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

Abstract 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 (m 1 ψ), 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 (m 5 C) 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 m 5 C‐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