Compositions and methods for protein production
View Patent ↗The present invention provides a method of selecting a mRNA for production of a polypeptide of interest comprising: a) producing an array of individual mRNA sequences comprising different nucleotide sequences encoding the polypeptide of interest; b) determining one or more or two or more of the following parameters for each individual mRNA sequence of (a): (i) minimum free energy (MFE) RNA secondary structure; (ii) ensemble free energy (EFE); (iii) frequency of the minimum free energy (FMFE) RNA secondary structure in a thermodynamic ensemble; and (iv) ensemble diversity (ED); c) ranking the individual mRNA sequences of the array according the parameters determined in step (b); and d) selecting a mRNA sequence from the ranked array of step (c), wherein the selected mRNA produces the polypeptide of interest. The present invention further provides a method of selecting a mRNA for enhanced and reduced production of a polypeptide of interest.
1. A method of selecting a mRNA for expression of a polypeptide of interest in a plant or a fungus, the method comprising:
a) producing an array of individual mRNA sequences comprising different nucleotide sequences encoding the polypeptide of interest, wherein the individual mRNA sequences comprise nucleotides from positions −5 through 50 relative to the start codon and the array is generated by back-translation of the polypeptide of interest;
b) determining minimum free energy (MFE) RNA secondary structure for each individual mRNA sequence of (a);
c) ranking the individual mRNA sequences of the array according to the MFE RNA secondary structure determined in (b) from highest MFE RNA secondary structure to lowest MFE RNA secondary structure;
d) selecting a mRNA sequence from the ranked array of step (c);
e) synthesizing said mRNA; and
f) introducing said synthesized mRNA into a plant or a fungus, thereby expressing the mRNA and producing the polypeptide of interest in said plant or fungus, wherein at least steps a) to c) are carried out on a sufficiently programmed processor, wherein, for enhanced production of a polypeptide of interest, the mRNA is selected from the group of mRNAs having an MFE in the range from about 0 kcal/mol to about −2 kcal/mol, and, for reduced production of a polypeptide of interest, the mRNA is selected from the group of mRNAs having an MFE in the range from about −9 kcal/mol to about −18 kcal/mol.
2. A method of selecting a mRNA for expression of a polypeptide of interest in a plant or a fungus, wherein the polypeptide of interest is a leader peptide, the method comprising:
a) producing an array of individual mRNA sequences comprising different nucleotide sequences encoding the polypeptide of interest, wherein the individual mRNA sequences comprise nucleotides from positions −20 through 50 relative to the start codon and the array is generated by back-translation of the polypeptide of interest;
b) determining minimum free energy (MFE) RNA secondary structure for each individual mRNA sequence of (a);
c) ranking the individual mRNA sequences of the array according to the MFE RNA secondary structure determined in (b) from highest MFE RNA secondary structure to lowest MFE RNA secondary structure;
d) selecting a mRNA sequence from the ranked array of step (c);
e) synthesizing said mRNA; and
f) introducing said synthesized mRNA into a plant or a fungus, thereby expressing the mRNA and producing the polypeptide of interest in said plant or a fungus, wherein at least steps a) to c) are carried out on a sufficiently programmed processor, wherein, for enhanced production of a polypeptide of interest, the mRNA is selected from the group of mRNAs having an MFE in the range from about 0 kcal/mol to about −2 kcal/mol, and, for reduced production of a polypeptide of interest, the mRNA is selected from the group of mRNAs having an MFE in the range from about −9 kcal/mol to about −18 kcal/mol.