Methods of making modified branched rnas

Abstract

Disclosed herein are branched RNA oligonucleotides and methods of making the same. The branched RNA oligonucleotides may be made by using a brancher phosphoramidite. Also provided are compositions comprising one or more of the branched RNA oligonucleotides provided herein, and methods of using said compositions for therapeutic applications.

Description

Atty. Docket No.114203-1801 METHODS OF MAKING MODIFIED RNAS CROSS-REFERENCE RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional application No.63 / 664,001, filed on June 25, 2024, the entire disclosure of which is incorporated by reference herein. BACKGROUND

[0002] The following discussion is merely provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.

[0003] Messenger RNA (mRNA) technology is an emerging alternative to conventional small molecule, DNA, and protein therapeutics and conventional vaccine approaches because it is potent, programmable, and capable of rapid production of mRNA with desired sequences. mRNA therapy is a rapidly developing field and has been used for the expression of therapeutic proteins, ranging from vascular regeneration factors (e.g., vascular endothelial growth factor A (VEGF-A), erythropoietin (EPO), GATA Binding Protein 4 (GATA4), Myocyte Enhancer Factor 2C (MEF2C), T-Box Transcription Factor 5 (TBX5), Myocardin (MYOCD)), to vaccines for COVID-19, influenza, and Zika virus. Despite recent clinical successes, mRNA therapy still faces challenges of instability, toxicity, short-term efficacy, and potential undesired immunological responses. Increasing the stability and translation efficiency of mRNAs to enhance their efficiency in vivo remains an important problem that must be solved to increase the feasibility of mRNA therapeutics for clinical applications. SUMMARY

[0004] Chemical modifications to the 5′ cap of mRNA have well-characterized biochemical functions. Chemical modifications providing multiple 5′ caps of branched mRNA have well- characterized biochemical functions such as increased protein expression in comparison to conventional linear mRNA. Previous strategies to synthesize multi-capped branched mRNA involved the preparation of branched 5′ oligonucleotide through click chemistry and ligation with an in-vitro transcribed mRNA. However, the synthesis scale and structural diversity of the branched oligonucleotide was restricted by the relatively long synthetic route including solid -1- 4935-1210-1197.1Atty. Docket No.114203-1801 phase synthesis of two linear oligonucleotides, click handle labeling of the oligonucleotides, click chemistry-based conjugation and HPLC purification.

[0005] Thus, provided herein are multi-capped branched mRNAs to improve the protein expression and stability of the mRNAs in cells and in vitro translation systems, thereby enhancing protein production, as well as scalable methods of making and using such modified mRNAs.

[0006] Accordingly, the present disclosure provides, in some aspects, a method of producing a branched RNA oligonucleotide, comprising synthesizing the branched RNA oligonucleotide with a brancher phosphoramidite including a first protected hydroxyl group and a second protected hydroxyl group, creating a branching point in the branched RNA oligonucleotide with the brancher phosphoramidite; wherein the step of synthesizing is performed on a solid-phase support.

[0007] In any embodiment, at least one of the first protected hydroxyl group and the second protected hydroxyl group of the brancher phosphoramidite may include dimethoxytrityl (DMTr), levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS). The first protected hydroxyl group and the second protected hydroxyl group of the brancher phosphoramidite may be deprotected with different treatments. The first protected hydroxyl group of the brancher phosphoramidite may include dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite may include levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS). The first protected hydroxyl group of the brancher phosphoramidite may include dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite may include levulinyl. The brancher phosphoramidite may have a structure according to Formula I -2- 4935-1210-1197.1Atty. Docket No.114203-1801 whereinR1is dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite comprises levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS); R2is tert-butyldimethylsilyl ether, halogen, alkyl, or hydrogen; X is a nitrogenous nucleotide base; and n is 1 to 12.

[0008] The brancher phosphoramidite may have a structure according to Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X, or Formula XI-3- 4935-1210-1197.1Atty. Docket No.114203-1801m n are some Formula IX, Formula X, or Formula XI are, respectively: -4- 4935-1210-1197.1Atty. Docket No.114203-1801 orbranch with the brancher phosphoramidite; (b) deprotecting the second protected hydroxyl group on the brancher phosphoramidite to form a hydroxyl group on the first oligonucleotide branch; (c) synthesizing a second oligonucleotide branch at the free hydroxyl group on the first oligonucleotide branch, thereby forming the branched RNA oligonucleotide.

[0010] The brancher phosphoramidite may be a first brancher phosphoramidite and the branching point is a first branching point, and synthesizing the second oligonucleotide branch comprises synthesizing the second oligonucleotide branch with a second brancher phosphoramidite.

[0011] The method may further include (d) deprotecting a protecting group on the second brancher phosphoramidite to form a hydroxyl group on the second oligonucleotide branch; (e) synthesizing a third oligonucleotide branch at the free hydroxyl group on the second oligonucleotide branch to form a second branching point. -5- 4935-1210-1197.1Atty. Docket No.114203-1801

[0012] The first oligonucleotide branch of the branched RNA oligonucleotide may include 1 nucleotide to 40 nucleotides (e.g., 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, 32, 33, 34, 35, 36, 37, 38, 39, or 40) between the branching point in the branched RNA oligonucleotide and a terminus of the first oligonucleotide branch. The first oligonucleotide branch of the branched RNA oligonucleotide may include 6 nucleotides. The first oligonucleotide branch of the branched RNA oligonucleotide may include 8 nucleotides. The first oligonucleotide branch of the branched RNA oligonucleotide may include 10 nucleotides.

[0013] The second oligonucleotide branch of the branched RNA oligonucleotide may include 1 nucleotide to 40 nucleotides between the branching point in the branched RNA oligonucleotide and a terminus of the second oligonucleotide branch. The second oligonucleotide branch of the branched RNA oligonucleotide may include 7 nucleotides. The second oligonucleotide branch of the branched RNA oligonucleotide may include 10 nucleotides. The second oligonucleotide branch of the branched RNA oligonucleotide may include 13 nucleotides.

[0014] The second oligonucleotide branch of the branched RNA oligonucleotide may include a spacer at the branching point of the branched RNA oligonucleotide. The spacer may include C1-C12alkyl or (CH2CH2O)nwhere n is 1-4.

[0015] The solid-phase support may include glass beads or polystyrene beads. The method may further include leaving the branched RNA oligonucleotide from the solid-phase support.

[0016] The method may further include capping phosphorylated 5’ terminuses of the branched RNA oligonucleotide, producing a capped branched RNA oligonucleotide. The method may further include purifying the capped branched RNA oligonucleotide, producing a purified capped branched RNA oligonucleotide. The step of purifying may include performing high- performance liquid chromatography (HPLC).

[0017] The capping may be performed chemically. Chemical capping may be performed through an anhydrous reaction between the first 5’-phosphorylated RNA oligonucleotide, the second 5’-phosphorylated RNA oligonucleotide, and capping nucleotides conjugated to imidazole in the presence of 1-methylimidazole. -6- 4935-1210-1197.1Atty. Docket No.114203-1801

[0018] The second oligonucleotide branch may include a 5’ untranslated region (5’ UTR) comprising an unstructured region. The unstructured region may include at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more consecutive adenosine, cytosine, guanine, or uridine nucleotides. The unstructured region may include no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10 consecutive adenosine, cytosine, guanine, or uridine nucleotides. The unstructured region may include between 5 and 60, between 10 and 55, between 15 and 50, between 20 and 45, between 25 and 40, or between 30 and 35 consecutive adenosine, cytosine, guanine, or uridine nucleotides.

[0019] The first oligonucleotide branch may be between 3 and 160, between 5 and 150, between 7 and 140, between 9 and 130, between 11 and 120, between 13 and 110, between 15 and 100, between 17 and 90, between 19 and 80, between 21 and 70, between 23 and 60, between 25 and 50, between 27 and 40, or between 30 and 35 nucleotides in length. The first oligonucleotide branch may be between 25 and 35 nucleotides in length. The second oligonucleotide branch may be between 3 and 160, between 5 and 150, between 7 and 140, between 9 and 130, between 11 and 120, between 13 and 110, between 15 and 100, between 17 and 90, between 19 and 80, between 21 and 70, between 23 and 60, between 25 and 50, between 27 and 40, or between 30 and 35 nucleotides in length. The second oligonucleotide branch may be between 25 and 35 nucleotides in length.

[0020] The first oligonucleotide branch may include one or more modified nucleotides. The second oligonucleotide branch may include one or more modified nucleotides. The one or more modified nucleotides may include a modified sugar. The modified sugar may be selected from the group consisting of 2′-deoxy fluoro (2FA), L-adenosine (LA), 2′-deoxyadenosine (dA), locked nucleic acid (LNA), 2′-methoxy (2OMe), 2′-methoxyethoxy (2MOE), 2′-thioribose, 2′,3′- dideoxyribose, 2′-amino-2′-deoxyribose, 2′ deoxyribose, 2′-azido-2′-deoxyribose, 2′-fluoro-2′- deoxyribose, 2′-O-methylribose, 2′-O-methyldeoxyribose, 3′-amino-2′,3′-dideoxyribose, 3′- azido-2′,3′-dideoxyribose, 3′-deoxyribose, 3′-O-(2-nitrobenzyl)-2′-deoxyribose, 3′-O- -7- 4935-1210-1197.1Atty. Docket No.114203-1801 methylribose, 5′-aminoribose, 5′-thioribose, 5-nitro-1-indolyl-2′-deoxyribose, 5′-biotin-ribose, 2′- O,4′-C-methylene-linked, 2′-O,4′-C-amino-linked ribose, and 2′-O,4′-C-thio-linked ribose.

[0021] The first oligonucleotide branch may include between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 75, between 75 and 100, between 100 and 125, between 125 and 150, or between 135 and 160 modified sugars. The first oligonucleotide branch may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, or more modified sugars. The second oligonucleotide branch may include between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified sugars. The second oligonucleotide branch may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified sugars.

[0022] The one or more modified nucleotides comprises a modified phosphate. The modified phosphate may be selected from the group consisting of phosphorothioate (PS), thiophosphate, 5′-O-methylphosphonate, 3′-O-methylphosphonate, 5′-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate. The first oligonucleotide branch may include between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 75, between 75 and 100, between 100 and 125, between 125 and 150, or between 135 and 160 modified phosphates. The first oligonucleotide branch may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, or more modified phosphates. The second oligonucleotide branch may include between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 -8- 4935-1210-1197.1Atty. Docket No.114203-1801 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified phosphates. The second oligonucleotide branch may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified phosphates.

[0023] The one or more modified nucleotides may include a modified nucleobase. The modified nucleobase may be selected from the group consisting of inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6- methyladenosine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4- thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3- Indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5- bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5- methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5- propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6- chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7- propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8- chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16-aminoallylcytosine, biotin-16- aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1- methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1- propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, -9- 4935-1210-1197.1Atty. Docket No.114203-1801 thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl- thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis- hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0024] The first oligonucleotide branch may include between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 75, between 75 and 100, between 100 and 125, between 125 and 150, or between 135 and 160 modified nucleobases. The first oligonucleotide branch may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, or more modified nucleobases. The second oligonucleotide branch may include between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified nucleobases. The second oligonucleotide branch may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified nucleobases. The one or more modified nucleotides may include one or more modified sugars, one or more modified phosphates, one or more modified nucleobases, or any combination thereof.

[0025] The capped branched RNA oligonucleotide may include a 5’ cap selected from the group consisting of 7-methylguanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’- guanosine (m7G-3’m-ppp-G), N7,2’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7Gm-ppp-G), 7-benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G-LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4- -10- 4935-1210-1197.1Atty. Docket No.114203-1801 chlorophenoxyethyl)-guanosine, 7-ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7-(carboxymethyl) guanosine (cm7G), 7-(2-phenylethyl) guanosine [7-(2- PhEt)G], 7-(1-phenylethyl) guanosine [7-(1-PhEt)G], m7GpppBH3G (D1 and D2 stereoisomers), m7GppBH3G (D1 and D2 stereoisomers), m7GpBH3G (D1 and D2 stereoisomers), m7GppBH3pm7G, m27,2’-OGpppBH3G (D1 and D2 stereoisomers), m27,2’-OGppBH3pG (D1 and D2 diastereomers), m27,2’-OGppSpG (D1 and D2 diastereomers), N-Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, 1-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, cap1, cap2, cap3, cap4, ARCA, modified ARCA, inosine, N1- methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0026] The second oligonucleotide branch further may include a poly-A tail. The poly-A tail comprises between 25 and 500 nucleotides. The poly-A tail may include between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 or 500 nucleotides. The poly-A tail may include 10 or more adenosine nucleotides. 25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96- 100%, 97-100%, 98-100%, or 99-100% of nucleotides of the poly-A tail may be adenosine nucleotides.100% of the nucleotides of the poly-A tail may be adenosine nucleotides. The poly- A tail may include one or more modified nucleotides.

[0027] The second oligonucleotide branch further may include a therapeutic nucleic acid. The therapeutic nucleic acid may be chosen from the group consisting of an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA. The branched RNA oligonucleotide may include a promoter sequence. -11- 4935-1210-1197.1Atty. Docket No.114203-1801

[0028] In another aspect, disclosed herein is a capped RNA transcript produced by any one of the methods disclosed herein.

[0029] In another aspect, disclosed herein is a branched RNA oligonucleotide having a structure according to Formula XII or XIII Formula XIII whereinR3is phosphate or 1-20 nucleotides; R4is phosphate or 1-500 nucleotides; R5is absent, alkyl, alkyl phosphate, or alkylene oxide; R6is phosphate or 1-1000 nucleotides; R7is OH, alkyl, halogen, or hydrogen; X and B are each independently a nitrogenous nucleotide base; and n is 1-12.

[0030] R3may include 1-10 nucleotides. R3may include 6 nucleotides. R3may include 8 nucleotides. R3may include 10 nucleotides. R4may include a nucleotide conjugated to a solid- phase support.

[0031] R5may include C1-C12alkyl, (CH2)xOPOOH(CH2)y, (CH2CH2O)z; wherein x and y are independently 1-12, and z is 1-2. R6may include 1-20 nucleotides. R6may include 6 nucleotides. R6may include 9 nucleotides. R6may include 12 nucleotides. R6may include 5’ untranslated region (5’ UTR) comprising an unstructured region. -12- 4935-1210-1197.1Atty. Docket No.114203-1801

[0032] The unstructured region may include at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more consecutive adenosine, cytosine, guanine, or uridine nucleotides. The unstructured region may include no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10 consecutive adenosine, cytosine, guanine, or uridine nucleotides. The unstructured region may include between 5 and 60, between 10 and 55, between 15 and 50, between 20 and 45, between 25 and 40, or between 30 and 35 consecutive adenosine, cytosine, guanine, or uridine nucleotides.

[0033] R4may include between 1 and 160, between 3 and 150, between 5 and 140, between 7 and 130, between 9 and 120, between 11 and 110, between 13 and 100, between 15 and 90, between 17 and 80, between 19 and 70, between 21 and 60, between 23 and 50, between 25 and 40, or between 20 and 35 nucleotides in length.

[0034] R6may include between 3 and 160, between 5 and 150, between 7 and 140, between 9 and 130, between 11 and 120, between 13 and 110, between 15 and 100, between 17 and 90, between 19 and 80, between 21 and 70, between 23 and 60, between 25 and 50, between 27 and 40, or between 30 and 35 nucleotides in length. R6may be between 25 and 35 nucleotides in length.

[0035] R3may include one or more modified nucleotides. R4may include one or more modified nucleotides. R6may include one or more modified nucleotides. The one or more modified nucleotides may include a modified sugar. The modified sugar may be selected from the group consisting of 2′-deoxy fluoro (2FA), L-adenosine (LA), 2′-deoxyadenosine (dA), locked nucleic acid (LNA), 2′-methoxy (2OMe), 2′-methoxyethoxy (2MOE), 2′-thioribose, 2′,3′- dideoxyribose, 2′-amino-2′-deoxyribose, 2′ deoxyribose, 2′-azido-2′-deoxyribose, 2′-fluoro-2′- deoxyribose, 2′-O-methylribose, 2′-O-methyldeoxyribose, 3′-amino-2′,3′-dideoxyribose, 3′- azido-2′,3′-dideoxyribose, 3′-deoxyribose, 3′-O-(2-nitrobenzyl)-2′-deoxyribose, 3′-O- methylribose, 5′-aminoribose, 5′-thioribose, 5-nitro-1-indolyl-2′-deoxyribose, 5′-biotin-ribose, 2′- O,4′-C-methylene-linked, 2′-O,4′-C-amino-linked ribose, and 2′-O,4′-C-thio-linked ribose. -13- 4935-1210-1197.1Atty. Docket No.114203-1801

[0036] The one or more modified nucleotides may include a modified phosphate. The modified phosphate may be selected from the group consisting of phosphorothioate (PS), thiophosphate, 5′-O-methylphosphonate, 3′-O-methylphosphonate, 5′-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0037] The one or more modified nucleotides may include a modified nucleobase. The modified nucleobase may be selected from the group consisting of inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6- methyladenosine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4- thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3- Indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5- bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5- methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5- propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6- chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7- propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8- chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16-aminoallylcytosine, biotin-16- aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1- methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1- propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, -14- 4935-1210-1197.1Atty. Docket No.114203-1801 thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl- thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis- hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0038] The one or more modified nucleotides may include one or more modified sugars, one or more modified phosphates, one or more modified nucleobases, or any combination thereof.

[0039] R3may include a capping nucleotide; R4may include the capping nucleotide; and R6may include the capping nucleotide.

