-modified cyclic polyribonucleotide to a subject; and Expression of one or more corresponding expression sequences in a fully modified circular polyribonucleotide in tissues and comparing to obtain increased expression of one or more expression sequences. In one embodiment, the first portion comprises an IRES. In one embodiment, the first portion comprises about 5 to 1000 consecutive unmodified nucleotides. In one embodiment, the first No more than 5% of the nucleotides in the segment are modified nucleotides.

[0065] The present disclosure provides a method for increasing the stability of a cyclic polyribonucleotide in a subject, comprising: providing a hybrid circular polyribonucleotide, The polyribonucleotides are composed of at least one modified polyribonucleotide and 5% or less of a polyribonucleotide. a first portion of consecutive nucleotides having a modified nucleotide; and administering a modified cyclic polyribonucleotide to a subject; and detecting a nucleotide sequence in a cell or tissue of the subject. The hybrid modified circular polyribonucleotides are more potent than the corresponding unmodified circular polyribonucleotides. and obtaining increased stability for the nucleotide. In the present invention, a method for increasing the stability of a cyclic polyribonucleotide in a subject includes: providing a hybrid modified cyclic polyribonucleotide, The ribonucleotides contain at least one modified nucleotide and no more than 5% modified nucleotides. a first portion comprising at least about 5 to 1000 contiguous nucleotides having a sequence; administering a hybrid modified cyclic polyribonucleotide to a subject; and hybrid-modified cyclic polyribonucleotides compared to the corresponding unmodified cyclic polyribonucleotides in tissues. and obtaining increased stability for the cyclic polyribonucleotide. In the present invention, a method for increasing the stability of a cyclic polyribonucleotide in a subject includes: providing a hybrid modified circular polyribonucleotide, The nucleotides are composed of at least one modified polyribonucleotide and consecutive unmodified nucleotides. and subjecting the hybrid modified cyclic polyribonucleotide to a step of and administering the corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. to obtain increased stability for hybrid modified cyclic polyribonucleotides compared to In some embodiments, the first portion comprises an IRES. In the present invention, the first portion contains at least about 5 to 1000 consecutive unmodified nucleotides. In an embodiment, the first portion lacks 5'-methylcytidine or pseudouridine. In one embodiment, 5% or less of the nucleotides in the first portion are modified nucleotides. It's Chido.

[0066] Methods of Using Modified Cyclic Polyribonucleotides In one aspect, the invention described herein provides a hybrid modified circular polyribonucleic acid (HPA)-based Methods for Using Compositions of Nucleotides and Delivery of Hybrid Modified Cyclic Polyribonucleotides In some embodiments, the hybrid modified cyclic polyribonucleotide comprises a The present invention provides a method for delivering cyclic ribonucleotides to a subject in a manner similar to that described herein, compared to the corresponding unmodified cyclic polyribonucleotide. The administration of the hybrid modified cyclic polyribonucleotides described in Decreased or reduced immunogenicity, increased translation efficiency (e.g., hybrid modifications) in increased expression of one or more expression sequences in a modified circular polyribonucleotide, or increased may result in improved stability compared to fully modified cyclic polyribonucleotide equivalents. Administration of the hybrid modified cyclic polyribonucleotides described herein may be used to treat cells of a subject. Increased translation efficiency in cells or tissues (e.g., hybrid modified circular polyribonucleoside) In some embodiments, the expression of one or more expression sequences in the target gene may result in increased expression of the target gene. Thus, the hybrid modified circular polyribonucleotide comprises at least one modified polyribonucleotide. In one embodiment, the present invention comprises a first portion of a nucleotide and a second portion of contiguous unmodified nucleotides. The present invention relates to a method for using hybrid modified cyclic polyribonucleotides, The modified cyclic polyribonucleotide comprises at least one modified nucleotide and a first moiety. wherein the first portion comprises at least about 5 consecutive unmodified nucleotides. In one embodiment, the hybrid circular polyribonucleotide comprises one or more expression sequences Includes.

[0067] In one embodiment, the first portion has at least 5% or less modified nucleotides. In one embodiment, the first portion comprises at least about 5 to 1000 contiguous nucleotides. In one embodiment, the first portion comprises at least about 5 to 1000 consecutive unmodified nucleotides. minutes, 5'-methylcytidine, pseudouridine, or N1-methyl-pseudouridine In one embodiment, no more than 5% of the nucleotides in the first portion are modified. In some embodiments, 0%, 1%, 2%, 3%, 4%, or 5% of the first portion The following nucleotides are modified: is unmodified. In one embodiment, the first portion is an IRES. In one embodiment, the first portion is an IRES containing 5% or less modified nucleotides. In some embodiments, the first portion does not include modified nucleotides (e.g., unmodified nucleotides). In one embodiment, the first portion is an IRES (containing only unmodified nucleotides). In one embodiment, the first portion is an IRES consisting of unmodified nucleotides. The nucleotide sequence of the nucleotide sequence is a sequence that binds to proteins, peptides, biomolecules, DNA, RNA, or cellular targets. In one embodiment, the hybrid modified cyclic poly In some embodiments, the ribonucleotides are translatable. The ribonucleotides may be incorporated into pharmaceutical compositions further comprising a pharmaceutically acceptable carrier or excipient. be.

[0068] The hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide and The modified nucleotides may comprise a first portion comprising consecutive unmodified nucleotides. The modified polyribonucleic acid of the hybrid modified circular polyribonucleotide is The nucleotides are the natural unmodified nucleotides adenosine (A), uridine (C), and ribonucleotides represented by the chemical formulas in Table 1. unmodified amino acids such as uridine (U), guanine (G), cytidine (C), and monophosphate Analogs or derivatives having one or more chemical modifications to the chemical composition of natural ribonucleotides The chemical modification of the modified ribonucleotide may be at the sugar, the nucleobase, or the internucleoside linkage. For any one or more functional groups of a ribonucleotide (e.g., a linking The modification may be to the phosphate / phosphodiester bond / phosphodiester backbone. In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Modified nucleotides of the polyribonucleotide or hybrid cyclic polyribonucleotide are Gilbert, WV, et al. Science, incorporated herein by reference. .Identified in 2016 Jun17;352(6292):1408-1412 The modification may be any modification known by a person skilled in the art, such as those described herein or in this document. For example, the modifications can be as set forth in Table 2.

[0069] [Table 1]

[0070] [Table 2]

[0071] In certain embodiments, the modified nucleotide is N(6) methyladenosine (m6A), 5'-methylcytidine (5mC), pseudouridine, 2'-O-methyl, 2'-O-methyl Toxoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T- Deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O- Dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl ( 2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAP) EOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (L NA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'-anhydroheteronucleotides xylitol nucleic acid (HNA), morpholino, methylphosphonate nucleotides, thiophospho N3-P5'-phosphoramidites, and 2'-fluoro N3-P5'-phosphoramidites In certain embodiments, the modified nucleotide is selected from the group consisting of Gilbert,WV,et al.Science.2016 Jun 17;352(6292):1408-1412, or Any modified nucleotide known by one of skill in the art, such as those in the literature.

[0072] The first portion of the hybrid modified cyclic polyribonucleotide described herein may comprise at least one In one embodiment, the first portion comprises at least about 5 to 1000 contiguous nucleotides. , at least about 5-1000, 10-1000, 20-1000, 50-1000, 10 0~1000, 200~1000, 300~1000, 400~1000, 500~10 00, 600~1000, 700~1000, 800~1000, 900~1000, or The hybrid modified cyclic nucleotides described herein comprise 900 to 2000 consecutive nucleotides. The first portion of the polyribonucleotide is a contiguous nucleotide sequence having 5% or less modified nucleotides. In some embodiments, the first portion may contain 0%, 1%, 2%, 3%, In some embodiments, the contiguous nucleotides include no more than 4% or 5% modified nucleotides. In one embodiment, the first portion is an IRES. In one embodiment, the first portion is 5% or more In one embodiment, the first portion is an IRES containing the modified nucleotides: It is an IRES that does not contain modified nucleotides (e.g., contains only unmodified nucleotides). In one embodiment, the first portion is an IRES consisting of unmodified nucleotides. The first portion of the hybrid modified cyclic polyribonucleotide described herein is a non-contiguous The first portion may contain at least about 5 consecutive unmodified nucleotides. In certain embodiments, the first portion may comprise at least about 5 to 1000 consecutive unmodified amino acids. In one embodiment, the first portion comprises at least about 5 to 1000 nucleotides. , 10~1000, 20~1000, 50~1000, 100~1000, 200~10 00, 300~1000, 400~1000, 500~1000, 600~1000, 7 00~1000, 800~1000, 900~1000, or 900~2000 continuous non-corrected In some embodiments, the first portion comprises at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 90 0, or 1000, or any number in between, contiguous unmodified nucleotides. In embodiments, the first portion may be 5, 10, 20, 50, 100, 200, 300, 40 0, 500, 600, 700, 800, 900, 1000, or any number in between The first portion may comprise consecutive unmodified nucleotides. The first portion may comprise an IRES. wherein the first moiety is 5'-methylcytidine, pseudouridine, or N1-methyl- In one embodiment, the first moiety lacks N(6) methyl uridine. Denosine (m6A), 5'-methylcytidine (5mC), pseudouridine, 2'-O- Methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O -AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminoethyl Dimethylaminopropyl (2'-O-DMAP), TO-Dimethylaminoethyloxyethyl 2'-O-DMAEOE, 2'-ON-methylacetamide (2'-O-NMA ), locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1' ,5'-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleic acid Thiophosphonate nucleotides, and 2'-fluoro N3-P5'-phosphoro In one embodiment, the first Portions are incorporated herein by reference. Science.2016 Jun17;352(6292):1408-1412 Nucleotides known by those skilled in the art, such as those identified in the literature, or in this document. It lacks the modifier.

[0073] The hybrid modified cyclic polyribonucleotides described herein comprise, in addition to a first portion, The amino acid moiety may include 5'-methylcytidine, pseudouridine, or a combination thereof. The hybrid-modified cyclic polyribonucleotide is N(6) methyladenosine (m6A), 5' -Methylcytidine (5mC), pseudouridine, 2'-O-methyl, 2'-O-methox Diethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethyl 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2' -O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEO E), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA ), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'-anhydrohexyl Toll nucleic acid (HNA), morpholino, methylphosphonate nucleotides, thiophosphonate from the group consisting of 2'-fluoro N3-P5'-phosphoramidites, The modified nucleotides may include a selected modification, where the modified nucleotides are outside of the first portion. In embodiments, the modified nucleotides outside the first portion are selected from the group consisting of nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2 Gilbert, WV, et al. Science. 2016 Jun1 7;352(6292):1408-1412, or as otherwise specified in this document. The modified nucleotide may be any modified nucleotide known by those skilled in the art, such as those in the references.

[0074] Reduced immunogenicity The method for reducing or decreasing the immunogenicity of a cyclic polyribonucleotide in a subject includes the steps of: providing a hybrid modified cyclic polyribonucleotide, The ribonucleotides may be composed of at least one modified polyribonucleotide and consecutive unmodified ribonucleotides. a first portion of the modified cyclic polyribonucleotide, and a second portion of the modified cyclic polyribonucleotide. administering the compound to an animal and detecting the corresponding unmodified circular polyribonucleic acid in cells or tissues of the subject. and (iii) obtaining reduced or decreased immunogenicity for modified cyclic polyribonucleotides compared to cyclic polyribonucleotides. In certain embodiments, the first portion may comprise at least about 5, 10, 20 , 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive unmodified nucleotides. In some embodiments, no more than 1%, 2% or more of the nucleotides in the first portion are modified. In some embodiments, no more than 3%, 4%, or 5% of the nucleotides are modified. In one embodiment, the first portion is an IRE fragment, and the nucleotides in the first portion are unmodified. In one embodiment, the first portion is a protein-specific nucleotide sequence consisting of unmodified nucleotides. configured to bind to proteins, peptides, biomolecules, DNA, RNA, or cellular targets In one embodiment, the first portion contains no more than 5% modified nucleotides. In one embodiment, the first portion is an IRES that does not include modified nucleotides. In one embodiment, the first portion is an IRES. In one embodiment, the modified cyclic polyribonucleotide is an IRES. The reduced immunogenicity is due to the presence of the corresponding unmodified circular polyribonucleic acid in the cells or tissues of the subject. Compared to Ochid, at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2 , 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4 0.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9 .0, 9.5, or 10.0 times lower.

[0075] In certain embodiments, the hybrid modified circular polyribonucleoside disclosed herein The modified cyclic polyribonucleotides, after administration to a subject, exhibit a reduced or no activity compared to the corresponding unmodified cyclic polyribonucleotide. In certain embodiments, the hybrids disclosed herein have reduced immunogenicity. The modified cyclic polyribonucleotides are converted to corresponding unmodified cyclic polyribonucleotides after administration to a subject. than leotide, at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2 .2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5 , 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 , 9.5, or 10.0 times less immunogenic. The immunogenicity described in is obtained after administration of a hybrid modified cyclic polyribonucleotide to a subject. , RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS , OASL, PKR, and IFN-β. is assessed by the level of activation.

[0076] Increased translation efficiency The present disclosure provides a method for expressing one or more expression sequences in a subject, the method comprising: a first portion of contiguous unmodified nucleotides, and one or more providing a hybrid modified circular polyribonucleotide comprising an expression sequence; administering a modified cyclic polyribonucleotide to a subject; and and comparing the expression of one or more corresponding expression sequences of fully modified circular polyribonucleotide equivalents in and obtaining increased expression of one or more expression sequences in comparison to the control. In embodiments, the first portion is at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive unmodified nucleic acids In one embodiment, no more than 5% of the nucleotides in the first portion are modified. In some embodiments, 1%, 2%, 3%, 4%, or 5% or more of the first portion is In one embodiment, the nucleotide in the first portion is In one embodiment, the first portion is an IRES. wherein the first portion is a protein, peptide, or biomolecule consisting of unmodified nucleotides. , DNA, RNA, or a binding moiety configured to bind to a cellular target. In some embodiments, the first portion is an IRES containing 5% or less modified nucleotides. In an embodiment, the first portion is an IRES that does not contain modified nucleotides. In some embodiments, the first portion is an IRES consisting of unmodified nucleotides. In some embodiments, the hybrid modified circular polyribonucleotide contains one or more expression sequences. nothing.

[0077] In one embodiment, expression of one or more hybrid modified circular polyribonucleotides The increased expression of the sequence may be achieved by expressing one or more of the fully modified circular polyribonucleotide equivalents of the sequence. In some embodiments, the hybrid modified cyclic poly(L-lysine) The increased expression of one or more expression sequences of polyribonucleic acid is achieved by the fully modified circular polyribonucleic acid. at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or In one embodiment, one or more of the hybrid modified cyclic polyribonucleotides The increased expression of the above expression sequences is similar to that of the corresponding unmodified cyclic polyribonucleotides. In some embodiments, the hybrid modified circular polyribonucleic acid Increased expression of one or more expression sequences of a nucleic acid fragment is achieved by the addition of a corresponding unmodified circular polyribonucleic acid fragment. than Chid, at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1 0.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher.

[0078] In one embodiment, one or more sequences of hybrid modified circular polyribonucleotides The increased expression of the cyclic polyribonucleotides is at least about 10 times greater than the fully modified cyclic polyribonucleotide equivalent. 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 5 5%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 70%, 800%, 900%, 1000%, 2000% , 5000%, 10000%, 100000% or more higher. and increasing expression of one or more sequences of hybrid modified circular polyribonucleotides. is at least about 10% higher than the fully modified circular polyribonucleotide equivalent. In some embodiments, the expression of one or more sequences of hybrid modified circular polyribonucleotides is increased. The resulting expression is at least about 20% higher than the fully modified circular polyribonucleotide equivalent. In some embodiments, the expression of one or more sequences of hybrid modified circular polyribonucleotides is The increased expression of the gene is at least about 50% greater than the fully modified circular polyribonucleotide equivalent. In one embodiment, one or more of the hybrid modified cyclic polyribonucleotides The increased expression of the sequence is less than that of the corresponding unmodified cyclic polyribonucleotide. At least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 70%, 800%, 900%, 1000%, 2000%, 5000%, 10000%, 100000% or even higher. In one embodiment, an increase in expression of one or more sequences of hybrid modified circular polyribonucleotides. The increased expression is at least about 10% greater than that of the corresponding unmodified cyclic polyribonucleotide. In one embodiment, one or more of the hybrid modified cyclic polyribonucleotides The increased expression of the sequence is less than that of the corresponding unmodified circular polyribonucleotide. In one embodiment, the hybrid modified circular polyribonucleic acid Increased expression of one or more sequences of the corresponding unmodified cyclic polyribonucleotide is In one embodiment, the hybrid modified cyclic poly Increased expression of one or more sequences of ribonucleotides is observed 1 day, 2 days after administration to a subject. Sun, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 1 4th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 28th, or or any date thereafter.

[0079] In one embodiment, expression of one or more hybrid modified circular polyribonucleotides Expression of the sequence is determined by the expression of one or more fully modified circular polyribonucleotide equivalents after administration to a subject. It has a translation efficiency similar to or higher than that of the expressed sequence. One or more expression sequences of the hybrid modified cyclic polyribonucleotide are fully expressed after administration to a subject. from one or more expressed sequences of all modified circular polyribonucleotide equivalents, at least about 0.7 ,0.8,0.9,1.0,1.1,1.2,1.3,1.5,1.6,1.8,2,2 In one embodiment, the hybridization product has a translation efficiency that is 0.2, 2.5, 2.8, or 3 times higher. Expression of one or more expression sequences of the lid-modified circular polyribonucleotide after administration to a subject is have translation efficiencies similar to or higher than the corresponding unmodified cyclic polyribonucleotides. In one embodiment, one or more of the hybrid modified cyclic polyribonucleotides The expressed sequence, after administration to a subject, is at least as efficient as the corresponding unmodified cyclic polyribonucleotide. Also about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1. 8, 2, 2.2, 2.5, 2.8, or 3 times higher translation efficiency.

[0080] In one embodiment, expression of one or more hybrid modified circular polyribonucleotides Expression of the sequence was measured on days 1, 2, 3, 4, 5, 6, 7, 8, and 9 after administration to the subject. Sun, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th 20, 21, 28, or more days, or any day in between, Similar to or identical to one or more of the expressed sequences of the fully modified circular polyribonucleotide equivalents In one embodiment, the hybrid modified circular polyribonucleic acid The expression sequence of one or more of the protease inhibitors is expressed on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3 Sun, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 1 7 days, 18 days, 19 days, 20 days, 21 days, 28 days, or more, or any period in between At any given time point, one or more expressed sequences of fully modified circular polyribonucleotide equivalents , at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1 0.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher translation efficiency. In the form of one or more expression sequences of hybrid modified circular polyribonucleotides are similar to or superior to the corresponding unmodified cyclic polyribonucleotide after administration to a subject. In one embodiment, the hybrid modified circular polyribonucleic acid The expression sequence of one or more of the protease inhibitors is expressed on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3 Sun, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 1 7 days, 18 days, 19 days, 20 days, 21 days, 28 days, or more, or any period in between At any given time point, the nucleotide sequence of the corresponding unmodified cyclic polyribonucleotide is at least about 0.7 times that of the corresponding unmodified cyclic polyribonucleotide. ,0.8,0.9,1.0,1.1,1.2,1.3,1.5,1.6,1.8,2,2 0.2, 2.5, 2.8, or 3 times higher translation efficiency.

