Modified mRNA for Efficient and Safe Protein Expression

Example 6

Modified nucleotides can increase the translational efficiency and reduce cellular toxicity caused by the immunogenic response to exogenous mRNA. Here, 4 different representative modified nucleotide compositions (FIG. 30) can be included in the cell-selective mRNA. Each of the modified nucleotides can be used as a complete substitute for the unmodified nucleotides to achieve the maximum effect. Modified and unmodified mRNA can be synthesized with a 5′ cap using an ARCA cap analog (TriLink) and PolyA tail using reagents to increase the stability of the mRNAs (TriLink).

To determine if modified nucleic acids affect target site recognition, modified and unmodified mRNA encoding green fluorescent protein (GFP) or luciferase (Luc) followed by 4 target sequences (TS) for miR-126, miR-143 or control scrambled sequences will be transcribed in vitro by T7 RNA polymerase (FIG. 5).