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US 2022/0162659 A1Published application
Cell-free production of ribonucleic acid
AssigneeGreenLight Biosciences, Inc.
InventorsAndrey J. ZARUR, Drew S. CUNNINGHAM, James R. ABSHIRE, Rachit JAIN, Michael E. HUDSON
Life of this patent
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Abstract
This invention relates to in vitro production of nucleic acids, particularly RNAs and specifically messenger RNAs (mRNA).
Claims101
A cell-free reaction method for synthesizing a eukaryotic messenger ribonucleic acid (mRNA), the method comprising:
- (a) incubating in a reaction mixture cellular RNA and one or more enzymes that depolymerize RNA under conditions wherein the cellular RNA is substantially depolymerized, wherein the resulting first reaction mixture comprises 5′ nucleoside monophosphates;
- and
- (b) treating said reaction mixture under conditions wherein the RNA depolymerizing enzymes are eliminated or inactivated;
- and
- (c) incubating said reaction mixture comprising nucleoside monophosphates with a second reaction mixture comprising i) at least one polyphosphate (PPK) kinase and a phosphate donor;
- and optionally ii) at least one cytidine monophosphate (CMP) kinase, iii) at least one uridine monophosphate (UMP) kinase, iv) at least one guanosine monophosphate (GMP) kinase, v) at least one nucleoside-diphosphate (NDP) kinase, under conditions wherein nucleotide triphosphates are produced and further wherein vi) at least one RNA polymerase, vii) at least one DNA template encoding an mRNA with a polyA tail, viii) one or more capping reagents are added under conditions that produce mRNA, and further wherein, optionally ix) at least one deoxyribonuclease is added under conditions that digest the DNA following RNA production.
3 more elements
A cell-free reaction method for synthesizing a eukaryotic messenger ribonucleic acid (mRNA), the method comprising:
- (a) incubating in a reaction mixture cellular RNA and one or more enzymes that depolymerize RNA under conditions wherein the cellular RNA is substantially depolymerized, wherein the resulting first reaction mixture comprises 5′ nucleoside monophosphates;
- and
- (b) treating said reaction mixture under conditions wherein the RNA depolymerizing enzymes are eliminated or inactivated;
- and
- (c) incubating said reaction mixture comprising nucleoside monophosphates with a second reaction mixture comprising i) at least one polyphosphate (PPK) kinase and a phosphate donor and optionally ii) at least one cytidine monophosphate (CMP) kinase, iii) at least one uridine monophosphate (UMP) kinase, iv) at least one guanosine monophosphate (GMP) kinase, v) at least one nucleoside-diphosphate (NDP) kinase, under conditions wherein nucleotide triphosphates are produced and further wherein vi) at least one RNA polymerase, vii) at least one DNA template encoding an mRNA, and viii) one or more capping reagents are added under conditions that produce capped RNA;
- and further wherein optionally ix) at least one deoxyribonuclease is added under conditions that digest the DNA following RNA production;
- and
- d) (i) further incubating said reaction mixture produced in step (c) in the presence of a polyA polymerase and ATP, under conditions that produce mRNA
- or
- (ii) removing the RNA polymerase from the reaction mixture of step (c) by inactivation or physical separation followed by producing mRNA by further incubating the reaction mixture in the presence of polyA polymerase and ATP.
7 more elements
A cell-free reaction method for synthesizing a eukaryotic messenger ribonucleic acid (mRNA), the method comprising:
- (a) incubating in a reaction mixture cellular RNA and one or more enzymes that depolymerize RNA under conditions wherein the cellular RNA is substantially depolymerized, wherein the resulting first reaction mixture comprises 5′ nucleoside monophosphates;
- and
- (b) treating said reaction mixture under conditions wherein the RNA depolymerizing enzymes are eliminated or inactivated;
- and
- (c) incubating said reaction mixture comprising nucleoside monophosphates with a second reaction mixture comprising i) at least one polyphosphate (PPK) kinase and a phosphate donor;
- and optionally ii) at least one cytidine monophosphate (CMP) kinase, iii) at least one uridine monophosphate (UMP) kinase, iv) at least one guanosine monophosphate (GMP) kinase, v) at least one nucleoside-diphosphate (NDP) kinase, under conditions wherein nucleotide triphosphates are produced and further wherein vi) at least one RNA polymerase vii) at least one DNA template encoding an mRNA with a poly A tail, are added under conditions that produce uncapped RNA, and further wherein optionally viii) at least one deoxyribonuclease is added under conditions that digest the DNA following RNA production;
- and
- (d) exchanging the buffer from said reaction mixture from step (c) and incubating said reaction mixture in the presence of capping enzymes, GTP, and a methyl donor to produce mRNA.