[0081] In one embodiment, expression of one or more hybrid modified circular polyribonucleotides Increased expression of the sequence is observed at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 2 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th 19th, 20th, 21st, 28th, or more days, or any day in between. At any one time point, more than 5% or at least 10%, 20%, 30%, 40%, 50%, 60%, 7 having a first portion containing 0%, 80%, 90%, or 100% modified nucleotides; Similar to or higher than the fully modified circular polyribonucleotide equivalent. In this embodiment, the increased expression of one or more of the hybrid modified circular polyribonucleotide sequences The onset of symptoms occurred on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 after administration to the subject. 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 5% on the 20th, 21st, 28th, or more days, or any day in between. More than or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, or 100% modified nucleotides. polyribonucleotide equivalents, at least about 1.1, 1.2, 1.3, 1.5, 1. 6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4. 0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8. 0, 8.5, 9.0, 9.5, or 10.0 times higher.

[0082] In one embodiment, expression of one or more hybrid modified circular polyribonucleotides Increased expression of the sequence may be greater than 5%, or at least 10%, 20%, 30%, 40%, 50%, or %, 60%, 70%, 80%, 90%, or 100% modified nucleotides. a fully modified circular polyribonucleotide equivalent having a moiety of In one embodiment, one or more nucleotides of the hybrid modified circular polyribonucleotide are The increased expression of the target sequence may be greater than 5%, or at least 10%, 20%, 30%, 40%, 5% or more. containing 0%, 60%, 70%, 80%, 90%, or 100% modified nucleotides. from a fully modified circular polyribonucleotide equivalent having a portion of at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times higher.

[0083] In one embodiment, expression of one or more hybrid modified circular polyribonucleotides Increased expression of the sequence is observed at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 2 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th 19th, 20th, 21st, 28th, or more days, or any day in between. At any one time point, more than 5% or at least 10%, 20%, 30%, 40%, 50%, 60%, 7 having a first portion containing 0%, 80%, 90%, or 100% modified nucleotides; Similar to or higher than the fully modified circular polyribonucleotide equivalent. In this embodiment, the increased expression of one or more of the hybrid modified circular polyribonucleotide sequences The onset of symptoms occurred on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 after administration to the subject. 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 5% on the 20th, 21st, 28th, or more days, or any day in between. More than or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, or 100% modified nucleotides. polyribonucleotide equivalents, at least about 1.1, 1.2, 1.3, 1.5, 1. 6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4. 0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8. 0, 8.5, 9.0, 9.5, or 10.0 times higher.

[0084] In one embodiment, one or more sequences of hybrid modified circular polyribonucleotides The increased expression of ,8th,9th,10th,11th,12th,13th,14th,15th,16th,17th,1 8th, 19th, 20th, 21st, 28th, or more days, or any day in between At this point, more than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, having a first portion comprising 70%, 80%, 90%, or 100% modified nucleotides; at least about 5%, 10%, 1% or more of the fully modified circular polyribonucleotide equivalent 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 6 5%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150 %, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500 %, 600%, 70%, 800%, 900%, 1000%, 2000%, 5000%, 1 0,000%, 100,000%, or even higher. Increased expression of one or more sequences of the modified cyclic polyribonucleotide may be achieved by administration to a subject. After, 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th , 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 28th , or more, or at any time on any day therebetween, more than 5% or at least 10% , 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100 % of a fully modified circular polyribonucleotide equivalent having a first portion comprising modified nucleotides In one embodiment, the hybrid modified cyclic polysaccharide is at least about 10% higher than that of the original. The increased expression of one or more sequences of ribonucleotides is observed 1 day after administration to the subject. 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 28th, or or more than 5%, or at least 10%, 20% at any time on any day therebetween. , 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% modification a fully modified circular polyribonucleotide equivalent having a first portion comprising a nucleotide In one embodiment, the hybrid modified cyclic polyribonucleic acid Increased expression of one or more sequences of nucleotides may occur within 1 day, 2 days, 3 days, or 4 days after administration to a subject. Sunday, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 28th or more , or at any time on any day therebetween, more than 5%, or at least 10%, 20%, or 30% , 40%, 50%, 60%, 70%, 80%, 90%, or 100% modified nucleosides a fully modified circular polyribonucleotide equivalent having a first portion containing a nucleotide; At least about 50% higher.

[0085] As described herein, in certain embodiments, translation efficiency or increased expression refers to high a hybrid-modified cyclic polyribonucleotide or the corresponding unmodified cyclic polyribonucleo Cells or in vitro translation systems containing modified or fully modified circular polyribonucleotide equivalents. (e.g., rabbit reticulocyte lysate).

[0086] Increased stability The present disclosure provides a method for increasing the stability of a cyclic polyribonucleotide in a subject, comprising: providing a hybrid-modified cyclic polyribonucleotide, The modified cyclic polyribonucleotide comprises at least one modified polyribonucleotide and consecutive non- a first portion of modified nucleotides and a hybrid modified circular polyribonucleic acid administering a corresponding unmodified cyclic polynucleotide to a subject and detecting the corresponding unmodified cyclic polynucleotide in cells or tissues of the subject. increased for hybrid-modified cyclic polyribonucleotides compared to polyribonucleotides. and achieving stability. In one embodiment, the first portion is at least At least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, In some embodiments, the amino acid sequence comprises 700, 800, 900, or 1000 consecutive unmodified nucleotides. In one embodiment, no more than 5% of the nucleotides in the first portion are modified. No more than 1%, 2%, 3%, 4%, or 5% of the nucleotides in the first portion are modified. In some embodiments, the nucleotides in the first portion are unmodified. In one embodiment, the first portion is an IRES. In another embodiment, the first portion is an unmodified nucleotide. Proteins, peptides, biomolecules, DNA, RNA, or cellular targets In one embodiment, the first portion comprises a binding site configured to bind to the In one embodiment, the first portion is an IRES comprising the following modified nucleotides: In one embodiment, the first portion is an IRES that does not contain modified nucleotides. In one embodiment, the hybrid modified nucleotide IRES is The increased stability of the ribonucleotide-like structure is related to the corresponding unmodified structure in the cells or tissues of interest. at least about 1.1, 1.2, 1.3, 1.5 compared to the decorated circular polyribonucleotide , 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8 , 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 , 8.0, 8.5, 9.0, 9.5, or 10.0 times higher.

[0087] In certain embodiments, the hybrid modified circular polyribonucleoside disclosed herein The amount of cyclic polyribonucleotides in the ribonucleotides is increased after administration to a subject, compared to the corresponding unmodified cyclic polyribonucleotides. In certain embodiments, the disclosed hybrid modified circular polyribonucleic acid has excellent stability. The nucleotides are less likely to react with the corresponding unmodified cyclic polyribonucleotides after administration to a subject. All are approximately 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8 ,3,3.2,3.3,3.5,3.8,4.0,4.2,4.5,4.8,5.0,5 .5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10. 0-fold increased stability.

[0088] In certain embodiments, the hybrid modified circular polyribonucleoside disclosed herein After administration to the subjects, the dose was measured on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 1st days. 0th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 2nd On the 0th, 21st, 28th, or more days, or any day in between, the corresponding In some embodiments, the modified cyclic polyribonucleotide has increased stability compared to the unmodified cyclic polyribonucleotide. In the present invention, the hybrid modified cyclic polyribonucleotides disclosed herein are At 14 days after administration, increased safety was observed compared to the corresponding unmodified cyclic polyribonucleotide. In certain embodiments, the disclosed hybrid modified circular polyribonucleic acid Otide was administered to the subjects on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, On the 20th, 21st, 28th, or more days, or any day in between, at least about 1.1, 1.2, 1.3, 1.4, 1.6, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2. 5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3. 8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7. 5, 8.0, 8.5, 9.0, 9.5, or 10.0 times greater increased stability. In some embodiments, the disclosed hybrid modified cyclic polyribonucleotides are administered to a subject. at least about 14 days after administration of the compound of formula (I) or (II) of formula (II ... formula (II) 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times higher It has increased stability.

[0089] In certain embodiments, the hybrid modified cyclic polyribonucleotide is administered to a subject. After the modification, the cyclic polyribonucleotide has a longer half-life than the corresponding unmodified cyclic polyribonucleotide. In the present invention, the hybrid modified cyclic polyribonucleotide is converted to a corresponding unmodified cyclic polyribonucleotide after administration to a subject. At least about 1.1, 1.2, 1.3, 1.5, 1. 6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4. 0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8. In some embodiments, the half-life is 0, 8.5, 9.0, 9.5, or 10.0 times longer. Therefore, the half-life is determined for hybrid modified cyclic polyribonucleotides or the corresponding unmodified cyclic polyribonucleotides. Ribonucleotides are introduced into cells, and the hybrid modified circular polynucleotides introduced into the cells are and measuring the levels of cyclic polyribonucleotides or the corresponding unmodified cyclic polyribonucleotides. and is measured by.

[0090] In certain embodiments, the hybrid modified cyclic polyribonucleotide is administered to a subject. After conversion, the resulting polyribonucleotide has a half-life at least that of the corresponding unmodified circular polyribonucleotide. In some embodiments, the hybrid modified cyclic polyribonucleotide, after administration to a subject, It has an increased half-life relative to that of the corresponding unmodified cyclic polyribonucleotide. In some forms, the half-life is about 5%, 10%, 15%, 20%, 25%, or In some embodiments, the increase is 30%, 35%, 40%, 45%, 50%, or more. wherein the hybrid modified cyclic polyribonucleotide has a cytotoxicity of at least about 100% after administration to a subject. hours to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th 1, 28 days, 29 days, 30 days, 60 days, or more, or any period in between In certain embodiments, the half-life or persistence within the cell is The hybrid-modified cyclic polyribonucleotide is administered to a subject within about 10 minutes to about 7 days. or approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours , 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours , 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours , 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days or less, or any period therebetween In certain embodiments, the half-life or persistence within the cell is determined over any period of time. After administration to a subject, the hybrid-modified cyclic polyribonucleotide acts as a cytoplasmic receptor agonist while the cells are dividing. In one embodiment, the hybrid modified cyclic polynucleotide has a half-life or persistence in cells. Ribonucleotides have a half-life or persistence within cells after administration to a subject and after division. In certain embodiments, the hybrid modified cyclic polyribonucleotide is administered to a subject. More than about 10 minutes to about 30 days, or at least about 1 hour, 2 hours, 3 hours, or 4 hours after administration , 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours , 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days days, 30 days, 60 days, or more, or any time in between, It has a half-life or persistence within dividing cells.

[0091] In certain embodiments, the hybrid modified cyclic polyribonucleotide is administered to a subject. In one embodiment, the hybrid modified cyclic polynucleotide is After administration to a subject, the ribonucleotides persist in daughter cells after mitosis. In the method, the hybrid modified cyclic polyribonucleotide is complexed in cells after administration to a subject. In one embodiment, the cells are produced and passaged into daughter cells. 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% Efficiently pass on at least one hybrid modified circular polyribonucleotide to daughter cells. In certain embodiments, cells undergoing meiosis are at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% Efficiently, the hybrid modified circular polyribonucleotide is passed to daughter cells. In this embodiment, at least 25%, 50%, or more of the cells undergoing mitosis are isolated from the target tissue after administration to the subject. %, 60%, 70%, 80%, 85%, 90%, 95%, or 99% efficiency of hybridization The red-modified circular polyribonucleotide is passed to daughter cells.

[0092] Modified cyclic polyribonucleotides Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid Brid-modified cyclic polyribonucleotides are used in the methods described herein. In one embodiment, the hybrid modified cyclic polyribonucleotide comprises at least one In one embodiment, the modified polyribonucleotide comprises a first portion of contiguous unmodified nucleotides. In some embodiments, the hybrid modified cyclic polyribonucleotides described herein may be at least one modified nucleotide and a first portion, wherein the first portion comprises at least All are approximately 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 70 In some embodiments, the amino acid sequence comprises 0, 800, 900, or 1000 consecutive unmodified nucleotides. In one embodiment, no more than 5% of the nucleotides in the first portion are modified. No more than 0%, 1%, 2%, 3%, 4%, or 5% of the nucleotides in the portion are modified. In some embodiments, the nucleotides in the first portion are unmodified. In one embodiment, the first portion is an IRES. In another embodiment, the first portion is 5% or less In one embodiment, the first portion is an IRES comprising modified nucleotides of the formula: In one embodiment, the first portion is an unmodified IRES that does not contain any nucleotides. In one embodiment, the first portion is an IRES consisting of unmodified nucleotides. Nucleotides, proteins, peptides, biomolecules, DNA, RNA, or cellular targets In one embodiment, the first portion comprises a binding site configured to bind to 5 Lacking '-methylcytidine, pseudouridine, or N1-methyl-pseudouridine In certain embodiments, the hybrid modified cyclic polyribonucleotide is In a pharmaceutical composition further comprising an acceptable carrier or excipient. The hybrid modified cyclic polyribonucleotide is delivered to the subject. Hybrid modified cyclic polyribonucleotides are equivalent to fully modified cyclic polyribonucleotides. Reduced or decreased immunogenicity, increased translation efficiency (e.g., hybridization) compared to the increased expression of one or more expression sequences in a modified circular polyribonucleotide, or The composition may have added stability.

[0093] The first portion comprises 5% or less modified nucleotides in the hybrid modified circular polyribonucleotide. In one embodiment, the first portion comprises 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, No more than 1%, 2%, 3%, 4%, or 5% of the consecutive nucleotides are modified. , comprising consecutive unmodified nucleotides in a hybrid modified circular polyribonucleotide. may comprise at least about 5 consecutive unmodified nucleotides. The first portion has no more than 0%, 1%, 2%, 3%, 4%, or 5% modified nucleotides. at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 6 In some embodiments, the sequence comprises 0, 700, 800, 900, or 1000 contiguous nucleotides. wherein the first portion contains no more than 0%, 1%, 2%, 3%, 4%, or 5% modified nucleotides. In one embodiment, the amino acid sequence comprises at least about 5 to 1000 consecutive nucleotides having a sequence The first portion has no more than 0%, 1%, 2%, 3%, 4%, or 5% modified nucleotides. at least about 5-1000, 10-1000, 20-1000, 50-1000, 100~1000, 200~1000, 300~1000, 400~1000, 500~ 1000, 600-1000, 700-1000, 800-1000, 900-1000 or 900 to 2000 contiguous nucleotides. In one embodiment, the first portion , at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 60 0, 700, 800, 900, or 1000 consecutive unmodified nucleotides. In one embodiment, the first portion comprises at least about 5 to 1000 consecutive unmodified nucleotides. In some embodiments, the first portion is at least about 5-1000, 10-1000, 2 0~1000, 50~1000, 100~1000, 200~1000, 300~100 0, 400~1000, 500~1000, 600~1000, 700~1000, 80 Contains 0 to 1000, 900 to 1000, or 900 to 2000 consecutive unmodified nucleotides In some embodiments, the first portion is 5, 10, 20, 50, 100, 200, 30 0, 400, 500, 600, 700, 800, 900, 1000, or any value in between The first portion may comprise any number of consecutive unmodified nucleotides. The first portion may comprise an IRES. In some embodiments, the first portion comprises a binding site. Configured to bind to peptides, proteins, biomolecules, DNA, RNA, or cellular targets The nucleotide sequence includes a binding site.

[0094] In one embodiment, the hybrid modified circular polyribonucleotide is an IRES element. or a binding moiety configured to bind to a protein, DNA, RNA, or cellular target. Modified nucleotides, e.g., 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 'Methylcytidine and pseudouridine. In these cases, hybrid modified cyclic Polyribonucleotides have a lower cyclicity compared to the corresponding unmodified cyclic polyribonucleotides. In these cases, the hybrid modified cyclic polyribonucleotide has low immunogenicity. The corresponding cyclic polyribonucleotide containing no methylcytidine or pseudouridine In one embodiment, the hybrid modified cyclic polynucleotide has lower immunogenicity compared to the hybrid modified cyclic polynucleotide. The polyribonucleotides are at least about 1 times more cyclic than the corresponding unmodified cyclic polyribonucleotides. .1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3 .2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6 0.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times In one embodiment, the immunogenicity described herein is GI, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OA Expression, signal transduction, or activity of at least one of SL, PKR, and IFN-β In one embodiment, the hybrid modified circular polyribonucleic acid is evaluated by The nucleotides are the corresponding unmodified cyclic polyribonucleotides, e.g., 5' methylcytidine and It has a longer half-life than the corresponding cyclic polyribonucleotide that does not contain pseudouridine. In certain embodiments, the hybrid modified cyclic polyribonucleotide is a corresponding unmodified from cyclic polyribonucleotides, at least about 1.1, 1.2, 1.3, 1.5, 1.6 , 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0 , 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 , 8.5, 9.0, 9.5, or 10.0 times longer half-lives. In this embodiment, the half-life is measured using a hybrid modified cyclic polyribonucleotide or the corresponding cyclic polyribonucleotide. The hybrid modified cyclic ribonucleotides introduced into the cell are By measuring the levels of polyribonucleotides or the corresponding cyclic polyribonucleotides Therefore, it is measured.

[0095] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid A hybrid-modified cyclic polyribonucleotide contains at least one modified nucleotide. The hybrid modified cyclic polyribonucleotides described herein may be obtained by sequentially unmodified The first portion may comprise a nucleotide and at least one modified nucleotide. The nucleotides are outside the first portion. Modification of fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides Modified polyribonucleotides are naturally occurring unmodified nucleotides as shown herein. Unmodified amino acids such as adenosine (A), uridine (U), guanine (G), and cytidine (C) Analogs or derivatives having one or more chemical modifications to the chemical composition of natural ribonucleotides The chemical modification of the modified ribonucleotide may be at the sugar, nucleobase, or internucleoside. to any one or more functional groups of a ribonucleotide, such as a bond (e.g., to link phosphate / phosphodiester bond / phosphodiester backbone) modifications .