[0040] The capped nucleotide may include a 5’ cap. The 5’ cap may be selected from the group consisting of 7-methylguanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’- guanosine (m7G-3’m-ppp-G), N7,2’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7Gm-ppp-G), 7-benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G-LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4- chlorophenoxyethyl)-guanosine, 7-ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7-(carboxymethyl) guanosine (cm7G), 7-(2-phenylethyl) guanosine [7-(2- PhEt)G], 7-(1-phenylethyl) guanosine [7-(1-PhEt)G], m7GpppBH3G (D1 and D2 stereoisomers), m7GppBH3G (D1 and D2 stereoisomers), m7GpBH3G (D1 and D2 stereoisomers), m7GppBH3pm7G, m27,2’-OGpppBH3G (D1 and D2 stereoisomers), m27,2’-OGppBH3pG (D1 and D2 diastereomers), m27,2’-OGppSpG (D1 and D2 diastereomers), N-Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, 1-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, -15- 4935-1210-1197.1Atty. Docket No.114203-1801 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, cap1, cap2, cap3, cap4, ARCA, modified ARCA, inosine, N1- methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0041] R6may include a poly-A tail. The poly-A tail may include between 25 and 500 nucleotides. The poly-A tail may include between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 or 500 nucleotides. The poly-A tail may include 10 or more adenosine nucleotides.25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100% of nucleotides of the poly-A tail may be adenosine nucleotides.100% of the nucleotides of the poly-A tail may be adenosine nucleotides. The poly-A tail may include one or more modified nucleotides.

[0042] R6may include a therapeutic nucleic acid. The therapeutic nucleic acid may be chosen from the group consisting of an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.

[0043] In an aspect, disclosed herein is a delivery agent comprising the capped RNA transcript disclosed herein or the branched RNA oligonucleotide disclosed herein, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.

[0044] The nanoparticle or microparticle may be a lipid nanoparticle or microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

[0045] In an aspect, disclosed herein is a cell comprising the capped RNA transcript disclosed herein or the branched RNA oligonucleotide disclosed herein. The cell may be a mammalian cell.

[0046] In an aspect, disclosed herein is a composition comprising the capped RNA transcript of disclosed herein, the branched RNA oligonucleotide disclosed herein, the delivery agent disclosed herein, or the cell disclosed herein. -16- 4935-1210-1197.1Atty. Docket No.114203-1801

[0047] The composition may further include an additional agent. The additional agent may be an agent which has a therapeutic effect when administered to a subject. The additional agent may be chosen from the list consisting of a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a small molecule, an aptamer, a lipid, or a carbohydrate. The additional agent may be a shRNA, a siRNA, or an antisense oligonucleotide (ASO). The additional agent may be an antigen or adjuvant. The composition may be a pharmaceutical composition, wherein the pharmaceutical composition may include a pharmaceutically acceptable excipient.

[0048] In an aspect, disclosed herein is a method of preventing or treating a disease in a subject, comprising introducing an effective amount of the capped RNA transcript disclosed herein, the branched RNA oligonucleotide disclosed herein, the delivery agent disclosed herein, the cell disclosed herein, or the composition disclosed herein to the subject. The subject may be a human.

[0049] In an aspect, disclosed herein is the capped RNA transcript disclosed herein, the branched RNA oligonucleotide disclosed herein, the delivery agent disclosed herein, the cell disclosed herein, or the composition disclosed herein for use in preventing or treating a disease in a subject.

[0050] In an aspect, disclosed herein is a kit comprising the capped RNA transcript disclosed herein, the branched RNA oligonucleotide disclosed herein, the delivery agent disclosed herein, the cell disclosed herein, or the composition disclosed herein. The kit may further include an RNA ligase.

[0051] In an aspect, disclosed herein is a kit comprising the capped RNA transcript disclosed herein, the branched RNA oligonucleotide disclosed herein, the delivery agent disclosed herein, the cell disclosed herein, or the composition disclosed herein, a device for administering the composition to a subject, and instructions for administering the composition to the subject.

[0052] In an aspect, disclosed herein is a use of the capped RNA transcript disclosed herein, the branched RNA oligonucleotide disclosed herein, the delivery agent disclosed herein, the cell disclosed herein, or the composition disclosed herein for the manufacture of a medicament for treatment of a disease in a subject. -17- 4935-1210-1197.1Atty. Docket No.114203-1801

[0053] In an aspect, disclosed herein is the capped RNA transcript disclosed herein, the branched RNA oligonucleotide disclosed herein, the delivery agent disclosed herein, the cell disclosed herein, or the composition disclosed herein for use in the treatment of a disease in a subject.

[0054] The foregoing general description and following detailed description are examples and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.

[0055] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are provided as being part of the inventive subject matter disclosed herein and may be employed in any combination to achieve the benefits described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG.1 shows a general synthesis workflow of synthesis of different branched oligonucleotides using a brancher phosphoramidite.

[0057] FIG.2 shows a workflow for the solid phase synthesis of branched oligonucleotide with functional handles.

[0058] FIGS.3A-3B show an evaluation of different lengths of the first oligonucleotide branch before the branching point. FIG.3A illustrates a branched oligonucleotide with an arrow noting the first oligonucleotide branch length that was evaluated. FIG.3B is a graph of relative luminescence for different branched oligonucleotides with different first oligonucleotide branch lengths.

[0059] FIGS.4A-4B show an evaluation of different lengths of the second oligonucleotide branch. FIG.4A illustrates a branched oligonucleotide with an arrow noting the second oligonucleotide branch length that was evaluated. FIG.4B is a graph of relative luminescence for different branched oligonucleotides with different second oligonucleotide branch lengths. -18- 4935-1210-1197.1Atty. Docket No.114203-1801

[0060] FIGS.5A-5B how an evaluation of different types of spacers at the branching point. FIG.5A illustrates a branched oligonucleotide with an arrow noting the spacer and different spacer structures that were evaluated. FIG.5B is a graph of relative luminescence for different branched oligonucleotides with the different spacers in FIG.5A. DETAILED DESCRIPTION

[0061] Provided herein are modified mRNAs comprising multiple 5’ cap regions comprising one or more oligonucleotide branches in order to improve stability and / or translation efficiency of the modified mRNA in cells and thereby enhance the production of encoded gene products, such as proteins. Also provided are methods of making the modified mRNAs described herein by using a brancher phosphoramidite to synthesize branched mRNAs. Also provided are methods of screening for altered mRNA stability and / or translation efficiency conferred by structure and number of branches. Furthermore, provided herein are methods of producing a composition of RNA transcripts wherein at least 95% of the RNA transcripts in the composition comprise multiple 5’ cap.

[0062] RNA transcripts may include multiple branches that each include a modified 5’ cap region comprises a 5’ cap region comprising one or more modified nucleobases, phosphodiester linkages, sugar backbones, and / or 5’ caps. Particularly, dual, or multiple branched oligonucleotide can be produced if one or multiple phosphoramidite branchers are introduced during stem strand synthesis, while incorporation of phosphoramidite branchers during branch strand synthesis can produce a heterozygous multi-branched oligonucleotide.

[0063] These methods offer several advantages, including improved scalability: each batch of 1 µmol synthesis can generate 0.25 µmol of branched oligonucleotide (about 2 mg for branched oligonucleotides in 20 nucleotides), which provides large scale preparation; simplified workflow: using orthogonal protecting groups on the phosphoramidite brancher for oligonucleotide synthesis and solid phase extraction for purification provides a one-step preparation of branched oligonucleotide with high purity (>90%); the synthesis methods provide the preparation of branched oligonucleotides with greater topological diversity (e.g., multi- branching or ramified branching). These synthesis methods provide improved scalability and structural expansibility, and therefore can be used to construct and test different branched -19- 4935-1210-1197.1Atty. Docket No.114203-1801 oligonucleotides for mRNA translation and stability regulation. Additionally, these synthesis methods facilitate the exploration of mRNA modification due to compatibility with phosphoramidite derivatives. Equivalents

[0064] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0065] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. -20- 4935-1210-1197.1Atty. Docket No.114203-1801 Definitions

[0066] In the claims, as well as in the specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

[0067] In the claims, as well as in the specification, recitation of the phrase “between X and Y,” wherein X and Y are two separate values, it should be appreciated that these ranges include the use of these end values. For example, if a claim recites a range of between 1 and 10, this includes the values of 1, 10, and any value in between (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, etc.).

[0068] A “messenger RNA” (“mRNA”) as used herein refers to a nucleic acid comprising an open reading frame (ORF) encoding a gene product, such as a protein. An mRNA may comprise a poly-A region that is 3’ to the ORF. An mRNA may also comprise a 5’ untranslated region (5’ UTR) that is 5’ to (upstream of) the ORF, and a 3’ untranslated region (3’ UTR) that is 3’ to (downstream of) the ORF. A mRNA may also comprise a 5’ cap at the 5’ end of the mRNA.

[0069] An “open reading frame” (“ORF”), such as an ORF encoding a protein, as used herein refers to a nucleic acid sequence comprising a coding sequence that leads to the production of the protein when the ORF is translated. The nucleic acid sequence may be an RNA sequence, in which case translation of the RNA sequence produces a polypeptide with the amino acid sequence of the protein. The nucleic acid sequence may be a DNA sequence, in which case the protein is produced when an RNA polymerase uses the DNA sequence to transcribe an RNA -21- 4935-1210-1197.1Atty. Docket No.114203-1801 molecule comprising an RNA sequence that is complementary to the DNA sequence, and translation of the RNA sequence produces a polypeptide with the amino acid sequence of the protein. An ORF typically begins with a START codon, such as AUG in the RNA sequence (ATG in the DNA sequence), and ends with a STOP codon, such as UAG, UAA, or UGA in the RNA sequence (TAG, TAA, or TGA in the DNA sequence), with the number of bases between the G of the start codon and the T or U of the STOP codon being a multiple of 3 (e.g., 3, 6, 9, 12, etc.).

[0070] With reference to numbering of the nucleotide positions within a nucleic acid molecule, a position of +1 refers to the first nucleotide of the nucleic acid molecule (e.g., of the RNA molecule), +2 is the second nucleotide, +3 is the third nucleotide, and so on.

[0071] In some embodiments of the modified mRNAs provided herein, the mRNA comprises a 5′ untranslated region (5′ UTR) and a 3′ untranslated region (3′ UTR).5′ and 3′ UTRs are sequences within an mRNA that do not encode amino acids of the protein encoded by the mRNA, and are thus not part of the open reading frame. The 5′ UTR is 5′ to (upstream of) the open reading frame. The 3′ UTR is 3′ to (downstream of) the open reading frame. In some embodiments, the 3′ UTR comprises one or more nucleotides that are 3′ to the open reading frame and 5′ to (upstream of) the poly-A region of the mRNA.

[0072] In some embodiments of the modified mRNAs provided herein, the mRNA comprises, in 5’-to-3’ order: 1) a 5’ cap, optionally modified; 2) a modified 5’ UTR; 3) an open reading frame (ORF); 4) a 3’ UTR; and 5) a poly-A region. In some embodiments, the first nucleotide of the 5’ UTR is 3’ to (downstream of) the 5’ cap, and the last nucleotide of the 5’ UTR is 5’ to (upstream of) the first nucleotide of the ORF. In some embodiments, the first nucleotide of the ORF is 3’ to (downstream of) the last nucleotide of the 5’ UTR, and the last nucleotide of the ORF is 5’ to (upstream of) the first nucleotide of the 3’ UTR. In some embodiments, the ORF is between the last nucleotide of the 5’ UTR and the first nucleotide of the 3’ UTR. In some embodiments, the first nucleotide of the 3’ UTR is 3’ to (downstream of) the last nucleotide of the ORF, and the last nucleotide of the 3’ UTR is 5’ to (upstream of) the first nucleotide of the poly-A region. In some embodiments, the 5’ UTR is between the 5’ cap and the first nucleotide of the ORF. In some embodiments, the 3’ UTR is between the ORF and -22- 4935-1210-1197.1Atty. Docket No.114203-1801 the poly-A region. In some embodiments, the 5’ cap is 5’ to (upstream of) the first nucleotide of the 5’ UTR. In some embodiments, the first nucleotide of the poly-A region is 3’ to (downstream of) the last nucleotide of the 3’ UTR.

[0073] In some embodiments, the RNA is a linear RNA. A linear RNA is an RNA with a 5′ terminal nucleotide and a 3′ terminal nucleotide. The 5′ terminal nucleotide of a linear RNA is covalently bonded to only one adjacent nucleotide of the RNA, with the adjacent nucleotide occurring 3′ to the 5′ terminal nucleotide in the nucleic acid sequence of the RNA. The 3′ terminal nucleotide of a linear RNA is covalently bonded to only one adjacent nucleotide of the RNA, with the adjacent nucleotide occurring 5′ to the 3′ terminal nucleotide in the nucleic acid sequence of the RNA. In a nucleic acid sequence comprising every nucleotide of a linear RNA in 5′-to-3′ order, the 5′ terminal nucleotide is the first nucleotide in the sequence, and the 3′ terminal nucleotide is the last nucleotide in the sequence.

[0074] An RNA molecule that can be translated is referred to as a messenger RNA, or mRNA. A DNA or RNA sequence encodes a gene through codons. A codon refers to a group of three nucleotides within a nucleic acid, such as DNA or RNA, sequence. An anticodon refers to a group of three nucleotides within a nucleic acid, such as a transfer RNA (tRNA), that are complementary to a codon, such that the codon of a first nucleic acid associates with the anticodon of a second nucleic acid through hydrogen bonding between the bases of the codon and anticodon. For example, the codon 5′-AUG-3′ on an mRNA has the corresponding anticodon 3′-UAC-5′ on a tRNA. During translation, a tRNA with an anticodon complementary to the codon to be translated associates with the codon on the mRNA, generally to deliver an amino acid that corresponds to the codon to be translated, or to facilitate termination of translation and release of a translated polypeptide from a ribosome.

[0075] Translation is the process in which the RNA coding sequence is used to direct the production of a polypeptide. The first step in translation is initiation, in which a ribosome associates with an mRNA, and a first transfer RNA (tRNA) carrying a first amino acid associates with the first codon, or START codon. The next phase of translation, elongation, involves three steps. First, a second tRNA with an anticodon that is complementary to codon following the START codon, or second codon, and carrying a second amino acid, associates with the mRNA. -23- 4935-1210-1197.1Atty. Docket No.114203-1801 Second, the carbon atom of terminal, non-side chain carboxylic acid moiety of the first amino acid reacts with the nitrogen of the terminal, non-side chain amino moiety of the second amino acid carried, forming a peptide bond between the two amino acids, with the second amino acid being bound to the second tRNA, and the first amino acid bound to the second amino acid, but not the first tRNA. Third, the first tRNA dissociates from the mRNA, and the ribosome advances along the mRNA, such that the position at which the first tRNA associated with the ribosome is now occupied by the second tRNA, and the position previously occupied by the second tRNA is now free for an additional tRNA carrying an additional amino acid to associate with the mRNA. These three steps of 1) association of a tRNA carrying amino acid, 2) formation of a peptide bond, which adds an additional amino acid to a growing polypeptide, and 3) advancement of the ribosome along the mRNA, continue until the ribosome reaches a STOP codon, which results in termination of translation. Generally, tRNAs that associate with STOP codons do not carry an amino acid, so the association of a tRNA that does not carry an amino acid during the elongation step results in cleavage of the bond between the polypeptide and the tRNA carrying the final amino acid in the polypeptide, such that the polypeptide is released from the ribosome. Alternatively, ribosomes may dissociate from the mRNA and release the polypeptide if no tRNA associates with the STOP codon.

[0076] A “nucleic acid,” or “polynucleotide,” as used herein, refers to an organic molecule comprising two or more covalently bonded nucleotides. A “nucleotide,” as used herein, refers to an organic molecule comprising a 1) a nucleoside comprising a sugar covalently bonded to a nitrogenous base (nucleobase); and 2) a phosphate group that is covalently bonded to the sugar of the nucleoside. Nucleotides in a polynucleotide are typically joined by a phosphodiester bond, in which the 3′ carbon of the sugar of a first nucleotide is linked to the 5′ carbon of the sugar of a second nucleic acid by a bridging phosphate group. Typically, the bridging phosphate comprises two non-bridging oxygen atoms, which are bonded only to a phosphorus atom of the phosphate, and two bridging oxygen atoms, each of which connects the phosphorus atom to either the 3′ carbon of the first nucleotide or the 5′ carbon of the second nucleotide. In a nucleic acid sequence describing the order of nucleotides in a nucleic acid, a first nucleotide is said to be 5′ to (upstream of) a second nucleotide if the 3′ carbon of first nucleotide is connected to the 5′ carbon of the second nucleotide. Similarly, a second nucleotide is said to be 3′ to (downstream of) a first nucleotide if the 5′ carbon of the second nucleotide is connected to the 3′ carbon of the first -24- 4935-1210-1197.1Atty. Docket No. 114203-1801 nucleotide. Nucleic acid sequences are typically read in 5′->3′ order, starting with the 5′ nucleotide and ending with the 3′ nucleotide.

[0077] A “nitrogenous nucleotide base” or “nitrogenous base” is a nitrogen-containing biological compound that forms the base portion of a nucleoside. Included within the scope of these terms are naturally occurring nucleobases, such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), as well as variants or derivatives of A, G, C, T, and U, such as 5- methylcytosine, pseudouridine, dihydrouracil, xanthine, hyoxanthine, and 7-methylguanine.

[0078] A “modified nucleotide,” as used herein, refers to a nucleotide with a structure that is not the canonical structure of an adenosine nucleotide, cytidine nucleotide, guanine nucleotide, or uracil nucleotide. A canonical structure of a molecule refers to a structure that is generally known in the art to be the structure referred to by the name of the molecule. A canonical structure of an adenosine nucleotide, which comprises an adenine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of adenosine monophosphate: .also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3′ oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0079] The canonical structure of a cytosine nucleotide which comprises a cytosine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of cytidine monophosphate: -25- 4935-1210-1197.1Atty. Docket No. 114203-1801 . The canonical structure of CMP also refers to structures in which of the phosphate and / or one or more hydroxyl groups of the sugarin which an oxygen atom of the phosphate and / or the 3′ oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0080] The canonical structure of a guanine nucleotide which comprises a guanine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of guanosine monophosphate: . The canonical structure of GMP also refers to structures in of the phosphate and / or one or more hydroxyl groups of thesugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3′ oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0081] The canonical structure of an uracil nucleotide which comprises an uracil base, ribose sugar, and one or more phosphate groups, is shown below, in the form of uridine monophosphate: . The canonical structure of UMP also refers to structures in which onethe phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3′ oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence. -26- 4935-1210-1197.1Atty. Docket No. 114203-1801

[0082] The structure of a modified nucleotide may differ from the structure of a canonical nucleotide due to one or more modifications in the sugar, nitrogenous base, or phosphate of the nucleotide. In some embodiments, the modified nucleotide comprises a modified nucleoside that is not the canonical structure of an adenine nucleoside, cytosine nucleoside, guanine nucleoside, or uracil nucleoside.

