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-12258369-B2
Publication Date
2025-03-25
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 initiating capped oligonucleotide primers for synthesizing 5′-capped RNAs. The primers include Cap 0, Cap 1, Cap 2, or TMG-Cap-like structures and an m7Gppp[N2′Ome]n[N]m-form general description within a defined structural framework. The primers further include variable nucleoside bases B1 through B10, with adjustable structural parameters for sugar/linkage substituents and terminal substituents.
A defined Formula I structure characterizes the initiating capped oligonucleotide primer, including binary/flag parameters M and L, q1 through q9 values, and substituent sets X1 through X13, Y1 through Y13, Z0 through Z22, and terminal substituents R1 through R12. B1 is complementary to a nucleoside base on a nucleic acid template at transcription template position +1 from the transcription start site, and subsequent bases are complementary to template nucleotides at increasing positions. The last nucleoside base of the initiating capped oligonucleotide primer is complementary to the last hybridized transcription template nucleotide.
The document also discloses RNA products containing these initiating capped oligonucleotide primers, as well as cells containing the RNA and pharmaceutical compositions comprising such RNAs. Transcription methods are described using a polynucleotide template and an RNA polymerase, intended to initiate RNA synthesis with improved capping behavior compared with approaches that rely on enzymatic capping and 2′-O-methylation.
Claims Coverage
The document provides two independent claims. Independent claim 1 covers the initiating capped oligonucleotide primer defined by a structured variable-base and substituent framework with template-position complementarity rules for transcription initiation. Independent claim 29 covers a primer-DNA template complex with a transcription start site defining nucleotides at template positions +1 to +3 and requiring hybridization at least across +1, +2, and +3.
Initiating capped oligonucleotide primer with variable bases and template-position complementarity
An initiating capped oligonucleotide primer comprising a structure with each of B1 through B10 being 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; each of R1 is independently H or methyl; R2 and R3 are independently H, OH, alkyl, O-alkyl, halogen, a linker or a detectable marker; 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; wherein B1 is complementary to a nucleoside base on a nucleic acid template at transcription template position +1 from the transcription start site; B2 through B9, if present, are complementary to the respective nucleoside bases on the nucleic acid template at transcription template position +2 and so on from the transcription start site; and B10 is complementary to the last hybridized transcription template nucleotide.
Primer-DNA template complex with transcription start site at +1 to +3
A complex comprising an initiating capped oligonucleotide primer and a DNA template, wherein the initiating capped oligonucleotide primer comprises a structure with B1 being guanine; B2 and B3 being independently a natural, a modified, or an unnatural nucleoside base; and R1, R2, R3 and R4 being independently OH or O-methyl; wherein the DNA template comprises a promoter region comprising a transcriptional start site having a first nucleotide at nucleotide position +1, a second nucleotide at nucleotide position +2, and a third nucleotide at nucleotide position +3; and wherein the initiating capped oligonucleotide primer is hybridized to the DNA template at least at nucleotide positions +1, +2, and +3.
Across the two independent claims, the inventive coverage centers on an initiating capped oligonucleotide primer defined by constrained, variable structural parameters and base complementarity to transcription template positions, and on a primer-DNA template complex where the promoter region defines nucleotides at +1, +2, and +3 and hybridization spans at least those positions.
Stated Advantages
Avoidance of expensive and inefficient enzymatic capping and 2′-O-methylation.
Reduction of reverse initiation.
Improved yield and improved capping fidelity.
Documented Applications
Generating or using luciferase mRNA in an assay to assess translational activity associated with the disclosed capped RNA approaches.
Cells containing the RNA, including Huh-7 cells and THP-1 cells (differentiated).
Using LC-MS to evaluate capping efficiency, including comparisons of template fidelity with deoxy residues at +1/+2.
Transcription using a polynucleotide template plus RNA polymerase, including embodiments consistent with T7 RNA polymerase.
Pharmaceutical compositions comprising the disclosed RNAs.
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