[0096] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) polyribonucleotides or hybrid modified cyclic polyribonucleotides) are one or more post-transcriptional Modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequences, Methylation, acylation, phosphorylation, methylation, acetylation of lysine and arginine residues, and One or more post-transcriptional modifications include: Any of the over 100 different nucleoside modifications identified in RNA (Rozenski, J., Crain, P., and McClo skey, J. (1999). The RNA Modification Datab ase:1999 update.Nucl Acids Res 27:196-19 7). In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) are used. Polyribonucleotides (or hybrid modified cyclic polyribonucleotides) are pyridine-4 -one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio Auridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy Uridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyluridine 5-propynyl-uridine, 1-propynyl-pseudouridine 5-Taurinomethyluridine, 1-Taurinomethyl-pseudouridine, 5-Taurin 1-Taurinomethyl-2-thio-uridine, 1-Taurinomethyl-4-thio-uridine, 5-Methyl -uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine , 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydrouridine 2-thio-dihydrouridine, 2-thio-dihydropseudouridine 2-Methoxyuridine, 2-Methoxy-4-thiouridine, 4-Methoxy-pseudo-uridine Douridine, and 4-methoxy-2-thio-pseudouridine In one embodiment, the modified cyclic polyribonucleic acid comprises at least one nucleoside. The oxidases are 5-aza-cytidine, pseudocytidine, 3-methyl-cytidine, N4-acetamide, Cytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine Cytidine, 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine , 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudocytidine , 4-thio-1-methyl-pseudocytidine, 4-thio-1-methyl-1-deaza-pseudocytidine Idocytidine, 1-methyl-1-deaza-pseudocytidine, Zebularine, 5-azase Zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zeb Larin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy - pseudocytidine, and 4-methoxy-1-methyl-pseudocytidine In one embodiment, the modified cyclic polynucleotide comprises at least one nucleoside selected from the group consisting of methyl, ... Ribonucleotides are 2-aminopurine, 2,6-diaminopurine, and 7-deaza-adenosine. 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8 -aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza 2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- Isopentenyl adenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2 -Methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinyl Glycinylcarbamoyl adenosine, N6-threonyl Carbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and and 2-methoxy-adenine. In one embodiment, the mRNA contains inosine, 1-methyl-inosine, 1-iosine, , wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thiazolinone O-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza -guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl Guanosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo N-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine N2,N2-dimethyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine It contains at least one nucleoside.

[0097] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are N(6) methylated cyclic polyribonucleotides. adenosine (m6A), 5'-methylcytidine (5mC), pseudouridine, or N1- Methyl-pseudouridine, 2'-O-methyl, 2'-O-methoxyethyl (2'-O- MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro , 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2 '-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T -O-Dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON- Methylacetamide (2'-O-NMA), locked nucleic acid (LNA), ethylene nucleic acid (E NA), peptide nucleic acid (PNA), 1',5'-anhydrohexitol nucleic acid (HNA) , morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, and 2'-fluoro N3-P5'-phosphoramidites, In one embodiment, the modified cyclic polyribonucleotide comprises one modified nucleotide. (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Otide) is described in Gilbert, WV, et al., which is incorporated herein by reference. .Science.2016 Jun17;352(6292):1408-1412 Nucleic acids known by those skilled in the art, such as those identified in the literature, or in this document, may be used. Contains oxide modifications.

[0098] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid A bridge-modified cyclic polyribonucleotide is a polyribonucleotide that has a modified sugar, nucleobase, or internucleoside linkage. etc. (e.g., phosphate / phosphodiester bond / phosphodiester The pyrimidine nucleobase may contain any useful modification (to the backbone). One or more atoms of the pyrimidine nucleobase may be: 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., For example, one or more modifications) are present on each of the sugar and internucleoside linkages. Oxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), Ribonucleic acid (ribonucleic acid (ribonucleotides)) to peptide nucleic acid (PNA), locked nucleic acid (LNA) or hybrids thereof Further modifications are described herein.

[0099] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) increase translation efficiency In some embodiments, the amino acid sequence includes at least one N(6) methyl adenosine (m6A) modification. In this state, the N(6) methyladenosine (m6A) modification is nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) This may reduce or decrease the immunogenicity of the compound (e.g., methicillin-3-phosphate dehydrogenase).

[0100] In certain embodiments, the modification may include a chemical modification or a cell-derived modification. Some non-limiting examples of intracellular RNA modifications are given in Nat Reviews Mol Cel Biol, 2017, 18:202-210, from “RNA modification ons and structures cooperate to guide RN A-protein interactions” by Lewis and Pan It is stated as follows.

[0101] "Pseudouridine" refers, in another embodiment, to m 1 acp 3 Ψ(1-methyl-3- (3-amino-3-carboxypropyl)pseudouridine. This term is used in 1 In another embodiment, In another embodiment, the term refers to Ψm (2'-O-methylpseudouridine). In another embodiment, the term refers to m5D (5-methyldihydrouridine). This term is m 3 In another embodiment, this refers to Ψ(3-methylpseudouridine). The term refers to a pseudouridine moiety that is not further modified. The term refers to the monophosphate, diphosphate, or refers to triphosphate. In another embodiment, the term is used in the art to refer to refers to any other pseudouridine known. Each possibility represents a separate embodiment of the present invention. .

[0102] In certain embodiments, chemical modifications to the ribonucleotides of the cyclic polyribonucleotide are Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) can promote immune evasion. Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are incorporated herein by reference. "Current protocols in nucleic acid acid chemistry”, Beaucage, SLet al. (Eds. ), John Wiley & Sons, Inc., New York, NY, USA They can be synthesized and synthesized by methods well established in the art, such as those described in Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, etc.). Oxidation (mono-, di-, and tri-phosphorylation), conjugation, inversion bond, etc.), 3'-end modification (conjugation, D NA nucleotides, inverted bonds, etc.), base modifications (e.g., stabilizing bases, destabilizing bases, or substitution by a base that base-pairs with a wide repertoire of partners), removal of a base (abasid nucleotide), or conjugate base. Modified ribonucleotide bases include 5-methyl They may also include cytidine and pseudouridine. In certain embodiments, the base modifications are cyclic polynucleotides. Some of the functional effects of ribonucleotides include expression, immune response, stability, and cell In some embodiments, the modifications include biorthogonal modifications. (unnatural bases) nucleotides, e.g., unnatural bases. Kimoto et al., Chem Commun (Cambridge), 2, incorporated herein by reference. 017,53:12309,DOI:10.1039 / c7cc06661a sea ​​bream.

[0103] In one embodiment, the sugars of one or more ribonucleotides of the cyclic polyribonucleotide Modifications (e.g., at the 2' or 4' positions) or sugar substitutions, as well as backbone modifications, may be Modified cyclic polyribonucleotides (e.g., complete polyribonucleotides) include modifications or substitutions of the phosphodiester bond. Specific examples of modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides Examples include, but are not limited to, modifications or substitutions of phosphodiester bonds in nucleosides. Cyclic polyribonucleotides containing modified backbones, such as interside modifications, or non-natural internucleoside linkages Modified cyclic polyribonucleotides with modified backbones (e.g., fully modified cyclic polyribonucleotides) As the cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide, In particular, those that do not have a phosphorus atom in the skeleton are included. As sometimes referred to in the art, nucleoside derivatives having a phosphorus atom in their internucleoside backbone are Modified RNAs that do not have the amino acid sequence may also be considered to be oligonucleosides. In addition, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or half-modified cyclic polyribonucleotides) can be used. Hybrid modified cyclic polyribonucleotides (polyribonucleotides) contain phosphorus atoms in their internucleoside backbone. It contains ribonucleotides having the following structure:

[0104] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid Examples of bridge-modified cyclic polyribonucleotide backbones include phosphorothioate, Chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyls methyl phosphotriesters, 3'-alkylene phosphonates and chiral phosphonates methyl and other alkyl phosphonates, phosphinates, 3'-amino phosphoramidates and phosphoramidates such as aminoalkylphosphoramidates, thionophosphoramidates, dates, thionoalkylphosphonates, thionoalkylphosphotriesters, and Boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs, and reverse polar nucleotides in which 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 In certain embodiments, the cyclic polyribonucleotide has a negative charge or a positive charge. It can carry an electric charge.

[0105] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid Modified nucleotides that can be incorporated into the hybrid-modified cyclic polyribonucleotide are nucleotides The polysaccharide may be modified at the interosidic bond (e.g., the phosphate backbone). With respect to the nucleotide backbone, the terms "phosphate" and "phosphodiester" refer to The backbone phosphate group can be modified by replacing one or more of the oxygen atoms with various substituents. Additionally, modified nucleosides and nucleotides may be modified by the methods described herein. This may involve extensive replacement of the unmodified phosphate moiety with another internucleoside linkage. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphorosele nate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoroamine Phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters Phosphorodithioates are those in which both non-linking oxygens are replaced with sulfur. The phosphate linker is a nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate) oates), and also by replacement of the linking oxygen with a carbon (bridging methylene-phosphonates). It can be modified.

[0106] Non-natural phosphorothioate backbone linkages provide stability to RNA and DNA polymers. A-thio substituted phosphate moieties are provided for the purpose of phosphorothioate DNA and R NAs have increased nuclease resistance and therefore a longer half-life within the cellular environment Phosphorothioates linked to cyclic polyribonucleotides are essential for the regulation of cellular innate immune molecules. Weaker binding / activation is predicted to reduce the innate immune response.

[0107] In certain embodiments, modified nucleosides include α-thio-nucleosides (e.g., For example, 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-guanidine (α-thiocytidine), 5'-O-(1-thiophosphate)-guanidine 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-uridine Examples include phosphate-pseudouridine.

[0108] Other nucleoside linkages that may be used in accordance with the present invention, including internucleoside linkages that do not contain a phosphorus atom, include Internucleoside linkages are described herein.

[0109] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) contain one or more cytotoxic For example, the cytotoxic nucleoside may include a cyclic nucleoside, such as a bifunctional modification. Cytotoxic nucleosides include, but are not limited to, nucleosides that can be incorporated into polyribonucleotides. However, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclo Pentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside 1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cyano Tosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine A combination of tabine, tegafur and uracil, tegafur ((RS)-5-fluoro-1- (tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), Xacitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC) Further examples include fludarabine phosphene, N4-behenoyl-1-β-D-arabinofuranosylcytosine, N4-octadecyl N4-palmitoyl-1-(2-C-cytosine) Ano-2-deoxy-β-D-arabino-pentofuranosyl)cytosine, and P-405 5 (cytarabine 5'-elaidate).

[0110] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid A hybrid-modified cyclic polyribonucleotide may be uniformly modified along the entire length of the molecule or For example, one or more or all types of nucleotides (e.g., Natural nucleotides, purines or pyrimidines, or any one of A, G, U, C, I, pU One or more or all of the ribonucleotides may be a cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide). nucleotide or hybrid modified cyclic polyribonucleotide) or in that given It may or may not be uniformly modified in a given sequence region. In addition, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or half-modified cyclic polyribonucleotides) can be used. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises pseudouridine. In the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide), or hybrid modified cyclic polyribonucleotides) contain inosine, which is a nucleotide that stimulates the immune system characterized cyclic polyribonucleotides as endogenous to viral RNA. Inosine incorporation may also result in improved RNA stability / reduced degradation. See, for example, Yu, Z. et al. (2015), which is incorporated by reference in its entirety. RNA editing by ADAR1 marks dsRNA as “sel See Cell Res. 25, 1283-1284.

[0111] In some embodiments, the given sequence region (e.g., the first portion or the unmodified portion) All nucleotides in the hybrid modified cyclic polyribonucleotide in In certain embodiments, the modifications may increase expression and / or weaken the immune response. m6A, which may weaken the immune response; inosine, which may increase RNA stability or translational readthrough. Pseudouridine (stagger element) which may increase the stability and / or enhance immune response. m5C, which may weaken the response; and 2,2,7-trimethylbenzyl methyltransferase, which promotes intracellular translocation (e.g., nuclear localization). Methylguanosine is an example.

[0112] Various sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures ) can be present at various positions in the cyclic polyribonucleotide. Nucleotide analogs or other modifications may be used so that the function of the ribonucleotide is not substantially reduced. The decoration may be a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or It is understood that the nucleotide sequence may be located at any position of the hybrid modified circular polyribonucleotide. The modifications may also be modifications of non-coding regions. nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) nucleotides) are about 1% to about 100% modified nucleotides (relative to the total nucleotide content) or one or more types of nucleotides, i.e., A, G, U, or C For one or more) or any percentage in between (e.g., 1% to 20% > 1% ~25%, 1%~50%, 1%~60%, 1%~70%, 1%~80%, 1%~90%, 1%~95%, 10%~20%, 10%~25%, 10%~50%, 10%~60%, 1 0%~70%, 10%~80%, 10%~90%, 10%~95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~95%, 20%~100%, 50%~60%, 50%~70% , 50%~80%, 50%~90%, 50%~95%, 50%~100%, 70%~80 %, 70%~90%, 70%~95%, 70%~100%, 80%~90%, 80%~9 5%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%) may include:

[0113] In some embodiments, the cyclic polyribonucleotide is a fully modified cyclic polyribonucleotide. nucleotides or fully modified circular polyribonucleotides, all or substantially all modified adenosine residues, all or substantially all modified uridine residues, all or substantially all modified adenosine residues, Qualitatively all modified guanine residues, all or substantially all modified cytidine residues, or In one embodiment, the circular RNA comprises at least 10% Contains 20%, 30%, 40%, 50%, 60%, 70%, or 80% modified nucleotides In some embodiments, the fully modified circular RNA is substantially entirely (e.g., 80% , 85%, 90%, 95%, 97%, 98%, or 99%, or greater than about 100% In certain embodiments, the modified circular polynucleotides provided herein include modified nucleotides. Polyribonucleotides are hybrid modified cyclic polyribonucleotides. The modified cyclic polyribonucleotide may have at least one modified nucleotide and may have consecutive It may have a portion of unmodified nucleotides (e.g., first portion / unmodified portion). This unmodified portion of the de-modified cyclic polyribonucleotide may be at least about 5, 10, 15, or 16. or 20 consecutive unmodified nucleotides, or any number therebetween. In some embodiments, the unmodified portion of the hybrid modified cyclic polyribonucleotide is at least about 30, 40, 40, 60, 70, 80, 90, 100, 110, 120, 130, 140 , 150, 160, 180, 200, 220, 250, 280, 300, 320, 350 , 380, 400, 420, 450, 500, 550, 600, 650, 700, 750 , 800, 850, 900, or 1000 consecutive unmodified nucleotides, or any combination thereof In some embodiments, the hybrid modified circular polyribonucleic acid The denominator may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodified moieties. In one embodiment, the hybrid modified cyclic polyribonucleotide comprises at least one , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 70 , 80, 100, 120, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 or more modified nucleotides. In embodiments, the hybrid modified cyclic polyribonucleotide comprises at least 1%, 2% ,5%,7%,8%,10%,12%,15%,18%,20%,25%,30%,35 %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95 %, or 99%, but less than 100%, of the modified nucleotides. In some embodiments, the unmodified portion comprises a binding site. Configured to bind to peptides, proteins, biomolecules, DNA, RNA, or cellular targets In one embodiment, the unmodified portion comprises an IRES.

[0114] In some cases, the hybrid modified cyclic polyribonucleotides described herein compared to the corresponding fully modified but otherwise identical cyclic polyribonucleotide, For example, the 5' methylated nucleotides have similar immunogenicity to those of the IRES element. A hybrid modified cyclic polyribonucleotide having cytidine and pseudouridine is 5'-methylcytidine and pseudouridine throughout, and unmodified cytidine and and uridines, but the corresponding cyclic polyribonucleotide is the same, In one embodiment, the IRES may have similar or less immunogenicity. Hybrids with 5' methylcytidine and pseudouridine throughout except for the element The red-modified cyclic polyribonucleotide contains 5' methylcytidine and pseudocytidine throughout. The corresponding uridine-containing cytidines are identical except that they have no unmodified cytidines or uridines. Has a translation efficiency similar to or higher than that of cyclic polyribonucleotides .

[0115] In certain embodiments, the hybrid modified cyclic polyribonucleotide is unmodified. In some embodiments, the hybridization site has no modified nucleotides, e.g., no modified nucleotides. A bridge-modified cyclic polyribonucleotide may be unmodified, e.g., a ... a bond configured to bind to a protein, DNA, RNA, or cellular target, In some embodiments, the hybrid modified cyclic polyribonucleotide has a unmodified, e.g., an internal ribosome entry site (IRES) with no modified nucleotides In some embodiments, the hybrid modified cyclic polyribonucleotide has an internal Fewer than 5% of the nucleotides in the internal ribosome entry site (IRES) are modified nucleotides In some embodiments, the nucleotides in the IRES are unmodified. In the form of 0%, 1%, 2%, 3%, 4%, or 5% or less of the nucleotides in the IRES In some embodiments, the hybrid modified cyclic polyribonucleotide is , with modified nucleotides throughout except for the binding site. Hybrid modified cyclic polyribonucleotides are suitable for peptides, proteins, biomolecules, and DNA. A, RNA, or a binding site configured to bind to a cellular target. In some embodiments, the hybrid modified circular polyribonucleic acid The fragment has modified nucleotides throughout, except for the IRES element. In one embodiment, the hybrid modified circular polyribonucleotide comprises an IRES element and one or more It has modified nucleotides throughout, except for the other portions above. While not wishing to be bound by any particularity, the unmodified IRES element may be a hybrid modified circular polynucleotide. The modified nucleotides are then converted to corresponding nucleotides that are not translatable, e.g., For one or more expressed sequences similar to or higher than polyribonucleotides The translation efficiency is to be

[0116] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) The polyribonucleotide or hybrid modified cyclic polyribonucleotide) has at least one linear Equivalents, such as linear expression sequences or linear circular polyribonucleotides, have half-lives. In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) are used. Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are the linear equivalents of In certain embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more increase In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) are used. polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises at least Approximately 1 hour to approximately 30 days, or at least approximately 2 hours, 6 hours, 12 hours, 18 hours, or 24 hours 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 day, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 1 9 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 2 7 days, 28 days, 29 days, 30 days, 60 days, or more, or any period in between It has a half-life or persistence within the cell over any period of time. , modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid The hybrid-modified cyclic polyribonucleotide is , 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 Hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours Hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours for up to 1 hour, 4 days, 5 days, 6 days, 7 days or less, or any time in between. In one embodiment, the modified circular polyribonucleic acid has a half-life or persistence in cells. nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Ribonucleotides) have a half-life or persistence within the cell while the cell is dividing. In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used in the cell after division. In certain embodiments, modified cyclic polyribonucleotides ( For example, fully modified circular polyribonucleotides or hybrid modified circular polyribonucleo- nucleotides. The duration of the stimuli is from about 10 minutes to about 30 days, or at least about 1 hour, 2 hours, 3 hours, or 4 hours. 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13:00 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 1 3 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 2 1 day, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 2 For 9 days, 30 days, 60 days, or more, or any time in between. , have a half-life or persistence within dividing cells.

[0117] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used to express one or more expression sequences. The nucleic acid sequence comprises a sequence and is configured for sustained expression in the cells of a subject in vivo. In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) may be used. Polyribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) are expressed as 1 in cells at later time points. The expression of one or more expression sequences is configured to be equal to or greater than that at the previous time point. In such embodiments, expression of one or more expression sequences is maintained at a relatively stable level. The expression of the expression sequence may be relatively stable over an extended period of time. For example, in some cases, at least 7, 8, 9, 10, 12, 14 , 16, 18, 20, 22, 23 days or more in cells The expression of the above expression sequences is 50%, 45%, 40%, 35%, 30%, 25%, 20%, 1 5%, 10%, or less than 5% reduction. The expression of one or more expression sequences in the 50%, 45%, 40%, 35% for 8, 20, 22, 23 or more days; Maintained at a level that does not change by more than 30%, 25%, 20%, 15%, 10%, or 5% can be.