[0083] An example of a canonical structure of adenosine, an adenine nucleoside, is reproduced below: (adenosine). The canonical structure of adenosine also refers to structures inhydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5′ carbon is bound to a 5′ phosphate in a nucleic acid sequence, and structures in which a 3′ oxygen atom is bound to a 5′ phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0084] An example of a canonical structure of cytidine, a cytosine nucleoside, is reproduced below: (cytidine). The canonical structure of cytidine also refers to structures in whichgroups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5′ carbon is bound to a 5′ phosphate in a nucleic acid sequence, and structures in which a 3′ oxygen atom is bound to a 5′ phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0085] An example of a canonical structure of guanosine, a guanine nucleoside, is reproduced below: -27- 4935-1210-1197.1Atty. Docket No. 114203-1801 (guanosine). The canonical structure of guanosine also refers to structures hydroxyl groups of the phosphate and / or one or more hydroxyl groups ofthe sugar are deprotonated, structures in which the 5′ carbon is bound to a 5′ phosphate in a nucleic acid sequence, and structures in which a 3′ oxygen atom is bound to a 5′ phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0086] An example of a canonical structure of uridine, an uracil nucleoside, is reproduced below: (uridine). The canonical structure of uridine also refers to structures in which onegroups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5′ carbon is bound to a 5′ phosphate in a nucleic acid sequence, and structures in which a 3′ oxygen atom is bound to a 5′ phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0087] A “ligase,” as used herein, refers to an enzyme that is capable of forming a covalent bond between two nucleotides, and the process of “ligation” refers to the formation of the covalent bond between the two nucleotides.

[0088] A “poly-A tail,” as used herein, refers to a nucleic acid sequence comprising adenosine nucleotides that is attached to the 3′ end of a nucleic acid, such as an RNA. A poly-A tail or poly-A region may consist of nucleotides that are 25-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99- 100% adenosine nucleotides. As used herein, the terms “poly-A tail” and “poly-A region” are used interchangeably. The adenosine nucleotides comprised by a poly-A tail may be canonical adenosine nucleotides or modified (non-canonical) adenosine nucleotides. -28- 4935-1210-1197.1Atty. Docket No.114203-1801

[0089] A “5′ cap,” as used herein, refers to one or more nucleotides that are covalently attached to the 5′ end of a nucleic acid, such as an RNA molecule. A “5′ cap region,” as used herein, refers to a nucleic acid comprising a 5′ nucleotide cap and one or more modified nucleotides. A 5′ cap may comprise a 5′ capping nucleotide that is attached to the 5′ end of a mRNA by a 5′ to 5′ triphosphate internucleotide linkage. In some embodiments, a nucleotide attached to a mRNA by a 5′ to 5′ triphosphate internucleotide linkage is referred to as a “native” 5′ capping nucleotide. In some embodiments, a native 5′ capping nucleotide is a 7- methylguanosine (m7G) nucleotide. In some embodiments, a 5′ cap is a modified 5′ cap, comprising one or more modified nucleotides, such as the 5′ capping nucleotide, or one or more modified internucleotide modifications, such as modifications to the 5′ to 5′ triphosphate internucleotide linkage. In some embodiments, a 5′ cap comprises one or more nucleotides with a sugar modification, such as 2′-O-methylation.

[0090] An example of a canonical structure of 7-methylguanosine (m7G) attached to a ribonucleic acid sequence (e.g., a mRNA) by a 5′ to 5′ triphosphate internucleotide linkage is reproduced below:

[0091] A “counterion” or “anionic counterion” is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality. In some embodiments, an anionic counterion is monovalent (e.g., including one formal negative charge). An anionic counterion may also be multivalent (e.g., including more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F–, Cl–, Br–, I–), NO3–, ClO4–, OH–, H2PO4–, HCO3−, HSO4–, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p–toluenesulfonate, benzenesulfonate, 10–camphor sulfonate, naphthalene–2–sulfonate, naphthalene–1–sulfonic acid–5–sulfonate, ethan–1–sulfonic acid–2– sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, -29- 4935-1210-1197.1Atty. Docket No.114203-1801 tartrate, glycolate, gluconate, and the like), BF4−, PF4–, PF6–, AsF6–, SbF6–, B[3,5-(CF3)2C6H3]4]–, B(C6F5)4−, BPh4–, Al(OC(CF3)3)4–, and carborane anions (e.g., CB11H12–or (HCB11Me5Br6)–). Exemplary counterions which may be multivalent include CO32−, HPO42−, PO43−, B4O72−, SO42−, S2O32−, carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes.

[0092] Use of the phrase “at least one instance” refers to 1, 2, 3, 4, or more instances, but also encompasses a range, e.g., for example, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive. Synthesis of Branched mRNAs

[0093] FIG.1 shows a general synthesis workflow of synthesis of different branched oligonucleotides using a brancher phosphoramidite. The synthesis may be used to form oligonucleotides with a single branch 110 (as in a fork structure), multiple branches stemming off of a primary oligonucleotide 120 (as in a comb structure), ramified multi-branched oligonucleotides 130 with branches stemming off of other branches (“heterozygous”), or multi- branched oligonucleotides with different branch nucleotide sequences 140.

[0094] As shown in FIG.1, a brancher phosphoramidite is used to introduce single or multiple branching points during the solid phase oligo synthesis with solid-phase support (e.g., CPG). After the synthesis of a stem strand, the levulinyl group on the phosphoramidite brancher may be selectively deprotected with hydrazine solution to expose free hydroxyl groups. Branch strand synthesis may then be performed based on the free hydroxyl groups and the desired branched oligo product may be deprotected and cleaved from solid-phase support (e.g., CPG). The 5′ multi-capped oligonucleotide may be obtained after chemical capping and HPLC purification. For example, dual 110 and multiple 120 branched oligonucleotides may be produced if one or multiple branchers, respectively, are introduced during stem strand synthesis, while incorporation of a phosphoramidite brancher during branch strand synthesis may produce the heterozygous multi-branched oligonucleotide 130. Multi-branched oligonucleotide 140 with different branch nucleotide sequences may be synthesized using sequential branching pointing introduction. After the introduction of the first brancher phosphoramidite, the 5′ of the -30- 4935-1210-1197.1Atty. Docket No.114203-1801 oligonucleotide may be protected by dimethoxytrityl (DMTr) group. Then, the blocking group on the brancher may be removed and the branch strand may be extended. Following the same process, the DMTr may be removed, followed by second brancher synthesis. After deblocking, the second branch strand may be be synthesized with different sequences.

[0095] The methods of producing a branched RNA oligonucleotide disclosed herein include synthesizing the branched RNA oligonucleotide with a brancher phosphoramidite. The brancher phosphoramidite includes a first protected hydroxyl group and a second protected hydroxyl group. The brancher phosphoramidite creates a branching point in the RNA oligonucleotide. Synthesis may be performed on a solid-phase support. The solid-phase support may be, for example, glass beads or polystyrene beads.

[0096] The brancher phosphoramidite may have multiple protected hydroxyl groups. The brancher phosphoramidite may include 2, 3, or 4 protected hydroxyl groups. Brancher phosphoramidites with 2 protected hydroxyl groups provide 1 branch in the oligonucleotide. Brancher phosphoramidites with 3 protected hydroxyl groups provide 2 branches in the oligonucleotide. Brancher phosphoramidites with 4 protected hydroxyl groups provide 3 branches in the oligonucleotide. The protecting groups of the brancher phosphoramidite may be deprotected with the same treatment or may be deprotected with different, orthogonal treatments. Where protecting groups are deprotected with the same treatment, the different branches may have the same sequence. A brancher phosphoramidite with orthogonal protecting groups may be used to synthesize different sequences on each branch of the oligonucleotide. For example a brancher phosphoramidite with orthogonal protecting groups may include one protecting group that is dimethoxytrityl (DMTr) deprotected with trichloroacetic acid (TCA), ad another that is a levulinyl protecting group that is deprotected with hydrazine hydrate.

[0097] At least one of the first protected hydroxyl group and the second protected hydroxyl group of the brancher phosphoramidite may include a protecting group selected from one of dimethoxytrityl (DMTr), levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS). The first protected hydroxyl group and the second protected hydroxyl group of the brancher phosphoramidite may be orthogonal, meaning that the protected hydroxyl groups are deprotected with different treatments. For example, the first protected -31- 4935-1210-1197.1Atty. Docket No.114203-1801 hydroxyl group of the brancher phosphoramidite may include dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite may include levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS). As another example, the first protected hydroxyl group of the brancher phosphoramidite may include dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite may include levulinyl.

[0098] The brancher phosphoramidite may have a structure according to Formula I whereinR1is dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite comprises levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS); R2is tert-butyldimethylsilyl ether, halogen, alkyl, or hydrogen; X is a nitrogenous nucleotide base; and n is 1 to 12.

[0099] The brancher phosphoramidite may have a structure according to Formula IAwherein -32- 4935-1210-1197.1Atty. Docket No.114203-1801 R1is dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite comprises levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS); X is adenine, uracil, thymine, guanine, or cytosine in substituted or unsubstituted form; and n is 1 to 12.

[0100] For example, the brancher phosphoramidite may have a structure according to Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X, or Formula XIAtty. Docket No.114203-1801 , whereVIII, Formula IX, Formula X, or Formula XI are, respectively: ,-34- 4935-1210-1197.1Atty. Docket No.114203-1801 ormay include first synthesizing a first oligonucleotide branch with the brancher phosphoramidite, then deprotecting the second protected hydroxyl group on the brancher phosphoramidite to form a hydroxyl group on the first oligonucleotide branch; and then synthesizing a second oligonucleotide branch at the free hydroxyl group on the first oligonucleotide branch, thereby forming the branched RNA oligonucleotide.

[0102] In any embodiment, the brancher phosphoramidite may be a first brancher phosphoramidite and the branching point may be a first branching point, and synthesizing the second oligonucleotide branch may include synthesizing the second oligonucleotide branch with a second brancher phosphoramidite. Synthesis may further include deprotecting a protected group on the second brancher phosphoramidite to form a hydroxyl group on the second oligonucleotide branch, then synthesizing a third oligonucleotide branch at the free hydroxyl group on the second oligonucleotide branch to form a second branching point.

[0103] FIG.2 shows a workflow for the solid phase synthesis of branched oligonucleotide with functional handles. A brancher phosphoramidite may be introduced to oligonucleotide during the stem strand synthesis. After removing the blocking group, the photocleavable linker and purification handle (e.g., fluorous modifier) may be incorporated into the branch strand. The product may be obtained after global deprotection and the resulting oligonucleotides may be used for chemical capping or enzymatic ligation. The purification handle on the oligonucleotide -35- 4935-1210-1197.1Atty. Docket No.114203-1801 may facilitate the purification process of mRNA synthesis, and the purification handle may be subsequently removed upon the exposure to UV light. The functional handle may be any suitable structure with available phosphoramidite monomers, for example purification handles (fluorous modifier or biotin). Branched mRNAs

[0104] In some aspects, the present disclosure provides branched mRNAs comprising one or multiple branching oligonucleotides. The branched RNA oligonucleotide may have a structure according to Formula XII or XIII Formula XIII whereinR3is phosphate or 1-20 nucleotides; R4is phosphate or 1-500 nucleotides; R5is absent, alkyl, alkyl phosphate, or alkylene oxide; R6is phosphate or 1-1000 nucleotides; R7is OH, alkyl, halogen, or hydrogen; X and B are each independently a nitrogenous nucleotide base; and n is 1-12.

[0105] The branched RNA oligonucleotide may have a structure according to Formula XIIA or XIIIA -36- 4935-1210-1197.1Atty. Docket No.114203-1801 Formula XIIIA whereinR3is phosphate or 1-20 nucleotides; R4is phosphate or 1-500 nucleotides; R5is absent, alkyl, alkyl phosphate, or alkylene oxide; R6is phosphate or 1-1000 nucleotides; B is a nitrogenous nucleotide base; and n is 1-12.

[0106] R3of the branched RNA oligonucleotide may include 1-20 nucleotides. R3of the branched RNA oligonucleotide may include 1-10 nucleotides. For example, R3of the branched RNA oligonucleotide may include 6 nucleotides, 8 nucleotides, or 10 nucleotides.

[0107] R4of the branched RNA oligonucleotide may include a nucleotide conjugated to a solid-phase support. The solid-phase support may include controlled-pore glass (CPG) or polystyrene (PS). R4of the branched RNA oligonucleotide may include 1-20 nucleotides. R4of the branched RNA oligonucleotide may include 1-10 nucleotides.

[0108] R5of the branched RNA oligonucleotide may include C1-C12 alkyl, (CH2)xOPOOH(CH2)y, or (CH2CH2O)z; wherein x and y are independently 1-12, and z is 1-2. -37- 4935-1210-1197.1Atty. Docket No.114203-1801

[0109] R6of the branched RNA oligonucleotide may include 1-40 nucleotides, 1-20 nucleotides, or 1-10 nucleotides. R6may include 6 nucleotides, 9 nucleotides, or 12 nucleotides.

[0110] In some aspects, the present disclosure provides modified branched mRNAs comprising multiple 5’ cap regions, wherein the 5’ cap region comprises a 5’ nucleotide cap and one or more modified nucleotides. A modified branched mRNA may be a modified linear branched mRNA.

[0111] The modified branched mRNA may include one or more open reading frames (ORFs). The “5′ cap region”, as used herein, may refer to a region of a branched mRNA that is 5′ to (upstream of) the ORF.

[0112] In some embodiments, the 5’ cap region may include a 5’ untranslated region (5’ UTR). In some embodiments, the 5’ cap region comprises a 5’ cap. In eukaryotic cells, mRNAs possess a cap structure in which an N7-methylguanine (m7G) moiety is linked to the first transcribed nucleotide by a 5’-5’-triphosphate bridge. The 5′ cap plays multiple roles in pre- mRNA splicing, mRNA export, RNA stability through blocking degradation by the 5’-3’ exoribonuclease (ExoN), escaping recognition of the cellular innate immune system, and the production of proteins encoded by mRNAs. The presence of a 5′ cap in an mRNA facilitates the initiation of translation (see, e.g., Gallie. Genes & Dev.1991.5:2108–2116, and Munroe et al. Mol Cell Biol.1990.10(7):3441–3455). The 5′ cap is added by a 5′ capping enzyme, such as mRNA guanylyltransferase. Translation initiation is a rate-limiting step of mRNA translation and heavily depends on the 5’ N7-methylguanosine (m7G) cap and its interaction with eukaryotic translation initiation factors (eIFs), including the cap-binding eIF4E protein. Chemical modification on or near the 5’ cap influence binding of eIFs and decapping enzymes, which subsequently impact downstream mRNA translation and stability. For example, the presence of 2’ O-methyl (2’OMe) groups on the first and second transcribed nucleotides (known as Cap- 0 / 1 / 2, referring to zero, one, or two 2’OMe groups) reduces mRNA immunogenicity and increases protein expression. Additionally, N6-methyladenosine (m6A) on the first base controls mRNA stability through increased resistance to decapping by Dcp2. Furthermore, the 5′ cap stabilizes the mRNA by protecting the ORF from the activity of exonucleases, such as polynucleotide phosphorylase (PNPase), which can remove 3′ and 5′ nucleotides from an -38- 4935-1210-1197.1Atty. Docket No.114203-1801 mRNA. As an exonuclease removes nucleotides, the mRNA becomes progressively shorter, and once all the nucleotides downstream of the open reading frame are removed, the nucleotides removed by the exonuclease are nucleotides of the ORF. Removal of nucleotides from the ORF prevents translation of the encoded protein. Additionally, the association of an exonuclease with the mRNA near the ORF can inhibit translation by sterically hindering ribosomes and tRNAs from associating with the mRNA. The composition of a 5′ cap typically comprises a 5′ m7G attached to the mRNA by a 5′ to 5′ triphosphate internucleotide linkage.

[0113] In some embodiments of the modified branched mRNAs provided herein, the modified branched mRNA comprises one or more modified nucleotides in the 5′ cap region of the mRNA. In some embodiments, the 5′ cap region includes one or more nucleotides that are not canonical adenosine, cytidine, guanosine, or uridine nucleotides. In some embodiments, the 5′ cap region comprises between 1 and 3, between 3 and 5, between 5 and 7, or between 7 and 10 5′ caps. In some embodiments, the 5′ cap region comprises between 10–500 nucleotides. In some embodiments, the 5′ cap region comprises between 10 and 15, between 15 and 20, between 20 and 25, between 25 and 50, between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 and 500 nucleotides. Chemical synthesis of 5’ cap regions

[0114] Existing methods for preparing capped linear mRNA do not accommodate modifications that are not tolerated by RNA polymerase or capping enzymes, nor modifications that extend beyond the first two bases, creating a screening bias due to differing cap incorporation efficiencies. To overcome these challenges, the capping process was decoupled from mRNA synthesis as described herein.

[0115] In some embodiments of the methods provided herein, the method comprises first synthesizing a 5’-phosphorylated branched RNA oligonucleotide with a specific sequence and / or predetermined modifications. In the methods described herein, the synthesized 5’- phosphorylated branched RNA oligonucleotide defines the 5’ UTR when ligated to an RNA transcript. Thus, as used herein, the terms “5’-phosphorylated branched RNA oligonucleotide,” -39- 4935-1210-1197.1Atty. Docket No.114203-1801 “5’-phosphorylated branched oligonucleotide,” and “5’-phosphorylated branched UTR” are used interchangeably. In some embodiments, the 5’-phosphorylated branched RNA oligonucleotide comprises one or more modified nucleotides which may affect RNA translation and / or stability.

[0116] In some embodiments, the 5’-phosphorylated branched oligonucleotide comprises a modified phosphate, resulting in a modified internucleotide linkage. Modified phosphates used in the present invention may be, but are not limited to, phosphorothioate (PS), thiophosphate, 5′-O- methylphosphonate, 3′-O-methylphosphonate, 5′-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate. In some embodiments, more than one modified phosphate is used. In some embodiments, the 5’- phosphorylated oligonucleotide comprises 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 or more modified phosphates. In some embodiments, the 5’-phosphorylated oligonucleotide comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified phosphates. In some embodiments, the modified phosphates of the 5’-phosphorylated oligonucleotide comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total phosphates in the 5’-phosphorylated oligonucleotide.

