A new approach to RNA synthesis: immobilization of stably and functionally co-tethered promoter DNA and T7 RNA polymerase.
Nucleic Acids Res
; 52(17): 10607-10618, 2024 Sep 23.
Article
in En
| MEDLINE
| ID: mdl-39011885
ABSTRACT
Current approaches to RNA synthesis/manufacturing require substantial (and incomplete) purification post-synthesis. We have previously demonstrated the synthesis of RNA from a complex in which T7 RNA polymerase is tethered to promoter DNA. In the current work, we extend this approach to demonstrate an extremely stable system of functional co-tethered complex to a solid support. Using the system attached to magnetic beads, we carry out more than 20 rounds of synthesis using the initial polymerase-DNA construct. We further demonstrate the wide utility of this system in the synthesis of short RNA, a CRISPR guide RNA, and a protein-coding mRNA. In all cases, the generation of self-templated double stranded RNA (dsRNA) impurities are greatly reduced, by both the tethering itself and by the salt-tolerance that local co-tethering provides. Transfection of the mRNA into HEK293T cells shows a correlation between added salt in the transcription reaction (which inhibits RNA rebinding that generates RNA-templated extensions) and significantly increased expression and reduced innate immune stimulation by the mRNA reaction product. These results point in the direction of streamlined processes for synthesis/manufacturing of high-quality RNA of any length, and at greatly reduced costs.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Viral Proteins
/
DNA-Directed RNA Polymerases
/
RNA, Messenger
/
Promoter Regions, Genetic
Limits:
Humans
Language:
En
Journal:
Nucleic Acids Res
/
Nucleic acids res
/
Nucleic acids research
Year:
2024
Type:
Article
Affiliation country:
United States