[0118] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be used in mammals, e.g. In certain embodiments, the modified cyclic polyribonucleotide is non-immunogenic in humans. (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Ochids are cells derived from aquaculture animals (fish, crabs, shrimp, oysters, etc.), mammalian cells , such as pet or zoo animals (cats, dogs, lizards, birds, lions, tigers, and Cells derived from livestock or working animals (horses, cows, pigs, chickens, etc.) Cells derived from the human genome, human cells, cultured cells, primary cells or cell lines, stem cells, progenitor cells, transformed cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any of these In one embodiment, the nucleotide sequence is capable of replicating or replicating in any combination. The present invention relates to modified cyclic polyribonucleotides described herein (e.g., fully modified cyclic Polyribonucleotides or hybrid modified circular polyribonucleotides) Here, the cells may be derived from aquaculture animals (fish, crab, shrimp, oyster, etc.), mammalian cells, or the like. Animal cells, such as pet or zoo animals (cats, dogs, lizards, birds, lions, Cells derived from livestock or working animals (horses, cows, pigs, chickens, etc.) Cells derived from the human genome, human cells, cultured cells, primary cells or cell lines, stem cells, progenitor cells, etc. cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells or In some embodiments, the cells are nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) The compound is modified to contain riboflavin.

[0119] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) can enhance cellular functions, e.g. In certain embodiments, the modulation is for at least about 1 hour to about 30 minutes. days or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days , 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or more, or any time in between Cellular functions, such as regulation, that persist over time are stably altered. For example, about 30 minutes or less to about 7 days, or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours , 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours , 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days A cellular function, such as regulation, that lasts for less than or any time in between is considered transient. is changed to.

[0120] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) polyribonucleotide or hybrid modified cyclic polyribonucleotide) is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, Each of the nucleotides is about 50 nucleotides, at least about 75 nucleotides, and at least about 100 nucleotides. nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least About 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides , at least about 6,000 nucleotides, at least about 7,000 nucleotides, At least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 1 0,000 nucleotides, at least about 12,000 nucleotides, at least about 14, 000 nucleotides, at least about 15,000 nucleotides, at least about 16,00 0 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides Nucleotides, at least about 19,000 nucleotides or at least about 20,000 nucleotides In some embodiments, modified cyclic polyribonucleotides (e.g., completely Modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) are The modified polypeptide may be of a size sufficient to accommodate a binding site for a polypeptide. Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid The maximum size of the modified cyclic polyribonucleotide ( For example, fully modified circular polyribonucleotides or hybrid modified circular polyribonucleo- nucleotides. and / or modified circular polyribonucleotides (e.g., fully modified circular polyribonucleotides). technical restrictions using ribonucleotides or hybrid modified cyclic polyribonucleotides It can be understood that the magnitude can be within the approximate range. Although not confined, multiple segments of RNA form a "string" of DNA and RNA. The 5' and 3' free ends of the 5' and 3' fragments were annealed to form the 1' fragment. can be circularized when only one 5' free end and one 3' free end remain) In some embodiments, modified cyclic polyribonucleotides (e.g., For example, fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides. The maximum size of a nucleoprotein (NUCL) may be limited by its ability to package and deliver RNA to a target. In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) are The size of the ribonucleotide or hybrid modified cyclic polyribonucleotide) is useful It is long enough to encode a polypeptide and therefore has at least 20,000 nucleotides. Nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides At least 7,500 nucleotides or at least 5,000 nucleotides, At least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 0 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, At least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides A nucleic acid sequence of at least 100 nucleotides in length can be useful.

[0121] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides are described elsewhere herein. In one embodiment, the element comprises one or more of the elements described above. In one embodiment, the element comprises a spacer sequence. In one embodiment, the elements may be separated from one another by a single ribonucleic acid or a linker. nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 40 0 nucleotides, approximately 500 nucleotides, approximately 600 nucleotides, approximately 700 nucleotides , about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, and up to about 1 kb, at least about 1000 nucleotides, and any amount of nucleotides therebetween In some embodiments, one or more elements may be spaced apart, e.g., by a spacer element. In some embodiments, the modified cyclic polyribonucleotides are contiguous with each other, lacking One or more elements in the In certain embodiments, the modified cyclic polynucleotide is Polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) may be used to provide one or more desired functions or properties as described herein. corresponding, e.g., binding sites for ribosomes, e.g., translation, e.g., rolling sites It contains secondary or tertiary structure corresponding to circle-type translation.

[0122] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) have specific sequence properties. For example, modified circular polyribonucleotides (e.g., fully modified circular polyribonucleo- Polyribonucleotides (polyribonucleotides) are cyclic ribonucleotides with specific nucleotide compositions. In some such embodiments, modified cyclic polyribonucleotides (e.g., Fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) , may contain one or more purine-rich regions (adenine or guanosine). In embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) are nucleotides that contain one or more purine-rich In some embodiments, the modified circular polyribonucleotide may contain a region (adenine or guanosine). Nucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) A ribonucleotide may contain one or more AU-rich regions or elements (AREs). In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) A modified cyclic polyribonucleotide (a nucleotide or hybrid modified cyclic polyribonucleotide) is a polyribonucleotide that contains one or more adenine nucleotides. The region may include a tch region.

[0123] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides are described elsewhere herein. The repeating elements may include one or more of the repeating elements described above.

[0124] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides are described elsewhere herein. It may contain one or more of the modifications described above.

[0125] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid A hybrid modified cyclic polyribonucleotide (a polyribonucleotide with a modified nucleotide sequence) is a polyribonucleotide that contains one or more substitutions, insertions, or modifications relative to the reference sequence. and / or additions, deletions, and covalent modifications, particularly when the parent polyribonucleotide is is included in the range.

[0126] Expression sequence peptide or polypeptide In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are peptides or polynucleotides. The peptides include at least one expressible sequence encoding a peptide, such as: Non-limiting examples include small peptides, peptidomimetics (e.g., peptoids), amino acids, and amino acid analogs. Peptides can be linear or branched. Such peptides have a molecular weight of less than about 5,000 grams per mole and a molecular weight of less than about 2,000 grams per mole. Molecular weight less than about 1,000 grams per mole, Molecular weight less than about 1,000 grams per mole Molecular weights of less than about 500 grams, as well as salts, esters and other pharmaceutically acceptable salts of such compounds. Such peptides may have a form that is acceptable to the human body. substances, hormones, drugs, toxins, viral or microbial particles, synthetic molecules and their agonists Examples include inhibitors and antagonists.

[0127] The polypeptide may be linear or branched. 00 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, approximately 200 to approximately 10,000 amino acids, approximately 500 to approximately 5,000 amino acids The length may be about 1,000 to about 2,500 amino acids, or any range therebetween. In certain embodiments, the polypeptide is less than about 40,000 amino acids, about 35,000 less than 0 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, Less than 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids 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 2,0 less than about 1,000 amino acids, less than about 1,500 amino acids, less than about 1,000 amino acids, less than about 9 less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids Acid or shorter lengths may be useful.

[0128] Some examples of peptides or polypeptides include, but are not limited to, those with fluorescent tags or Markers, antigens, peptide therapeutics, synthetic or analogue peptides derived from natural bioactive peptides peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides peptides, bicyclic peptides, targeting or cytotoxic peptides, degradative or self-immolative peptides and and multiple degrading or self-immolating peptides. Peptides useful in this regard include antigen-binding peptides, e.g., antigen-binding antibodies or antibody-like fragments. Also included are antibodies, such as single chain antibodies, nanobodies (see, e.g., Steeland et al. al.2016.Nanobodies as therapeutics:big o Portunities for small antibodies.Drug D (See Iscov Today:21(7):1076-113.) The antigen-binding peptide may bind to a cytoplasmic antigen, a nuclear antigen, or an intraorganellar antigen.

[0129] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides (or hybrid modified cyclic polyribonucleotides) are composed of one or more RNA The polypeptides may include expression sequences, each of which may encode a polypeptide. Thus, a polypeptide is any protein that can be produced. The polypeptide may be secreted from the cell or may be present in the cytoplasm, nucleus, or The polypeptide may be a polypeptide that can be confined to a membrane compartment. Although not specified, at least part of the viral envelope protein, metabolic regulatory enzymes (e.g. For example, regulating lipid or steroid production), antigens, tolerogens, cytokines, toxins, An enzyme whose absence is associated with disease and which is active within an animal (e.g., in the animal's intestine) until cleaved. Non-protein-binding polypeptides and hormones are also included.

[0130] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used to prepare proteins, e.g. For example, the expression sequences described herein include those encoding therapeutic proteins. The modified cyclic polyribonucleotides shown (e.g., fully modified cyclic polyribonucleotides or Therapeutic proteins that can be expressed from the hybrid modified cyclic polyribonucleotide include: Antioxidant activity, binding, cargo receptor activity, catalytic activity, molecular carrier activity, molecular function regulator, Molecular transducer activity, nutrient storage activity, protein tag, structural molecule activity, toxin activity, translocation Some examples of therapeutic proteins include those that have transcriptional, translational, or transport activity. Examples include, but are not limited to, enzyme replacement proteins, replacement proteins, protein vaccines, etc. vaccination, antigens (e.g., tumor antigens, viruses, bacteria), hormones, cytokines, antibodies, immune Immunotherapy (e.g., cancer), reprogramming / transdifferentiation factors, transcription factors, chimeric antigen receptors , transposases or nucleases, immune effectors (e.g., immune response / signaling affecting susceptibility to death), regulated death effector proteins (e.g., apo inducers of necrosis or necrosis), non-lytic inhibitors of tumors (e.g., inhibitors of oncoproteins) ), epigenetic modifiers, epigenetic enzymes, transcription factors, DNA or proteins Protein-modifying enzymes, DNA intercalators, efflux pump inhibitors, nuclear receptor activators or inhibitors, Proteasome inhibitors, competitive inhibitors for enzymes, protein synthesis effectors or inhibitors Agents, nucleases, protein fragments or domains, ligands or receptors and CRISPR systems or components thereof.

[0131] In certain embodiments, the modified cyclic polyribonucleotides disclosed herein (e.g., , fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) Exemplary proteins that can be expressed from the Proteins, hormones, growth factors, growth factor receptor modulators and regenerative proteins (e.g. , proteins involved in proliferation and differentiation, e.g., therapeutic proteins for wound healing) In certain embodiments, the modified circular polyribonucleic acid disclosed herein is nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) An exemplary protein that can be expressed from a nucleotide is EGF (epidermal growth factor). In certain embodiments, the modified circular polyribonucleic acid disclosed herein may be nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Exemplary proteins that can be expressed from a ribozyme include enzymes, such as oxidoreductases, Tase enzymes, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, AT In some embodiments, the enzymes disclosed herein include P-independent enzymes and desaturases. The modified cyclic polyribonucleotides shown (e.g., fully modified cyclic polyribonucleotides or are exemplary proteins that can be expressed from hybrid modified cyclic polyribonucleotides. Examples include intracellular or cytoplasmic proteins. Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrids) Lid-modified cyclic polyribonucleotide) is used to synthesize NanoLuc® luciferase In one embodiment, the modified cyclic polynucleotides disclosed herein express (nLuc). Polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular Exemplary proteins that can be expressed from a polyribonucleotide include secreted proteins, In some cases, modified cyclic polyribonucleotides ( For example, fully modified circular polyribonucleotides or hybrid modified circular polyribonucleo- nucleotides. Tides) express secreted proteins that may have a short therapeutic half-life in the blood, or or a protein having a subcellular localization signal or a secretory signal peptide In some embodiments, modified cyclic polyribonucleotides (e.g., , fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) In some cases, the modified cyclic polynucleotide expresses Gaussia luciferase (gLuc). Ribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic Polyribonucleotides (polyribonucleotides) are used to bind non-human proteins, e.g., fluorescent proteins, energy transfer proteins, Express a target receptor or a protein tag such as Flag, Myc, or His. In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Exemplary polyribonucleotides that can be expressed from ribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) include: Suitable proteins include GFP. In one embodiment, modified circular polyribonucleotides are Nucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) A ribonucleotide) is a nucleotide that is used to create a tagged protein, e.g., a fusion protein containing a protein tag. proteins or modified proteins, such as chitin-binding protein (CBP), maltose-binding protein (MBP), Protein (MBP), Fc tag, glutathione-S-transferase (GST), A viTag (GLNDIFEAQKIEWHE), calmodulin-tag (KRRWKK NFIAVSAANRFKKISSSGAL); polyglutamate tag (EEEEEE) ;E-tag (GAPVPYPDPLEPR); FLAG-tag (DYKDDDDK), H A-tag (YPYDVPDYA); His-tag (HHHHHH); Myc-tag (EQ KLISEEDL);NE-tag(TKENPRSNQEESYDDNES);S-tag (KETAAAKFERQHMDS);SBP-tag(MDEKTTGWRGGHVVE GLAGELEQLRARLEHHPQGQREP);Softag 1(SLAELL NAGLGGS);Softag 3(TQDPSRVG);Spot-tag(PDRV RAVSHWSS); Strep-tag (Strep-tag II: WSHPQFEK); TC tag (CCPGCC); Ty tag (EVHTNQDPLD); V5 tag (GKPIP NPLLGLDST); VSV-tag (YTDIEMNRLGK); or Xpress-tag It expresses the gene (DLYDDDDK).

[0132] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be used to bind antibodies, e.g. In one embodiment, the modified circular polyribonucleic acid is expressed as a polypeptide fragment, or a portion thereof. nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Antibodies expressed by ribonucleotides include IgA, IgD, IgE, IgG, and Ig In one embodiment, the modified cyclic polynucleotides can be of any isotype, such as M. Polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular Polyribonucleotides) are parts of antibodies, such as light chains, heavy chains, Fc fragments, CDRs (complementarity determining region), Fv fragment, or Fab fragment, further portions thereof In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) are expressed. Polyribonucleotides (cyclic or hybrid modified cyclic polyribonucleotides) are used as part of antibodies. For example, a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide) may be expressed. A cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) is a polyribonucleotide consisting of two or more Each of these expresses a portion of an antibody, and the collection of these may be In some cases, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) may be used to construct antibodies. Modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides are used in antibodies It comprises one expressed sequence encoding the heavy chain of the antibody and another expressed sequence encoding the light chain of the antibody. In some cases, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) are expressed in cells or in a cell-free environment. When combined, the light and heavy chains undergo the appropriate modification, folding, or other translation to form a functional antibody. It can be subject to post-translational modification.

[0133] Adjustment element In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be used as regulatory elements, e.g. The modified circular polyribonucleotide comprises a sequence that regulates the expression of the expression sequence.

[0134] Regulatory elements can include sequences located adjacent to an expressed sequence that encodes an expression product. The element may be operably linked to an adjacent sequence. In addition, one may increase the amount of expressed product compared to the amount of product expressed in the absence of a The regulatory elements increase the amount of product expressed for multiple expression sequences linked in tandem. Thus, one regulatory element can promote the expression of more than one expression sequence. A number of regulatory elements are known to those of skill in the art.

[0135] The regulatory elements provided herein can include alternative translation sequences. As used herein, the term "alternative translation sequence" refers to a modified circular polyribonucleotide, e.g. For example, a specific riboswitch aptazyme can selectively initiate or activate translation of an expressed sequence. Regulatory elements may also include selective degradation sequences. As used herein, the term "selectively degradable sequence" refers to a modified cyclic polyribonucleotide or It may refer to a nucleic acid sequence that initiates degradation of the expression product of a modified circular polyribonucleotide. Potential selective degradation sequences may include riboswitch aptazymes and miRNA binding sites.

[0136] In certain embodiments, the regulatory element is a translational modulator. The translation of the expressed sequence in the modified circular polyribonucleotide may be modulated. In some embodiments, the modified cyclic polynucleotide may be a translation enhancer or suppressor. Polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotide) is a nucleic acid sequence comprising at least one translational sequence adjacent to at least one expressed sequence. In some embodiments, the modified cyclic polyribonucleotide (e.g., Fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) In one embodiment, the translation modulator flanks each expression sequence. The data may be present on one or both sides of each expression sequence and may be used to encode the expression product, e.g., a peptide and / or This results in the separation of the polypeptides.

[0137] In certain embodiments, the translation initiation sequence may function as a regulatory element. In one embodiment, the translation initiation sequence comprises an AUG codon. , AUG, CUG, GUG, UUG, ACG, AUC, AUU, AAG, AUA or AG In one embodiment, the translation initiation sequence includes any eukaryotic start codon, such as G. In some embodiments, translation is regulated by selective conditions, e.g., stress-induced Under certain conditions, it will initiate from an alternative translation initiation sequence, for example, a translation initiation sequence other than the AUG codon. Illustrative examples include modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides). The translation of the nucleotides (nucleotides or hybrid modified circular polyribonucleotides) is different from that of other translations such as ACG. Another non-limiting example is a modified cyclic polyribonucleotide (e.g., For example, a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide. Translation can begin at an alternative translation initiation sequence, CTG / CUG. Examples include modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides). or hybrid modified circular polyribonucleotide) translation can be initiated by an alternative translation initiation sequence, GTG / As yet another non-limiting example, a modified cyclic polyribonucleotide may start with GUG. (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Otides are short stretches of repeating RNA, e.g., CGG, GGGGCC, CAG, CTG. Translation is initiated from a repeat-associated non-AUG(RAN) sequence, such as an alternative translation initiation sequence containing obtain.

[0138] adjacent to the codon that initiates translation, including but not limited to the initiation codon or another initiation codon. The adjacent nucleotides may affect the translation efficiency, length and / or structure of the modified circular polyribonucleotide. (See, e.g., the entire contents of which are incorporated herein by reference. See Matsuda and Mauro, PLoS ONE, 2010, 5:11. (See references below.) Masking any of the nucleotides adjacent to the codon that initiates translation and the position of translation initiation, translation efficiency, length and / or can change the structure.

[0139] In one embodiment, the masking agent is attached to the masked start codon or to an alternative start codon. To mask or hide codons to reduce the likelihood of translation initiation in the They may be used near the start codon or an alternative start codon. Non-limiting examples of masking agents include antisense locked nucleic acid (LNA) oligonucleotides and exon junction complexes (EJC) (see, for example, Mat. suda and Mauro describing masking agents LNA oligonucleotides and EJCs(PLoS ONE, 2010 5:11). In another embodiment, the masking agent is To increase the likelihood that the gene will start at an alternative start codon, a modified circular polyribonucleic acid nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) It can be used to mask the start codon of the ribonucleotide (ribonucleotide).

[0140] In one embodiment, translation is achieved by, but not limited to, the transcription of GRSF-1 in the presence of GRSF-1. Under selective conditions, such as virus-induced selection, modified cyclic polyribonucleotides (e.g., For example, fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides. ) contains the GRSF-1 binding site, see for example http: / / jvi.asm.org / c Please refer to content / 76 / 20 / 10417.full.

[0141] Translation initiation sequence In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides are used to form polypeptides. In some embodiments, the translation initiation sequence may include a translation initiation sequence, e.g., a start codon. The sequence may comprise a Kozak or Shine-Dalgarno sequence. Polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified The modified circular polyribonucleotide (PCP) is a translation initiation sequence adjacent to the expression sequence, e.g., a Kozak sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In embodiments, a translation initiation sequence, e.g., a Kozak sequence, is located on one or both sides of each expression sequence. In one embodiment, the modified circular polyribonucleic acid nucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) The polynucleotides contain at least one translation initiation sequence adjacent to the expressed sequence. In embodiments, the translation initiation sequence provides conformational flexibility to the modified cyclic polyribonucleotide. In certain embodiments, the translation initiation sequence is substantially the same as that of the modified circular polyribonucleotide. within the single-stranded region.

