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-10519189-B2
Publication Date
2019-12-31
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 a complex comprising an initiating capped oligonucleotide primer and a DNA template. The DNA template includes a promoter region with a transcriptional start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2, and the initiating capped oligonucleotide primer is hybridized to the DNA template at least at nucleotide positions +1 and +2.
The initiating capped oligonucleotide primer comprises a capped oligonucleotide primer structure in which B1 and B2 independently are a natural, a modified, or an unnatural nucleoside base, and R1, R2, and R3 independently are OH or O-methyl. The disclosure further covers 5′-capped RNA molecules formed using initiating capped oligonucleotide primers, including primers having cap-containing structures such as m7Gppp[N2′Ome]n[N]m and/or Formula I.
The document describes cap contexts including Cap 0, Cap 1, Cap 2, and TMG-Cap, and provides embodiments in which optional 2′-O-methylation is present at early transcript positions consistent with co-transcriptional capping.
Claims Coverage
Independent claim coverage centers on defining a complex of an initiating capped oligonucleotide primer hybridized to a DNA template at the promoter transcription start site, with the primer’s base identities (B1, B2) and substituent states (R1, R2, R3 as OH or O-methyl) constrained to coordinate with nucleotide positions +1 and +2. The inventive features are primarily specified for this primer/template alignment and cap-related substituent configuration.
Initiating capped oligonucleotide primer hybridized at +1 and +2
An initiating capped oligonucleotide primer hybridized to a DNA template at least at nucleotide positions +1 and +2, where the DNA template comprises a promoter region with a transcriptional start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2.
Capped primer structure with variable bases B1 and B2
A primer in which B1 and B2 are independently a natural, a modified, or an unnatural nucleoside base within the initiating capped oligonucleotide primer structure.
Cap-related substituent constraints R1, R2, and R3 as OH or O-methyl
A primer in which R1, R2, and R3 independently are OH or O-methyl as part of the initiating capped oligonucleotide primer structure.
Across the independent-claim framework, inventive coverage is directed to aligning an initiating capped oligonucleotide primer with the promoter start site at nucleotide positions +1 and +2, while defining primer nucleoside base variability (B1, B2) and cap-related substituent states (R1, R2, R3 as OH or O-methyl) to produce co-transcriptional 5′-capped RNAs.
Stated Advantages
Improved transcription yields and high capping efficiency/fidelity compared with ARCA-like approaches, as described in the disclosure.
Efficient co-transcriptional Cap formation consistent with cap contexts (including Cap 1/TMG-Cap), as described in the disclosure.
Translation activity in cell assays consistent with efficient co-transcriptional Cap formation, as described in the disclosure.
Documented Applications
Formation of 5′-capped RNAs using the initiating capped oligonucleotide primer/DNA template complex during transcription under promoter control (not limited to T7), as described in the disclosure.
Cell assays using luciferase mRNA and evaluation in Huh-7 and THP-1 cells consistent with efficient co-transcriptional Cap formation, as described in the disclosure.
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