Reengineering mRNA primary structure for enhanced protein production
Described herein are rules to modify natural mRNAs or to engineer synthetic mRNAs to increase their translation efficiencies. These rules describe modifications to mRNA coding and 3′ UTR sequences intended to enhance protein synthesis by: 1) decreasing ribosomal diversion via AUG or non-canonical initiation codons in coding sequences, and/or 2) by evading miRNA-mediated down-regulation by eliminating one or more miRNA binding sites in coding sequences.
1. A method of improving full-length protein expression efficiency comprising:
a) providing a polynucleotide comprising:
i) a coding sequence for the full-length protein;
ii) a primary initiation codon that is upstream of the coding sequence of the full-length protein, said primary initiation codon encoding the first amino acid of the coding sequence of the full-length protein; and
iii) one or more secondary initiation codons located within the coding sequence of the full-length protein downstream of the primary initiation codon; and
b) mutating the one or more secondary initiation codons located within the coding sequence of the full-length protein downstream of the primary initiation codon, wherein the mutation results in a decrease in initiation of protein synthesis at the one or more secondary initiation codons,
thereby increasing expression efficiency of the full-length protein initiated at the primary initiation codon,
wherein mutating the one or more secondary initiation codons located within the coding sequence of the full-length protein downstream of the primary initiation codon comprises mutating one or more nucleotides such that the amino acid sequence of the protein remains unaltered.