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Updated: May 21, 2025

Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP
Published on: September 8, 2023
A novel replicase-mediated self-amplifying RNA amplification mechanism of the SARS-CoV-2 replication-transcription
Hsien-Lin Liu1, Sam Lin2, William Hung2
1WJWU and LYNN Institute for Stem Cell Research, La Puente, CA, 91744, USA.
Abstract:
A novel self-amplifying mRNA (samRNA) amplification mechanism was first discovered in the SARS-CoV-2 replication-transcription system and named replicase cycling reaction (RCR). In principle, RCR is a replicase-mediated transcription reaction driven by the SARS-CoV-2 RNA-dependent RNA polymerases (RdRPs), which amplify a specific samRNA construct consisting of an RNA/mRNA sequence flanked by a 5'-end RdRP-reverse-promoter (5'-RdRP-RP) and a 3'-end RdRP-forward-promoter (3'-RdRP-FP) on both sides. Based on this samRNA composition, we had not only successfully established the first in-vitro RCR reaction for directly amplifying the SARS-CoV-2 genomic and subgenomic RNAs but also further used it in a combined in-vitro-transcription and RCR (IVT-RCR) protocol to identify new functions of the SARS-CoV-2 NSP7, NSP8, and NSP12 proteins, leading to a fast diagnostic assay for measuring the SARS-CoV-2 RdRP activity. These findings may shed a new light on the molecular mechanisms of SARS-CoV-2 replication and transcription. As a result, in addition to the previously found primer-dependent RNA synthesis activity of the coronaviral RdRP complexes, we herein reported another new 5'/3'-promoter-dependent, primer-independent samRNA synthesis mechanism mediated by the SARS-CoV-2 RdRP complex. Based on this novel RCR mechanism, the associated samRNA composition is conceivably useful for facilitating the design and development of next-generation RNA/mRNA medicines and vaccines.
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