[0142] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid The hybrid-modified cyclic polyribonucleotide may include, but is not limited to, at least two At least three, at least four, at least five, at least six, at least seven, at least At least 8, at least 9, at least 10, at least 11, at least 12, at least at least 13, at least 14, at least 15, at least 16, at least 17, at least At least 18, at least 19, at least 20, at least 25, at least 30, at least at least 35, at least 40, at least 50, at least 60 or more than 60 start codons The transcription factor may contain two or more initiation codons, such as: Or it may start downstream of the first start codon.

[0143] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) polyribonucleotide or hybrid modified cyclic polyribonucleotide) is It may start with a codon that is not a nucleotide, such as AUG. Fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) Translations include, but are not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG , ATA / AUA, ATT / AUU, TTG / UUG, and other alternative translation initiation sequences. (Touriol et al., the entire contents of each of which are incorporated herein by reference). l.Biology of the Cell 95(2003)169-178 and M See Atsuda and Mauro, PLoS ONE, 2010 5:11 In some embodiments, translation is selectively regulated under selective conditions, e.g., stress-induced conditions. Non-limiting examples include modified cyclic polyribonucleotides (e.g., For example, a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide. Translation of the nucleotide sequence (nucleotide sequence) can be initiated from an alternative translation initiation sequence, such as ACG. , modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid (bridge-modified circular polyribonucleotide) translation is initiated from an alternative translation initiation sequence, CTG / CUG. As yet another non-limiting example, modified cyclic polyribonucleotides (e.g., , fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) Translation can begin at an alternative translation initiation sequence, GTG / GUG. and modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or Hybrid modified cyclic polyribonucleotides) are RNAs containing repeating RNAs, e.g., CGG, GGGG Repeat-associated non-A sequences, such as alternative translation initiation sequences containing short stretches of CC, CAG, or CTG Translation can begin at the UG(RAN) sequence.

[0144] In one embodiment, translation is mediated by eukaryotic initiation factor 4A ( eIF4A) processing (translation is initiated by blocking 43S scanning) is repressed by early upstream transcription from transcripts with RocA-eIF4A target sequences This leads to translation initiation and reduced protein expression, as seen for example in www.nature.co (See m / articles / nature17978).

[0145] IRES In some embodiments, modified cyclic polyribonucleotides described herein (e.g., , fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) contains an internal ribosome entry site (IRES) element. For example, fully modified circular polyribonucleotides or hybrid modified circular polyribonucleo- nucleotides. Suitable IRES elements for inclusion in the ribosome are those capable of engaging a eukaryotic ribosome. In one embodiment, the IRES element comprises an RNA sequence of 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 4 0 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 embodiments, the IRES element may be any of a variety of genes, including, but not limited to, viral, mammalian, and phenotypic genes. They are derived from the DNA of organisms including the fruit fly (Drosophila). The DNA includes, but is not limited to, encephalomyocarditis virus (EMCV) cDNA and poliovirus The vector may be derived from picornavirus complementary DNA (cDNA) as well as from a picornavirus cDNA. In this state, the Drosophila DNA from which the IRES element is derived Examples include, but are not limited to, Drosophila melanogaster An example is the antennapedia gene derived from P. anogaster.

[0146] In one embodiment, the IRES element is derived at least in part from a virus, e.g., For example, it is ABPV_IGRpred, AEV, ALPV_IGRpred, BQCV _IGRpred, BVDV1_1-385, BVDV1_29-391, CrPV_5 NCR, CrPV_IGR, crTMV_IREScp, crTMV_IRESmp75 , crTMV_IRESmp228, crTMV_IREScp, crTMV_IRES cp, CSFV, CVB3, DCV_IGR, EMCV-R, EoPV_5NTR, ER AV_245-961, ERBV_162-920, EV71_1-748, FeLV- Notch2, FMDV_type_C, GBV-A, GBV-B, GBV-C, gyp sy_env, gypsyD5, gypsyD2, HAV_HM175, HCV_typ e_1a, HiPV_IGRpred, HIV-1, HoCV1_IGRpred, HR V-2, IAPV_IGRpred, idefix, KBV_IGRpred, LINE -1_ORF1_-101_to_-1, LINE-1_ORF1_-302_to_- 202, LINE-1_ORF2_-138_to_-86, LINE-1_ORF1_ -44_to_-1, PSIV_IGR, PV_type1_Mahoney, PV_t ype3_Leon, REV-A, RhPV_5NCR, RhPV_IGR, SINV1 _IGRpred, SV40_661-830, TMEV, TMV_UI_IRESmp 228, viral IRES such as TRV_5NTR, TrV_IGR, or TSV_IGR In one embodiment, the IRES element can be derived from AML1 / RUNX1, A ntp-D, Antp-DE, Antp-CDE, Apaf-1, Apaf-1, AQP 4, AT1R_var1, AT1R_var2, AT1R_var3, AT1R_var 4, BAG1_p36delta236nt, BAG1_p36, BCL2, BiP_- 222_-3, c-IAP1_285-1399, c-IAP1_1313-1462, c-jun, c-myc, Cat-1_224, CCND1, DAP5, eIF4G, e IF4GI-ext, eIF4GII, eIF4GII-long, ELG1, ELH, FGF1A, FMR1, Gtx-133-141, Gtx-1-166, Gtx-1-1 20, Gtx-1-196, hairless, HAP4, HIF1a, hSNM1, H sp101, hsp70, hsp70, Hsp90, IGF2_leader2, Kv1 .4_1.2, L-myc, LamB1_-335_-1, LEF1, MNT_75-2 67, MNT_36-160, MTG8a, MYB, MYT2_997-1152, n- MYC, NDST1, NDST2, NDST3, NDST4L, NDST4S, NRF_ -653_-17, NtHSF1, ODC1, p27kip1, p53_128-269 , PDGF2 / c-sis, Pim-1, PITSLRE_p58, Rbm3, reap er, Scamper, TFIID, TIF4631, Ubx_1-966, Ubx_3 73-961, UNR, Ure2, UtrA, VEGF-A_-133_-1, XIAP _5-464, XIAP_305-466, or YAP1 In one embodiment, the IRES element is derived in part from a synthetic IRES, such as (G AAA)16, (PPT19)4, KMI1, KMI1, KMI2, KMI2, KMIX , including X1 or X2.

[0147] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) polyribonucleotide or hybrid modified cyclic polyribonucleotide) At least one IR adjacent to the expressed sequence (e.g., 2, 3, 4, 5, or more). In one embodiment, the IRES comprises at least one (e.g., two, three, four) IRES. In one embodiment, the modified circular Polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified The resulting polyribonucleotides are present on one or both sides of each expression sequence. It contains one or more IRES sequences that provide for the isolation of peptides and / or polypeptides.

[0148] Termination element In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used to express one or more expression sequences. Each expression sequence may or may not have a termination element. In the form of a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleoside) a polyribonucleotide (a nucleotide or hybrid modified circular polyribonucleotide) containing one or more expression sequences, The expression sequence may be a modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide). The termination element is used to ensure that the polyribonucleotide (polyribonucleotide or hybrid modified circular polyribonucleotide) is continuously translated. The elimination of termination elements allows for the expression product to be expressed without ribosome stalling or dropping off. For example, rolling circle translation or continuous expression of peptides or polypeptides. In such an embodiment, rolling circle translation may occur through each expressed sequence. In certain other embodiments, the termination element of the expression sequence is It may be part of a stagger element. In certain embodiments, a modified cyclic polyribonucleotide (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) One or more of the expression sequences in the modified circular polyribonucleotides (nucleotides) contain a termination element. Nucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in Rolling circle translation or expression occurs. In such cases, the ribosome binds to the termination element. When a stop codon is encountered, for example, and translation is terminated, the expression product falls off the ribosome. In certain embodiments, translation occurs at least one of the ribosomes, e.g. is terminated while remaining in contact with the modified circular polyribonucleotide.

[0149] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used to express one or more expression sequences. In one embodiment, one or more expression sequences may be arranged in a sequence of two or more sequences, each of which may contain a termination element at the end of the sequence. In such an embodiment, translation is terminated and a rolling circle is formed. In one embodiment, the ribosome is transformed into a modified circular polyribonucleic acid. In some such embodiments, the modified circular polyribonucleoside is completely released from the nucleotide. nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) The generation of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the Before the start of translation, ribosomes react with modified circular polyribonucleotides (e.g., fully modified circular polyribonucleotides). ribonucleotide or hybrid modified cyclic polyribonucleotide) Generally, a termination element may require three in-frame nucleotides to indicate the end of translation. In one embodiment, the modified circular polyribonucleotides include a set of nucleotides, e.g., UAA, UGA, UAG. Nucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) One or more termination elements in the ribonucleotides (including, but not limited to, ribonucleotides) can terminate translation. Off-frame or -1 and +1 shifted reading frames (e.g., hidden stops) ) and other frameshift termination elements. Frameshift termination elements are located at the second and third loci of an expression sequence. and the nucleotide triplets TAA, TAG, and TG appearing in the third reading frame. A. Frameshift termination elements cause misreading of mRNA, which is often harmful to the cell. This can be important in preventing

[0150] Stagger element In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified circular polyribonucleotides) are used to flank the expression sequence. In one embodiment, the modified circular polyribonucleic acid comprises at least one staggered element. nucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) In one embodiment, the polynucleotides comprise staggered elements flanking each expressed sequence. The stagger element may be present on one or both sides of each expression sequence to enhance the expression product, e.g., peptide. In one embodiment, the staggered element provides separation of the nucleotides and / or polypeptides. In one embodiment, the modified cyclic polyribonucleotide is part of one or more expression sequences. (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) The polypeptide comprises one or more expression sequences, each of which is a modified circular polypeptide. It is separated from the subsequent expression sequence by a staggered element of ribonucleotides. In embodiments, the staggered elements are derived from (a) two translations of a single expressed sequence; or (b) ) Prevent the production of a single polypeptide from more than one translation of two or more expression sequences. In some embodiments, the stagger element is a sequence separated from one or more expressed sequences. In one embodiment, the staggered element comprises a portion of one or more expressed sequences. .

[0151] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) contain a staggered element. While maintaining rolling circle translation, continuous expression products, such as peptides or The stagger element induces ribosome pausing during translation to avoid polypeptide production. In one embodiment, a stagger element may be included to enhance the expression of one or more of the expressed sequences. The stagger element is located at the 3' end of at least one of the modified circular polyribonucleotides. Stagger elements can be configured to stall ribosomes during ring circle translation. Examples include, but are not limited to, 2A-like or CHYSEL (cis-acting hydrolase element) In one embodiment, the staggered element has the sequence: X1X2X3EX5NPG P (wherein X1 is absent or is G or H, and X2 is absent or or D or G, X3 is D, V, I, S, or M, and X5 is It encodes a sequence having a C-terminal consensus sequence of , any amino acid. In certain embodiments, the sequence is a non-conserved sequence of amino acids with a strong alpha helix propensity, followed by The consensus sequence is -D(V / I)ExNPG P (where x = any amino acid) Some non-limiting examples of stagger elements include GDVESNPGP, G DIEENPGP, VEPNPGP, IETNPGP, GDIESNPGP, GDVEL NPGP, GDIETNPGP, GDVENPGP, GDVEENPGP, GDVEQN PGP, IESNPGP, GDIELNPGP, HDIETNPGP, HDVETNPG P, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPG P and DSEFNPGP.

[0152] In some embodiments, the stagger elements described herein are The expression product is cleaved, for example, between the G and P of the consensus sequence. , modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid The hybrid-modified cyclic polyribonucleotide is a nucleotide sequence that cleaves the expression product. In some embodiments, the modified cyclic polyribonucleotide (e.g., , fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a stagger element flanking at least one expression sequence. Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrids) The lid-modified circular polyribonucleotide contains a stagger element after each expression sequence. In embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) The nucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) contain staggered elements and are Tagger elements are present on one or both sides of each expressed sequence to identify individual peptides from each expressed sequence. This results in the translation of the peptide and / or polypeptide.

[0153] In one embodiment, the stagger element comprises one or more ribosomes that induce pausing of the ribosome during translation. The non-natural nucleotides include the above modified nucleotides or non-natural nucleotides. Nucleic Acids (PNA), Morpholino and Locked Nucleic Acids (LNA) and Glycol Nucleic Acids (GNA Examples such as these may include threose nucleic acid (THN) and threose nucleic acid (TNA). Exemplary modifications include sugars, nucleobases, nucleotides, and for interosidic bonds (e.g., phosphate / phosphodiester bonds / phospho Any modifications (to the diester backbone) and those that can induce ribosome pausing during translation Some of the exemplary modifications provided herein may be used in combination with other modifications. These are described elsewhere in the Specification.

[0154] In some embodiments, the stagger element is an otherwise modified cyclic polyribonucleotide ( For example, fully modified circular polyribonucleotides or hybrid modified circular polyribonucleo- nucleotides. For example, in one exemplary modified cyclic polyribonucleotide, The tagger element is a termination element of the first expression sequence and a first A nucleotide spacer separating the termination element from the first translation initiation sequence following the expression sequence In some instances, the first staggered element of the first expression sequence comprises a modified circular The first expression sequence in the polyribonucleotide is followed by the first translation initiation sequence upstream (5' In some cases, the first expression sequence and the expression sequence following the first expression sequence are located at the end of the first expression sequence. are two separate expression sequences in a modified circular polyribonucleotide. The distance between the first expressed sequence and the first translation initiation sequence is the contiguous length of the first expressed sequence and the subsequent expressed sequence. In one embodiment, the first stagger element may include a stop element. and separates the expression product of the first expression sequence from the expression product of the subsequent expression sequence, thereby In some cases, modified cyclic polyribonucleotides (e.g., Fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) a modified circular polyribonucleotide containing a first staggered element upstream of the first translation initiation sequence of the subsequent sequence of Nucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) The first expressed sequence following the second expressed sequence is translated sequentially. The corresponding modified circular polynucleotides contain a staggered element of the second expression sequence upstream of the translation initiation sequence of the second expression sequence. Polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified In some cases, modified cyclic polyribonucleotides (cyclic polyribonucleotides) are not translated continuously. There is only one expression sequence in the ribonucleotide, and the first expression sequence and its subsequent expression sequences The sequences are the same expression sequences. The tagger element is a first termination element and a first expression sequence in the modified circular polyribonucleotide. It contains a nucleotide spacer sequence that separates the termination element from the downstream translation initiation sequence. In such an example, the first stagger element is the first nucleotide in the modified circular polyribonucleotide. It is located upstream (5' end) of the first translation initiation sequence of the target sequence. The distance between the tagger element and the first translation initiation sequence is determined by the distance between the first expression sequence and any subsequent expression sequences. In one embodiment, the first stagger element is a first the expression product of one round of the expression sequence from the expression product of the next round of the first expression sequence, thereby In some cases, modified cyclic polyribonucleotides (e.g., , fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) a modified circular polypeptide containing a first stagger element upstream of a first translation initiation sequence of a first expression sequence in Polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified The cyclic polyribonucleotides (CPNPs) are translated continuously, while the corresponding modified CPNPs are nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) a staggered element upstream of the second translation initiation sequence of the second expression sequence in the ribonucleotide sequence corresponding modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) or hybrid modified circular polyribonucleotides) are not translated continuously. In the method, the distance between the second stagger element and the second translation initiation sequence is Nucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) The distance between the first stagger element in the ribonucleotide sequence and the first translation start site is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold In some cases, the first staggered element and the first translational opening The distance between the start and end of the sequence must be at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, or 8nt. t, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt , 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt , 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt or the like In one embodiment, the distance between the second stagger element and the second translation start is greater than At least 2nt, 3nt, 4nt greater than the distance between the first stagger element and the first translation start t, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13n t, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25n t, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65n In some embodiments, the modified cyclic polynucleotide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, Ribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic A polyribonucleotide (ribonucleotide-like structure) contains two or more expression sequences.

[0155] regulatory nucleic acids In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) encode regulatory nucleic acids. and, for example, one or more expression sequences that regulate the expression of endogenous and / or exogenous genes. In certain embodiments, the modified circular polyribonucleic acid provided herein comprises nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) The expression sequences of nucleotides include, but are not limited to, tRNA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, sn Non-coding RNAs such as exRNA, scaRNA, Y RNA, and hnRNA The nucleic acid may include a sequence that is antisense to a regulatory nucleic acid such as a target RNA.

[0156] In one embodiment, the regulatory nucleic acid targets a host gene. International Patent Publication No. WO 2019118919A, which is incorporated herein by reference. Regulatory nucleic acids described in

[0177] and

[0181] to

[0189] of Pamphlet No. 1 One of the following may be mentioned.

[0157] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) The guide RNA (g) is a polyribonucleotide or hybrid modified cyclic polyribonucleotide. In one embodiment, the modified cyclic polyribonucleotide comprises a regulatory nucleic acid such as a modified cyclic polyribonucleotide (e.g., a modified cyclic polyribonucleotide). (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) The gRNA fragment (or fragments) contains or encodes a guide RNA. The RNA contains the "scaffold" sequence and genomic target required to bind to the defective effector moiety. It consists of a user-defined targeting sequence of approximately 20 nucleotides for RNA sequences generally consist of 17 to 24 nucleotides (e.g., 19, 20, or 21 nucleotides). The custom nucleic acid sequence is designed to be complementary to the target nucleic acid sequence. gRNA Generators and Algorithms to Use in Designing Effective Guide RNAs Gene editing mimics the natural crRNA-tracrRNA complex and racrRNA (to bind to the nuclease) and at least one crRNA (to edit (to guide the nuclease to the sequence targeted for A chimeric "single guide RNA" ("sgRNA"), a (synthetic) single RNA molecule Chemically modified sgRNAs have also been demonstrated to be effective for genome editing. For example, Hendel et al. (2015) Nature Biotechno See l., 985-991.

[0158] gRNAs encode specific DNA sequences (e.g., promoters, enhancers, silencers, etc.) that encode genes. The gene may recognize a sequence adjacent to or within a promoter or repressor.

[0159] In one embodiment, the gRNA is part of a CRISPR system for gene editing. For gene editing, modified circular polyribonucleotides (e.g., fully modified The modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide may be and the target DNA sequence of the target gene can be designed to include one or more guide RNA sequences corresponding to the target DNA sequence of the target gene, For example, Cong et al. (2013) Science, 339:819-823; Ran et al.(2013)Nature Protocols,8:2281- See 2308. At least about 16 or 17 nucleotides of the gRNA sequence are is required by Cas9 for cleavage to occur; for Cpf1, the gRNA sequence At least about 16 nucleotides of the sequence are required to achieve detectable DNA cleavage. do.