[0117] In some embodiments, the 5’-phosphorylated branched oligonucleotide comprises a modified sugar. Modified sugars used in the present invention may be, but are not limited to, 2′- deoxy fluoro (2FA), L-adenosine (LA), 2′-deoxyadenosine (dA), locked nucleic acid (LNA), 2′- methoxy (2OMe), 2′-methoxyethoxy (2MOE), 2′-thioribose, 2′,3′-dideoxyribose, 2′-amino-2′- deoxyribose, 2′ deoxyribose, 2′-azido-2′-deoxyribose, 2′-fluoro-2′-deoxyribose, 2′-O- methylribose, 2′-O-methyldeoxyribose, 3′-amino-2′,3′-dideoxyribose, 3′-azido-2′,3′- dideoxyribose, 3′-deoxyribose, 3′-O-(2-nitrobenzyl)-2′-deoxyribose, 3′-O-methylribose, 5′- aminoribose, 5′-thioribose, 5-nitro-1-indolyl-2′-deoxyribose, 5′-biotin-ribose, 2′-O,4′-C- methylene-linked, 2′-O,4′-C-amino-linked ribose, and 2′-O,4′-C-thio-linked ribose. L-adenosine (LA) refers to the enantiomer of D-adenosine. A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2’ -40- 4935-1210-1197.1Atty. Docket No.114203-1801 and 4’ carbons. This structure effectively “locks” the ribose in the 3’-endo structural conformation. In some embodiments, the 5’-phosphorylated branched oligonucleotide comprises 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 or more modified sugars. In some embodiments, the 5’-phosphorylated branched oligonucleotide comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified sugars. In some embodiments, the modified sugars of the 5’-phosphorylated branched oligonucleotide comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total sugars in the 5’-phosphorylated oligonucleotide.

[0118] In some embodiments, the 5’-phosphorylated branched oligonucleotide comprises a modified nucleobase. Modified nucleobases used in the present invention may be, but are not limited to, inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2-thiocytosine, 2- thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6- dihydrouracil, 5-[(3-Indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5- aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5- carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5- hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5- iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5- methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16- aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- 6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1- -41- 4935-1210-1197.1Atty. Docket No.114203-1801 ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl- thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis- hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, the 5’-phosphorylated branched oligonucleotide comprises 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 or more modified nucleobases. In some embodiments, the 5’-phosphorylated branched oligonucleotide comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified nucleobases. In some embodiments, the modified nucleobases of the 5’-phosphorylated branched oligonucleotide comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total nucleobases in the 5’-phosphorylated branched oligonucleotide.

[0119] In some embodiments, the 5’-phosphorylated branched oligonucleotide is synthesized on a solid-phase support. In some embodiments, the solid support is controlled-pore glass (CPG) or polystyrene (PS). In some embodiments, the 5’-phosphorylated branched oligonucleotide is synthesized via phosphoramidite oligonucleotide synthesis. In some embodiments, the 5’- phosphorylated oligonucleotide is synthesized in a solvent system comprising a nonpolar counterion. In some embodiments, the nonpolar counterion used in oligonucleotide synthesis is ammonium. In some embodiments, the nonpolar counterion used in oligonucleotide synthesis is ammonium.

[0120] In some embodiments, a 5’ cap is added to the 5’-phosphorylated branched oligonucleotide to produce multiple 5’-capped oligonucleotide (i.e., a multi-5’-capped UTR). A -42- 4935-1210-1197.1Atty. Docket No.114203-1801 5’ cap can be added to a branched RNA oligonucleotide via enzymatic or chemical reactions. In some embodiments, caps are added to the 5’-phosphorylated branched oligonucleotide through chemical capping methods. Chemical capping may be performed by any method known in the art. Preferably, the chemical capping reaction is performed through an anhydrous reaction between the 5’-phosphorylated branched RNA oligonucleotide and a capping nucleotide conjugated to imidazole in the presence of 1-methylimidazole (see Abe et al., “Complete Chemical Synthesis of Minimal Messenger RNA by Efficient Chemical Capping Reaction” ACS Chem. Biol.2022, 17:1308-1314). In this method, the cap of interest is first conjugated to imidazole. A chemical reaction is then performed between the imidazole-conjugated capping oligonucleotide and a branch terminus of the 5’-phosphorylated branched oligonucleotide under anhydrous conditions and in the presence of 1-methylimidazole. This chemical reaction may be repeated for each branch of the 5’-phosphorylated branched oligonucleotide. In some embodiments, the capping reaction is performed in dimethyl sulfoxide (DMSO). The desired product of this reaction is a branched oligonucleotide with multiple caps on its 5’ ends with the cap of interest.

[0121] In some embodiments, the 5’ cap used in the present invention may be, but is not limited to, 7-methylguanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’- guanosine (m7G-3’m-ppp-G), N7,2’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7Gm-ppp-G), 7-benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G-LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4- chlorophenoxyethyl)-guanosine, 7-ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7-(carboxymethyl) guanosine (cm7G), 7-(2-phenylethyl) guanosine [7-(2- PhEt)G], 7-(1-phenylethyl) guanosine [7-(1-PhEt)G], m7GpppBH3G (D1 and D2 stereoisomers), m7GppBH3G (D1 and D2 stereoisomers), m7GpBH3G (D1 and D2 stereoisomers), m7GppBH3pm7G, m27,2’-OGpppBH3G (D1 and D2 stereoisomers), m27,2’-OGppBH3pG (D1 and D2 diastereomers), m27,2’-OGppSpG (D1 and D2 diastereomers), N-Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, 1-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic -43- 4935-1210-1197.1Atty. Docket No.114203-1801 moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, cap1, cap2, cap3, cap4, ARCA, modified ARCA, inosine, N1- methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0122] Thus, in some embodiments, a 5’ cap region provided herein comprises a 5’-capped oligonucleotide (i.e., a 5’-capped UTR) synthesized as described above. In some embodiments, the 5’ cap region comprises a modified 5’ cap, one or more modified phosphates, one or more modified sugars, and / or one or more modified nucleobases. The 5’ cap region may comprise any combination of modifications. In some embodiments, the 5’ cap region is between 5 and 50, between 10 and 45, between 15 and 40, between 20 and 35, between 25 and 30, or more than 30 nucleotides in length. In some embodiments, the 5’ cap region comprises 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 or more modified nucleotides. In some embodiments, the 5’ cap region comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified nucleotides. In some embodiments, the modified nucleotides of the 5’ cap region comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total nucleotides in the 5’ cap region.

[0123] In some embodiments, a capped RNA transcript as provided herein comprises more than one poly-A tail. Nucleotide modifications

[0124] In some embodiments, as disclosed and recognized herein it is beneficial to alter the type of nucleotide / nucleotide identity, specifically incorporation of adenosine (A), guanosine (G), 6-methyladenosine (m6A), or the non-canonical inosine (I) in the mRNA, preferably, at the +1 position, increases translation efficiency. In some embodiments, substitution of some or all uridine residues to N1-methylpseudouridine (m1Ψ) in the mRNA also boosts the translation. The nucleotides are numbered according to their position immediately downstream of the cap structure. For example, the cap structure found at the 5′ end of eukaryotic mRNAs consists of a -44- 4935-1210-1197.1Atty. Docket No.114203-1801 7-methylguanosine (m7G) moiety linked to the first nucleotide (+1 position) of the transcript via a 5′–5′ triphosphate bridge.

[0125] Other modified nucleotides include, but are not limited to, pseudouridine, 5- methylcytidine, 2-thiouridine, 5-methoxyuridine, 4-acetylcytidine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-Indolyl)propionamide-N-allyl]uracil, 5- aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5- carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5- hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5- iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5- methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16- aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- 6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1- ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl- thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis- hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine -45- 4935-1210-1197.1Atty. Docket No.114203-1801 (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A) have also been contemplated at +1 and other positions.

[0126] In some embodiments, the modified phosphate backbone can be phosphorothioate (PS), thiophosphate, 5′-O-methylphosphonate, 3′-O-methylphosphonate, 5-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, or guanidinopropyl phosphoramidate.

[0127] In some embodiments, introduction of locked nucleic acid (LNA), 2’-methoxyribose (2-OMe), and 2-methoxyethoxy (2-MOE) into the ribose sugar backbone increases mRNA translation. Addition of multiple 2-OMe and 2-MOE modified bases increases translation further. LNA specifically increased expression at the +1 position.

[0128] In some embodiments, the modified sugar can be 2-thioribose, 2,3-dideoxyribose, 2- amino-2-deoxyribose, 2’ deoxyribose, 2’-azido-2’-deoxyribose, 2’-fluoro-2’-deoxyribose, 2’-O- methylribose, 2’-O-methyldeoxyribose, 3’-amino-2’, 3’-dideoxyribose, 3’-azido-2,3- dideoxyribose, 3’-deoxyribose, 3’-O-(2-nitrobenzyl)-2’-deoxyribose, 3’-O-methylribose, 5’- aminoribose, 5’-thioribose, 5-nitro-1-indolyl-2’-deoxyribose, 5’-biotin-ribose, 2’-O,4’-C- methylene-linked, 2’-O,4’-C-amino-linked ribose, or 2’-O,4’-C-thio-linked ribose.

[0129] In these backbone modifications, stereoisomer structures are also considered since they have been shown to impact the RNA’s nuclease-resistance properties (Iwamoto et al., 2017, Nat. Biotech.35: 845-851; Jahns et al., 2022, Nucleic Acids Res.50(3): 1221-1240). Purification of capped branched RNA

[0130] In some embodiments, the nucleic acids described herein are purified by any method known in the art to remove undesired components from IVT or associated reactions (including unincorporated rNTPs, protein enzymes, salts, metal ions, etc.). Techniques for the isolation of RNA molecules are well known in the art. Well-known procedures include phenol / chloroform extraction and or precipitation with alcohol (ethanol, isopropanol) in the presence of monovalent -46- 4935-1210-1197.1Atty. Docket No.114203-1801 cations or lithium chloride. Additional non-limiting examples of purification procedures which can be used include size exclusion chromatography (Lukavsky, P.J. and Puglisi, J.D., 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, et al. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v.941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012). Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek). Techniques to remove contaminants, such as dsDNA, have been developed and are known in the art including but not limited to scalable HPLC purification (see, e.g., Kariko, et al., 2011, Nucl Acid Res, v.39 e142; Weissman, et al., 2012, Synthetic Messenger RNA and Cell Metabolism Modulation v.969 (Rabinovich, P.H. Ed)). In a preferred embodiment, the capped RNA described herein is purified through HPLC, as HPLC-purified RNA has been reported to be translated at much greater levels compared to other purification methods, particularly in primary cells and in vivo.

[0131] In some aspects, synthesis of the capped branched oligonucleotides is performed on a solid phase support and purification may include washing the solid phase support after synthesis of the capped branched oligonucleotides to remove unreacted reagents and byproducts, followed by cleavage of the capped branched oligonucleotides from the solid phase support. Further purification after cleavage may include ethanol precipitation or column purification to remove residual contaminants. In this way, using the solid phase support provides a synthesis method that does not require the use of high-performance liquid chromatography (HPLC) for purification, thereby providing faster synthesis and scalability.

[0132] In some aspects, the capped RNA oligonucleotides provided herein (e.g., comprising one or more modified oligonucleotides) may be purified by high-performance liquid chromatography (HPLC). In some embodiments, the capped RNA oligonucleotide is purified by reverse-phase HPLC (RP-HPLC). The addition of a counterion to the mobile phase of a HPLC setup can improve separation of the desired product from unwanted products. In a preferred embodiment, HPLC gradients used to isolate the capped RNA oligonucleotides comprise hydrophobic hexylammonium ions. In some embodiments, the gradient is chosen from ethyl -47- 4935-1210-1197.1Atty. Docket No.114203-1801 ammonium, diethyl ammonium, triethyl ammonium, propyl ammonium, dipropyl ammonium, hexyl ammonium, dihexyl ammonium, octyl ammonium, dioctyl ammonium, etc. The number and lengths of carbon chains may be altered based on the lengths of the oligonucleotide to be captured and the desired feature for separation. In some embodiments, the concentration of hydrophobic ions (e.g., hexylammonium ions) used for HPLC purification of RNA oligonucleotides is between 10 mM to 200 mM. In some embodiments, the concentration of hydrophobic ions is between 10 mM and 20 mM, between 20 mM and 30 mM, between 30 mM and 40 mM, between 40 mM and 50 mM, between 50 mM and 60 mM, between 60 mM and 70 mM, between 70 mM and 80 mM, between 80 mM and 90 mM, between 90 mM and 100 mM, between 100 mM and 110 mM, between 110 mM and 120 mM, between 120 mM and 130 mM, between 130 mM and 140 mM, between 140 mM and 150 mM, between 150 mM and 160 mM, between 160 mM and 170 mM, between 170 mM and 180 mM, between 180 mM and 190 mM, or between 190 mM and 200 mM. In some embodiments, the concentration of hydrophobic ions is greater than 200 mM. Compositions comprising capped branched RNA transcripts

[0133] In some aspects, the present disclosure provides a delivery reagent comprising any of the capped branched RNA transcripts provided herein. In some embodiments, any of the capped branched RNA transcripts provided herein are conjugated to a delivery agent. Any of the capped branched RNA transcripts provided herein may be conjugated to a delivery agent that includes, for example, a lipid, a peptide, a protein, an antibody, or a carbohydrate. Lipids used in the conjugation and delivery of branched mRNAs are generally known in the art, and include, for example, cholesterol. Peptides, proteins, antibodies, and carbohydrates used in the conjugation and delivery of branched mRNAs are generally known in the art and include, for example, any peptide, protein, antibody, or carbohydrate known to bind specifically to a moiety (e.g., a protein) on the surface of a target cell type. Methods for conjugating a lipid, peptide, protein, antibody, or carbohydrate to a capped branched RNA transcript include, for example, methods of conjugating a lipid, peptide, protein, antibody, or carbohydrate to a capped branched RNA transcript at a 5’ or 3’ terminus, and are generally known in the art. -48- 4935-1210-1197.1Atty. Docket No.114203-1801

[0134] In some embodiments, any of the capped branched RNA transcripts provided herein are conjugated to or encapsulated by a delivery agent that includes, for example, a nanoparticle, a microparticle, or an exosome. A nanoparticle refers to a particle having a diameter between approximately 10 nm and 1000 nm. A microparticle is defines as a particle having a diameter greater than 1000 nm (1 µm), such as a particle having a diameter between approximately 1 µm and 100 µm. In some embodiments, a nanoparticle or microparticle is approximately spherical. In some embodiments, a nanoparticle or microparticle is hollow, comprising an internal core. In some embodiments, a nanoparticle or microparticle is a lipid nanoparticle or lipid microparticle, respectively. A lipid nanoparticle or lipid microparticle refers to a composition comprising one or more lipids that form an aggregate of lipids, or an enclosed structure with an interior surface and an exterior surface. In some embodiments, a lipid nanoparticle or lipid microparticle comprises a lipid bilayer that encloses an aqueous core. Lipids used in the formulation of lipid nanoparticles and lipid microparticles for delivering RNAs are generally known in the art, and include, but are not limited to, ionizable amino lipids, non-cationic lipids, sterols, and polyethylene glycol-modified lipids. See, e.g., Buschmann et al. Vaccines.2021.9(1):65. In some embodiments, the capped RNA transcript is surrounded by the lipids of the lipid nanoparticle or the lipid microparticle and are present in the interior of the lipid nanoparticle or lipid microparticle. In some embodiments, the capped branched RNA transcript is dispersed throughout the lipids of the lipid nanoparticle or lipid microparticle. In some embodiments, the lipid nanoparticle or lipid microparticle comprises an ionizable amino lipid, a non-cationic lipid, a sterol, and / or a polyethylene glycol (PEG)-modified lipid. Lipid nanoparticles and lipid microparticles comprising branched mRNAs may be prepared by any means generally known in the art, such as, for example, detergent dialysis, emulsion, centrifugation, evaporation, thin film hydration, or ethanol dilution. See, e.g., Barba et al. Pharmaceutics.2019.11(8):360. An exosome refers to a type of lipid nanoparticle produced by eukaryotic cells as a result of the inward budding of vesicles within multivesicular bodies and are generally between 30 nm and 150 nm in diameter. Exosomes comprise a heterogenous mixture of endogenous lipids, such as phospholipids, membrane-anchored proteins, and carbohydrates present in eukaryotic cells, and enclose an aqueous core. Exosomes may have beneficial features that are difficult to achieve with synthetically produced lipid nanoparticles, such as, for example, the ability to pass through the blood brain barrier and deliver capped branched RNA transcripts to tissues within the brain. -49- 4935-1210-1197.1Atty. Docket No.114203-1801 Exosomes comprising capped branched RNA transcripts may be produced by any means generally known in the art, such as, for example, by sonicating or electroporating isolated exosomes in the presence of a capped branched RNA transcript, or mixing exosomes with a lipid-conjugated capped RNA transcript, such as, for example, a capped branched RNA transcript that has been conjugated to cholesterol. See, e.g., Roberts et al. Nat Rev Drug Discov. 2020.19(10):673-694.

[0135] In some embodiments, a nanoparticle or microparticle is a polymeric nanoparticle or polymeric microparticle, respectively. A polymeric nanoparticle or polymeric microparticle refers to a nanoparticle or microparticle composition, respectively, comprising one or more polymers that form an aggregate of polymers, or an enclosed structure with an interior surface and an exterior surface. In some embodiments, a polymeric nanoparticle or polymeric microparticle comprises a polymeric layer that encloses an aqueous core. Polymers used in the formulation of polymeric nanoparticles and polymeric microparticles for delivering RNA are generally known in the art, and include cationic polymers such as, but are not limited to, polyethylenimine (PEI), poly-amido-amine (PAA), poly-beta amino-esters (PBAEs), polylysine (PLL), spermine, chitosan, polyurethane, and derivatives thereof (e.g., PEI stearic acid (PSA) copolymer). See, e.g., Liu et al. Front Bioeng Biotechnol.2021.9:718753. In some embodiments, the capped branched RNA transcript is surrounded by the polymers of the polymeric nanoparticle or the polymeric microparticle and are present in the interior of the polymeric nanoparticle or polymeric microparticle. In some embodiments, the capped branched RNA transcript is dispersed throughout the polymers of the polymeric nanoparticle or polymeric microparticle.