5 more elements
A cell-free reaction method for synthesizing a eukaryotic messenger ribonucleic acid (mRNA), the method comprising:
- (a) incubating in a reaction mixture cellular RNA and one or more enzymes that depolymerize RNA under conditions wherein the cellular RNA is substantially depolymerized, wherein the resulting first reaction mixture comprises 5′ nucleoside monophosphates;
- and
- (b) treating said reaction mixture under conditions wherein the RNA depolymerizing enzymes are eliminated or inactivated;
- and
- (c) incubating said reaction mixture comprising nucleoside monophosphates with a second reaction mixture comprising i) at least one polyphosphate (PPK) kinase and a phosphate donor;
- and optionally ii) at least one cytidine monophosphate (CMP) kinase, iii) at least one uridine monophosphate (UMP) kinase, iv) at least one guanosine monophosphate (GMP) kinase, v) at least one nucleoside-diphosphate (NDP) kinase, under conditions wherein nucleotide triphosphates are produced and further wherein vi) at least one RNA polymerase vii) at least one DNA template encoding an mRNA, are added under conditions that produce uncapped, untailed RNA, and further wherein, optionally viii) at least one deoxyribonuclease is added under conditions that digest the DNA following RNA production;
- and
- d) (i) further incubating said reaction mixture produced in step (c) in the presence of a polyA polymerase and ATP, under conditions that produce uncapped RNA;
- or
- (ii) removing the RNA polymerase from the reaction mixture of step (c) by inactivation or physical separation followed by producing uncapped RNA by further incubating the reaction mixture in the presence of polyA polymerase and ATP;
- and
- e) exchanging the buffer from said reaction mixture from step (c) and incubating said reaction mixture in the presence of capping enzymes, GTP, and a methyl donor to produce mRNA.
9 more elements
A cell-free reaction method for synthesizing a eukaryotic messenger ribonucleic acid (mRNA), the method comprising:
- (a) incubating in a reaction mixture cellular RNA and one or more enzymes that depolymerize RNA under conditions wherein the cellular RNA is substantially depolymerized, wherein the resulting first reaction mixture comprises nucleoside diphosphates;
- (b) treating said reaction mixture under conditions wherein the RNA depolymerizing enzymes are eliminated or inactivated;
- and
- (c) incubating said reaction mixture comprising nucleoside diphosphates with a second reaction mixture comprising i) at least one polyphosphate (PPK) kinase and a phosphate donor;
- and optionally ii) at least one nucleoside-diphosphate (NDP) kinase, and optionally iii) at least one cytidine monophosphate (CMP) kinase, iv) at least one uridine monophosphate (UMP) kinase, v) at least one guanosine monophosphate (GMP) kinase, under conditions wherein nucleotide triphosphates are produced and further wherein vi) at least one RNA polymerase, vii) at least one DNA template encoding an mRNA with a polyA tail, viii) one or more capping reagents are added under conditions that produce mRNA, and further wherein optionally ix) at least one deoxyribonuclease is added under conditions that digest the DNA following RNA production.
