Compositions and methods for synthesizing 5′-Capped RNAS

Inventors

Hogrefe, Richard I. • Lebedev, Alexandre • McCaffrey, Anton P. • Shin, Dongwon

Assignees

Trilink Biotechnologies LLC

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 disclosure describes a complex comprising an initiating capped oligonucleotide primer and a DNA template. The DNA template comprises a promoter region having a transcriptional start site with a first nucleotide at nucleotide position +1, a second nucleotide at nucleotide position +2, and a third nucleotide at nucleotide position +3, and the capped oligonucleotide primer is hybridized to the DNA template at least at nucleotide positions +1, +2, and +3.

The initiating capped oligonucleotide primer includes a structure in which B1, B2, and B3 are independently a natural, a modified, or an unnatural nucleoside base, and R1, R2, R3, and R4 are independently OH or O-methyl. The primer structure provides cap features associated with Cap 0, Cap 1, Cap 2, and TMG-cap-like features, including m7G and guanosine analogs, and optional 2′-O-methylated nucleosides.

The disclosure emphasizes that initiating capped oligonucleotide primers with the general form m7Gppp[N2′Ome]n[N]m and/or Formula I structures are used to improve initiation fidelity and capping efficiency/yield while reducing the need for post-transcriptional capping and 2′-O-methylation steps. The disclosure also reports compatibility with T7 transcription systems and provides example primer sequences and performance results using assays such as co-transcriptional capping efficiency via LC-MS and translation activity in cells.

Claims Coverage

The independent claim covers a complex defined by an initiating capped oligonucleotide primer and a DNA template promoter region with defined transcriptional start-site positions +1 to +3, with the primer hybridized at those positions. The claim language includes three inventive features: hybridization at +1, +2, and +3; configurable nucleoside base identity at B1 to B3; and OH or O-methyl substituent selection at R1 to R4.

Across the independent claim and its refinements, the patent focuses on a complex in which a structured initiating capped oligonucleotide primer is hybridized to a DNA template promoter transcriptional start site at positions +1 to +3, while allowing defined variability in nucleoside base identity and substituent chemistry.

Stated Advantages

Documented Applications