Synthesizing Modified mRNA Constructs

As described previously (Nelson et al., 2021 (link)), plasmids were cloned to encode an inactivated T7 promoter followed by a 5′ untranslated region (UTR), Kozak sequence, coding sequences of PE2 or MLH1dn, and a 3′ UTR. T7 promoter inactivation prevents potential transcription from circular plasmid template during mRNA generation. These components together were PCR amplified with Phusion U Green Multiplex Master Mix (Thermo Fisher Scientific) using primers that correct T7 promoter inactivation and append a 119-nt poly(A) tail to the 3′ UTR. The resulting PCR product was purified with the QIAquick PCR Purification Kit (Thermo Fisher Scientific) and served as a template for subsequent in vitro transcription. PE2 and MLH1dn mRNAs were transcribed from these templates using the HiScribe T7 High-Yield RNA Synthesis Kit (New England BioLabs) with co-transcriptional capping by CleanCap AG (TriLink Biotechnologies) and full replacement of UTP with N¹-Methylpseudouridine-5′-triphosphate (TriLink Biotechnologies). Transcribed mRNAs were precipitated in 2.5 M lithium chloride (Thermo Fisher Scientific), washed twice in 70% ethanol, then dissolved in nuclease-free water. The resulting PE2 and MLH1dn mRNA was quantified with a NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific) and was stored at −80°C.