Self-amplifying RNA (saRNA) derived from alphavirus replicons enables robust intracellular RNA amplification and high-level protein expression at substantially lower doses than nonreplicating messenger RNA (mRNA) platforms. Venezuelan equine encephalitis virus (VEEV)-based replicons lacking the viral structural genes are among the most extensively characterized saRNA backbones, combining efficient cytoplasmic replication with a favorable safety profile. However, incorporating chemical nucleoside modifications such as N1-methylpseudouridine (mψ), now standard in conventional mRNA workflows, can directly impact replicase activity and hinder RNA amplification, necessitating backbone-specific optimization. Recent studies have shown that incorporation of 5-methylcytidine (mC) preserves VEEV saRNA replication while reducing innate immune activation and improving expression durability. In an independent study, phosphatase treatment to remove residual 5'-triphosphates was shown to enhance saRNA functionality. Building on these findings, here we describe a streamlined protocol for generating mC-modified VEEV-based saRNA using a single-step in vitro transcription (IVT) strategy. This protocol utilizes PCR-generated DNA templates with an encoded poly(A) tail, CleanCap AU for co-transcriptional capping, and post-transcriptional phosphatase treatment to minimize immunostimulatory RNA species. Finally, the RNA was purified using the phenol-chloroform-isoamyl alcohol method and functionally evaluated by transfection into HEK293T cells. This protocol provides a reproducible framework for producing capped, tailed, and chemically modified saRNA suitable for downstream functional and translational studies. © 2026 Wiley Periodicals LLC. Basic Protocol 1: IVT and purification of saRNA with modified nucleotides Basic Protocol 2: Functional assessment of IVT-generated saRNA in HEK293T Cells. 1 5 5