[0136] In some embodiments, a nanoparticle or microparticle is a protein nanoparticle or protein microparticle, respectively. A protein nanoparticle or protein microparticle refers to a nanoparticle or microparticle composition, respectively, comprising one or more proteins that form an aggregate of proteins, or an enclosed structure with an interior surface and an exterior surface. In some embodiments, a protein nanoparticle or protein microparticle comprises a protein layer that encloses an aqueous core. Proteins used in the formulation of protein nanoparticles and protein microparticles for delivering RNA are generally known in the art, and include but are not limited to, viral coat proteins and ferritin. See, e.g., Wang et al. Nat -50- 4935-1210-1197.1Atty. Docket No.114203-1801 Nanotechnol.2020.15(5):406-416. In some embodiments, the capped branched RNA transcript is surrounded by the proteins of the protein nanoparticle or the protein microparticle and are present in the interior of the protein nanoparticle or protein microparticle. In some embodiments, the capped branched RNA transcript is external to the proteins of the protein nanoparticle or the protein microparticle and are attached to the exterior surface of the protein nanoparticle or protein microparticle. In some embodiments, the capped branched RNA transcript is conjugated to proteins of the protein nanoparticle or protein microparticle through a covalent linkage, such as, for example, that formed by a click chemistry reaction, or by fusing the capped branched RNA transcript and protein each to a protein or peptide of a protein / peptide pair known to react to form a covalent linkage.

[0137] In some embodiments, a nanoparticle or microparticle is a solid nanoparticle or solid microparticle. A solid nanoparticle or solid microparticle refers to a nanoparticle or microparticle composition, respectively, comprising one or more materials that form a solid structure, which has an external surface and may or may not comprise an internal surface. A solid nanoparticle or solid microparticle may comprise any suitable material that is generally known in the art, such as, for example, gold, silver, or silicon dioxide (silica). In some embodiments, a capped branched RNA transcript is conjugated to the external surface of a solid nanoparticle or solid microparticle. Solid nanoparticles and solid microparticles comprising capped branched RNA transcripts may be produced by any means generally known in the art, such as, for example, by linking the capped branched RNA transcripts to the surface of the solid nanoparticle or solid microparticle through thiol linkages (e.g., modifying the DNA to comprise cyclic disulfide- anchoring groups), or by modifying the external surface of the solid nanoparticle or solid microparticle with one or more cationic materials (e.g., PEI) within which capped branched RNA transcripts are present. See, e.g., Roberts et al. Nat Rev Drug Discov.2020.19(10):673-694, Lee et al. Nano Lett.2007, 7(7):2112–2115, and Paris and Vallet-Regi. Pharmaceutics.2020, 12(6):526.

[0138] In some aspects, the present disclosure provides cells comprising any of the capped RNA branched transcripts provided herein. In some embodiments, the cell is a human cell comprising any one of the capped RNA transcripts provided herein. A “cell” is the basic structural and functional unit of all known independently living organisms. It is the smallest unit -51- 4935-1210-1197.1Atty. Docket No.114203-1801 of life that is classified as a living thing. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as plants, fungi, and animals, including cattle, horses, chickens, turkeys, sheep, swine, dogs, cats, and humans, are multicellular. In some embodiments, the half-life of the capped branched RNA transcript in the cell is 15–900 minutes. In some embodiments, the half-life of the capped branched RNA transcript in the cell is 30–600 minutes. In some embodiments, the half-life of the capped branched RNA transcript in the cell is 60–300 minutes. In some embodiments, the half-life of the capped branched RNA transcript is at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 minutes. In some embodiments, the half-life of the capped branched RNA transcript in the cell is at least 30, at least 60, at least 90, at least 120, at least 150, at least 180, at least 210, at least 240, at least 270, at least 300, at least 330, at least 360, at least 390, at least 420, at least 450, at least 480, at least 510, at least 540, at least 570, at least 600, at least 630, at least 660, at least 690, at least 720, at least 750, at least 780, at least 810, at least 840, or at least 870 minutes. In some aspects, the present disclosure provides compositions comprising any of the modified mRNAs, delivery agents, or cells provided herein. In some embodiments, the composition further comprises one or more additional agents, such as a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a small molecule, an aptamer, a lipid, or a carbohydrate. In some embodiments, the additional agent has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is an agent for use in modulating the expression and / or activity of one or more gene products (e.g., proteins) in a subject. In some embodiments, the additional agent is a nucleic acid for use in decreasing the expression and / or activity of one or more gene products (e.g., proteins), such as a short hairpin RNA (shRNA), small interfering RNA (siRNA), or an antisense oligonucleotide (ASO). In some embodiments, the additional agent is an inhibitor for decreasing the activity of one or more gene products (e.g., proteins). In some embodiments, the agent is a small molecular inhibitor. In some embodiments, the additional agent is an agent for enhancing an immune response in a subject. In some embodiments, the additional agent is an antigen, such as a nucleic acid antigen, a protein antigen, or a phospholipid antigen. In some embodiments, the additional agent is an adjuvant, such as, for example, aluminum hydroxide or potassium aluminum sulfate (alum), monophosphoryl lipid A (MPL), an oil-in-water emulsion (e.g., a squalene emulsion), a cytosine phosphoguanine (CpG) oligodeoxynucleotide, or another adjuvant that is known in the art. See, e.g., Di Pasquale, A et al. -52- 4935-1210-1197.1Atty. Docket No.114203-1801 Vaccines.2015.3(2):320–343. In some embodiments, the composition is a pharmaceutical composition comprising any one of the capped branched RNA transcripts, delivery agents, or cells provided herein, and a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients, carriers, buffers, stabilisers, isotonicising agents, preservatives or antioxidants, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g., parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal.

[0139] In some aspects, the present disclosure provides a method of administering to a subject any of the capped branched RNA transcripts, delivery agents, cells, compositions, or pharmaceutical compositions provided herein. In some embodiments, the subject is a human. In some embodiments, the administration is parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal. In some embodiments, the composition is to be stored below 50 °C, below 40 °C, below 30 °C, below 20 °C, below 10 °C, below 0 °C, below -10 °C, below -20 °C, below -30 °C, below -40 °C, below -50 °C, below -60 °C, below -70 °C, or below -80 °C, such that the nucleic acids are relatively stable over time. In some embodiments, the capped branched RNA transcript is introduced into a cell in a subject by in vivo electroporation. In vivo electroporation is the process of introducing nucleic acids or other molecules into a cell of a subject using a pulse of electricity, which promote passage of the nucleic acids or other molecules through the cell membrane and / or cell wall. See, e.g., Somiari et al. Molecular Therapy., 2000.2(3):178–187. The capped branched RNA transcript to be delivered is administered to the subject, such as by injection, and a pulse of electricity is applied to the injection site, whereby the electricity promotes entry of the nucleic acid into cells at the site of administration. In some embodiments, the capped branched RNA transcript is delivered to and taken up by cells of the subject (e.g., cells local to the site of administration or throughout the subject) via a delivery agent that is associated with (e.g., conjugated to) the capped branched RNA transcript. In some embodiments, the capped branched RNA transcript is administered with other elements, such as buffers and / or excipients, which increase the efficiency of electroporation. -53- 4935-1210-1197.1Atty. Docket No.114203-1801

[0140] In some aspects, the present disclosure provides a kit comprising any of the capped branched RNA oligonucleotides, RNA precursors, or capped branched RNA transcripts provided herein. The capped branched RNA oligonucleotide and RNA precursor can be combined in the presence of an RNA ligase to produce a capped branched RNA transcript, such as one of the capped branched RNA transcripts provided herein. In some embodiments, the kit comprises a ligase. In some embodiments, the kit comprises an RNA ligase. In some embodiments, the kit comprises a T4 RNA ligase. In some embodiments, a kit comprises a T4 RNA ligase 1. In some embodiments, a kit comprises a T4 RNA ligase 2. In some embodiments, the kit comprises an RtcB RNA ligase. In some embodiments, the kit further comprises a buffer for carrying out the ligation. In some embodiments, the kit further comprises a nucleotide triphosphate, such as ATP, to provide energy required by the ligase. In some embodiments, the kit is to be stored below 50 °C, below 40 °C, below 30 °C, below 20 °C, below 10 °C, below 0 °C, below -10 °C, below -20 °C, below -30 °C, below -40 °C, below -50 °C, below -60°C, below -70 °C, or below -80 °C, such that the nucleic acids are relatively stable over time.

[0141] In some aspects, the present disclosure provides a kit comprising any of the pharmaceutical compositions provided herein and a delivery device. A delivery device refers to machine or apparatus suitable for administering a composition to a subject, such as a syringe or needle. In some embodiments, the kit is to be stored below 50 °C, below 40 °C, below 30 °C, below 20 °C, below 10 °C, below 0 °C, below -10 °C, below -20 °C, below -30 °C, below -40 °C, below -50 °C, below -60°C, below -70 °C, or below -80 °C, such that the nucleic acids of the pharmaceutical composition are relatively stable over time. In some embodiments, the kit further comprises instructions for administering any of the pharmaceutical compositions provided herein to a subject. Pharmaceutical compositions for delivery and methods therefore

[0142] This invention provides pharmaceutical compositions comprising capped branched RNA molecules of the disclosure, particularly linear and circularized mRNA molecules. In certain embodiments pharmaceutical compositions of the invention further comprise pharmaceutically acceptable excipients and in certain other embodiments comprise one or more additional therapeutics agents. -54- 4935-1210-1197.1Atty. Docket No.114203-1801

[0143] In some embodiments, the compositions are suitable to be administered to a human subject in need thereof. In the context of the present disclosure, “active ingredient” refers generally to the capped branched RNA molecules described herein, particularly linear branched mRNA molecules as well as any additional therapeutic agents provided therewith.

[0144] It is generally understood by a person of ordinary skill in the art that the compositions described herein are also suitable for administration to any non-human subjects as well. A person of ordinary skill in the veterinary arts will understand that pharmaceutical compositions described herein can be suitable for administration to mammals including but not limited to primates, cattle, pigs, horses, sheep, goats, cats, dogs, mice, rats, whales, and other mammals. A person of ordinary skill in the veterinary arts also will understand that pharmaceutical compositions described herein can be suitable for administration to birds including by not limited to chickens, ducks, geese, turkey, and other domesticated birds, as well as wild birds particularly endangered species of such birds. Additionally, a person of ordinary skill in the veterinary arts will understand that pharmaceutical compositions described herein can be suitable for administration to a wide variety of fish including commercial or wild salmon, tuna, cod, sardine, zebra fish, shark, or the like.

[0145] Pharmacological compositions described herein can be prepared by any method known or developed in the art of pharmacology, immunology, virology, or in biotechnology in general.

[0146] In some embodiments, the formulations of a pharmacological composition described herein can comprise a unit dose of at least one RNA, in addition to at least one other pharmaceutically acceptable excipient. Such excipients can include but are not limited to, solvents, dispersions, buffers, diluents, surfactants, emulsifiers, isotonic agents, preservatives, thickeners, lubricating agents, oils, or the like.

[0147] In some embodiments, the pharmacological composition can comprise a delivery mechanism further comprising a lipid nanoparticle. The size of the lipid nanoparticle can be altered to counteract immunogenic response from the subject, or to allow for increased potency and pharmacological activity. -55- 4935-1210-1197.1Atty. Docket No.114203-1801

[0148] In other embodiments, the pharmacological composition can comprise a delivery mechanism further comprising a lipidoid as previously described in the art. See Akinc et al., 2008, Nat Biotechnol. 26:561-596; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920; Akinc et al., 2009, Mol Ther.17:872-879; Love et al., 2010. Proc Natl Acad Sci USA 107:1864-1869; Leuschner et al., 2011, Nat Biotechnol. 29:1005-1010, all of which is incorporated herein in their entirety. Lipidoids refers broadly to lipid nanoparticles, liposomes, lipid emulsions, lipid micelles and the like. Lipidoids containing the pharmacological composition comprising the derivatized RNA can be administered parenterally by means including but not limited to, intravenous injection, intramuscular injection, subcutaneous injection, via dialysate, intrathecal injection, or intracranial injection.

[0149] A person of ordinary skill in the art would also recognize that other nucleotide delivery mechanisms exist such as the use of viral like, or viral derived particles. See Rohovie et al., 2016, Bioengineering & Translational Med.2(1): 43-57. Virus like particles can include coat proteins or viral capsids of a virus. Such particles can be PEGylated or further annealed to compounds that avoid phagocytotic clearance. Additionally, the surface of the virus like particle can be further functionalized to provide cellular specific targeting, facilitate extravasation, facilitate radio labeling, improve permeability across cellular boundaries, or to transcytose the blood-brain barrier. The virus like particles can be derived for animal viruses, bacteriophages, or plant viruses. Examples of suitable virus for derivation of a virus like particle delivery mechanism include but are not limited to cowpea chlorotic mottle virus, cowpea mosaic virus, hepatitis B virus (core), enterobacteria phage MS2, Salmonella typhimurium P22, enterobacteria phage Qβ amongst other suitable viruses. Derivatized RNA payloads can be loaded into the virus like particles by electrostatic adsorption or any other suitable method known to a person of ordinary skill in the art.

[0150] Various exemplary embodiments of compositions and methods according to this invention are now described in the following non-limiting Examples. The Examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims. -56- 4935-1210-1197.1Atty. Docket No.114203-1801 EXAMPLES OF EMBODIMENTS

[0151] The following examples are given to illustrate the present disclosure. It should be understood, however, that the disclosure is not to be limited to the specific conditions or details described in these examples.

[0152] Embodiment 1: A method of producing a branched RNA oligonucleotide, comprising: synthesizing the branched RNA oligonucleotide with a brancher phosphoramidite including a first protected hydroxyl group and a second protected hydroxyl group, creating a branching point in the branched RNA oligonucleotide with the brancher phosphoramidite; wherein the step of synthesizing is performed on a solid-phase support.

[0153] Embodiment 2: The method of embodiment 1, wherein at least one of the first protected hydroxyl group and the second protected hydroxyl group of the brancher phosphoramidite comprises dimethoxytrityl (DMTr), levulinyl, benzyl (Bn), tert- butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS).

[0154] Embodiment 3: The method of embodiment 1, wherein the first protected hydroxyl group and the second protected hydroxyl group of the brancher phosphoramidite are deprotected with different treatments.

[0155] Embodiment 4: The method of embodiment 3, wherein the first protected hydroxyl group of the brancher phosphoramidite comprises dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite comprises levulinyl, benzyl (Bn), tert- butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS).

[0156] Embodiment 5: The method of embodiment 4, wherein the first protected hydroxyl group of the brancher phosphoramidite comprises dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite comprises levulinyl.

[0157] Embodiment 6: The method of embodiment 1 or 2, wherein the brancher phosphoramidite has a structure according to Formula I -57- 4935-1210-1197.1Atty. Docket No.114203-1801 whereinR1is dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite comprises levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS); R2is tert-butyldimethylsilyl ether, halogen, alkyl, or hydrogen; X is a nitrogenous nucleotide base; and n is 1 to 12.

[0158] Embodiment 7: The method of embodiment 1 or 2, wherein the brancher phosphoramidite has a structure according to Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X, or Formula XI-58- 4935-1210-1197.1Atty. Docket No.114203-1801m n are some Formula IX, Formula X, or Formula XI are, respectively: -59- 4935-1210-1197.1Atty. Docket No.114203-1801 orthe step of synthesizing comprises (a) synthesizing a first oligonucleotide branch with the brancher phosphoramidite; (b) deprotecting the second protected hydroxyl group on the brancher phosphoramidite to form a hydroxyl group on the first oligonucleotide branch; and (c) synthesizing a second oligonucleotide branch at the free hydroxyl group on the first oligonucleotide branch, thereby forming the branched RNA oligonucleotide.

[0160] Embodiment 9: The method of embodiment 8, wherein the brancher phosphoramidite is a first brancher phosphoramidite and the branching point is a first branching point, and synthesizing the second oligonucleotide branch comprises synthesizing the second oligonucleotide branch with a second brancher phosphoramidite.

[0161] Embodiment 10: The method of embodiment 9, further comprising: (d) deprotecting a protected group on the second brancher phosphoramidite to form a hydroxyl group on the second oligonucleotide branch; (e) synthesizing a third oligonucleotide branch at the free hydroxyl group on the second oligonucleotide branch to form a second branching point. -60- 4935-1210-1197.1Atty. Docket No.114203-1801

[0162] Embodiment 11: The method of any one of embodiments 1-8, wherein the first oligonucleotide branch of the branched RNA oligonucleotide comprises 1 nucleotide to 40 nucleotides between the branching point in the branched RNA oligonucleotide and a terminus of the first oligonucleotide branch.

[0163] Embodiment 12: The method of embodiment 11, wherein the first oligonucleotide branch of the branched RNA oligonucleotide comprises 6 nucleotides.

[0164] Embodiment 13: The method of embodiment 11, wherein the first oligonucleotide branch of the branched RNA oligonucleotide comprises 8 nucleotides.

[0165] Embodiment 14: The method of embodiment 11, wherein the first oligonucleotide branch of the branched RNA oligonucleotide comprises 10 nucleotides.

[0166] Embodiment 15: The method of any one of embodiments 1-8 or 11-14, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises 1 nucleotide to 40 nucleotides between the branching point in the branched RNA oligonucleotide and a terminus of the second oligonucleotide branch.

[0167] Embodiment 16: The method of embodiment 15, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises 7 nucleotides.

[0168] Embodiment 17: The method of embodiment 15, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises 10 nucleotides.

[0169] Embodiment 18: The method of embodiment 15, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises 13 nucleotides.

[0170] Embodiment 19: The method of any one of embodiments 1-18, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises a spacer at the branching point of the branched RNA oligonucleotide.