[0160] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid A hybrid-modified cyclic polyribonucleotide (BMP) is a nucleotide that regulates the expression of RNA encoded by a gene. Multiple genes may share some degree of sequence homology with each other, so in some instances In embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) are used. modified cyclic polyribonucleotides) have sufficient sequence homology In some embodiments, the modified cyclic polynucleotides can be designed to target specific classes of genes. Ribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic Polyribonucleotides (polyribonucleotides) can be shared between different gene targets or specific to a specific gene target. In certain embodiments, the modified ring may contain a sequence that is complementary to a sequence that is unique to the modified ring. cyclic polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid Modified cyclic polyribonucleotides (Cyclic Polyribonucleotides) are RNA sequences that share homology among several genes. Targeting conserved regions, thereby identifying gene families (e.g., different gene isoforms) It is designed to target several genes in the In some embodiments, modified cyclic polyribonucleotides (e.g., completely Modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) are They can be designed to target sequences unique to a particular RNA sequence of a gene.

[0161] In one embodiment, the expressed sequence is less than 5000 bp (e.g., about 5000 bp, 4 000bp, 3000bp, 2000bp, 1000bp, 900bp, 800bp, 7 00bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp , 50bp, 40bp, 30bp, 20bp, 10bp or less) In one embodiment, the expression sequences may independently or additionally comprise more than 10 bp ( For example, at least about 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 5 00bp, 600bp, 700bp, 800bp, 900bp, 1000kb, 1.1k b, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1. 8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2. 5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3. 2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, 4.9kb, 5kb or more) Has.

[0162] In certain embodiments, the expression sequence exhibits one or more of the characteristics described herein, e.g., one or more of the characteristics described herein. one or more peptide or protein coding sequences, one or more regulatory elements, one or more regulatory Nucleic acids, such as one or more non-coding RNAs, other expressed sequences, and any combination thereof Includes combinations.

[0163] Translation Efficiency In some embodiments, the modified cyclic polyribonucleotides provided herein ( For example, fully modified circular polyribonucleotides or hybrid modified circular polyribonucleo- nucleotides. The translation efficiency of the polypeptides (nucleotides) is measured using a reference, e.g., linear equivalents, linear expression sequences, linear modified circular polyribonucleic acids, nucleotide, or a fully modified circular polyribonucleotide equivalent. In the present invention, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) are cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used in the translation of the reference Translation efficiency is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300% ,350%,400%,450%,500%,600%,70%,800%,900%, 1000%, 2000%, 5000%, 10000%, 100000% or higher In some embodiments, modified cyclic polyribonucleotides (e.g., Fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) In one embodiment, the modified cyclic Polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified Modified circular polyribonucleotides (PCNs) have a translation efficiency 300% higher than that of their linear counterparts In some embodiments, the hybrid modified cyclic polyribonucleotide is a fully modified cyclic polyribonucleotide. It has a translation efficiency 10% higher than that of its polyribonucleotide equivalent. In this case, the hybrid modified cyclic polyribonucleotide is a fully modified cyclic polyribonucleotide. In one embodiment, the hybrid has a translation efficiency that is 300% higher than that of its counterpart. The modified cyclic polyribonucleotides have less cleavage than the corresponding cyclic polyribonucleotides. At most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 5 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1 00%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 4 00%, 450%, 500%, 600%, 70%, 800%, 900%, 1000%, 2 000%, 5000%, 10000%, 100000% or more translation efficiency In some embodiments, the hybrid modified cyclic polyribonucleotide is It has a translation efficiency at least about 10% higher than that of cyclic polyribonucleotides. In embodiments, the hybrid modified cyclic polyribonucleotide is a corresponding cyclic polyribonucleotide. The translation efficiency is at least about 20% higher than that of nucleotides. Thus, the hybrid modified cyclic polyribonucleotide is a corresponding cyclic polyribonucleotide. The translation efficiency is at least about 50% higher than that of

[0164] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used to express stoichiometric ratios Rolling circle translation produces a series of expression products in essentially equal proportions. In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified Cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) are expressed In some embodiments, the translation efficiency is stoichiometric, such that the products are produced in substantially equal proportions. In the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide), or hybrid modified cyclic polyribonucleotides) can be used to produce multiple expression products, e.g., , 4, 5, 6, 7, 8, 9, 10, 11, 12, or more expressed sequences. It has stoichiometric translation efficiency.

[0165] Rolling circle translation In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Once translation of the polyribonucleotide or hybrid modified circular polyribonucleotide is initiated , modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid Ribosomes bound to the hybrid-modified cyclic polyribonucleotide (GPP) react with the modified cyclic polyribonucleotide. Before completing at least one translation of a nucleotide, a modified circular polyribonucleotide (e.g. For example, a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide. In one embodiment, the modified circular polyribonucleic acid described herein does not leave the nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) ribonucleotides) are capable of rolling circle translation. During rolling circle translation, modified circular polyribonucleotides (e.g., fully modified circular Polyribonucleotides or hybrid modified circular polyribonucleotides) are used to initiate translation. Once the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or The ribosome bound to the hybrid modified cyclic polyribonucleotide (a modified cyclic polyribonucleotide) at least 2 times, at least 3 times, at least 4 times, at least 5 times at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 1 0 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, At least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, At least 100 times, at least 150 times, at least 200 times, at least 250 times, At least 500 times, at least 1000 times, at least 1500 times, at least 200 0 times, at least 5000 times, at least 10000 times, at least 10 5 Once or a few times At most 10 6 Before completing translation, modified circular polyribonucleotides (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) do not have.

[0166] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Rolling circle of polyribonucleotides or hybrid modified circular polyribonucleotides The type of translation may involve modified circular polyribonucleotides (e.g., fully modified circular polyribonucleotides). or hybrid modified circular polyribonucleotides) This results in the production of a peptide product (a "continuous" expression product). Cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified circular polyribonucleotide) contains a staggered element and is a modified circular polyribonucleo nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Rolling circle translation of modified circular polyribonucleotides (e.g., Fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) Polypeptide products generated from a single translation or from less than a single translation ("distinct" expression products) In certain embodiments, modified cyclic polyribonucleotides (e.g., complete polyribonucleotides) are produced. fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrids) All polyribonucleotides (lid-modified circular polyribonucleotides) produced during rolling circle translation At least 10%, 20%, 30%, 40%, 50%, or less of the polypeptide (mol / mol) at least 60%, at least 70%, at least 80%, at least 90%, at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99%, or In one embodiment, all polypeptides are composed of 100% distinct polypeptides. The ratio of the amounts of different products across the polypeptides is tested in an in vitro translation system. In an embodiment, the in vitro translation system used to test the dosage ratio is rabbit reticulocyte lysate. In some embodiments, the ratio of amounts of eukaryotic or prokaryotic cells, cultured cells, or The test is carried out in an in vivo translation system, such as a cell within an organism.

[0167] Untranslated region In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified circular polyribonucleotides are used in the untranslated region (UT) The UTRs of genomic regions containing genes can be transcribed but are not translated. In one embodiment, the UTR is upstream of the translation start sequence of the expressed sequence described herein. In one embodiment, the UTR may be included downstream of the expression sequence described herein. In some cases, one UTR for a first expression sequence may be included in a second expression sequence. It is the same as, contiguous with, or overlaps with another UTR for the sequence. In certain embodiments, the intron is a human intron. In this case, the intron is a full-length human intron, such as ZKSCAN1.

[0168] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) polyribonucleotide or hybrid modified cyclic polyribonucleotide) embedded in These AU links include UTRs with one or more stretches of adenosines and uridines. The ch signature may increase the turnover rate of the expression product.

[0169] The introduction, removal, or modification of UTR AU-rich elements (AREs) results in modified circular polyribonucleic acid (MPE) fragments. Certain modified circular polyribonucleotides may be useful for modulating the stability or immunogenicity of the peptide. When the nucleotide is modified, one or more copies of the ARE are inserted into the modified circular polyribonucleic acid. nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) The ARE may be introduced into a target gene (e.g., a nucleic acid fragment) and copies of the ARE may regulate translation and / or production of the expression product. Similarly, AREs can be modified circular polyribonucleotides (e.g., fully modified circular polyribonucleotides). and removed or modified in the These proteins are altered to regulate intracellular stability and thus affect translation and production of the resulting protein. Can be given.

[0170] Any UTR from any gene may be flanked by modified circular polyribonucleotides. It should be understood that the modified rings provided herein may be incorporated into the regions. cyclic polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid Exemplary UTRs that can be used in modified cyclic polyribonucleotides include those that are International Patent Publication No. WO 2019118919A1, which is incorporated herein by reference. Examples include those listed in issue brochures

[0200] to

[0201] .

[0171] Poly(A) sequence In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides contain poly-A sequences. In certain embodiments, the length of the poly-A sequence is greater than 10 nucleotides in length. In one embodiment, the poly-A sequence is greater than 15 nucleotides in length (e.g., at least Also about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80 , 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,80 0, 1,900, 2,000, 2,500, and 3,000 nucleotides or more In one embodiment, the poly-A sequence is International Patent Publication Number WO 2019118919A1 Brochure

[0202] ~ Designed according to the description of the poly-A sequence in

[0204] .

[0172] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) polyribonucleotide or hybrid modified cyclic polyribonucleotide) contains poly A or Modulating one or more properties of polyA-depleted or modified cyclic polyribonucleotides In one embodiment, the poly-A-deficient or modified poly-A-containing modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or half-modified cyclic polyribonucleotides) that Hybrid modified cyclic polyribonucleotides (Hybrid modified cyclic polyribonucleotides) can be modified to have one or more functional properties, e.g., immunogenicity. , half-life, expression efficiency, etc.

[0173] RNA binding In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides (or hybrid modified cyclic polyribonucleotides) are composed of one or more RNA The microRNA (or miRNA) binds to the 3'UTR of a nucleic acid molecule. downregulating gene expression by reducing nucleic acid molecule stability or inhibiting translation modified circular polyribonucleotides (e.g., complete polyribonucleotides) Modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) is 1 containing one or more microRNA target sequences, microRNA sequences, or microRNA seeds Such sequences are described in U.S. Patent Application No. 2005 / 0129994, the entire contents of which are incorporated herein by reference. Publication No. 2005 / 0261218, U.S. Patent Application Publication No. 2005 / 005900 Specification No. 5 and International Patent Publication No. WO 2019118919A1 pamphlet Any known microRNA, such as those taught in

[0207] to

[0215] of It can correspond to A.

[0174] Protein binding In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used to prepare proteins, e.g. For example, one or more tags that allow a ribosome to bind to an internal site in the RNA sequence. Protein binding sites, such as ribosome binding sites, can be modified into cyclic polynucleotides. By engineering modified cyclic polyribonucleotides (e.g., Fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) , modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) from components of the host's immune system. masking polyribonucleotides or hybrid modified cyclic polyribonucleotides thereby evading the host's immune system or having reduced detection by the host's immune system. The protein may have regulated degradation or regulated translation.

[0175] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be used to inhibit, for example, immune responses. For example, at least one immune target may be added to avoid a CTL (cytotoxic T lymphocyte) response. In certain embodiments, the immune protein binding site comprises an immune protein. Binding to a protein, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) modified cyclic polyribonucleotides) as exogenous In one embodiment, the immunoglobulin is a nucleotide sequence that serves to identify an immunoglobulin. The moiety binds to an immunity protein and is a modified cyclic polyribonucleotide (e.g., a fully modified cyclic Polyribonucleotides (cyclic or hybrid modified cyclic polyribonucleotides) are exogenously or It is a nucleotide sequence that serves to mask it as foreign.

[0176] The conventional mechanism of ribosome engagement on linear RNA is the capped 5' end of the RNA. The ribosome binds to the 5' end of the ribosome, which then moves to the initiation codon. The first peptide bond is then formed. According to the present invention, the modified cyclic polyribonucleotide (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Internal initiation of translation (i.e., cap-independent) of the nucleotide sequence (nucleotide) can occur at free or capped ends. Rather, the ribosome binds to an internal, non-capped site and This allows the ribosome to begin polypeptide elongation at the initiation codon. In the present invention, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or (hybrid modified cyclic polyribonucleotide) is a ribosome binding site, e.g., an initiation codon. It contains one or more RNA sequences containing donors.

[0177] Natural 5'UTRs have characteristics that play a role in translation initiation. It is generally known that the ribosomal protein is involved in the process of initiating the translation of many genes. The Kozak sequence has a signature similar to that of the consensus CCR(A / G)CCAUGG, where R is a purine ( The 5'UTR is an elongation factor binding site. It is also known to be involved in secondary structures involved in binding.

[0178] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are bound to proteins In one embodiment, the protein binding sequence encodes a protein binding sequence that: Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrids) Lid-modified cyclic polyribonucleotides) to target or localize them to specific targets. In one embodiment, the protein binding sequence is located in an arginine-rich region of the protein. Specific binding.

[0179] In one embodiment, the protein binding site includes, but is not limited to, ACIN1 , AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, C APRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CST F2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DG CR8, EIF3A, EIF4A3, EIF4G2, ELAVL1, ELAVL3, FA M120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR 2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, H NRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2 BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, L IN28A, LIN28B, m6A, MBNL2, METTL3, MOV10, MSI1 , MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2 , POLR2A, PRPF8, PTBP1, RBFOX2, RBM10, RBM22, R BM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SRSF1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1, TNRC 6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF 1, WDR33, XRN2, YBX1, YTHDC1, YTHDF1, YTHDF2, Y WHAG, ZC3H7B, PDK1, AKT1 and any other proteins that bind to RNA Examples include binding sites for proteins such as proteins.

[0180] Encryptogen As described herein, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides (ribonucleotides or hybrid-modified cyclic polyribonucleotides) are essential for the regulation of cellular innate immunity. In one embodiment, the compound includes an encryptogen to reduce, avoid, or prevent a response. When delivered to a cell, the reference compound, e.g., the modified circular polyribonucleic acid described nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) ribonucleotide), the corresponding unmodified cyclic polyribonucleotide, lacking the encryptogen modification Responses evoked by linear polynucleotides corresponding to circular polyribonucleotides Modified cyclic polyribonucleotides ( For example, fully modified circular polyribonucleotides or hybrid modified circular polyribonucleo- nucleotides. In one embodiment, a modified circular polyribonucleic acid (e.g., a ribonucleotide) is provided herein. nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) nucleotides) are less immunogenic than their counterparts lacking the encryptogen.

[0181] In certain embodiments, the encryptogen enhances stability. There is increasing evidence for the regulatory role that UTRs play in the stability of genes. The nodal features are encoded to improve the stability of the modified cyclic polyribonucleotide. It can be included in Gen.

[0182] In one embodiment, the 5' or 3' UTR is an amino acid sequence of an amino acid sequence in a modified circular polyribonucleotide. For example, removal or modification of UTR AU-rich elements (AREs) The decoration may be useful for modulating the stability or immunogenicity of the modified cyclic polyribonucleotide. do.

[0183] In one embodiment, expressed sequences, such as AU-rich elements (ARs) in translatable regions, E) Removal of the modification modulates the stability or immunogenicity of the modified cyclic polyribonucleotide. It may be useful to

[0184] In one embodiment, the encryptogen is a miRNA binding site or any other non- For example, the modified circular polynucleotides described herein contain a binding site for a coding RNA. Polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular Incorporation of miR-142 into polyribonucleotides (pNPs) inhibits expression in hematopoietic cells. Proteins encoded in modified circular polyribonucleotides, as well as those that regulate The immune response to the virus can be reduced or eliminated.

[0185] In one embodiment, the encryptogen is a protein, e.g., an immunity protein, It contains one or more protein binding sites that allow it to bind to the NA sequence. The binding site can be engineered into the modified circular polyribonucleotide to Nucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) Polyribonucleotides are modified cyclic polyribonucleotides ( For example, fully modified circular polyribonucleotides or hybrid modified circular polyribonucleo- nucleotides. By masking the host's immune system, the In some embodiments, the target gene may have reduced detection, regulated degradation, or regulated translation. In the form of a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleoside) The ribonucleotides (tide or hybrid modified cyclic polyribonucleotides) can be used to inhibit, for example, immune responses, e.g., C It contains at least one immunity protein binding site to avoid a TL response. In this embodiment, the immunity protein binding site binds to the immunity protein and binds to the modified circular polyribonucleic acid. nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) ribonucleotides) as being exogenous. be.

[0186] In certain embodiments, the encryptogen comprises one or more modified nucleotides. Exemplary modifications include modifications to the sugar, nucleobase, and internucleoside linkage (e.g., phosphate linkages). / phosphodiester bond / phosphodiester backbone) and any modifications and modified cyclic poly and any combination thereof that can prevent or reduce the immune response to ribonucleotides. Some exemplary modifications provided herein are described in detail below. .

[0187] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) polyribonucleotide or hybrid modified cyclic polyribonucleotide) is a reference compound, e.g. For example, modified circular polyribonucleotides lacking modifications (e.g., fully modified circular polyribonucleo- nucleotides ). and the response evoked by the ribonucleotides (tide or hybrid modified cyclic polyribonucleotides). In comparison, one of the methods described elsewhere herein may be used to reduce the immune response from the host. The above modifications are included. In particular, the addition of one or more inosines is endogenous to the virus. It has been shown that RNA is distinct from ribosomal RNA. Yu,Z.et al.(2015)RNA editing by ADAR1 ma rks dsRNA as “self”.Cell Res.25,1283-128 Please refer to 4.

[0188] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) polyribonucleotides or hybrid-modified circular polyribonucleotides) for shRNA or an RNA sequence that can be processed into siRNA, NA or siRNA targets RIG-1 and reduces RIG-1 expression. -1 can sense exogenous circular RNA and results in the degradation of the exogenous circular RNA. Therefore, for RIG-1 targeting shRNA, siRNA or any other regulatory nucleic acid The circular polynucleotide having the sequence may be used to induce immunity against modified circular polyribonucleotides, e.g. For example, it may reduce host cellular immunity.

[0189] In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) stimulate the innate immune response of cells. In reducing, avoiding, or preventing the reaction, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) In some such embodiments, the modified cyclic Polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified A modified circular polyribonucleotide (CPN) consists of a poly(A) sequence, a 5' end, a 3' end, a phosphate group, and a hydrochloride group. The hydroxyl group may be absent, or any combination thereof.

[0190] Riboswitches In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are polyribonucleotides that contain one or more ribonucleotides. Including Itch.

[0191] Riboswitches are typically modified circular polynucleotides that can directly bind to small target molecules. Ribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic It is believed to be part of a polyribonucleotide complex, and its target binding is essential for RNA translation and expression. affecting product stability and activity (Tucker BJ, Breaker RR) 2005), Curr Opin Struct Biol 15(3):342-8) Therefore, modified circular polyribonucleotides containing riboswitches (e.g., fully modified circular polyribonucleotides) can be used. cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) directly involved in regulating its own activity in response to the presence or absence of a molecule. In the present study, riboswitches have regions of affinity, like aptamers, for distinct molecules. Therefore, in the broader context of the present invention, any application contained within a non-coding nucleic acid The tamers can be used to sequester molecules from bulk volume. Downstream reporting of events by the

[0192] In certain embodiments, the riboswitch may be used to inhibit, but is not limited to, transcription termination, translation initiation, or other transcriptional events. gene expression, including damage, mRNA self-cleavage, and alterations of splicing pathways in eukaryotes Riboswitches can affect gene expression by binding or removing a trigger molecule. Thus, modified circular polyribonucleosides containing riboswitches can function to regulate the function of the riboswitch. nucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) nucleotide) to conditions that activate, deactivate, or block the riboswitch, thereby Expression can be altered by, for example, terminating transcription or blocking ribosome binding to RNA. The binding of the trigger molecule or its analogue can alter the properties of the riboswitch. Depending on the gene, the expression of the RNA molecule may be reduced or prevented, or the expression of the RNA molecule may be enhanced. Some examples of riboswitches are described herein.

