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-11878991-B2
Publication Date
2024-01-23
Expiration Date
Abstract
Provided herein are methods and compositions for synthesizing 5′Capped RNAs wherein the initiating capped oligonucleotide primers have the general form m7 Gppp[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 compositions and methods for synthesizing a fully templated RNA molecule by introducing an initiating capped oligonucleotide primer, or a salt thereof, into a mixture comprising a polynucleotide template and an RNA polymerase and incubating for a time sufficient to allow for transcription. The initiating capped oligonucleotide primer is defined by an m7Gppp[N2′Ome]n[N]m architecture and further characterized by a structured set of nucleoside base and substituent options for positions that enable Cap 0, Cap 1, Cap 2, and TMG-Cap-like structures.
The capped oligonucleotide primer architecture includes defined sets of sulfur/oxygen linkers, defined sugar or base substituent options, and additional positional substituent sets that govern allowable chemical embodiments of the initiating capped primer. The primer further includes modifiable substituent positions that can be H, OH, O-methyl, or a detectable marker, enabling different primer embodiments within the initiating capped oligonucleotide primer structure.
In synthesis, the initiating capped oligonucleotide primer is introduced with base-complementarity constraints to the promoter sequence of the polynucleotide template, including complementarity at position +1 in the promoter sequence. The primer is used with the polynucleotide template under conditions conducive to transcription by the RNA polymerase to generate fully templated RNA molecules containing the initiating capped primer at the 5′ end.
Claims Coverage
The independent claims are directed to methods for synthesizing fully templated RNA molecules by transcription using an initiating capped oligonucleotide primer with defined structural constraints. Across the independent claims, several inventive features are recited, centered on the initiating capped oligonucleotide primer structure and its complementarity relationship to promoter positions, together with defined primer base and substituent limitations.
Initiating capped oligonucleotide primer for templated RNA synthesis
A method comprising introducing an initiating capped oligonucleotide primer, or a salt thereof, of a structure in which each of B1 through B10 is independently a natural, modified or unnatural nucleoside base, M is 0 or 1, L is 0 or 1, q1 is 1 and each of q2 through q9 is independently 0 or 1, R1 is H or methyl, R2 and R3 are each OH, each of X1 through X13 is independently O or S, each of Y1 through Y13 is independently OH, SH, BH3, aryl, alkyl, O-alkyl or O-aryl, each of Z0 through Z22 is independently O, S, NH, CH2, C(halogen)2 or CH(halogen), and each of R4 through R12 is independently H, OH, O-methyl or a detectable marker into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription, and incubating said mixture for a time sufficient to allow for transcription of said template.
Complementarity to promoter +1 position in fully templated RNA synthesis
Introducing an initiating capped oligonucleotide primer, or a salt thereof, wherein B1 in the initiating capped oligonucleotide primer is complementary to the base at position +1 in the promoter sequence of the polynucleotide template, into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template, and incubating said mixture for a time sufficient to allow for transcription of said template.
Specific initiating capped oligonucleotide primer primer substituent constraints
Introducing an initiating capped oligonucleotide primer, or a salt thereof, wherein B1 is adenine or N6-methyladenine, B10 is uracil, R1 is H or methyl, R2 and R3 are each OH, and R4 is H, OH, O-methyl or a detectable marker into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template, and incubating said mixture for a time sufficient to allow for transcription of said template.
Specific initiating capped oligonucleotide primer with B1 guanine constraint
Introducing an initiating capped oligonucleotide primer, or a salt thereof, wherein B1 is guanine, B10 is a natural, modified, or unnatural nucleoside base, R1 is H or methyl, R2 and R3 are each OH, and R4 is H, OH, O-methyl or a detectable marker into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template, and incubating said mixture for a time sufficient to allow for transcription of said template.
Specific initiating capped oligonucleotide primer with B1 cytosine and B10 guanine constraints
Introducing an initiating capped oligonucleotide primer, or a salt thereof, wherein B1 is cytosine, B10 is guanine, R1 is H or methyl, R2 and R3 are each OH, and R4 is H, OH, O-methyl, or a detectable marker into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template, and incubating said mixture for a time sufficient to allow for transcription of said template.
Initiating capped oligonucleotide primer with constrained B1, B2, B10 and substituent options
Introducing an initiating capped oligonucleotide primer, or a salt thereof, wherein B1, B2, and B10 are independently a natural, modified or unnatural nucleoside base, R1 is H or methyl, R2 and R3 are each OH, and R4 and R5 are independently H, OH, O-methyl, or a detectable marker into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template, and incubating said mixture for a time sufficient to allow for transcription of said template.
The independent claims collectively cover transcription-based synthesis of fully templated RNA molecules using an initiating capped oligonucleotide primer having a defined capped primer structure with constrained base and substituent options, and a complementarity constraint to the promoter sequence at position +1. Other independent claims narrow the primer composition by fixing specific identities at B1 and/or B10 and limiting particular substituent positions such as R1 and R4, and in one case R4 and R5.
Stated Advantages
Improved capping efficiency.
Higher translational activity in cells compared to ARCA-based Cap 0 initiation.
Documented Applications
RNA molecules containing the initiating capped oligonucleotide primer architecture, including mRNA.
Pharmaceutical and cellular uses of RNA molecules synthesized by the claimed methods.
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