Effects of Various Poly(A) Tails on Luciferase Expression

Abstract

Synthesis of RNA by in vitro transcription has many applications in the production of vaccines, anticancer and gene therapy medicines. Despite several advantages of mRNA molecules over DNA in terms of transient transgene expression, rapid degradation of RNA is the limiting factor for the efficacy of mRNA-based therapeutics. Translational efficiency and stability of mRNA depends on the sequence of 5′ and 3′ untranslated regions, cap structure, coding sequence (CDS), and the poly(A) tail. So far, the role of the poly(A) tail sequence in mRNA stability and translational efficiency remains poorly investigated. There are few studies evaluating the influence of poly(A) tails containing certain non-A nucleotides or segments of non-A nucleotides on the efficacy of mRNA therapeutics. In our work, we conducted a comprehensive assessment of the effects of mRNA poly(A) tail on luciferase expression in cell lines HEK293 and DC2.4 and in BALB/c mice. Our results showed that mRNAs with a poly(A) tail consisting of 100 adenines separated by a segment of 10 nucleotides (50A-GCAUAUGACU-50A) have the best translational efficiency both in vitro (in cell lines) and in vivo (in BALB/c mice). Nonetheless, the results were inconsistent and depended both on cell lines and on the coding sequence (NanoLuc or firefly luciferase). Thus, effects of a mRNA poly(A) tail on luciferase expression may depend on the sequence of the gene in question and on specific features of the expression of RNA-binding proteins in cell lines.