[0193] Cyclic di-GMP riboswitch, FMN riboswitch (also called RFN element), gl mS riboswitch, glutamine riboswitch, glycine riboswitch, lysine riboswitch L-box (also called L-box), PreQ1 riboswitch (e.g., PreQ1-l riboswitch) switch and PreQ1-ll riboswitch), purine riboswitch, SAH riboswitch Chi, SAM riboswitch, SAM-SAH riboswitch, tetrahydrofolate riboswitch , theophylline-binding riboswitch, thymine pyrophosphate-binding riboswitch, T. tengukon T. tengcongensis glmS catalytic riboswitch, TPP riboswitch switch (also called THI-box), Moco riboswitch, or adenine-sensing add-A riboswitches, each of which is incorporated herein by reference in its entirety. This is described in International Patent Publication No. WO 2019118919A1, brochure

[0235] to

[0252] .

[0194] Aptazymes In certain embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides) Polyribonucleotides or hybrid modified cyclic polyribonucleotides) contain aptazymes. Aptazymes are catalytic R&D products in which the aptamer domain is used as an allosteric control element. It is a switch for conditional expression that is bound to a region of NA ("ribozyme" described below). In certain embodiments, the aptazymes are active in cell-type specific translation. In some embodiments, the aptazyme is used to induce cell state-specific translation, e.g., in virally infected cells. or is active in the presence of viral nucleic acid or viral protein.

[0195] Ribozymes (derived from ribonucleic acid enzymes, also called RNA enzymes or catalytic RNAs) are It is an RNA molecule that catalyzes chemical reactions.

[0196] Some non-limiting examples of ribozymes include hammerhead ribozymes, VL ribozymes, and the like. Ribozymes, leadzymes, hairpin ribozymes, and the like, all of which are incorporated herein by reference in their entirety. Other ribozymes include those described in paragraphs

[0254] to

[0259] of International Patent Publication No. WO 2019118919A1.

[0197] In certain embodiments, the modified circular RNAs described herein are useful for transcription and replication of RNA. For example, circular RNAs can be used to identify non-coding RNAs, lncRNAs, mRNAs, and iRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRN In one embodiment, the circular RNA can be used to encode an antisense After transcription, such circular RNAs can be transformed into functional linear miRNAs. iRNA can be generated. Non-limiting examples of circular RNA expression and regulatory applications are listed in Table 3. do.

[0198] [Table 3]

[0199] target binding In one embodiment, modified circular RNA (e.g., fully modified circular polyribonucleotides) or hybrid modified cyclic polyribonucleotides) bind to one or more targets. In embodiments, the circular RNA binds to both DNA and protein targets, e.g. In another embodiment, a circular RNA (e.g., a fully modified circular polyribonucleotide) is used to mediate transcription. Polyribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) form protein complexes. In another embodiment, the circular RNA (e.g., For example, fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides. ) bind to two or more different targets, such as proteins, and bind these proteins, e.g. In one embodiment, the pharmaceutical composition comprises a hybrid modified cyclic polylysine. wherein the hybrid modified cyclic polyribonucleotide comprises at least a first target, e.g., RNA, DNA, protein, or a first portion comprising a first binding site configured to bind to a first binding portion of a cellular target; wherein the first binding moiety is a first circular polyribonucleoside consisting of unmodified nucleotides; a nucleotide sequence that is ... The modified cyclic polyribonucleotide forms a complex. In some embodiments, the pharmaceutical composition The article comprises a hybrid modified cyclic polyribonucleotide, wherein the hybrid modified cyclic The polyribonucleotide may contain at least one modified nucleotide; a first target, e.g., configured to bind to a first binding moiety of an RNA, DNA, protein, or cellular target; a first portion comprising a first binding site (wherein the first binding site is a non-modified nucleotide) a first cyclic polyribonucleotide (cyclic RNA) binding motif consisting of: a second binding site configured to bind to a second binding moiety of the target (wherein the second binding site is the first binding moiety comprises a second circular RNA-binding motif, and the second binding moiety comprises a The first target, the second target, and the hybrid modified circular polyribonucleoside are distinct from the binding moiety. The peptide forms a complex, and the first target or the second target is not a microRNA. In one embodiment, a pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, , the hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding moiety of a first target; wherein the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding moiety. and a second binding moiety configured to bind to a second binding moiety of a second target. a binding site (wherein the second binding moiety is a second circular RNA-binding motif), The first binding moiety is different from the second binding moiety, and both the first target and the second target are In one embodiment, the pharmaceutical composition is a hybrid modified circular polyribonucleotide. Nucleotides are proteins, peptides, biomolecules, and DNAs that consist of unmodified nucleotides. A, RNA, or a first portion comprising a binding site configured to bind to a cellular target. In some embodiments, the first portion described herein is composed of unmodified nucleotides. The compound may be a protein, peptide, biomolecule, DNA, RNA, or cellular target. The binding site is composed of:

[0200] In one embodiment, modified circular RNA (e.g., fully modified circular polyribonucleotides) or hybrid modified cyclic polyribonucleotides) are used to bind DNA, RNA, and proteins. and thereby modulating a cellular process (e.g., protein expression). In some embodiments, synthetic modified circular RNA (e.g., fully modified circular Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are selected Interaction with at least one moiety, e.g., a binding moiety, of DNA, RNA, or protein and thereby competes for binding with the endogenous counterpart.

[0201] In one embodiment, synthetic modified circular RNA (e.g., fully modified circular polyribonucleic acid The nucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) bind and / or sequester miRNAs. In another embodiment, synthetic modified circular RNA (e.g., fully modified circular polyribonucleic acid) is used. Polyribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) bind proteins and In another embodiment, the synthetic, modified circular RNA binds and / or sequesteres mRNA. In another embodiment, synthetic modified circular RNA (e.g., fully modified circular polynucleotides) is used. Ribonucleotides or hybrid modified cyclic polyribonucleotides) bind ribosomes In another embodiment, synthetic modified circular RNA (e.g., fully modified Cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides are In another embodiment, the synthetic modified circular RNA binds and / or sequesteres the long RNA. strand non-coding RNA (lncRNA) or any other non-coding RNA, e.g. For example, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, long chain Binds and / or sequesters non-coding RNAs and shRNAs. Binding and / or sequestration part In addition to the cleavage site, the modified circular RNA may contain a degradation element, which may be bound and / or sequestered. This results in the degradation of RNA and / or proteins.

[0202] In one embodiment, modified circular RNA (e.g., fully modified circular polyribonucleotide or (hybrid modified cyclic polyribonucleotide) is a molecule that encodes lncRNA or a sequence of lncRNA. For example, modified circular RNAs may be naturally occurring, non-circular lncRNAs or fragments thereof. In one embodiment, the lncRNA or the sequence of the lncRNA comprises a spacer. Circularization with or without a sequence can be used to produce synthetic modified circular RNAs (e.g., fully modified circular RNAs). The resulting polyribonucleotides are then cyclic or hybrid modified cyclic polyribonucleotides.

[0203] In one embodiment, modified circular RNA (e.g., fully modified circular polyribonucleotide or The hybrid modified cyclic polyribonucleotide has ribozyme activity. In some embodiments, modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid Modified cyclic polyribonucleotides (RbPs) act as ribozymes to bind pathogenic or endogenous RbPs. In one embodiment, the nucleic acid fragment can be used to cleave NA, DNA, small molecules, or proteins. , modified circular RNA (e.g., fully modified circular polyribonucleotides or hybrid modified circular In one embodiment, the synthetic modified cyclic R NA (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) nucleotide) can specifically recognize and cleave RNA (e.g., viral RNA). In another embodiment, modified circular RNA (e.g., fully modified circular polyribonucleic acid) can be used. Polyribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) specifically recognize proteins. and cleavage. In another embodiment, modified circular RNA (e.g., fully modified Cyclic polyribonucleotides (or hybrid modified cyclic polyribonucleotides) are small molecules can be specifically recognized and decomposed.

[0204] In one embodiment, modified circular RNA (e.g., fully modified circular polyribonucleotide or (hybrid modified cyclic polyribonucleotides) are immolating or is a self-cleaving or cleavable modified circular RNA. In one embodiment, the modified circular RNA (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Otides) are RNAs, e.g., miRNAs, tRNAs, rRNAs, snoRNAs, ncRs can be used to deliver RNA, siRNA, long non-coding RNA, and shRNA In one embodiment, synthetic modified circular RNA (e.g., fully modified circular polyribonucleoside) is used. (i) a self-cleavable element (e.g., a tide or hybrid modified cyclic polyribonucleotide) (2) cleavage recruitment sites (e.g., ADA R), (3) a degradable linker (glycerol), (4) a chemical linker, and / or (5 ) microRNAs separated by spacer sequences. In this regard, synthetic modified circular RNAs may be prepared by: (1) incorporating a self-cleavable element (e.g., hammerhead, spindle, or septum) splicing element), (2) cleavage recruitment site (e.g., ADAR), and (3) degradable linker (glycerol), (4) a chemical linker, and / or (5) a spacer sequence. It is composed of siRNAs spaced apart.

[0205] In one embodiment, the modified circular RNA is a transcribable / replicable modified circular RNA. modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular The polyribonucleotide may encode any type of RNA. Thus, the synthetic modified circular RNA comprises an antisense miRNA and a transcription element. In this study, after transcription, linear functional miRNAs are converted into modified circular RNAs (e.g., fully modified circular polymerases). Polyribonucleotides are produced from cyclic ribonucleotides or hybrid modified cyclic polyribonucleotides.

[0206] In one embodiment, modified circular RNA (e.g., fully modified circular polyribonucleotide or is a hybrid modified circular polyribonucleotide) in combination with a translation element to achieve the above attributes It has one or more of the following.

[0207] target Modified circular RNA (e.g., fully modified circular polyribonucleotides or hybrid modified circular The polyribonucleotide contains at least one binding site for a target binding moiety. Targets include, but are not limited to, nucleic acids (e.g., RNA, DNA, RNA-DNA hybrids, etc.). hybrids), small molecules (e.g., drugs), aptamers, polypeptides, proteins, lipids , carbohydrates, antibodies, viruses, virus particles, membranes, multicomponent complexes, cells, other cellular parts, Any fragment thereof, and any combination thereof (e.g., Fredriksson et al.,(2002)Nat Biotech 20: 473-77;Gullberg et al.,(2004)PNAS, 101:84 20-24). For example, the target may be a single-stranded RNA, a double-stranded RNA, a single-stranded DNA, or a DNA, double-stranded DNA, DNA containing one or more double-stranded regions and one or more single-stranded regions, or RNA, RNA-DNA hybrids, small molecules, aptamers, polypeptides, proteins , lipids, carbohydrates, antibodies, antibody fragments, antibody mixtures, virus particles, membranes, multicomponent It may be a complex, a cell, a cell part, any fragment thereof, or any combination thereof.

[0208] In certain embodiments, the target is a polypeptide, a protein, or any fragment thereof. For example, the target may be a purified polypeptide, an isolated polypeptide, a fusion-tagged polypeptide, or a polypeptides bound to or spanning the membrane of a cell, virus, or virion Desmosomes, cytoplasmic proteins, intracellular proteins, extracellular proteins, kinases, phosphatases enzymes, aromatases, helicases, proteases, oxidoreductases, reductases, Transferases, hydrolases, lyases, isomerases, glycosylases, extracellular Matrix proteins, ligases, ion transporters, channels, pores, apoptosis Proteins, cell adhesion proteins, pathogenic proteins, abnormally expressed proteins, transcription factors molecules, transcription factors, translation proteins, chaperones, secreted proteins, ligands, hormones , cytokines, chemokines, nuclear proteins, receptors, transmembrane receptors, signal transduction molecules , antibodies, membrane proteins, integral membrane proteins, peripheral membrane proteins, cell wall proteins, Globular proteins, fibrous proteins, glycoproteins, lipoproteins, chromosomal proteins , any fragment thereof, or any combination thereof. In one embodiment, the target is a heterologous polypeptide. The protein is overexpressed in cells using molecular techniques such as immunoprecipitation. In some embodiments, the target is a recombinant polypeptide. For example, the target may be a recombinant polypeptide of a bacterium (e.g., a bacterial Samples produced from E. coli, yeast, mammalian, or insect cells (e.g., In one embodiment, the target is in a protein (e.g., a protein overexpressed by the organism). In some embodiments, the polypeptide has a mutation, insertion, deletion, or polymorphism. The target may confer immunity to the organism or generate an immune response in the organism, such as antibody production. An antigen, such as a polypeptide, is used to

[0209] In one embodiment, the target is an antibody. An antibody binds to a specific spatial and polar target of another molecule. Antibodies can be monoclonal, polyclonal, or recombinant. The antibody may be an antibody, and may be prepared by methods known in the art, such as immunizing a host and collecting serum (polyclonal). or by preparing continuous hybrid cell lines and secreting secreted proteins (monopolysaccharides). By collecting (clonal) antibodies, or at least the specific binding required for natural antibodies, Cloning a nucleotide sequence encoding an amino acid sequence or a mutant form thereof Natural antibodies are held together by disulfide bonds. A protein containing at least two interconnected heavy (H) chains and two light (L) chains. Each heavy chain may comprise a heavy chain variable region (V H ) and a heavy chain constant region. The common region consists of three domains: C H1, C H2 and C H3 Each light chain may be composed of Chain variable region (V L The light chain constant region can be composed of one domain. N, C L V H and V L The region is called the framework region (FR). hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions Each V H and V L The sequence is as follows: FR1, CDR1, FR2, C DR2, FR3, CDR3, and FR4, arranged from amino to carboxy terminus It can consist of three CDRs and four FRs. The constant region of an antibody interacts with various cells of the immune system. (e.g., effector cells) and the first component of the classical complement system (C1q). It can mediate the binding of immunoglobulins to major tissues or factors. Antibodies can be of any isotype ( IgA, IgM, IgD, IgA, and IgY), class (e.g., Ig G1, lgG2, lgG3, lgG4, lgA1 and lgA2), subclasses or modifications thereof Antibodies may include intact immunoglobulins or fragments thereof. An antibody fragment is a fragment of an antibody that retains the ability to specifically bind to a binding moiety such as an antigen. It may refer to one or more fragments. Furthermore, it may refer to a fragment that maintains binding affinity for a particular molecule. Aggregates, polymers, and conjugates of immunoglobulins or their fragments are also acceptable, provided that they are not Examples of antibody fragments include Fab fragments, V fragments, and L , V H , C L and C H1Monovalent fragments consisting of domains; F(ab)2 fragment, hinge region A bivalent fragment containing two Fab fragments linked by a disulfide bridge in the Fragment;V H and C H1 Fd fragment containing domain; V of a single arm of an antibody L and V H Fv fragment consisting of domains; V H Single domain antibodies ( dAb) fragment (Ward et al., (1989) Nature 341: 544-46); and isolated CDRs and V L and V H The domains pair to form monovalent molecules. Single-chain fragments (scFv) (also known as single-chain Fv (scFv); e.g. For example, Bird et al., (1988) Science 242:423-26; See Huston et al., (1988) PNAS 85:5879-83. Therefore, examples of antibody fragments include Fab, F(ab)2, , scFv, Fv, dAb, etc. Two domains V L and V H But separate legacies Although they are encoded by genes, they can be engineered using recombinant methods to produce single proteins. These can be linked by artificial peptide linkers that allow them to be made into protein chains. Single chain antibodies, such as: The fragments can be obtained using conventional techniques known to those skilled in the art, and may be derived from intact antibodies. Antibodies can be human, humanized, chimeric, It may be isolated, dog, cat, donkey, sheep, any plant, animal, or mammal.

[0210] In certain embodiments, the target is a ribonucleotide and / or a deoxyribonucleic acid. polymeric forms of amino acids (adenine, guanine, thymine, or cytosine), such as DNA or RNA (e.g., mRNA). DNA can be a linear DNA molecule (e.g., a restriction fragment). ), viruses, plasmids, and double-stranded DNA found in chromosomes. In the present invention, the polynucleotide target may be a single-stranded, double-stranded, small interfering RNA (siRNA), Messenger RNA (mRNA), transfer RNA (tRNA), chromosome, gene, non-co loading genomic sequences, genomic DNA (e.g., fragmented genomic DNA), purified polynucleotides nucleotides, isolated polynucleotides, hybridized polynucleotides, transcription factor binding site, mitochondrial DNA, ribosomal RNA, eukaryotic polynucleotide, prokaryote Polynucleotides, synthesized polynucleotides, ligated polynucleotides, Recombinant polynucleotides, polynucleotides containing nucleic acid analogs, methylated polynucleotides a nucleotide, a demethylated polynucleotide, any fragment thereof, or any of these In some embodiments, the target is a recombinant polynucleotide. In embodiments, the target is a heterologous polynucleotide. For example, the target may be a bacterium (e.g., For example, polynucleotides produced from E. coli, yeast, mammalian, or insect cells. In some embodiments, the polynucleotide is a heterologous polynucleotide (e.g., a polynucleotide heterologous to the organism). Thus, the target is a polynucleotide that has a mutation, insertion, deletion, or polymorphism.

[0211] In certain embodiments, the target is an aptamer. Aptamers have high specificity and affinity. An aptamer is an isolated nucleic acid molecule that binds to a binding moiety, such as a protein, in a manner that is compatible with its target. held in a specific conformation that provides chemical contacts for specific binding to a given target Aptamers are nucleic acid-based molecules, but there are also two types of aptamers: There is a fundamental difference between the structure of the nucleic acid and other nucleic acid molecules such as mRNA. encodes information through its linear base sequence, and thus this sequence provides the function of information storage. In stark contrast, aptamer function is based on specific binding of target molecules. does not depend entirely on conserved linear base sequences (non-coding sequences), It depends on the specific secondary / tertiary / quaternary structure. The potential is entirely fortuitous and is dependent on the binding of the aptamer to its cognate target. Aptamers are also naturally occurring nucleic acid sequences that bind to specific proteins. These latter sequences are not found in natural nucleic acids (e.g., nucleic acid binding sites). binds to a specific subgroup of proteins involved in transcription, translation, and transport of Aptamers, on the other hand, are short, isolated, naturally occurring sequences embedded within the genome of an organism. Aptamers that bind to nucleic acid-binding proteins can be identified. However, in most cases, such aptamers are based on natural nucleic acid binding proteins. More importantly, the sequence has little or no sequence identity to sequences recognized by In fact, aptamers are known to bind to virtually all proteins (not just nucleic acid binding proteins) and They can bind to almost any partner of interest, including small molecules, carbohydrates, peptides, etc. For most partners, even proteins, there is no natural nucleic acid sequence to which they bind. For a partner having such a sequence, for example, a nucleic acid binding protein, Such sequences are relatively weakly used in nature compared to tightly binding aptamers. As a result of their low binding affinity, they differ from aptamers, which bind to selected partners. and capable of specifically binding to a partner and modulating the activity or binding interaction of the partner, For example, through binding, aptamers can block the ability of their partners to function. The functional property of specific binding to a partner is an intrinsic property of an aptamer. Aptamers are 6-35 kDa in size (20-100 nucleotides) and are Binds to its partner with molar to subnanomolar affinity and closely related targets can distinguish between related proteins from the same gene family (e.g., aptamers can be selected for related proteins from the same gene family). Aptamers bind to specific partners through hydrogen bonds, static bonds, and other mechanisms. Use commonly found intermolecular interactions such as electrocomplementarity, hydrophobic contacts, and steric exclusion Aptamers have the advantages of high specificity and affinity, low immunogenicity, and high biological efficacy. and excellent pharmacokinetic properties, for use as therapeutic and diagnostic agents. Aptamers are hybrids of covalently linked complementary polynucleotides. Molecular stem and loop structures (e.g., hairpin loop structures) formed from dimerization The stem may comprise a hybridized polynucleotide and the loop may comprise two It is a region that covalently binds two complementary polynucleotides.

