Compositions and methods for synthesizing 5′-capped RNAs
Inventors
Hogrefe, Richard I. • Lebedev, Alexandre • McCaffrey, Anton P. • Shin, Dongwon
Assignees
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Publication Number
US-11414453-B2
Publication Date
2022-08-16
Expiration Date
Abstract
Provided herein are methods and compositions for synthesizing 5′Capped RNAs wherein the initiating capped oligonucleotide primers have the general form m7Gppp[N2′Ome]n[N]m wherein m7G is N7-methylated guanosine or any guanosine analog, N is any natural, modified or unnatural nucleoside, “n” can be any integer from 0 to 4 and “m” can be an integer from 1 to 9.
Core Innovation
The invention relates to 5′-capped RNA synthesis using an initiating capped oligonucleotide primer that includes a defined cap-containing structure and a nucleic acid backbone with at least one locked nucleic acid unit. The initiating capped oligonucleotide primer supports co-transcriptional formation of Cap 0, Cap 1, Cap 2, and TMG-cap RNAs using an RNA polymerase.
The initiating capped oligonucleotide primer is described with structural parameters that specify allowable nucleoside base identities, indices including M and L, and substituent options for groups designated R1 through R12 and atoms designated X1 through X13, Y1 through Y13, and Z0 through Z22. In certain embodiments, one or more of R2 or R4 through R12 includes a linker or a detectable marker, and the linker is related to a locked nucleic acid unit.
The background contrasts conventional capping approaches, including mCAP/ARCA competition, laborious post-processing, and orientation/capping inefficiency. The initiating capped oligonucleotide primers can outcompete NTP initiation and enable incorporation of Cap 1 and Cap 2 without extra enzymatic steps.
Claims Coverage
The independent claim covers an initiating capped oligonucleotide primer with a defined capped-primer structural configuration and a nucleic acid backbone including at least one locked nucleic acid unit. Dependent claims further narrow the primer by specifying linker placement and the relationship between the linker and the locked nucleic acid unit.
Initiating capped oligonucleotide primer with capped-primer structural configuration and locked nucleic acid unit backbone
An initiating capped oligonucleotide primer comprising a specified structural configuration with defined nucleoside base options, index constraints, substituent options, and linkage atom options, including that a nucleic acid backbone of the initiating capped oligonucleotide primer comprises at least one locked nucleic acid unit.
Linker among designated substituent positions on the initiating capped oligonucleotide primer
The initiating capped oligonucleotide primer has at least one linker among R2 or R4 through R12.
Linker includes an O-hydrocarbyl moiety
The initiating capped oligonucleotide primer has a linker that includes an O-hydrocarbyl moiety.
Linker is part of a locked nucleic acid unit
The capped oligonucleotide primer is configured such that the linker is part of a locked nucleic acid unit.
R2 is a linker
The initiating capped oligonucleotide primer specifies that R2 is a linker.
Overall, the claim set covers initiating capped oligonucleotide primers with a specified capped-primer structure and at least one locked nucleic acid unit, and it further narrows embodiments by requiring a linker at designated positions, including R2, and by requiring that the linker is part of a locked nucleic acid unit.
Stated Advantages
The initiating capped oligonucleotide primers can outcompete NTP initiation.
Cap 1 and Cap 2 incorporation is asserted to be achieved without extra enzymatic steps.
The background asserts improvements over conventional capping approaches described as having mCAP/ARCA competition, laborious post-processing, and orientation/capping inefficiency.
Documented Applications
Pharmaceutical compositions containing the resulting capped RNAs.
Cell-based protein expression using capped RNAs, including luciferase mRNA.
Use in cell contexts including Huh-7 cells and THP-1 cells, as described in the disclosure.
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