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-10913768-B2
Publication Date
2021-02-09
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 described compositions and methods provide initiating capped oligonucleotide primers for synthesizing 5′-capped RNAs. The initiating capped oligonucleotide primer includes an m7Gppp[N2′Ome]n[N]m framework, and a primer Formula I is specified with multiple variable groups that define nucleoside base options and substituents across the primer positions, including B1–B10 and R1–R4/R2–R3.
The primer structure is parameterized using M and L and binary constraints q1–q9, with q1 fixed to 1 and q2–q9 independently selectable as 0 or 1. Additional variable groups define heteroatom and atom types, including X1–X13 as O or S; Y1–Y13 as OH, SH, BH3, aryl, alkyl, O-alkyl, or O-aryl; and Z0–Z22 substituent categories including O, S, NH, CH2, C(halogen)2, or CH(halogen). The framework includes substituent positions R4–R12 selectable as H, OH, OMe, or a detectable marker.
The document further describes transcription to produce the templated RNA using an RNA polymerase system. In the transcription context, the initiating capped oligonucleotide primer is introduced into a mixture comprising a template and an RNA polymerase and incubated for a time sufficient to allow transcription of the template. The primer-based approach is positioned relative to traditional cap-dinucleotides, including mCAP/ARCA, and enzymatic capping and 2′-O-methylation.
Claims Coverage
The independent claims cover four inventive features: a parameterized initiating capped oligonucleotide primer structure, a method for synthesizing a fully templated RNA molecule using the primer, a DNA template and primer complex hybridized at promoter start positions, and a narrower initiating capped oligonucleotide primer variant with reduced variable constraints.
Parameterized initiating capped oligonucleotide primer structure
An initiating capped oligonucleotide primer comprising a structure in which B1 through B10 are independently a natural, modified or unnatural nucleoside base; M is 0 or 1; L is 0 or 1; q1 is 1 and q2 through q9 are independently 0 or 1; R1 is H or methyl; R2 and R3 are independently H, OH, alkyl, O-alkyl, halogen, a linker or a detectable marker; X1 through X13 are independently O or S; Y1 through Y13 are independently OH, SH, BH3, aryl, alkyl, O-alkyl or O-aryl; Z0 through Z22 are independently O, S, NH, CH2, C(halogen)2 or CH(halogen); and R4 through R12 are independently H, OH, OMe or a detectable marker.
Method for synthesizing a fully templated RNA molecule using the initiating capped oligonucleotide primer
A method comprising introducing the initiating capped oligonucleotide primer into a mixture comprising an RNA polymerase under conditions conducive to transcription of the polynucleotide template and incubating the mixture for a time sufficient to allow transcription of the template.
DNA template and initiating capped oligonucleotide primer complex hybridized at promoter start positions
A complex comprising a DNA template and an initiating capped oligonucleotide primer, wherein the DNA template comprises a promoter region comprising a transcriptional start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2, and wherein the initiating capped oligonucleotide primer is hybridized to the DNA template at least at nucleotide positions +1 and +2.
Narrow initiating capped oligonucleotide primer structure with reduced variable constraints
An initiating capped oligonucleotide primer wherein B1 and B2 are independently a natural, modified or unnatural nucleoside base; R1 is H or methyl; and R2 and R3 are independently H, OH, alkyl, or O-alkyl.
Collectively, the independent claims define a capped oligonucleotide primer structure with explicit parameterized chemical variability, use that primer in an RNA polymerase transcription reaction to synthesize fully templated RNA, and define a DNA template/promoter-based complex in which the primer is hybridized to specific transcription start positions. A narrower independent claim specifies a reduced-parameter initiating capped oligonucleotide primer variant.
Stated Advantages
Described advantages over traditional cap-dinucleotides, including mCAP/ARCA, and enzymatic capping and 2′-O-methylation.
Documented Applications
Co-transcriptional production of RNA with a described example using luciferase translation.
Performance support via described capping efficiency estimates and initiation fidelity comparisons.
Use of cells containing the RNAs, including THP-1 cells and Huh-7 cells.
Pharmaceutical composition contexts containing the RNAs.
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