Method and device for optimizing a nucelotide sequence for the purpose of expression in a protein

Abstract

The invention relates to a method for optimizing a nucleotide sequence for expression of a protein on the basis of the amino acid sequence of the protein, in which for a particular region there is specification of a test sequence with m optimization positions on which the codon occupation is varied, a quality function being used to ascertain the optimal codon occupation on these optimization positions, and one or more codons of this optimal occupation being specified as codons of the optimized nucleotide sequence. These steps are iterated, with the codons of the optimized nucleotide sequence which are specified in the preceding steps remaining unchanged in subsequent iteration steps. The invention additionally relates to a device for carrying out this method.

1 . A method for optimizing a nucleotide sequence for the expression of a protein on the basis of the amino acid sequence of the protein, which comprises the following steps carried out on a computer:
 generation of a first test sequence of n codons which correspond to n consecutive amino acids in the protein sequence, where n is a natural number and is less than or equal to N, the number of amino acids in the protein sequence,   specification of m optimiziation positions in the test sequence which correspond to the position of m codons at which the occupation by a codon, relative to the test sequence, is to be optimized, where m≦n and m<N,   generation of one or more further test sequences from the first test sequence by replacing at one or more of the m optimization positions a codon of the first test sequence by another codon which expresses the same amino acid,   assessment of each of the test sequences with a quality function and ascertaining the test sequence which is optimal in relation to the quality function,   specification of p codons of the optimal test sequence which are located at one of the m optimization positions, as result codons which form the codons of the optimized nucleotide sequence at the positions which corresponds to the position of said p codons in the test sequence, where p is a natural number and p≦m,   iteration of the preceding steps, where in each iteration step the test sequence comprises the appropriate result codon at the positions which correspond to positions of specified result codons.   
     
     
         2 . The method as claimed in  claim 1 , characterized in that in one or more iteration steps the m optimization positions of the test sequences directly follow one or more result codons which have been specified as part of the optimized nucleotide sequence. 
     
     
         3 . The method as claimed in  claim 1 , characterized in that in one or more iteration steps the p codons which are specified as result codons of the optimized nucleotide sequence are p consecutive codons. 
     
     
         4 . The method as claimed in  claim 1 , characterized in that in one iteration step test sequences with all possible codon occupations for the m optimization positions are generated from the first test sequence, and the optimal test sequence is ascertained from these test sequences. 
     
     
         5 . The method as claimed in  claim 1 , characterized by:
 assessment of each test sequence with a quality function, ascertaining of an extreme value within the values of the quality function for all partial sequences generated in an iteration step,   specification of p codons of the test sequence which corresponds to the extremal value of the weight function as result codons at the appropriate positions, where p is a natural number and p≦m.   
     
     
         6 . The method as claimed in  claim 5 , characterized in that the quality function takes account of one or more of the following criteria:
 codon usage for a predefined organism, GC content, repetitive sequences, secondary strutures, inverse complementary sequence repeats and sequence motifs.   
     
     
         7 . The method as claimed in  claim 6 , characterized in that the quality function is a function of various single terms which in each case assess one criterion from the following list of criteria:
 codon usage for a predefined organism, GC content, sequence motifs, repetitive sequences, secondary structures, inverse complementary sequence repeats.   
     
     
         8 . The method as claimed in  claim 1 , characterized in that the quality function takes account of one or more of the following criteria:
 exclusion of inverse complementary sequence identities of more than 20 nucleotides to the transcriptome of a predefined organism,   exclusion of homology regions of more than 100 base pairs to a predefine DNA sequence,   exclusion of homology regions with more than 90% similarity of the nucleotide sequence to a predefined DNA sequence.   
     
     
         9 . The method as claimed in  claim 1 , characterized by the step of synthesizing the optimized nucleotide sequence. 
     
     
         10 . The method as claimed in  claim 9 , characterized in that the step of synthesizing the optimized nucleotide sequence takes place in a device for automatic synthesis of nucleotide sequences which is controlled by the computer which optimizes the nucleotide sequence. 
     
