US 12,590,329 B2 · Replicase cycling reaction (RCR)

Abstract

This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.

Claims18

Dependent claims

An RNA replicase-mediated RNA amplification method, comprising:

(a) providing at least a linear RNA sequence, wherein said RNA sequence contains at least a 5′-end RdRp binding site and at least a 3′-end RdRp binding site, wherein said 5′-end RdRp binding site is SEQ ID NO:
2 and said 3′-end RdRp binding site is homologous to any one of SEQ ID NO:
8, 10, or 11;
(b) providing at least an RNA replicase, wherein said RNA replicase is isolated or modified from the RNA-dependent RNA polymerases (RdRp) of coronavirus;
and
(c) mixing the RNA sequence of (a) and the RNA replicase of (b) under a buffer condition, so as to elicit RNA replicase-mediated production and amplification of said RNA sequence, wherein said buffer condition contains ribonucleoside triphosphate molecules (rNTPs) required for RNA synthesis and is in a pH range from 6.0 to 8.0 as well as in a temperature range from 20° C. to 45° C., thereby producing amplified RNA sequences.

3 more elements

Dependencies are inferred from the claim text.

Replicase cycling reaction (RCR)

Life of this patent

Replicase cycling reaction (RCR)

Life of this patent

Abstract

Claims18