Process for designing diverged, codon-optimized large repeated DNA sequences
This disclosure concerns methods for the design of synthetic nucleic acid sequences that encode polypeptide amino acid repeat regions. This disclosure also concerns the use of such sequences to express a polypeptide of interest that comprises amino acid repeat regions, and organisms comprising such sequences.
1. A method for chemically synthesizing a nucleic acid molecule, the method comprising:
(i) isolating an amino acid sequence that provides a polypeptide of interest, wherein the polypeptide of interest comprises at least one amino acid repeat region;
(ii) extracting a nucleic acid sequence from the amino acid sequence and identifying at least one amino acid repeat region from the polypeptide of interest within the nucleic acid sequence;
(iii) deducing a plurality of sample codon-optimized nucleic acid sequences that each encodes the amino acid sequence;
(iv) aligning the plurality of sample codon-optimized nucleic acid sequences by sequence homology and assembling a neighbor-joining tree comprising the plurality of sample codon-optimized nucleic acid sequences;
(v) selecting one of the plurality of sample codon-optimized nucleic acid sequences as a diverged, codon-optimized nucleic acid sequence, wherein the selected sample codon-optimized nucleic acid sequence is from the most deeply branched section of the neighbor-joining tree; and
(vi) chemically synthesizing the nucleic acid sequence encoding the polypeptide of interest that includes the selected sample codon-optimized nucleic acid sequence.
2. The method according to claim 1 , wherein preparing the amino acid sequence that provides the polypeptide of interest comprises obtaining a nucleic acid sequence that encodes the amino acid sequence from a genome database or cloning from a source genome.
3. The method according to claim 1 , wherein deducing a plurality of sample codon-optimized nucleic acid sequences that each encodes the amino acid sequence comprises utilizing the codon-usage bias of an organism.
4. The method according to claim 1 , wherein the polypeptide of interest is expressed in an organism.
5. The method according to claim 1 , wherein the polypeptide of interest is a synthetic polypeptide that is not known to be expressed in an organism.
6. The method according to claim 1 wherein step (ii) through step (vi) are independently performed for at least some of the amino acid repeat regions in the polypeptide of interest.
7. The method according to claim 6 , wherein step (ii) through step (vi) are independently performed for each of the amino acid repeat regions in the polypeptide of interest.
8. The method according to claim 1 , wherein the chemically synthesized nucleic acid sequence in step (vi) comprises: (a) the nucleic acid sequence encoding the polypeptide of interest that includes the selected sample codon-optimized nucleic acid sequence, and (b) at least one regulatory element operably linked to the nucleic acid sequence.
9. The method according to claim 1 , wherein the nucleic acid sequence is suitable for introduction into a host organism.
10. The method according to claim 8 , wherein the nucleic acid sequence is suitable for introduction into a host organism.
11. The method according to claim 10 , wherein the nucleic acid sequence is an expression vector.