Streamlined In Vitro Transcription for Generating Self-Amplifying RNA With Modified Nucleotides

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Virology

Background:

• Self-amplifying RNA (saRNA) offers advantages over mRNA for protein expression.
• Venezuelan equine encephalitis virus (VEEV)-based saRNA is efficient but requires optimization for modified nucleotides.
• Chemical modifications like N1-methylpseudouridine can impede saRNA amplification.

Purpose of the Study:

• To develop a streamlined protocol for generating 5-methylcytidine (m5C)-modified VEEV-based saRNA.
• To optimize saRNA production for enhanced intracellular amplification and protein expression.
• To provide a reproducible method for creating chemically modified saRNA for research.

Main Methods:

• Utilized a single-step in vitro transcription (IVT) strategy.
• Employed PCR-generated DNA templates with encoded poly(A) tails.
• Incorporated CleanCap AU for co-transcriptional capping and post-transcriptional phosphatase treatment.
• Purified saRNA using the phenol-chloroform-isoamyl alcohol method.

Main Results:

• Successfully generated m5C-modified VEEV-based saRNA with enhanced functionality.
• Demonstrated reduced innate immune activation and improved expression durability.
• Validated the protocol through functional assessment in HEK293T cells.

Conclusions:

• The developed protocol provides a reproducible framework for producing capped, tailed, and chemically modified saRNA.
• This method is suitable for downstream functional and translational studies.
• Optimized saRNA production holds promise for advancing RNA-based therapeutics and research.