2 more elements
A cell-free reaction method for synthesizing a eukaryotic messenger ribonucleic acid (mRNA), the method comprising:
- (a) incubating in a reaction mixture cellular RNA and one or more enzymes that depolymerize RNA under conditions wherein the cellular RNA is substantially depolymerized, wherein the resulting first reaction mixture comprises nucleoside diphosphates;
- and
- (b) treating said reaction mixture under conditions wherein the RNA depolymerizing enzymes are eliminated or inactivated;
- and
- (c) incubating said reaction mixture comprising nucleoside diphosphates with a second reaction mixture comprising i) at least one polyphosphate (PPK) kinase and a phosphate donor and optionally ii) at least one nucleoside-diphosphate (NDP) kinase, and optionally iii) at least one cytidine monophosphate (CMP) kinase, iv) at least one uridine monophosphate (UMP) kinase, v) at least one guanosine monophosphate (GMP) kinase, under conditions wherein nucleotide triphosphates are produced and further wherein vi) at least one RNA polymerase vii) at least one DNA template encoding an mRNA;
- viii) one or more capping reagents are added under conditions that produce capped RNA, and further wherein optionally ix) at least one deoxyribonuclease is added under conditions that digest the DNA following RNA production;
- and
- d) (i) further incubating said reaction mixture produced in step (c) in the presence of a polyA polymerase and ATP, under conditions that produce mRNA
- or
- (ii) removing the RNA polymerase from the reaction mixture of step (c) by inactivation or physical separation followed by producing mRNA by further incubating the reaction mixture in the presence of polyA polymerase and ATP.
7 more elements
A cell-free reaction method for synthesizing a eukaryotic messenger ribonucleic acid (mRNA), the method comprising:
- (a) incubating in a reaction mixture cellular RNA and one or more enzymes that depolymerize RNA under conditions wherein the cellular RNA is substantially depolymerized, wherein the resulting first reaction mixture comprises nucleoside diphosphates;
- (b) treating said reaction mixture under conditions wherein the RNA depolymerizing enzymes are eliminated or inactivated;
- (c) incubating said reaction mixture comprising nucleoside diphosphates with a second reaction mixture comprising i) at least one polyphosphate (PPK) kinase and a phosphate donor;
- and optionally ii) at least one nucleoside-diphosphate (NDP) kinase, and optionally iii) at least one cytidine monophosphate (CMP) kinase, iv) at least one uridine monophosphate (UMP) kinase, v) at least one guanosine monophosphate (GMP) kinase, under conditions wherein nucleotide triphosphates are produced and further wherein vi) at least one RNA polymerase and vii) at least one DNA template encoding an mRNA with a poly A tail, are added under conditions that produce uncapped RNA;
- and further wherein optionally viii) at least one deoxyribonuclease is added under conditions that digest the DNA following RNA production;
- and
- (d) exchanging the buffer from said reaction mixture from step (c) and incubating said reaction mixture in the presence of capping enzymes, GTP, and a methyl donor to produce mRNA.
4 more elements
A cell-free reaction method for synthesizing a eukaryotic messenger ribonucleic acid (mRNA), the method comprising:
- (a) incubating in a reaction mixture cellular RNA and one or more enzymes that depolymerize RNA under conditions wherein the cellular RNA is substantially depolymerized, wherein the resulting first reaction mixture comprises nucleoside diphosphates;
- and
- (b) treating said reaction mixture under conditions wherein the RNA depolymerizing enzymes are eliminated or inactivated;
- and
- (c) incubating said reaction mixture comprising nucleoside diphosphates with a second reaction mixture comprising i) at least one polyphosphate (PPK) kinase and a phosphate donor;
- and optionally ii) at least one nucleoside-diphosphate (NDP) kinase, and optionally iii) at least one cytidine monophosphate (CMP) kinase, iv) at least one uridine monophosphate (UMP) kinase, v) at least one guanosine monophosphate (GMP) kinase, vi) under conditions wherein nucleotide triphosphates are produced and further wherein vii) at least one RNA polymerase and viii) at least one DNA template encoding an mRNA, are added under conditions that produce uncapped, untailed RNA, and further wherein optionally ix) at least one deoxyribonuclease is added under conditions that digest the DNA following RNA production;
- (d) (i) further incubating said reaction mixture produced in step (c) in the presence of a polyA polymerase and ATP, under conditions that produce uncapped RNA;
- or
- (ii) removing the RNA polymerase from the reaction mixture of step (c) by inactivation or physical separation followed by producing uncapped RNA by further incubating the reaction mixture in the presence of polyA polymerase and ATP;
- and
- e) exchanging the buffer from said reaction mixture from step (c) and incubating said reaction mixture in the presence of capping enzymes, GTP, and a methyl donor to produce mRNA.
8 more elements
Dependencies are inferred from the claim text.