Synthetic mRNA produced by in vitro transcription (ivt mRNA) is the active pharmaceutical ingredient of approved anti-COVID-19 vaccines and of many drugs under development. Such synthetic mRNA typically contains several hundred bases of non-coding “untranslated” regions (UTRs) that are involved in the stabilization and translation of the mRNA. However, UTRs are often complex structures, which may complicate the entire production process. To eliminate this obstacle, we managed to reduce the total amount of nucleotides in the UTRs to only four bases. In this way, we generate minimal ivt mRNA (“minRNA”), which is less complex than the usual optimized ivt mRNAs that are contained, for example, in approved vaccines. We have compared the efficacy of minRNA to common augmented mRNAs (with UTRs of globin genes or those included in licensed vaccines) in vivo and in vitro and could demonstrate equivalent functionalities. Our minimal mRNA design will facilitate the further development and implementation of ivt mRNA-based vaccines and therapies.

Synthetic mRNA produced by in vitro transcription is the active pharmaceutical ingredient of approved vaccines and of many drugs under development. It typically contains a 3’ poly-A tail required for optimal stabilization and translation of the mRNA. The average length of the poly-A tail in the produced mRNA cannot be precisely predicted and consequently must be measured using complicated technologies such as reverse transcription and sequencing or cleavage of the poly-A tail and analysis by capillary electrophoresis or mass spectrometry. We report an accelerated method to evaluate the average poly-A tail length in synthetic mRNA, which benefits from the fact that thiazole orange is fluorescent only when in close proximity of nucleic acid. A poly-dT(12) oligonucleotide having thiazole orange at its extremities emits a fluorescence signal proportional to the amount of poly-A sequence available. Using a titration curve made with known amounts of poly-A RNA oligonucleotide, the thiazole orange oligo dT probe can instantly indicate the average length of the poly-A tail in an mRNA sample. This fast and easy method can be used in any laboratory to determine the size of the poly-A tail in in vitro transcribed mRNA produced for research, pre-clinical studies and clinical trials.

Synthetic mRNA produced by in vitro transcription is the active pharmaceutical ingredient of approved vaccines and of many drugs under development. It typically contains a 3′ poly(A) tail required for optimal stabilization and translation of the mRNA. The average length of the poly(A) tail in the produced mRNA cannot be precisely predicted and consequently must be measured using complicated technologies such as reverse transcription and sequencing or cleavage of the poly(A) tail and analysis by capillary electrophoresis or mass spectrometry. We report an accelerated method to evaluate the average poly(A) tail length in synthetic mRNA, which benefits from the fact that thiazole orange is fluorescent only when in close proximity of nucleic acid. An oligo(dT)₁₂ oligonucleotide having thiazole orange at its extremities emits a fluorescence signal proportional to the amount of poly(A) sequence available. Using a titration curve made with known amounts of poly(A) RNA oligonucleotide, the thiazole orange oligo(dT)₁₂ oligonucleotide can instantly indicate the average length of the poly(A) tail in an mRNA sample. This fast and easy method can be used in any laboratory to determine the size of the poly(A) tail in in vitro-transcribed mRNA produced for research, pre-clinical studies and clinical trials.