     
         11 . A device for optimizing a nucleotide sequence for the expression of a protein on the basis of the amino acid sequence of the protein, which has a computer unit which comprises:
 a unit for generation of a first test sequence of n codons which correspond to n consecutive amino acids in the protein sequence, where n is a natural number and is less than or equal to N, the number of amino acids in the protein sequence,   a unit for specification of m optimiziation positions in the test sequence which correspond to the position of m codons at which the occupation by a codon, relative to the test sequence, is to be optimized, where m≦n and m<M,   a unit for generation of one or more further test sequences from the first test sequence by replacing at one or more of the m optimization positions a codon of the first test sequence by another codon which expresses the same amino acid,   a unit for assessment of each of the test sequences with a quality function and for ascertaining the test sequence which is optimal in relation to the quality function,   a unit for specification of p codons of the optimal test sequence which are located at one of the m optimization positions, as result codons which form the codons of the optimized nucleotide sequence at the positions which correspond to the positions of said p codons in the test sequence, where p is a natural number and p≦m,   a unit for iteration of the steps of generation of a plurality of test functions, of assessment of the test sequences and of specification of result codons, where in each iteration step the test sequence comprises the appropriate result codon at the positions which correspond to positions of specified result condons in the optimized nucleotide sequence, and the optimization positions are different from positions of result codons.   
     
     
         12 . The device as claimed in  claim 11 , characterized by a unit for carrying out the steps of a method as claimed in  claim 1 . 
     
     
         13 . The device as claimed in  claim 11 , characterized by a device for automatic synthesis of nucleotide sequences which is controlled by the computer in such a way that it synthesizes the optimized nucleotide sequence. 
     
     
         14 . A computer program which comprises program code which can be executed by a computer and which, when it is executed on a computer, causes the computer to carry out a method as claimed in  claim 1 . 
     
     
         15 . (canceled) 
     
     
         16 . A computer-readable data medium on which a program as claimed in  claim 14  is stored in computer-readable form. 
     
     
         17 . A nucleic acid which includes a nucleotide sequence coding for a protein and which is obtainable by a method as claimed in  claim 9 . 
     
     
         18 . The nucleic acid as claimed in  claim 17 , characterized in that the latter includes a nucleotide sequence which codes in a predefined organism for a protein, where said nucleotide sequence is not present in the naturally occurring genome of the organism. 
     
     
         19 . The nucleic acid as claimed in  claim 18 , characterized in that the organism is selected from the following group:
 viruses, especially vaccinia viruses,   prokaryotes, especially  Escherichia coli , caulobacter cresentus,  bacillus subtilis , mycobacterium spec.   yeasts, especially  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia angusta,      insects, especially  Sprodoptera frugiperda, Drosophila  spec.,   mammals, especially  Homo sapiens, Macaca mulata, Mus musculus, Bos taurus, Capra hircus, Ovis aries, Oryctolagus cuniculus, Rattus norvegicus, Chinese hamster ovary, monocotyledonous  plants, especially  Oryza sativa, Zea mays, Triticum aestivum , dicotyledonous plants, especially  Glycin max, Gossypium hirsutum, Nicotiana tabacum, Arabidopsis thaliana, Solanum tuberosum.      
     
     
         20 . The nucleic acid as claimed in  claim 1 , characterized in that the protein encoded by the nucleotide sequence is one of the following proteins and/or falls into one of the following protein classes:
 enzymes, especially polymerases, endonucleases, ligases, lipases, proteases, kinases, phosphatases, topoisomerases,   cytokines, chemokines, transcription factors, oncogenes,   proteins from thermophilic organisms, from cryophilic organisms, from halophilic organisms, from acidophilic organisms, from basophilic organisms,   proteins with repetitive sequence elements, especially structural proteins,   human antigens, especially tumor antigens, tumor markers, autoimmune antigens, diagnostic markers,   viral antigens, especially from HAV, HBV, HCV, HIV, SIV, HPV, rinoviruses, influenza viruses, herpesviruses, poliomaviruses, hendra virus, dengue virus, AAV, adenoviruses, HTLV, RSV,   antigens of disease-causing parasites, e.g. protozoa, especially those causing malaria, leishmania,  trypanosoma , toxoplasmas, amoeba,   antigens of disease-causing bacteria or bacterial pathogens, especially of the genera  Chlamydia, staphylococci, Klebsiella, Streptococcus, Salmonella, Listeria, Borrelia, Escherichia coli,      antigens of organisms of safety level L4, especially  Bacillus anthracis , Ebola virus, Marburg virus, poxviruses.   
     
     
         21 . The nucleic acid as claimed in  claim 18 , characterized in that the quality function takes account at least of one the following criteria:
 GC content,   codon usage of the predefined organism,   exclusion of inverse complementary sequence identities of more than 20 nucleotides to the transcriptome of a predetermined organism,   complete or substantial exclusion of homology regions of more than 100 base pairs to a predefined DNA sequence,   complete or substantial exclusion of homology regions with a similarity of more than 90% to a predefined DNA sequence.   
     
     
         22 - 28 . (canceled)