Synthetic nucleic acid molecule compositions and methods of preparation

Abstract

A method to prepare synthetic nucleic acid molecules having reduced inappropriate or unintended transcriptional characteristics when expressed in a particular host cell.

1 - 47 . (canceled)  
     
     
         48 . A method to prepare a synthetic nucleic acid molecule comprising an open reading frame, comprising: 
 a) altering a plurality of transcription regulatory sequences in a parent nucleic acid sequence which encodes a polypeptide having at least 100 amino acids to yield a synthetic nucleic acid molecule which has at least 3-fold fewer transcription regulatory sequences relative to the parent nucleic acid sequence, wherein the transcription regulatory sequences are selected from the group consisting of transcription factor binding sequences, intron splice sites, poly(A) addition sites, enhancer sequences and promoter sequences; and    b) altering greater than 25% of the codons in the synthetic nucleic acid sequence which has a decreased number of transcription regulatory sequences to yield a further synthetic nucleic acid molecule, wherein the codons which are altered do not result in an increased number of transcription regulatory sequences, wherein the further synthetic nucleic acid molecule encodes a polypeptide with at least 85% amino acid sequence identity to the polypeptide encoded by the parent nucleic acid sequence.    
     
     
         49 . A method to prepare a synthetic nucleic acid molecule comprising an open reading frame, comprising: 
 a) altering greater than 25% of the codons in a parent nucleic acid sequence which encodes a polypeptide having at least 100 amino acids to yield a codon-altered synthetic nucleic acid molecule, and    b) altering a plurality of transcription regulatory sequences in the codon-altered synthetic nucleic acid molecule to yield a further synthetic nucleic acid molecule which has at least 3-fold fewer transcription regulatory sequences relative to a synthetic nucleic acid molecule with a random selection of codons at the codons which differ, wherein the transcription regulatory sequences are selected from the group consisting of transcription factor binding sequences, intron splice sites, poly(A) addition sites, enhancer sequences and promoter sequences, and wherein the further synthetic nucleic acid molecule encodes a polypeptide with at least 85% amino acid sequence identity to the polypeptide encoded by the parent nucleic acid sequence.    
     
     
         50 . The method of  claim 48  or  49  wherein the parent nucleic acid sequence encodes a reporter molecule.  
     
     
         51 . The method of  claim 48  or  49  wherein the parent nucleic acid sequence encodes a luciferase.  
     
     
         52 . The method of  claim 48  or  49  wherein the synthetic nucleic acid molecule hybridizes under medium stringency hybridization conditions to the parent nucleic acid sequence.  
     
     
         53 . The method of  claim 48  or  49  wherein the codons which are altered encode the same amino acid as the corresponding codons in the parent nucleic acid sequence.  
     
     
         54 . (canceled)  
     
     
         55 . A method for preparing at least two synthetic nucleic acid molecules which are codon distinct versions of a parent nucleic acid sequence which encodes a polypeptide, comprising: 
 a) altering a parent nucleic acid sequence to yield a synthetic nucleic acid molecule having an increased number of a first plurality of codons that are employed more frequently in a selected host cell relative to the number of those codons in the parent nucleic acid sequence; and    b) altering the parent nucleic acid sequence to yield a further synthetic nucleic acid molecule having an increased number of a second plurality of codons that are employed more frequently in the host cell relative to the number of those codons in the parent nucleic acid sequence, wherein the first plurality of codons is different than the second plurality of codons, and wherein the synthetic and the further synthetic nucleic acid molecules encode the same polypeptide.    
     
     
         56 . The method of  claim 55  further comprising altering a plurality of transcription regulatory sequences in the synthetic nucleic acid molecule, the further synthetic nucleic acid molecule, or both, to yield at least one yet further synthetic nucleic acid molecule which has at least 3-fold fewer transcription regulatory sequences relative to the synthetic nucleic acid molecule, the further synthetic nucleic acid molecule, or both.  
     
     
         57 . The method of  claim 55  further comprising altering at least one codon in the first synthetic sequence to yield a first modified synthetic sequence which encodes a polypeptide with at least one amino acid substitution relative to the polypeptide encoded by the first synthetic nucleic acid sequence.  
     
     
         58 . The method of  claim 56  further comprising altering at least one codon in the second synthetic sequence to yield a second modified synthetic sequence which encodes a polypeptide with at least one amino acid substitution relative to the polypeptide encoded by the first synthetic nucleic acid sequence.  
     
     
         59 . The method of  claim 55  wherein the synthetic sequences encode a luciferase.  
     
     
         60 - 64 . (canceled)  
     
     
         65 . The method of  claim 48  or  49  further comprising altering the further synthetic nucleic acid molecule to encode a polypeptide having at least one amino acid substitution relative to the polypeptide encoded by the parent nucleic acid sequence.  
     
     
         66 . The method of  claim 48  or  49  wherein the altering of transcription regulatory sequences does not introduce amino acid substitutions to the polypeptide encoded by the synthetic nucleic acid molecule.