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US 2024/0181079 A1Published application
Production of circular polyribonucleotides in a eukaryotic system
AssigneeFlagship Pioneering Innovations Vii, LLC
InventorsBarry Andrew Martin, Swetha Srinivasa Murali, Yajie Niu, Derek Thomas Rothenheber, Michka Gabrielle Sharpe, Andrew McKinley Shumaker
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Abstract
The present disclosure relates, generally, to methods for producing, purifying, and using circular RNA from a eukaryotic system.
Claims51
A eukaryotic system for circularizing a polyribonucleotide, comprising a eukaryotic cell that comprises:
- (a) a linear polyribonucleotide having the formula 5′-(A)-(B)-(C)-(D)-(E)-3′, wherein the elements (A), (B), (C), (D), and (E) are operably linked, and wherein:
- (A) comprises a 5′ self-cleaving ribozyme;
- (B) comprises a 5′ annealing region comprising a 5′ complementary region;
- (C) comprises a polyribonucleotide cargo;
- (D) comprises a 3′ annealing region comprising a 3′ complementary region;
- and
- (E) comprises a 3′ self-cleaving ribozyme;
- wherein the 5′ complementary region and the 3′ complementary region have a free energy of binding of less than −5 kcal/mol, and/or wherein the 5′ complementary region and the 3′ complementary region have a Tm of binding of at least 10° C.;
- and
- (b) an RNA ligase;
- wherein cleavage of the 5′ self-cleaving ribozyme produces a free 5′-hydroxyl group on the 5′ end of the linear polyribonucleotide, and wherein cleavage of the 3′ self-cleaving ribozyme produces a free 2′,3′-cyclic phosphate group on the 3′ end of the linear polyribonucleotide, resulting in a ligase-compatible linear polyribonucleotide;
- and wherein the 5′ and 3′ ends of the ligase-compatible linear polyribonucleotide are ligated by the RNA ligase, thereby producing a circular polyribonucleotide.
9 more elements
A method for producing a circular RNA, comprising:
- (a) contacting in a eukaryotic cell:
- (i) a linear polyribonucleotide having the formula 5′-(A)-(B)-(C)-(D)-(E)-3′, wherein the elements (A), (B), (C), (D), and (E) are operably linked, and wherein:
- (A) comprises a 5′ self-cleaving ribozyme;
- (B) comprises a 5′ annealing region comprising a 5′ complementary region;
- (C) comprises a polyribonucleotide cargo;
- (D) comprises a 3′ annealing region comprising a 3′ complementary region;
- and
- (E) comprises a 3′ self-cleaving ribozyme;
- wherein the 5′ complementary region and the 3′ complementary region have a free energy of binding of less than −5 kcal/mol, and/or wherein the 5′ complementary region and the 3′ complementary region have a Tm of binding of at least 10° C.;
- wherein cleavage of the 5′ self-cleaving ribozyme produces a free 5′-hydroxyl group on the 5′ end of the linear polyribonucleotide, and wherein cleavage of the 3′ self-cleaving ribozyme produces a free 2′,3′-cyclic phosphate group on the 3′ end of the linear polyribonucleotide, resulting in a ligase-compatible linear polyribonucleotide;
- and
- (ii) an RNA ligase;
- whereby the 5′ and 3′ ends of the ligase-compatible linear polyribonucleotide are ligated by the RNA ligase, thereby producing a circular polyribonucleotide;
- and
- (b) optionally, purifying the circular polyribonucleotide.
12 more elements
A eukaryotic cell comprising:
- (a) a linear polyribonucleotide having the formula 5′-(A)-(B)-(C)-(D)-(E)-3′, wherein the elements (A), (B), (C), (D), and (E) are operably linked, and wherein:
- (A) comprises a 5′ self-cleaving ribozyme;
- (B) comprises a 5′ annealing region comprising a 5′ complementary region;
- (C) comprises a polyribonucleotide cargo;
- (D) comprises a 3′ annealing region comprising a 3′ complementary region;
- and
- (E) comprises a 3′ self-cleaving ribozyme;
- wherein the 5′ complementary region and the 3′ complementary region have a free energy of binding of less than −5 kcal/mol, and/or wherein the 5′ complementary region and the 3′ complementary region have a Tm of binding of at least 10° C.;
- wherein cleavage of the 5′ self-cleaving ribozyme produces a free 5′-hydroxyl group on the 5′ end of the linear polyribonucleotide, and wherein cleavage of the 3′ self-cleaving ribozyme produces a free 2′,3′-cyclic phosphate group on the 3′ end of the linear polyribonucleotide, resulting in a ligase-compatible linear polyribonucleotide;
- and
- (b) an RNA ligase, wherein the RNA ligase is capable of ligating the 5′ end and the 3′ end of the ligase-compatible linear polyribonucleotide to produce a circular RNA.
8 more elements
Dependencies are inferred from the claim text.