[0212] In certain embodiments, the target is a small molecule. For example, the small molecule may be a macrocycle, an inhibitor, or a In certain embodiments, the small molecule may be a small molecule having five or fewer hydrogen atoms. In some embodiments, the small molecule contains 10 or fewer hydrogen bond acceptors. In one embodiment, the small molecule has a molecular weight of 500 daltons or less. In some embodiments, the small molecule has a molecular weight of about 180 to 500 daltons. In embodiments, the small molecule has an octanol-water partition coefficient lop P of 5 or less. In some embodiments, the small molecule comprises a partition coefficient log P between -0.4 and 5.6. In some embodiments, the small molecule has a molar refractive index between 40 and 130. In some embodiments, the small molecule contains from about 20 to about 70 atoms. 40 angstroms 2 It has the following polar surface area:

[0213] In certain embodiments, the target is a cell. For example, the target may be an intact cell, a transformed cell, or a transformed cell. Cells treated with a compound (e.g., a drug), fixed cells, lysed cells, or any of these. In some embodiments, the target is a single cell. Thus, the target is a plurality of cells.

[0214] In some embodiments, a single target or multiple (e.g., two or more) targets may be used to generate multiple results. In one embodiment, the single target has 2, 3, 4, 5, 6, 7, 8, 9 In one embodiment, a mixture of targets or a label may be used. Two or more targets, such as a library, are present in a sample, and the sample contains two or more binding sites. In some embodiments, a single target or multiple targets may be associated with multiple different binding moieties. For example, the plurality of targets can include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 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, 19,0 The compound may comprise 0, 20,000, 25,000, or 30,000 binding moieties.

[0215] A target may comprise multiple binding moieties, including at least two different binding moieties. For example, The binding moiety is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 9 0, 100, 200, 300, 400, 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, 19,00 0, 20,000, 21,000, 22,000, 23,000, 24,000, or 2 It may contain multiple binding moieties, including 5,000 different binding moieties.

[0216] Binding Sites and Binding Moieties In some cases, modified circular RNA (e.g., fully modified circular polyribonucleotide or The hybrid modified cyclic polyribonucleotide contains one binding site. wherein the first portion is a protein, peptide, biomolecule, or the like, which is composed of unmodified nucleotides. The binding site is configured to bind to a molecule, DNA, RNA, or a cellular target. In embodiments, the first portion is a protein, peptide, or configured to bind to biomolecules, DNA, RNA, or cellular targets, or combinations thereof. In some cases, the modified circular RNA (e.g., a fully modified Cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) are at least For example, modified circular RNAs may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or later In some embodiments, the modified circular RNA described herein may comprise the above binding site. (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) A nucleotide (nucleotide) is a molecular scaffold that binds to one or more binding moieties of one or more targets. Each target is , including but not limited to, different or the same nucleic acid (e.g., RNA, DNA, RNA-DNA hybrids), small molecules (e.g., drugs), aptamers, polypeptides, proteins, lipids proteins, carbohydrates, antibodies, viruses, virus particles, membranes, multicomponent complexes, cells, cell parts, etc. It may be any of these fragments, and any combination thereof. In some embodiments, one or more binding sites bind to one or more binding moieties of the same target. In some embodiments, one or more binding sites bind to one or more binding moieties of different targets. In embodiments, the modified circular RNA comprises a linker for one or more binding moieties of one or more targets. In one embodiment, modified circular RNA (e.g., fully modified circular polyribonucleotides) acts as a target. nucleotide or hybrid modified cyclic polyribonucleotide) is a nucleotide that binds to one or more targets. In some embodiments, the antibody specifically binds to one or more binding moieties to modulate a cellular process. In the modified circular RNA described herein (e.g., fully modified circular polyribonucleic acid), modified cyclic polyribonucleotides) are substituted with one or more specific nucleotides of interest. In one embodiment, the modified circular RNA (e.g., a complete Modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) are This includes multiple binding sites or combinations of binding sites for each binding moiety of interest. For example, modifications Circular RNA (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Ribonucleotides) are binding sites for polynucleotide targets such as DNA or RNA. For example, modified circular RNA (e.g., fully modified circular polyribonucleotides or hybrids) The hybrid-modified cyclic polyribonucleotide contains a binding site for the mRNA target. For example, modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified The modified cyclic polyribonucleotide (RNA) contains a binding site for the rRNA target. For example, The circular RNA comprises a binding site for a tRNA target. For example, a modified circular RNA (e.g., , fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) contains a binding site for a genomic DNA target.

[0217] In some cases, modified circular RNA (e.g., fully modified circular polyribonucleotide or Hybrid modified cyclic polyribonucleotides) are used for binding moieties in single-stranded DNA. In some cases, modified circular RNA (e.g., a fully modified circular polyribonucleotide) Polyribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used in double-stranded DNA. In some cases, the modified circular RNA (e.g., Modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used as anti- In some cases, the modified circular RNA (e.g., For example, fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides. ) contains a binding site for a binding moiety on the viral particle. Circular RNA (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) Ribonucleotides contain binding sites for binding moieties on small molecules. In this case, modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified The modified cyclic polyribonucleotide (PCP) provides a binding site for a binding moiety within or on a cell. In some cases, modified circular RNA (e.g., fully modified circular polyribonucleotides) or hybrid modified cyclic polyribonucleotides) in RNA-DNA hybrids In some cases, the modified circular RNA (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) In some cases, the methylated polynucleotide comprises a binding site for a binding moiety in the polynucleotide. , modified circular RNA (e.g., fully modified circular polyribonucleotides or hybrid modified circular methylated polyribonucleotides) are used as a binding site for the binding moiety in unmethylated polynucleotides. In some cases, modified circular RNA (e.g., fully modified circular polyribonucleic acid) Polyribonucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used for binding in aptamers. In some cases, the modified circular RNA (e.g., a fully modified circular RNA) comprises a binding site for a moiety. Polyribonucleotides (cyclic or hybrid modified cyclic polyribonucleotides) are polypeptides In some cases, the modified circular RNA (e.g., For example, a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide. (Do) refers to polypeptides, proteins, protein fragments, tagged proteins, antibodies , antibody fragments, small molecules, viral particles (e.g., viral particles containing transmembrane proteins) The antibody or antibody fragment contains a binding site for a binding moiety in a cell.

[0218] In some cases, the linking moiety includes at least two amide bonds. In some cases, the linking moiety does not contain a phosphodiester bond. Not NA or RNA.

[0219] As provided herein, modified circular RNAs (e.g., fully modified circular polyribonucleoside) are The nucleotides (polyribonucleotides or hybrid modified cyclic polyribonucleotides) serve as binding moieties in the complex. The modified circular RNAs provided herein (e.g., For example, fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides. ) may contain one or more binding sites for a target to form a complex. The modified circular RNA (e.g., fully modified circular polyribonucleotide or cyclic polyribonucleotide) provided in Hybrid modified cyclic polyribonucleotides are modified circular RNAs (e.g., fully modified circular between a polyribonucleotide or hybrid modified cyclic polyribonucleotide) and a target In some embodiments, a modified circular RNA (e.g., a fully modified circular polynucleotide) can form a complex. Polyribonucleotides or hybrid modified cyclic polyribonucleotides) are single-targeted In one embodiment, a modified circular RNA (e.g., a fully modified circular polynucleotide) is formed. ribonucleotides or hybrid modified cyclic polyribonucleotides) can be used to bind two or more targets In one embodiment, the modified circular RNA (e.g., the fully modified Modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides are In one embodiment, two or more modified cyclic R NA (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) In one embodiment, two or more modified nucleotides form a complex with a single target. Modified circular RNA (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) In one embodiment, the ribonucleotide complex forms a complex with two or more targets. A first modified circular RNA (e.g., a fully modified circular polyribonucleotide or a hybrid modified The modified cyclic polyribonucleotide (a modified cyclic polyribonucleotide) binds a first binding moiety of a first target and a second binding moiety of a second target. In one embodiment, the first modified circular RNA (e.g., For example, a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide. The modified circular RNA (e.g., a modified circular RNA) forms a complex with a first binding moiety of a first target and binds a second modified circular RNA (e.g., a modified circular RNA) to the first target. , fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) forms a complex with a second binding moiety of a second target.

[0220] In one embodiment, modified circular RNA (e.g., fully modified circular polyribonucleotides) or hybrid modified cyclic polyribonucleotides) may be used to form one or more antibody-polypeptide complexes. Complex, polypeptide-polypeptide complex, polypeptide-DNA complex, polypeptide -RNA complex, polypeptide-aptamer complex, virus particle-antibody complex, virus virus particle-polypeptide complex, virus particle-DNA complex, virus particle-RNA complex body, virus particle-aptamer complex, cell-antibody complex, cell-polypeptide complex, Cell-DNA complex, cell-RNA complex, cell-aptamer complex, small molecule-polypeptide complex Tide complexes, small molecule-DNA complexes, small molecule-aptamer complexes, small molecule-cell complexes, For one or more binding moieties in small molecule-virus particle conjugates, and combinations thereof It may contain a binding site.

[0221] In some cases, the binding moiety is on a polypeptide, protein, or fragment thereof. In certain embodiments, the binding moiety is a domain, fragment, epitope, region The present invention also includes a region, or a portion of a polypeptide, protein, or fragment thereof. For example, The binding moiety may be a domain, fragment, epitope, region, or isolated polypeptide, cell The present invention also includes a portion of a polypeptide, purified polypeptide, or recombinant polypeptide. For example, a binding moiety may be a domain, fragment, epitope, region, or fragment of an antibody or fragment thereof. For example, a binding moiety may be a domain, fragment, epitope, or part of a For example, a binding moiety may be a domain, fragment, epitope, or portion of a transcription factor. A binding moiety may comprise a domain, fragment, or portion of a receptor. Binding moieties include domains, fragments, epitopes, regions, or portions of transmembrane receptors. an element, epitope, region, or portion of an isolated, purified, and / or recombinant polypeptide The binding moiety may be on or comprise a domain, fragment, epitope, or , region, or binding moiety on a portion of a mixture of analytes (e.g., lysate) or For example, a binding moiety may be a domain, fragment, epitope, region, or multiple fragments. The present invention relates to a method for detecting a cellular component of a leukocyte, comprising administering to a subject the leukocyte or a mammalian cell ... the lysate of which is derived from a leukocyte or a mammalian cell lysate.

[0222] In some cases, the binding moiety may be a domain, fragment, epitope, region, or The binding moiety is on or comprises a portion of a small molecule. For example, the binding moiety may be a domain, a flag on or including a portion of a ment, epitope, region, or agent, e.g. The binding moiety may be a domain, fragment, epitope, region, or portion of a compound. For example, the binding moiety may be a domain, fragment, epitope, or , region, or part of an organic compound. The binding moiety may be a domain, fragment, epitope, region, or fragment of less than 900 daltons. In some cases, the binding moiety is on or includes a portion of a small molecule having a molecular weight. is a domain, fragment, epitope, region, or molecular weight greater than 500 daltons The binding moiety may be, for example, a binding means A compound library generated by the compound diversity combinatorial library Combinatorial libraries are obtained from libraries of natural or synthetic molecules. Libraries and methods for their generation and screening are well known in the art. and are known in the art, and are disclosed in U.S. Patent Nos. 5,741,713 and 5,734,018. Specification; Specification No. 5,731,423; Specification No. 5,721,099; No. 5,7 Specification No. 08,153; Specification No. 5,698,673; Specification No. 5,688,997 Specification; Specification No. 5,688,696; Specification No. 5,684,711; Specification No. 5,6 Specification No. 41,862; Specification No. 5,639,603; Specification No. 5,593,853 Specification; Specification No. 5,574,656; Specification No. 5,571,698; Specification No. 5,5 Specification No. 65,324; Specification No. 5,549,974; Specification No. 5,545,568 Specification; Specification No. 5,541,061; Specification No. 5,525,735; Specification No. 5,4 Specification No. 63,564; Specification No. 5,440,016; Specification No. 5,438,119 and US Pat. No. 5,223,409, the disclosures of which are incorporated herein by reference. The present application is incorporated by reference.

[0223] A binding moiety may be a domain, fragment, epitope, region, or member of a specific binding pair. The binding moiety may be on or comprise a portion of a receptor (e.g., a ligand). A main, fragment, epitope, region, or monovalent (monoepitope) topical) or polyepitopic) moieties The binding moiety may be antigenic or haptenic. The portion may be a domain, fragment, epitope, region, or fragment containing at least one common epitope. on a single molecule or portions of multiple molecules that share a common tope or determinant, or The binding moiety may include a domain, fragment, epitope, region, or cell. on or in part of a part of a cell (e.g., a bacterial cell, a plant cell, or an animal cell) The binding moiety may be present in a natural environment (e.g., tissue), cultured cells, or microorganisms (e.g., can be either in a cell (e.g., bacteria, fungi, protozoa, or viruses) or in a lysed cell. The binding moiety may be modified (e.g., chemically) to include, but is not limited to, a dye (e.g., a fluorescent dye). a polypeptide modifying moiety such as a photopigment, a phosphate group, a carbohydrate group, or a polynucleotide such as a methyl group One or more additional binding sites, such as nucleotide modifying moieties, may be provided.

[0224] In some cases, the binding moiety may be a domain, fragment, or fragments found in a sample derived from a host. on or containing a site, epitope, region, or part of a molecule. Samples may be collected from body fluids (e.g., urine, blood, plasma, serum, saliva, semen, feces, saliva, cerebrospinal fluid, tears) The sample may be examined directly or may be modified to allow for more easily detectable binding moieties. A sample may be a quantity of material derived from an organism or a former organism. The sample may be natural, recombinant, synthetic, or non-natural. cell lysate or cell culture medium, in vitro translation sample, or sample (e.g., cell lysate In immunoprecipitation from a target gene, any of the above expressed naturally or recombinantly from cells It could be either one.

[0225] In some cases, the target binding moiety is expressed in a cell-free system or in vitro. In some cases, the binding portion of the target is in a cell extract. A template and transcription and translation reagents are in a cell extract. Exemplary sources of the substance include wheat germ, Escherichia coli, , rabbit reticulocytes, hyperthermophilic bacteria, hybridoma, Xenopus ) oocytes, insect cells, and mammalian cells (e.g., human cells). can be used to express peptides (e.g., to produce target polypeptides on an array) Exemplary cell-free methods include protein in situ array (PISA), multiplexed ELISA, and MIST, self-assembling mRNA translation, nucleic acid programmable protein assembly Arrays (NAPPA), Nanowell NAPPA, DNA arrays, and protein arrays (DA PA), membrane-free DAPA, nanowell copy and μIP-microintaglio print (microintaglio printing), and pMAC-protein microprinting Examples include lower leiomyography (Kilb et al., Eng. Life Sci. 2 (See 014,14,352-364).

[0226] In some cases, the target binding moiety is derived in situ (e.g., aligned) from the DNA template. In some cases, multiple binding moieties are synthesized in parallel or on a single substrate. In the reaction, the DNA is synthesized in situ from multiple corresponding DNA templates. Exemplary methods for target polypeptide expression in yeast include those described in Stevens, Structural Biology, Vol. ture 8(9):R177-R185(2000);Katzen et al., Trends Biotechnol.23(3):150-6.(2005);He et al.,Curr.Opin.Biotechnol.19(1):4-9.(2 008);Ramachandran et al.,Science 305(568 0):86-90.(2004);He et al., Nucleic Acids Res.29(15):E73-3(2001);Angenendt et al., Mol.Cell Proteomics 5(9):1658-66(2006);T ao et al, Nat Biotechnol 24(10):1253-4(20 06);Angenendt et al.,Anal.Chem.76(7):184 4-9(2004);Kinpara et al.,J.Biochem.136(2 ):149-54(2004);Takulapalli et al., J.Prot eome Res.11(8):4382-91(2012);He et al.,N at.Methods 5(2):175-7(2008);Chatterjee a nd J. LaBaer, ​​Curr Opin Biotech 17(4):334- 336(2006);He and Wang,Biomol Eng 24(4):3 75-80 (2007); and He and Taussig, J. Immunol. M Methods 274(1-2):265-70(2003) can be.

[0227] In some cases, the binding portion of the nucleic acid target is at least 6 nucleotides, e.g., At most 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, or 100 nucleosides In some cases, the binding moiety of the protein target comprises a range of nucleotides. In some cases, the binding moiety of the protein target comprises a nucleotide unsaturation. In some cases, the binding moiety of the nucleic acid target comprises a contiguous stretch of nucleotides in the nucleic acid sequence. Mutations or functional mutations, including deletions, additions, swaps, or truncations of Includes place.

[0228] In some cases, the binding portion of a protein target may be at least 6 amino acids, e.g., At least 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, or 100 amino acids In some cases, the binding moiety for a protein target may comprise a contiguous stretch of amino acids. In some cases, the binding portion of a protein target comprises a discontinuous stretch of amino acids. In some cases, the binding moiety for a protein target comprises an amino acid sequence The site of mutation or functional mutation includes deletion, addition, exchange, or truncation of an amino acid.

[0229] In certain embodiments, the binding moiety may be a domain, fragment, epitope, region, or or on or comprising a portion of a membrane-bound protein. Exemplary membrane-bound proteins Examples of the target molecules include, but are not limited to, GPCRs (e.g., adrenergic receptors, angiotensin receptors, acetylcholine receptors, cholecystokinin receptors, muscarinic acetylcholine receptors, neurotransmitters serotonin receptors, galanin receptors, dopamine receptors, opioid receptors, serotonin receptors receptors, somatostatin receptors, etc.), ion channels (e.g., nicotinic acetylcholine receptors, sodium channels, potassium channels, etc.), receptor tyrosine kinases, receptors Serine / threonine kinases, receptor guanylate cyclases, growth factor and hormone receptors receptors (e.g., epidermal growth factor (EGF) receptor), and others. Also, mutants of domains, fragments, epitop...

Claims

[Claim 1] The invention described in this specification.

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