Methods of making rna by splinted ligation and compositions thereof
AssigneeOrbital Therapeutics, Inc.
InventorsChristopher Robert CARLSON, David Eric WEINBERG, Gilles BESIN
Life of this patent
Estimating the term…
Abstract
The present invention provides, among other things, a method of making circular RNA by annealing a DNA splint having regions of homology to the 5′ and 3′ ends of linear RNA with an RNA ligase and subsequently removing the DNA splint. Also provided is a method of joining two linear RNA molecules by ligation using a DNA splint having regions of homology to the 3′ end of a first linear RNA and the 5′ end of a second linear RNA with a RNA ligase, and subsequently removing the DNA splints. The present invention provides a method of making circular RNA by joining two linear RNA molecules with an RNA ligase and two DNA splints, each comprising regions of homology to the 5′ end.
Claims96
A method of making circular RNA, the method comprising:
- (i) contacting a linear RNA comprising a 5′ end and a 3′ end with a RNA ligase and a DNA splint comprising a region complementary to the 5′ end of the linear RNA and a region complementary to the 3′ end of the linear RNA, wherein the DNA splint binds the linear RNA and the ligase joins the 5′ end and the 3′ end;
- and
- (ii) removing the DNA splint;
- thereby generating circular RNA.
A method of joining two linear RNA molecules by ligating with a DNA splint, the method comprising:
- (i) contacting the two linear RNA molecules, each comprising a 5′ end and a 3′ end with a RNA ligase and a DNA splint comprising a first region complementary to the 3′ end of the first linear RNA and a second region complementary to the 5′ end of the second linear RNA, wherein the DNA splint binds the linear RNA and the ligase joins the 3′ end of the first linear RNA and the 5′ end of the second linear RNA;
- and
- (ii) removing the DNA splint;
- thereby generating a single linear RNA.
The method of any one of the preceding claims, wherein the DNA splint is biotinylated.
The method of any one of the preceding claims, wherein the DNA splint is single-stranded.
The method of any one of the preceding claims, wherein the DNA splint comprises a gap.
The method of any one of the preceding claims, wherein the gap is between 1-20, 2-15, or 2-10, or 2-6 nucleotides in length, and wherein the splint anneals to the RNA to form overhangs comprising at least 1-20 non-complementary nucleotides at the 5′ and/or 3′ ends.
The method of any one of the preceding claims, wherein the gap is 4 nucleotides in length.
The method of any of the preceding claims, wherein the gap comprises adenine, thymine, uridine, guanine and/or cytosine nucleotides.
The method of any one of the preceding claims, wherein the gap is a homopolymeric stretch of nucleotides.
The method of any one of the preceding claims, wherein the gap is comprised of one or more modified nucleotides and/or abasic sites.
The method of any one of the preceding claims, wherein the ligase is an RNA ligase.
The method of any one of the preceding claims, wherein a region complementary to the 5′ end of the linear RNA and/or to the 3′ end of the linear RNA is at least 50 nucleotides in length.
The method of any one of the preceding claims, wherein the region complementary to the 5′ end of the linear RNA and/or to the 3′ end of the linear RNA is between 1% to 10% of the length of the linear RNA.
The method of any one of the preceding claims, wherein the region complementary to the 5′ end of the linear RNA and/or to the 3′ end of the linear RNA has a 5′ overhang and/or a 3′ overhang.
The method of any one of the preceding claims, wherein the method further comprises digesting the un-ligated RNA with an RNA exonuclease.
The method of any one of the preceding claims, wherein the linear RNA is synthetic RNA.
The method of any one of the preceding claims, wherein the linear RNA is in vitro transcribed RNA.
The method of any one of the preceding claims, wherein the 5′ end is a 5′ monophosphate.
The method of any one of the preceding claims, wherein the 5′ end is pre-adenylated.
The method of any one of the preceding claims, wherein the 3′ end is a 3′ hydroxyl.
The method of any one of the preceding claims, wherein the linear RNA is messenger RNA.
The method of any one of the preceding claims, wherein the linear RNA and/or circular RNA comprises one or more modified nucleotides.
The method of any one of the preceding claims, wherein the linear RNA and/or circular RNA is between 100 base pairs to 10 kilobases in length.
The method of any one of the preceding claims, wherein the linear RNA and/or circular RNA encodes a therapeutic protein.
The method of any one of the preceding claims, wherein the linear RNA and/or circular RNA is an RNA vaccine.
A circular RNA is generated by the method of any one of the preceding claims.
A method of manufacturing RNA comprising a step of circularizing RNA by the method of any one of the preceding claims.
A method of making circular RNA, the method comprising:
- (i) contacting at least two linear RNA molecules, each comprising a 5′ end and a 3′ end with an RNA ligase, and at least two DNA splints,
- wherein the first DNA splint comprises two regions of homology to the 3′ end of the first linear RNA and the 5′ end of the second linear RNA,
- wherein the second DNA splint comprises two regions of homology to the 3′ end of the second linear RNA and the 5′ end of the first linear RNA,
- such that the two DNA splints bind the linear RNA molecules and the ligase joins the ends of the linear RNA molecules;
- and
- (ii) removing the DNA splints;
- thereby generating a circular RNA.
A circular RNA molecule comprising an internal ribosome entry site (IRES) operably linked to at least one coding polynucleotide that encodes a protein of interest, wherein the IRES lacks N1-methylpseudouridine (N1m-ψ) modification.
A circular RNA comprising an internal ribosome entry site (IRES) operably linked to at least one coding polynucleotide that encodes a protein of interest, wherein the IRES comprises about 1-10% modified nucleotides.
Dependencies are inferred from the claim text.