Rational Design of 5’ UTRs for Optimized mRNA Expression and Cost-Effective, Rapid Analysis of Capped mRNA
Year: 2026
Type: preprint
Abstract: In vitro-transcribed (IVT) messenger RNA (mRNA) therapeutics require rigorous quality control of 5’ capping efficiency and 3’ poly(A) tail length, both of which constitute critical quality attributes (CQAs). Current analytical workflows for these CQAs rely on hybridization with nucleic acid probes and biotin-based affinity purification, which constrain throughput and increase per-assay cost. Here, we report a rationally designed 5’ untranslated region (5’ UTR) that sustains high translational efficiency while enabling probe-free, one-step purification for rapid quantification of capping efficiency and poly(A) tail length distribution. This approach exploits a strategically engineered 5’ UTR incorporating a purine-only RNase A-resistant fragment terminating at a single pyrimidine cleavage site, together with a translation-enhancing element comprising an N 6 -methyladenosine (m 6 A) deposition motif and an 18S rRNA complementarity sequence. This dual-function design accommodates GA-rich sequence segments without compromising mRNA translational output. Compared with conventional probe- and biotin-dependent workflows, the present method substantially reduces cost and processing time and is compatible with simultaneous detection of homopolymeric poly(A) tail. This integrated platform provides a simplified, probe-free and construct-universal mRNA analytical platform.
Institutions: Shanghai University, ShanghaiTech University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Award: G6427813561
OpenAlex ID: W7221480533