[0171] Embodiment 20: The method of embodiment 19, wherein the spacer comprises C1- C12 alkyl or (CH2CH2O)n where n is 1-4. -61- 4935-1210-1197.1Atty. Docket No.114203-1801

[0172] Embodiment 21: The method of any one of embodiments 1-20, wherein the solid- phase support comprises glass beads or polystyrene beads.

[0173] Embodiment 22: The method of embodiment 21, further comprising cleaving the branched RNA oligonucleotide from the solid-phase support.

[0174] Embodiment 23: The method of any one of embodiments 1-22, further comprising capping phosphorylated 5’ terminuses of the branched RNA oligonucleotide, producing a capped branched RNA oligonucleotide.

[0175] Embodiment 24: The method of embodiment 23, further comprising purifying the capped branched RNA oligonucleotide, producing a purified capped branched RNA oligonucleotide.

[0176] Embodiment 25: The method of embodiment 24, wherein the step of purifying comprises performing high-performance liquid chromatography (HPLC).

[0177] Embodiment 26: The method of any one of embodiments 23 to 25, wherein the capping is performed chemically.

[0178] Embodiment 27: The method of embodiment 26, wherein chemical capping is performed through an anhydrous reaction between the first 5’-phosphorylated RNA oligonucleotide, the second 5’-phosphorylated RNA oligonucleotide, and capping nucleotides conjugated to imidazole in the presence of 1-methylimidazole.

[0179] Embodiment 28: The method of any one of embodiments 1-27, wherein the second oligonucleotide branch comprises a 5’ untranslated region (5’ UTR) comprising an unstructured region.

[0180] Embodiment 29: The method of embodiment 28, wherein the unstructured region comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more consecutive adenosine, cytosine, guanine, or uridine nucleotides. -62- 4935-1210-1197.1Atty. Docket No.114203-1801

[0181] Embodiment 30: The method of embodiment 28, wherein the unstructured region comprises no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10 consecutive adenosine, cytosine, guanine, or uridine nucleotides.

[0182] Embodiment 31: The method of embodiment 28, wherein the unstructured region comprises between 5 and 60, between 10 and 55, between 15 and 50, between 20 and 45, between 25 and 40, or between 30 and 35 consecutive adenosine, cytosine, guanine, or uridine nucleotides.

[0183] Embodiment 32: The method of any one of embodiments 1-27, wherein the first oligonucleotide branch is between 3 and 160, between 5 and 150, between 7 and 140, between 9 and 130, between 11 and 120, between 13 and 110, between 15 and 100, between 17 and 90, between 19 and 80, between 21 and 70, between 23 and 60, between 25 and 50, between 27 and 40, or between 30 and 35 nucleotides in length.

[0184] Embodiment 33: The method of embodiment 32, wherein the first oligonucleotide branch is between 25 and 35 nucleotides in length.

[0185] Embodiment 34: The method of embodiment 32 or 33, wherein the second oligonucleotide branch is between 3 and 160, between 5 and 150, between 7 and 140, between 9 and 130, between 11 and 120, between 13 and 110, between 15 and 100, between 17 and 90, between 19 and 80, between 21 and 70, between 23 and 60, between 25 and 50, between 27 and 40, or between 30 and 35 nucleotides in length.

[0186] Embodiment 35: The method of embodiment 34, wherein the second oligonucleotide branch is between 25 and 35 nucleotides in length.

[0187] Embodiment 36: The method of any one of embodiments 1-35, wherein the first oligonucleotide branch comprises one or more modified nucleotides.

[0188] Embodiment 37: The method of any one of embodiments 1-36, wherein the second oligonucleotide branch comprises one or more modified nucleotides. -63- 4935-1210-1197.1Atty. Docket No.114203-1801

[0189] Embodiment 38: The method of embodiment 36 or 37, wherein the one or more modified nucleotides comprises a modified sugar.

[0190] Embodiment 39: The method of embodiment 38, wherein the modified sugar is selected from the group consisting of 2′-deoxy fluoro (2FA), L-adenosine (LA), 2′- deoxyadenosine (dA), locked nucleic acid (LNA), 2′-methoxy (2OMe), 2′-methoxyethoxy (2MOE), 2′-thioribose, 2′,3′-dideoxyribose, 2′-amino-2′-deoxyribose, 2′ deoxyribose, 2′-azido-2′- deoxyribose, 2′-fluoro-2′-deoxyribose, 2′-O-methylribose, 2′-O-methyldeoxyribose, 3′-amino- 2′,3′-dideoxyribose, 3′-azido-2′,3′-dideoxyribose, 3′-deoxyribose, 3′-O-(2-nitrobenzyl)-2′- deoxyribose, 3′-O-methylribose, 5′-aminoribose, 5′-thioribose, 5-nitro-1-indolyl-2′-deoxyribose, 5′-biotin-ribose, 2′-O,4′-C-methylene-linked, 2′-O,4′-C-amino-linked ribose, and 2′-O,4′-C-thio- linked ribose.

[0191] Embodiment 40: The method of any one of embodiments 36 to 39, wherein the first oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 75, between 75 and 100, between 100 and 125, between 125 and 150, or between 135 and 160 modified sugars.

[0192] Embodiment 41. The method of any one of embodiments 36 to 39, wherein the first oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, or more modified sugars.

[0193] Embodiment 42: The method of any one of embodiments 36 to 41, wherein the second oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified sugars.

[0194] Embodiment 43: The method of any one of embodiments 36 to 42, wherein the second oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least -64- 4935-1210-1197.1Atty. Docket No.114203-1801 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified sugars.

[0195] Embodiment 44: The method of any one of embodiments 36 to 43, wherein the one or more modified nucleotides comprises a modified phosphate.

[0196] Embodiment 45: The method of embodiment 44, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5′-O- methylphosphonate, 3′-O-methylphosphonate, 5′-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0197] Embodiment 46: The method of embodiment 44 or 45, wherein the first oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 75, between 75 and 100, between 100 and 125, between 125 and 150, or between 135 and 160 modified phosphates.

[0198] Embodiment 47: The method of embodiment 44 or 45, wherein the first oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, or more modified phosphates.

[0199] Embodiment 48: The method of any one of embodiments 44 to 47, wherein the second oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified phosphates.

[0200] Embodiment 49: The method of any one of embodiments 44 to 47, wherein the second oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least -65- 4935-1210-1197.1Atty. Docket No.114203-1801 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified phosphates.

[0201] Embodiment 50: The method of any one of embodiments 36 to 49, wherein the one or more modified nucleotides comprise a modified nucleobase.

[0202] Embodiment 51: The method of embodiment 50, wherein the modified nucleobase is selected from the group consisting of inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2- thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5- methyluracil, 5,6-dihydrouracil, 5-[(3-Indolyl)propionamide-N-allyl]uracil, 5- aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5- carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5- hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5- iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5- methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16- aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- 6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1- ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl- thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis- -66- 4935-1210-1197.1Atty. Docket No.114203-1801 hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0203] Embodiment 52: The method of embodiment 50 or 51, wherein the first oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 75, between 75 and 100, between 100 and 125, between 125 and 150, or between 135 and 160 modified nucleobases.

[0204] Embodiment 53: The method of embodiment 50 or 51, wherein the first oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, or more modified nucleobases.

[0205] Embodiment 54: The method of any one of embodiments 50 to 53, wherein the second oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified nucleobases.

[0206] Embodiment 55: The method of any one of embodiments 50 to 53, wherein the second oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified nucleobases.

[0207] Embodiment 56: The method of any one of embodiments 36 to 55, wherein the one or more modified nucleotides comprise one or more modified sugars, one or more modified phosphates, one or more modified nucleobases, or any combination thereof. -67- 4935-1210-1197.1Atty. Docket No.114203-1801

[0208] Embodiment 57: The method of any one of embodiments 23-55, wherein the capped branched RNA oligonucleotide comprises a 5’ cap selected from the group consisting of 7- methylguanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7G-3’m- ppp-G), N7,2’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7Gm-ppp-G), 7- benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G- LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4-chlorophenoxyethyl)-guanosine, 7- ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7- (carboxymethyl) guanosine (cm7G), 7-(2-phenylethyl) guanosine [7-(2-PhEt)G], 7-(1- phenylethyl) guanosine [7-(1-PhEt)G], m7GpppBH3G (D1 and D2 stereoisomers), m7GppBH3G (D1 and D2 stereoisomers), m7GpBH3G (D1 and D2 stereoisomers), m7GppBH3pm7G, m27,2’-OGpppBH3G (D1 and D2 stereoisomers), m27,2’-OGppBH3pG (D1 and D2 diastereomers), m27,2’-OGppSpG (D1 and D2 diastereomers), N-Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, 1-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, cap1, cap2, cap3, cap4, ARCA, modified ARCA, inosine, N1- methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0209] Embodiment 58: The method of any one of embodiments 1-57, wherein the second oligonucleotide branch further comprises a poly-A tail.

[0210] Embodiment 59: The method of embodiment 58, wherein the poly-A tail comprises between 25 and 500 nucleotides.

[0211] Embodiment 60: The method of embodiment 58, wherein the poly-A tail comprises between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 or 500 nucleotides. -68- 4935-1210-1197.1Atty. Docket No.114203-1801

[0212] Embodiment 61: The method of any one of embodiments 58 to 60, wherein the poly- A tail comprises 10 or more adenosine nucleotides.

[0213] Embodiment 62: The method of any one of embodiments 58 to 60, wherein 25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97- 100%, 98-100%, or 99-100% of nucleotides of the poly-A tail are adenosine nucleotides.

[0214] Embodiment 63: The method of any one of embodiments 58-62, wherein 100% of the nucleotides of the poly-A tail are adenosine nucleotides.

[0215] Embodiment 64: The method of any one of embodiments 58-62, wherein the poly-A tail comprises one or more modified nucleotides.

[0216] Embodiment 65: The method of any one of embodiments 1-64, wherein the second oligonucleotide branch further comprises a therapeutic nucleic acid.

[0217] Embodiment 66: The method of embodiment 65, wherein the therapeutic nucleic acid is chosen from the group consisting of an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.

[0218] Embodiment 67: The method of any one of embodiments 1-66, wherein the branched RNA oligonucleotide comprises a promoter sequence.

[0219] Embodiment 68: A capped RNA transcript produced by any one of embodiments 23- 67.

[0220] Embodiment 69: A branched RNA oligonucleotide having a structure according to Formula XII or XIII -69- 4935-1210-1197.1Atty. Docket No.114203-1801 Formula XIII whereinR3is phosphate or 1-20 nucleotides; R4is phosphate or 1-500 nucleotides; R5is absent, alkyl, alkyl phosphate, or alkylene oxide; R6is phosphate or 1-1000 nucleotides; R7is OH, alkyl, halogen, or hydrogen; X and B are each independently a nitrogenous nucleotide base; and n is 1-12.

[0221] Embodiment 70: The branched RNA oligonucleotide of embodiment 69, wherein R3comprises 1-10 nucleotides.

[0222] Embodiment 71: The branched RNA oligonucleotide of embodiment 69 or 70, wherein R3comprises 6 nucleotides.

[0223] Embodiment 72: The branched RNA oligonucleotide of embodiment 69 or 70, wherein R3comprises 8 nucleotides.

[0224] Embodiment 73: The branched RNA oligonucleotide of embodiment 69 or 70, wherein R3comprises 10 nucleotides.

[0225] Embodiment 74: The branched RNA oligonucleotide of any one of embodiments 69- 73, wherein R4comprises a nucleotide conjugated to a solid-phase support. -70- 4935-1210-1197.1Atty. Docket No.114203-1801

[0226] Embodiment 75: The branched RNA oligonucleotide of any one of embodiments 69- 74, wherein R5comprises C1-C12 alkyl, (CH2)xOPOOH(CH2)y, (CH2CH2O)z; wherein x and y are independently 1-12, and z is 1-2.

[0227] Embodiment 76: The branched RNA oligonucleotide of any one of embodiments 69- 75, wherein R6comprises 1-20 nucleotides.

[0228] Embodiment 77: The branched RNA oligonucleotide of any one of embodiments 69- 76, wherein R6comprises 6 nucleotides.

[0229] Embodiment 78: The branched RNA oligonucleotide of any one of embodiments 69- 76, wherein R6comprises 9 nucleotides.

[0230] Embodiment 79: The branched RNA oligonucleotide of any one of embodiments 69- 76, wherein R6comprises 12 nucleotides.

[0231] Embodiment 80: The branched RNA oligonucleotide of any one of embodiments 69- 80, wherein R6comprises 5’ untranslated region (5’ UTR) comprising an unstructured region.

[0232] Embodiment 81: The branched RNA oligonucleotide of embodiment 80, wherein the unstructured region comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more consecutive adenosine, cytosine, guanine, or uridine nucleotides.

[0233] Embodiment 82: The branched RNA oligonucleotide of embodiment 80, wherein the unstructured region comprises no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10 consecutive adenosine, cytosine, guanine, or uridine nucleotides.

[0234] Embodiment 83: The branched RNA oligonucleotide of embodiment 80, wherein the unstructured region comprises between 5 and 60, between 10 and 55, between 15 and 50, between 20 and 45, between 25 and 40, or between 30 and 35 consecutive adenosine, cytosine, guanine, or uridine nucleotides. -71- 4935-1210-1197.1Atty. Docket No.114203-1801

[0235] Embodiment 84: The branched RNA oligonucleotide of any one of embodiments 69- 83, wherein R4comprises between 1 and 160, between 3 and 150, between 5 and 140, between 7 and 130, between 9 and 120, between 11 and 110, between 13 and 100, between 15 and 90, between 17 and 80, between 19 and 70, between 21 and 60, between 23 and 50, between 25 and 40, or between 20 and 35 nucleotides in length.

[0236] Embodiment 85: The branched RNA oligonucleotide of any one of embodiments 69- 84, wherein R6comprises between 3 and 160, between 5 and 150, between 7 and 140, between 9 and 130, between 11 and 120, between 13 and 110, between 15 and 100, between 17 and 90, between 19 and 80, between 21 and 70, between 23 and 60, between 25 and 50, between 27 and 40, or between 30 and 35 nucleotides in length.

[0237] Embodiment 86: The branched RNA oligonucleotide of embodiment 85, wherein R6is between 25 and 35 nucleotides in length.

[0238] Embodiment 87: The branched RNA oligonucleotide of any one of embodiments 69- 86, wherein R3comprises one or more modified nucleotides.

[0239] Embodiment 88: The branched RNA oligonucleotide of any one of embodiments 69- 87, wherein R4comprises one or more modified nucleotides.

[0240] Embodiment 89: The branched RNA oligonucleotide of any one of embodiments 69- 88, wherein R6comprises one or more modified nucleotides.

[0241] Embodiment 90: The branched RNA oligonucleotide of any one of embodiments 87- 89, wherein the one or more modified nucleotides comprises a modified sugar.

[0242] Embodiment 91: The branched RNA oligonucleotide of embodiment 90, wherein the modified sugar is selected from the group consisting of 2′-deoxy fluoro (2FA), L-adenosine (LA), 2′-deoxyadenosine (dA), locked nucleic acid (LNA), 2′-methoxy (2OMe), 2′- methoxyethoxy (2MOE), 2′-thioribose, 2′,3′-dideoxyribose, 2′-amino-2′-deoxyribose, 2′ deoxyribose, 2′-azido-2′-deoxyribose, 2′-fluoro-2′-deoxyribose, 2′-O-methylribose, 2′-O- methyldeoxyribose, 3′-amino-2′,3′-dideoxyribose, 3′-azido-2′,3′-dideoxyribose, 3′-deoxyribose, 3′-O-(2-nitrobenzyl)-2′-deoxyribose, 3′-O-methylribose, 5′-aminoribose, 5′-thioribose, 5-nitro-1- -72- 4935-1210-1197.1Atty. Docket No.114203-1801 indolyl-2′-deoxyribose, 5′-biotin-ribose, 2′-O,4′-C-methylene-linked, 2′-O,4′-C-amino-linked ribose, and 2′-O,4′-C-thio-linked ribose.

[0243] Embodiment 92: The branched RNA oligonucleotide of any one of embodiments 87- 91, wherein the one or more modified nucleotides comprises a modified phosphate.

[0244] Embodiment 93: The branched RNA oligonucleotide of embodiment 92, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5′-O-methylphosphonate, 3′-O-methylphosphonate, 5′-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0245] Embodiment 94: The branched RNA oligonucleotide of any one of embodiments 87- 93, wherein the one or more modified nucleotides comprise a modified nucleobase.

[0246] Embodiment 95: The branched RNA oligonucleotide of embodiment 94, wherein the modified nucleobase is selected from the group consisting of inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-Indolyl)propionamide-N-allyl]uracil, 5- aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5- carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5- hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5- iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5- methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16- aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- -73- 4935-1210-1197.1Atty. Docket No.114203-1801 6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1- ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl- thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis- hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0247] Embodiment 96: The branched RNA oligonucleotide of any one of embodiments 87 to 95, wherein the one or more modified nucleotides comprise one or more modified sugars, one or more modified phosphates, one or more modified nucleobases, or any combination thereof.

[0248] Embodiment 97: The branched RNA oligonucleotide of any one of embodiments 69- 96, wherein R3comprises a capping nucleotide; R4comprises the capping nucleotide; and R6comprises the capping nucleotide.

[0249] Embodiment 98: The branched RNA oligonucleotide of embodiment 97, wherein the capped nucleotide comprises a 5’ cap.

[0250] Embodiment 99: The branched RNA oligonucleotide of embodiment 98, wherein the 5’ cap is selected from the group consisting of 7-methylguanosine (m7G), N7,3’-O-dimethyl- guanosine-5’-triphosphate-5’-guanosine (m7G-3’m-ppp-G), N7,2’-O-dimethyl-guanosine-5’- triphosphate-5’-guanosine (m7Gm-ppp-G), 7-benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G-LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4-chlorophenoxyethyl)-guanosine, 7-ethyl guanosine (e7G), 7-propyl -74- 4935-1210-1197.1Atty. Docket No.114203-1801 guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7-(carboxymethyl) guanosine (cm7G), 7-(2- phenylethyl) guanosine [7-(2-PhEt)G], 7-(1-phenylethyl) guanosine [7-(1-PhEt)G], m7GpppBH3G (D1 and D2 stereoisomers), m7GppBH3G (D1 and D2 stereoisomers), m7GpBH3G (D1 and D2 stereoisomers), m7GppBH3pm7G, m27,2’-OGpppBH3G (D1 and D2 stereoisomers), m27,2’-OGppBH3pG (D1 and D2 diastereomers), m27,2’-OGppSpG (D1 and D2 diastereomers), N- Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, 1-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L- nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, cap1, cap2, cap3, cap4, ARCA, modified ARCA, inosine, N1-methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0251] Embodiment 100: The branched RNA oligonucleotide of any one of embodiments 69 to 99, wherein R6comprises a poly-A tail.

