Purification and purity assessment of RNA molecules synthesized with modified nucleosides
View Patent ↗This invention provides purified preparations of an RNA, oligoribonucleotide, or polyribonucleotide comprising a modified nucleoside, and methods of assessing purity of purified preparations of an RNA, oligoribonucleotide, or polyribonucleotide comprising a modified nucleoside.
1. A method of preparing a purified preparation of RNA comprising at least one modified nucleoside selected from the group consisting of a 1-methyl-pseudouridine, m5C, m5U, m6A, s2U, Ψ, and 2′-O-methyl-U comprising the steps of:
a. producing a preparation of messenger RNA comprising at least one modified nucleoside;
b. subjecting the preparation of messenger RNA comprising at least one modified nucleoside to enzymatic digestion with 0.001 units of at least one enzyme selected from the group consisting of RNase III, RNase V1, Dicer, and Chipper; and
c. isolating the purified preparation of messenger RNA comprising at least one modified nucleoside.
2. A method of inducing a mammalian cell to produce a protein of interest, the method comprising the step of contacting the mammalian cell with the purified preparation of RNA prepared by the method of claim 1 , thereby inducing the mammalian cell to produce the protein of interest.
3. A method of preparing a purified preparation of RNA comprising at least one modified nucleoside selected from the group consisting of a 1-methyl-pseudouridine, m5C, m5U, m6A, s2U, Ψ, and 2′-O-methyl-U comprising the steps of:
a. producing a preparation of messenger RNA comprising at least one modified nucleoside;
b. subjecting the preparation of messenger RNA comprising at least one modified nucleoside to liquid chromatography using a linear gradient of 38% Buffer B (0.1 M triethylammonium acetate, 25% acetonitrile, pH 7.0) to 55% Buffer B in Buffer A (0.1 M triethylammonium acetate, pH 7.0) over 22 minutes; and
c. isolating the purified preparation of messenger RNA comprising at least one modified nucleoside.
4. A method of inducing a mammalian cell to produce a protein of interest, the method comprising the step of contacting the mammalian cell with the purified preparation of RNA prepared by the method of claim 3 , thereby inducing the mammalian cell to produce the protein of interest.
5. A method of preparing a purified preparation of RNA comprising at least one modified nucleoside comprising the steps of:
a. producing a preparation of messenger RNA comprising at least one modified nucleoside;
b. subjecting the preparation of messenger RNA comprising at least one modified nucleoside to at least one purification process selected from the group consisting of enzyme digestion with 0.001 units of at least one enzyme selected from the group consisting of RNase III, RNase V1, Dicer, and Chipper and liquid chromatography using a linear gradient of 38% Buffer B (0.1 M triethylammonium acetate, 25% acetonitrile, pH 7.0) to 55% Buffer B in Buffer A (0.1 M triethylammonium acetate, pH 7.0) over 22 minutes; and
c. isolating the purified preparation of messenger RNA comprising at least one modified nucleoside.
6. The method of claim 5 , wherein the at least one modified nucleoside is selected from the group consisting of 1-methyl-pseudouridine, m5C, m5U, m6A, s2U, Ψ, and 2′-O-methyl-U.
7. A method of inducing a mammalian cell to produce a protein of interest, the method comprising the step of contacting the mammalian cell with the purified preparation of RNA prepared by the method of claim 5 , thereby inducing the mammalian cell to produce the protein of interest.