[0252] Embodiment 101: The branched RNA oligonucleotide of embodiment 100, wherein the poly-A tail comprises between 25 and 500 nucleotides.

[0253] Embodiment 102: The branched RNA oligonucleotide of embodiment 100 wherein the poly-A tail comprises between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 or 500 nucleotides.

[0254] Embodiment 103: The branched RNA oligonucleotide of any one of embodiments 100 to 102, wherein the poly-A tail comprises 10 or more adenosine nucleotides.

[0255] Embodiment 104: The branched RNA oligonucleotide of any one of embodiments 100 to 102, wherein 25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90- 100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100% of nucleotides of the poly-A tail are adenosine nucleotides. -75- 4935-1210-1197.1Atty. Docket No.114203-1801

[0256] Embodiment 105: The branched RNA oligonucleotide of any one of embodiments 100 to 102, wherein 100% of the nucleotides of the poly-A tail are adenosine nucleotides.

[0257] Embodiment 106: The branched RNA oligonucleotide of any one of embodiments 100-102, wherein the poly-A tail comprises one or more modified nucleotides.

[0258] Embodiment 107: The branched RNA oligonucleotide of any one of embodiments 69-106, wherein R6comprises a therapeutic nucleic acid.

[0259] Embodiment 108: The branched RNA oligonucleotide of embodiment 107, wherein the therapeutic nucleic acid is chosen from the group consisting of an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.

[0260] Embodiment 109: A delivery agent comprising the capped RNA transcript of embodiment 68 or the branched RNA oligonucleotide of any one of embodiments 69-108, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.

[0261] Embodiment 110: The delivery agent of embodiment 109, wherein the nanoparticle or microparticle is a lipid nanoparticle or microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

[0262] Embodiment 111: A cell comprising the capped RNA transcript of embodiment 68 or the branched RNA oligonucleotide of any one of embodiments 69-108.

[0263] Embodiment 112: The cell of embodiment 111, wherein the cell is a mammalian cell.

[0264] Embodiment 113: A composition comprising the capped RNA transcript of embodiment 68, the branched RNA oligonucleotide of any one of embodiments 69-108, the delivery agent of embodiment 109 or 110, or the cell of embodiment 111 or 112.

[0265] Embodiment 114: The composition of embodiment 113 further comprising an additional agent. -76- 4935-1210-1197.1Atty. Docket No.114203-1801

[0266] Embodiment 115: The composition of embodiment 114, wherein the additional agent is an agent which has a therapeutic effect when administered to a subject.

[0267] Embodiment 116: The composition of embodiment 114 or 115, wherein the additional agent is chosen from the list consisting of a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a small molecule, an aptamer, a lipid, or a carbohydrate.

[0268] Embodiment 117: The composition of embodiment 116, wherein the additional agent is a shRNA, a siRNA, or an antisense oligonucleotide (ASO).

[0269] Embodiment 118: The composition of any one of embodiments 114-116, wherein the additional agent is an antigen or adjuvant.

[0270] Embodiment 119: The composition of any one of embodiments 113-118, wherein the composition is a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0271] Embodiment 120: A method of preventing or treating a disease in a subject, comprising introducing an effective amount of the capped RNA transcript of embodiment 68, the branched RNA oligonucleotide of any one of embodiments 69-108, the delivery agent of embodiment 109 or 110, the cell of embodiment 111 or 112, or the composition of any one of embodiments 113-119 to the subject.

[0272] Embodiment 121: The method of embodiment 120, wherein the subject is a human.

[0273] Embodiment 122: The capped RNA transcript of embodiment 68, the branched RNA oligonucleotide of any one of embodiments 69-108, the delivery agent of embodiment 109 or 110, the cell of embodiment 111 or 112, or the composition of any one of embodiments 113-119 for use in preventing or treating a disease in a subject.

[0274] Embodiment 123: A kit comprising the capped RNA transcript of embodiment 68, the branched RNA oligonucleotide of any one of embodiments 69-108, the delivery agent of embodiment 109 or 110, the cell of embodiment 111 or 112, or the composition of any one of embodiments 113-119. -77- 4935-1210-1197.1Atty. Docket No.114203-1801

[0275] Embodiment 124: The kit of embodiment 123, further comprising an RNA ligase.

[0276] Embodiment 125: A kit comprising the composition of embodiment 116, a device for administering the composition to a subject, and instructions for administering the composition to the subject.

[0277] Embodiment 126: Use of the capped RNA transcript of embodiment 68, the branched RNA oligonucleotide of any one of embodiments 69-108, the delivery agent of embodiment 109 or 110, the cell of embodiment 111 or 112, or the composition of any one of embodiments 113- 119 for the manufacture of a medicament for treatment of a disease in a subject.

[0278] Embodiment 127: The capped RNA transcript of embodiment 68, the branched RNA oligonucleotide of any one of embodiments 69-108, the delivery agent of embodiment 109 or 110, the cell of embodiment 111 or 112, or the composition of any one of embodiments 113-119 for use in the treatment of a disease in a subject. EXAMPLES Example 1: Methods for solid phase synthesis of branched oligonucleotides

[0279] Synthesis of branched oligonucleotides was performed in a stepwise process on solid support, allowing for controlled addition of nucleotide units. Synthesis was conducted with Mermade 6 Synthesizer (LGC Biosearch Technologies) at a 1-µmol scale. Reagents for synthesis included the 2'-O-tert-butyldimethylsilyl (2′-TBDMS) protected monomer phosphoramidites Bz- A-CE phosphoramidite, Ac-G-CE phosphoramidite, U-CE phosphoramidite, Ac-C-CE phosphoramidite, where Bz refers to a benzoyl group protecting the amino group, CE refers to a cyanoethyl group protecting the hydroxyl group, and Ac refers to an acetyl group protecting the amino group. The brancher phosphoramidite used to form branches in the oligonucleotides was 5-Me-dC brancher phosphoramidite having the structure -78- 4935-1210-1197.1Atty. Docket No. 114203-1801

[0280] Otheran oxidizer (0.02 M iodine in tetrahydrofuran / pyridine / water solution), deblock solution (3% trichloroacetic acid (TCA) in dichloromethane (DCM)), capping reagent (20% acetic anhydride in acetonitrile and 16% 1-methylimidazole in tetrahydrofuran), activator (0.25 M 5-ethylthio-1H-tetrazole (ETT) in anhydrous acetonitrile) and controlled pore glass (CPG).

[0281] FIG. 1 shows a general synthesis workflow of synthesis of different branched oligonucleotides using a brancher phosphoramidite. Oligonucleotides synthesis followed conventional phosphoramidite chemistry protocol with some modifications. Briefly, the synthesis cycle was started with 60 seconds 4,4'-dimethoxytrityl (DMTr) deblocking with deblock solution, followed by a coupling cycle (6 minutes repeated twice). Capping of the failed sequences was performed with capping reagent for 2 minutes and oxidization (2 minutes) was then performed with oxidizer to finish one coupling cycle. Particularly, chemical phosphorylating reagent (CPR II) was used to introduce 5′ phosphate and 5-Me-dC brancher phosphoramidite was incorporated into the oligonucleotide sequence at predetermined sites using the same coupling procedure. After the synthesis of a stem strand with DMTr-off mode, the 5′ free hydroxyl group was capped with capping reagent for 1 minute repeated 10 times to remove the 5′ DMTr protecting group. Then the 5′ free hydroxyl group was capped with capping reagent for 2 minutes 5 times to block the 5′ end of the stem strand. To remove the protecting group on the brancher phosphoramidite and perform branch strand synthesis, the CPG was washed with acetonitrile and the levulinyl group on the 5-Me-dC brancher was selectively deprotected by treating the CPG with 0.5 M hydrazine hydrate in 1:1 pyridine / acetic acid for 15 minutes to expose the hydroxyl. Predetermined branch strands were then synthesized following the same -79- 4935-1210-1197.1Atty. Docket No.114203-1801 coupling cycle described above to form the second branch of the oligonucleotide. The 5′ multi- capped oligonucleotide was obtained after chemical capping and HPLC purification. Example 2: Systematic interrogation of different lengths of the stem strand of branched capped mRNA 5' oligonucleotides synthesized through direct solid phase oligonucleotide synthesis

[0282] FIGS.3A-3B show an evaluation of different lengths of the stem strand (first oligonucleotide branch before the branching point). FIG.3A illustrates a branched oligonucleotide with an arrow noting the stem strand that was evaluated. FIG.3B is a graph of relative luminescence for different branched oligonucleotides with different stem strand lengths.

[0283] Dual-capped mRNA stem strand length was evaluated by ligating the synthetic branched oligonucleotide with Firefly luciferase reporter mRNA. Stem strands were 1, 6, 8, and 10 nucleotides in length. Protein expression of FLuc luminescence reporter was normalized to RLuc luminescence (transfection control) and compared with the single cap (monocap ctrl, monocap-m7G-rA) construct at 8 hours post transfection. n = 3, biological replicates. Mean ± SEM. Example 3: Systematic interrogation of different lengths of the branch strand of branched capped mRNA 5' oligonucleotides synthesized through direct solid phase oligonucleotide synthesis

[0284] FIGS.4A-4B how an evaluation of different lengths of the second oligonucleotide branch. FIG.4A illustrates a branched oligonucleotide with an arrow noting the second oligonucleotide branch length that was evaluated. FIG.4B is a graph of relative luminescence for different branched oligonucleotides with different second oligonucleotide branch lengths.

[0285] Dual-capped mRNA brancher strand length was evaluated by ligating the synthetic branched oligonucleotide with Firefly luciferase reporter mRNA. Branch strands were 1, 7, 10, and 13 nucleotides in length. Protein expression of FLuc luminescence reporter was normalized to RLuc luminescence (transfection control) and compared with the single cap (mono-m7G-rA) construct at 8 hours post transfection. n = 3, biological replicates. Mean ± SEM. -80- 4935-1210-1197.1Atty. Docket No.114203-1801 Example 4: Systematic interrogation of different lengths of the spacer of branched capped mRNA 5' oligonucleotides synthesized through direct solid phase oligonucleotide synthesis

[0286] FIGS.5A-5B how an evaluation of different types of spacers at the branching point. FIG.5A illustrates a branched oligonucleotide with an arrow noting the spacer and different spacer structures that were evaluated. FIG.5B is a graph of relative luminescence for different branched oligonucleotides with the different spacers in FIG.5A.

[0287] The spacer between branch strand and stem strand was evaluated by using different spacer phosphoramidites during solid phase synthesis. Protein expression of FLuc luminescence reporter was normalized to RLuc luminescence (transfection control) and compared with the single cap (mono-m7G-rA) construct at 8 hours post transfection. n = 3, biological replicates. Mean ± SEM. SEQUENCES

[0288] The following tables contain sequences of oligonucleotides used in the experiments shown in each referenced figure. Nucleotide modifications are denoted as follows: r = ribose sugar m7G = 7-methylguanosine ppp = triphosphate bridge Table 1. Sequences used in FIG.3 Identifier Sequence (5’ to 3’) Stem Strand Branch Strand SEQ ID NO:-81- 4935-1210-1197.1Atty. Docket No.114203-1801 / rArArArArArA 0 t 7G 7G AGAGAAAGAA 4a e . eque ces use . Identifier Sequence (5’ to 3’) Stem Strand Branch Strand SEQ ID NO:Table 3. Sequences used in FIG.5 Identifier Sequence (5’ to 3’) Stem Strand Branch Strand SEQ ID NO:-82- 4935-1210-1197.1Atty. Docket No.114203-1801 dC brancher phosphoramidite rArGrArGrArArA / hexane-diol / rArArArArArA spacer / / hexane-diol spacer / -83- 4935-1210-1197.1

Claims

Atty. Docket No.114203-1801 WHAT IS CLAIMED IS:

1. A method of producing a branched RNA oligonucleotide, comprising: synthesizing the branched RNA oligonucleotide with a brancher phosphoramidite including a first protected hydroxyl group and a second protected hydroxyl group, creating a branching point in the branched RNA oligonucleotide with the brancher phosphoramidite; wherein the step of synthesizing is performed on a solid-phase support.

2. The method of claim 1, wherein at least one of the first protected hydroxyl group and the second protected hydroxyl group of the brancher phosphoramidite comprises dimethoxytrityl (DMTr), levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert- butyldiphenylsilyl (TBDPS).

3. The method of claim 1, wherein the first protected hydroxyl group and the second protected hydroxyl group of the brancher phosphoramidite are deprotected with different treatments.

4. The method of claim 3, wherein the first protected hydroxyl group of the brancher phosphoramidite comprises dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite comprises levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS).

5. The method of claim 4, wherein the first protected hydroxyl group of the brancher phosphoramidite comprises dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite comprises levulinyl.

6. The method of claims 1 or 2, wherein the brancher phosphoramidite has a structure according to Formula I -84- 4935-1210-1197.1Atty. Docket No.114203-1801 whereinR1is dimethoxytrityl (DMTr) and the second protected hydroxyl group of the brancher phosphoramidite comprises levulinyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), or tert-butyldiphenylsilyl (TBDPS); R2is tert-butyldimethylsilyl ether, halogen, alkyl, or hydrogen; X is a nitrogenous nucleotide base; and n is 1 to 12.

7. The method of claims 1 or 2, wherein the brancher phosphoramidite has a structure according to Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X, or Formula XI-85- 4935-1210-1197.1Atty. Docket No.114203-1801m n are 8. The method of any one of claims 3-7, wherein the step of synthesizing comprises (a) synthesizing a first oligonucleotide branch with the brancher phosphoramidite; -86- 4935-1210-1197.1Atty. Docket No.114203-1801 (b) deprotecting the second protected hydroxyl group on the brancher phosphoramidite to form a hydroxyl group on the first oligonucleotide branch; and (c) synthesizing a second oligonucleotide branch at the free hydroxyl group on the first oligonucleotide branch, thereby forming the branched RNA oligonucleotide.

9. The method of claim 8, wherein the brancher phosphoramidite is a first brancher phosphoramidite and the branching point is a first branching point, and synthesizing the second oligonucleotide branch comprises synthesizing the second oligonucleotide branch with a second brancher phosphoramidite.

10. The method of claim 9, further comprising: (d) deprotecting a protected group on the second brancher phosphoramidite to form a hydroxyl group on the second oligonucleotide branch; (e) synthesizing a third oligonucleotide branch at the free hydroxyl group on the second oligonucleotide branch to form a second branching point.

11. The method of any one of claims 1-8, wherein the first oligonucleotide branch of the branched RNA oligonucleotide comprises 1 nucleotide to 40 nucleotides between the branching point in the branched RNA oligonucleotide and a terminus of the first oligonucleotide branch.

12. The method of claim 11, wherein the first oligonucleotide branch of the branched RNA oligonucleotide comprises 6 nucleotides.

13. The method of claim 11, wherein the first oligonucleotide branch of the branched RNA oligonucleotide comprises 8 nucleotides.

14. The method of claim 11, wherein the first oligonucleotide branch of the branched RNA oligonucleotide comprises 10 nucleotides.

15. The method of any one of claims 1-8 or 11-14, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises 1 nucleotide to 40 nucleotides between the branching point in the branched RNA oligonucleotide and a terminus of the second oligonucleotide branch. -87- 4935-1210-1197.1Atty. Docket No.114203-1801 16. The method of claim 15, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises 7 nucleotides.

17. The method of claim 15, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises 10 nucleotides.

18. The method of claim 15, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises 13 nucleotides.

19. The method of any one of claims 1-18, wherein the second oligonucleotide branch of the branched RNA oligonucleotide comprises a spacer at the branching point of the branched RNA oligonucleotide.

20. The method of claim 19, wherein the spacer comprises C1-C12alkyl or (CH2CH2O)nwhere n is 1-4.

21. The method of any one of claims 1-20, wherein the solid-phase support comprises glass beads or polystyrene beads.

22. The method of claim 21, further comprising cleaving the branched RNA oligonucleotide from the solid-phase support.

23. The method of any one of claims 1-22, further comprising capping phosphorylated 5’ terminuses of the branched RNA oligonucleotide, producing a capped branched RNA oligonucleotide.

24. The method of claim 23, further comprising purifying the capped branched RNA oligonucleotide, producing a purified capped branched RNA oligonucleotide.

25. The method of claim 24, wherein the step of purifying comprises performing high- performance liquid chromatography (HPLC).

26. The method of any one of claims 23 to 25, wherein the capping is performed chemically.

27. The method of claim 26, wherein chemical capping is performed through an anhydrous reaction between the first 5’-phosphorylated RNA oligonucleotide, the second 5’-phosphorylated -88- 4935-1210-1197.1Atty. Docket No.114203-1801 RNA oligonucleotide, and capping nucleotides conjugated to imidazole in the presence of 1- methylimidazole.

28. The method of any one of claims 1-27, wherein the second oligonucleotide branch comprises a 5’ untranslated region (5’ UTR) comprising an unstructured region.

29. The method of claim 28, wherein the unstructured region comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more consecutive adenosine, cytosine, guanine, or uridine nucleotides.

30. The method of claim 28, wherein the unstructured region comprises no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10 consecutive adenosine, cytosine, guanine, or uridine nucleotides.

31. The method of claim 28, wherein the unstructured region comprises between 5 and 60, between 10 and 55, between 15 and 50, between 20 and 45, between 25 and 40, or between 30 and 35 consecutive adenosine, cytosine, guanine, or uridine nucleotides.

32. The method of any one of claims 1-27, wherein the first oligonucleotide branch is between 3 and 160, between 5 and 150, between 7 and 140, between 9 and 130, between 11 and 120, between 13 and 110, between 15 and 100, between 17 and 90, between 19 and 80, between 21 and 70, between 23 and 60, between 25 and 50, between 27 and 40, or between 30 and 35 nucleotides in length.

33. The method of claim 32, wherein the first oligonucleotide branch is between 25 and 35 nucleotides in length.

34. The method of claims 32 or 33, wherein the second oligonucleotide branch is between 3 and 160, between 5 and 150, between 7 and 140, between 9 and 130, between 11 and 120, between 13 and 110, between 15 and 100, between 17 and 90, between 19 and 80, between 21 and 70, between 23 and 60, between 25 and 50, between 27 and 40, or between 30 and 35 nucleotides in length. -89- 4935-1210-1197.1Atty. Docket No.114203-1801 35. The method of claim 34, wherein the second oligonucleotide branch is between 25 and 35 nucleotides in length.

36. The method of any one of claims 1-35, wherein the first oligonucleotide branch comprises one or more modified nucleotides.

37. The method of any one of claims 1-36, wherein the second oligonucleotide branch comprises one or more modified nucleotides.

38. The method of claim 36 or 37, wherein the one or more modified nucleotides comprises a modified sugar.

39. The method of claim 38, wherein the modified sugar is selected from the group consisting of 2′-deoxy fluoro (2FA), L-adenosine (LA), 2′-deoxyadenosine (dA), locked nucleic acid (LNA), 2′-methoxy (2OMe), 2′-methoxyethoxy (2MOE), 2′-thioribose, 2′,3′-dideoxyribose, 2′-amino-2′-deoxyribose, 2′ deoxyribose, 2′-azido-2′-deoxyribose, 2′-fluoro-2′-deoxyribose, 2′- O-methylribose, 2′-O-methyldeoxyribose, 3′-amino-2′,3′-dideoxyribose, 3′-azido-2′,3′- dideoxyribose, 3′-deoxyribose, 3′-O-(2-nitrobenzyl)-2′-deoxyribose, 3′-O-methylribose, 5′- aminoribose, 5′-thioribose, 5-nitro-1-indolyl-2′-deoxyribose, 5′-biotin-ribose, 2′-O,4′-C- methylene-linked, 2′-O,4′-C-amino-linked ribose, and 2′-O,4′-C-thio-linked ribose.

40. The method of any one of claims 36 to 39, wherein the first oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 75, between 75 and 100, between 100 and 125, between 125 and 150, or between 135 and 160 modified sugars.

41. The method of any one of claims 36 to 39, wherein the first oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, or more modified sugars.

42. The method of any one of claims 36 to 41, wherein the second oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, -90- 4935-1210-1197.1Atty. Docket No.114203-1801 between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified sugars.

43. The method of any one of claims 36 to 42, wherein the second oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified sugars.

44. The method of any one of claims 36 to 43, wherein the one or more modified nucleotides comprises a modified phosphate.

45. The method of claim 44, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5′-O-methylphosphonate, 3′-O- methylphosphonate, 5′-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

46. The method of claim 44 or 45, wherein the first oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 75, between 75 and 100, between 100 and 125, between 125 and 150, or between 135 and 160 modified phosphates.

47. The method of claim 44 or 45, wherein the first oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, or more modified phosphates.

48. The method of any one of claims 44 to 47, wherein the second oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified phosphates. -91- 4935-1210-1197.1Atty. Docket No.114203-1801 49. The method of any one of claims 44 to 47, wherein the second oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified phosphates.

50. The method of any one of claims 36 to 49, wherein the one or more modified nucleotides comprise a modified nucleobase.

51. The method of claim 50, wherein the modified nucleobase is selected from the group consisting of inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2-thiocytosine, 2- thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6- dihydrouracil, 5-[(3-Indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5- aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5- carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5- hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5- iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5- methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16- aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- 6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1- ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, -92- 4935-1210-1197.1Atty. Docket No.114203-1801 thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl- thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis- hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

52. The method of claim 50 or 51, wherein the first oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 75, between 75 and 100, between 100 and 125, between 125 and 150, or between 135 and 160 modified nucleobases.

53. The method of claim 50 or 51, wherein the first oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, or more modified nucleobases.

54. The method of any one of claims 50 to 53, wherein the second oligonucleotide branch comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified nucleobases.

55. The method of any one of claims 50 to 53, wherein the second oligonucleotide branch comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified nucleobases. -93- 4935-1210-1197.1Atty. Docket No.114203-1801 56. The method of any one of claims 36 to 55, wherein the one or more modified nucleotides comprise one or more modified sugars, one or more modified phosphates, one or more modified nucleobases, or any combination thereof.

57. The method of any one of claims 23-55, wherein the capped branched RNA oligonucleotide comprises a 5’ cap selected from the group consisting of 7-methylguanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7G-3’m-ppp-G), N7,2’-O- dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7Gm-ppp-G), 7-benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G-LNA), chlorobenzyl-O- ethoxyguanosine (ClBnOEt7G), 7-(4-chlorophenoxyethyl)-guanosine, 7-ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7-(carboxymethyl) guanosine (cm7G), 7-(2- phenylethyl) guanosine [7-(2-PhEt)G], 7-(1-phenylethyl) guanosine [7-(1-PhEt)G], m7GpppBH3G (D1 and D2 stereoisomers), m7GppBH3G (D1 and D2 stereoisomers), m7GpBH3G (D1 and D2 stereoisomers), m7GppBH3pm7G, m27,2’-OGpppBH3G (D1 and D2 stereoisomers), m27,2’-OGppBH3pG (D1 and D2 diastereomers), m27,2’-OGppSpG (D1 and D2 diastereomers), N- Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, 1-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L- nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, cap1, cap2, cap3, cap4, ARCA, modified ARCA, inosine, N1-methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

58. The method of any one of claims 1-57, wherein the second oligonucleotide branch further comprises a poly-A tail.

59. The method of claim 58, wherein the poly-A tail comprises between 25 and 500 nucleotides. -94- 4935-1210-1197.1Atty. Docket No.114203-1801 60. The method of claim 58, wherein the poly-A tail comprises between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 or 500 nucleotides.

61. The method of any one of claims 58 to 60, wherein the poly-A tail comprises 10 or more adenosine nucleotides.

62. The method of any one of claims 58 to 60, wherein 25-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99- 100% of nucleotides of the poly-A tail are adenosine nucleotides.

63. The method of any one of claims 58-62, wherein 100% of the nucleotides of the poly-A tail are adenosine nucleotides.

64. The method of any one of claims 58-62, wherein the poly-A tail comprises one or more modified nucleotides.

65. The method of any one of claims 1-64, wherein the second oligonucleotide branch further comprises a therapeutic nucleic acid.

66. The method of claim 65, wherein the therapeutic nucleic acid is chosen from the group consisting of an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.

67. The method of any one of claims 1-66, wherein the branched RNA oligonucleotide comprises a promoter sequence.

68. A capped RNA transcript produced by any one of claims 23-67.

69. A branched RNA oligonucleotide having a structure according to Formula XII or XIII -95- 4935-1210-1197.1Atty. Docket No.114203-1801 Formula XIII whereinor R4is phosphate or 1-500 nucleotides; R5is absent, alkyl, alkyl phosphate, or alkylene oxide; R6is phosphate or 1-1000 nucleotides; R7is OH, alkyl, halogen, or hydrogen; X and B are each independently a nitrogenous nucleotide base; and n is 1-12.

70. The branched RNA oligonucleotide of claim 69, wherein R3comprises 1-10 nucleotides.

71. The branched RNA oligonucleotide of claim 69 or 70, wherein R3comprises 6 nucleotides.

72. The branched RNA oligonucleotide of claim 69 or 70, wherein R3comprises 8 nucleotides.

73. The branched RNA oligonucleotide of claim 69 or 70, wherein R3comprises 10 nucleotides.

74. The branched RNA oligonucleotide of any one of claims 69-73, wherein R4comprises a nucleotide conjugated to a solid-phase support. -96- 4935-1210-1197.1Atty. Docket No.114203-1801 75. The branched RNA oligonucleotide of any one of claims 69-74, wherein R5comprises C1-C12 alkyl, (CH2)xOPOOH(CH2)y, or (CH2CH2O)z; wherein x and y are independently 1-12, and z is 1-2.

76. The branched RNA oligonucleotide of any one of claims 69-75, wherein R6comprises 1- 20 nucleotides.

77. The branched RNA oligonucleotide of any one of claims 69-76, wherein R6comprises 6 nucleotides.

78. The branched RNA oligonucleotide of any one of claims 69-76, wherein R6comprises 9 nucleotides.

79. The branched RNA oligonucleotide of any one of claims 69-76, wherein R6comprises 12 nucleotides.

80. The branched RNA oligonucleotide of any one of claims 69-80, wherein R6comprises 5’ untranslated region (5’ UTR) comprising an unstructured region.

81. The branched RNA oligonucleotide of claim 80, wherein the unstructured region comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more consecutive adenosine, cytosine, guanine, or uridine nucleotides.

82. The branched RNA oligonucleotide of claim 80, wherein the unstructured region comprises no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10 consecutive adenosine, cytosine, guanine, or uridine nucleotides.

83. The branched RNA oligonucleotide of claim 80, wherein the unstructured region comprises between 5 and 60, between 10 and 55, between 15 and 50, between 20 and 45, between 25 and 40, or between 30 and 35 consecutive adenosine, cytosine, guanine, or uridine nucleotides. -97- 4935-1210-1197.1Atty. Docket No.114203-1801 84. The branched RNA oligonucleotide of any one of claims 69-83, wherein R4comprises between 1 and 160, between 3 and 150, between 5 and 140, between 7 and 130, between 9 and 120, between 11 and 110, between 13 and 100, between 15 and 90, between 17 and 80, between 19 and 70, between 21 and 60, between 23 and 50, between 25 and 40, or between 20 and 35 nucleotides in length.

85. The branched RNA oligonucleotide of any one of claims 69-84, wherein R6comprises between 3 and 160, between 5 and 150, between 7 and 140, between 9 and 130, between 11 and 120, between 13 and 110, between 15 and 100, between 17 and 90, between 19 and 80, between 21 and 70, between 23 and 60, between 25 and 50, between 27 and 40, or between 30 and 35 nucleotides in length.

86. The branched RNA oligonucleotide of claim 85, wherein R6is between 25 and 35 nucleotides in length.

87. The branched RNA oligonucleotide of any one of claims 69-86, wherein R3comprises one or more modified nucleotides.

88. The branched RNA oligonucleotide of any one of claims 69-87, wherein R4comprises one or more modified nucleotides.

89. The branched RNA oligonucleotide of any one of claims 69-88, wherein R6comprises one or more modified nucleotides.

90. The branched RNA oligonucleotide of any one of claims 87-89, wherein the one or more modified nucleotides comprises a modified sugar.

91. The branched RNA oligonucleotide of claim 90, wherein the modified sugar is selected from the group consisting of 2′-deoxy fluoro (2FA), L-adenosine (LA), 2′-deoxyadenosine (dA), locked nucleic acid (LNA), 2′-methoxy (2OMe), 2′-methoxyethoxy (2MOE), 2′-thioribose, 2′,3′- dideoxyribose, 2′-amino-2′-deoxyribose, 2′ deoxyribose, 2′-azido-2′-deoxyribose, 2′-fluoro-2′- deoxyribose, 2′-O-methylribose, 2′-O-methyldeoxyribose, 3′-amino-2′,3′-dideoxyribose, 3′- azido-2′,3′-dideoxyribose, 3′-deoxyribose, 3′-O-(2-nitrobenzyl)-2′-deoxyribose, 3′-O- -98- 4935-1210-1197.1Atty. Docket No.114203-1801 methylribose, 5′-aminoribose, 5′-thioribose, 5-nitro-1-indolyl-2′-deoxyribose, 5′-biotin-ribose, 2′- O,4′-C-methylene-linked, 2′-O,4′-C-amino-linked ribose, and 2′-O,4′-C-thio-linked ribose.

92. The branched RNA oligonucleotide of any one of claims 87-91, wherein the one or more modified nucleotides comprises a modified phosphate.

93. The branched RNA oligonucleotide of claim 92, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5′-O- methylphosphonate, 3′-O-methylphosphonate, 5′-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

94. The branched RNA oligonucleotide of any one of claims 87-93, wherein the one or more modified nucleotides comprise a modified nucleobase.

95. The branched RNA oligonucleotide of claim 94, wherein the modified nucleobase is selected from the group consisting of inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2- thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5- methyluracil, 5,6-dihydrouracil, 5-[(3-Indolyl)propionamide-N-allyl]uracil, 5- aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5- carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5- hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5- iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5- methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16- aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- 6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6- -99- 4935-1210-1197.1Atty. Docket No.114203-1801 propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1- ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl- thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis- hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

96. The branched RNA oligonucleotide of any one of claims 87 to 95, wherein the one or more modified nucleotides comprise one or more modified sugars, one or more modified phosphates, one or more modified nucleobases, or any combination thereof.

97. The branched RNA oligonucleotide of any one of claims 69-96, wherein R3comprises a capping nucleotide; R4comprises the capping nucleotide; and R6comprises the capping nucleotide.

98. The branched RNA oligonucleotide of claim 97, wherein the capped nucleotide comprises a 5’ cap.

99. The branched RNA oligonucleotide of claim 98, wherein the 5’ cap is selected from the group consisting of 7-methylguanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’- guanosine (m7G-3’m-ppp-G), N7,2’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7Gm-ppp-G), 7-benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G-LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4- chlorophenoxyethyl)-guanosine, 7-ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl -100- 4935-1210-1197.1Atty. Docket No.114203-1801 guanosine (cp7G), 7-(carboxymethyl) guanosine (cm7G), 7-(2-phenylethyl) guanosine [7-(2- PhEt)G], 7-(1-phenylethyl) guanosine [7-(1-PhEt)G], m7GpppBH3G (D1 and D2 stereoisomers), m7GppBH3G (D1 and D2 stereoisomers), m7GpBH3G (D1 and D2 stereoisomers), m7GppBH3pm7G, (D1 and D2 stereoisomers), m27,2’-OGppBH3pG (D1 and D2 diastereomers), (D1 and D2 diastereomers), N-Arylmethyl analogs, glyceryl, 4',5'-methylene (beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide,nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, cap1, cap2, cap3, cap4, ARCA, modified ARCA, inosine, N1- methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

100. The branched RNA oligonucleotide of any one of claims 69 to 99, wherein R6comprises a poly-A tail.

101. The branched RNA oligonucleotide of claim 100, wherein the poly-A tail comprises between 25 and 500 nucleotides.

102. The branched RNA oligonucleotide of claim 100 wherein the poly-A tail comprises between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 or 500 nucleotides.

103. The branched RNA oligonucleotide of any one of claims 100 to 102, wherein the poly-A tail comprises 10 or more adenosine nucleotides.

104. The branched RNA oligonucleotide of any one of claims 100 to 102, wherein 25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97- 100%, 98-100%, or 99-100% of nucleotides of the poly-A tail are adenosine nucleotides.

105. The branched RNA oligonucleotide of any one of claims 100 to 102, wherein 100% of the nucleotides of the poly-A tail are adenosine nucleotides. -101- 4935-1210-1197.1Atty. Docket No.114203-1801 106. The branched RNA oligonucleotide of any one of claims 100-102, wherein the poly-A tail comprises one or more modified nucleotides.

107. The branched RNA oligonucleotide of any one of claims 69-106, wherein R6comprises a therapeutic nucleic acid.

108. The branched RNA oligonucleotide of claim 107, wherein the therapeutic nucleic acid is chosen from the group consisting of an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.

109. A delivery agent comprising the capped RNA transcript of claim 68 or the branched RNA oligonucleotide of any one of claims 69-108, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.

110. The delivery agent of claim 109, wherein the nanoparticle or microparticle is a lipid nanoparticle or microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

111. A cell comprising the capped RNA transcript of claim 68 or the branched RNA oligonucleotide of any one of claims 69-108.

112. The cell of claim 111, wherein the cell is a mammalian cell.

113. A composition comprising the capped RNA transcript of claim 68, the branched RNA oligonucleotide of any one of claims 69-108, the delivery agent of claim 109 or 110, or the cell of claim 111 or 112.

114. The composition of claim 113 further comprising an additional agent.

115. The composition of claim 114, wherein the additional agent is an agent which has a therapeutic effect when administered to a subject.

116. The composition of claim 114 or 115, wherein the additional agent is chosen from the list consisting of a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a small molecule, an aptamer, a lipid, or a carbohydrate. -102- 4935-1210-1197.1Atty. Docket No.114203-1801 117. The composition of claim 116, wherein the additional agent is a shRNA, a siRNA, or an antisense oligonucleotide (ASO).

118. The composition of any one of claims 114-116, wherein the additional agent is an antigen or adjuvant.

119. The composition of any one of claims 113-118, wherein the composition is a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

120. A method of preventing or treating a disease in a subject, comprising introducing an effective amount of the capped RNA transcript of claim 68, the branched RNA oligonucleotide of any one of claims 69-108, the delivery agent of claim 109 or 110, the cell of claim 111 or 112, or the composition of any one of claims 113-119 to the subject.

121. The method of claim 120, wherein the subject is a human.

122. The capped RNA transcript of claim 68, the branched RNA oligonucleotide of any one of claims 69-108, the delivery agent of claim 109 or 110, the cell of claim 111 or 112, or the composition of any one of claims 113-119 for use in preventing or treating a disease in a subject.

123. A kit comprising the capped RNA transcript of claim 68, the branched RNA oligonucleotide of any one of claims 69-108, or the delivery agent of claim 109 or 110.

124. The kit of claim 123, further comprising an RNA ligase.

125. A kit comprising the composition of claim 116, a device for administering the composition to a subject, and instructions for administering the composition to the subject.

126. Use of the capped RNA transcript of claim 68, the branched RNA oligonucleotide of any one of claims 69-108, the delivery agent of claim 109 or 110, the cell of claim 111 or 112, or the composition of any one of claims 113-119 for the manufacture of a medicament for treatment of a disease in a subject. -103- 4935-1210-1197.1Atty. Docket No.114203-1801 127. The capped RNA transcript of claim 68, the branched RNA oligonucleotide of any one of claims 69-108, the delivery agent of claim 109 or 110, the cell of claim 111 or 112, or the composition of any one of claims 113-119 for use in the treatment of a disease in a subject. -104- 4935-1210-1197.1

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