Viral synthetic nucleic acid sequences and use thereof
Nucleic acid molecules comprising a coding sequence with at least one codon substituted to a synonymous codon, a modified form of a virus comprising the nucleic acid molecules of the invention, and methods for producing these nucleic acid molecules, and viruses, are provided.
1. A method for producing a nucleic acid molecule optimized or deoptimized for expression in a particular cellular context within a cell, the method comprising:
a. selecting a coding sequence,
b. selecting a reference set of genes expressed in a particular cellular context within a cell wherein said reference set is a subset of said cell's total genome or is a set of viral genes coordinately expressed in said cellular context and which are a subset of a virus's genome,
c. for each codon of said coding sequence, computing a parameter that effects translation rate for said codon and its synonymous codons over all sequences in the reference set, and
d. substituting at least one codon of said coding sequence with a synonymous codon with a faster rate of translation in said particular cellular context, in order to optimize said coding sequence or substituting at least one codon of said coding sequence with a synonymous codon with a slower rate of translation in said particular cellular context, in order to deoptimize said coding sequence,
thereby producing a nucleic acid molecule optimized or deoptimized for expression in a particular cellular context.
2. The method of claim 1 , wherein said parameter that effects translation is selected from the group consisting of: relative synonymous codons frequencies (RSCF), and relative codon-tRNA adaptation and codon typical decoding rate (TDR).
3. The method of claim 1 , wherein the particular cellular context is selected from the group consisting of: a specific period of a cell's life cycle, during cellular division, during cellular stress, during apoptosis, during viral infection, or during viral lysogeny of the cell.
4. A method for producing a modified virus, the method comprising:
a. selecting an endogenous viral sequence,
b. performing steps b-d of the method of claim 1 to optimize or deoptimize said endogenous viral sequence, thereby producing a reengineered viral sequence, wherein said selected coding sequence of steps b-d of the method of claim 1 is the selected endogenous viral sequence of step a, and
c. replacing the endogenous viral sequence with the reengineered viral sequence, thereby producing a modified virus.
5. A method for producing an attenuated virus, the method comprising the method of claim 4 wherein,
a. said viral sequence is an essential viral sequence, and
b. said synonymous codon with a different translation rate has a slower translation rate, thereby producing an attenuated virus.
6. The method of claim 1 , wherein said reference set of genes are genes whose mRNA is translated in said particular cellular context within a cell.
7. The method of claim 1 , wherein said reference set of genes is a set of viral genes coordinately expressed in a particular cellular context within a cell wherein said reference set is a subset of said virus's total genome.
8. The method of claim 7 , wherein said set of coordinately expressed viral genes is selected from the group consisting of: genes with a common structure, genes with common temporal expression during a virus's life cycle, genes encoding for proteins having common function, genes translated at a common cellular location and genes encoding for proteins having common cellular localization.
9. The method of claim 8 , wherein said temporal expression during a virus's life cycle is selected from the group consisting of: early expression in the virus life cycle, intermediate expressing in the virus life cycle, and late expression in the virus life cycle.
10. The method of claim 8 , wherein said parameter that effects translation rate is selected from the group consisting of: relative synonymous codons frequencies (RSCF), relative codon-tRNA adaptation, codon typical decoding rate (TDR), GC content, average repetitive substring index, codon pair bias, dinucleotide bias, nucleotide bias and amino acid bias.
11. A method for producing a nucleic acid molecule optimized or deoptimized for expression in a specific period of a cell's life cycle, the method comprising:
a. selecting a coding sequence,
b. selecting a reference set of genes expressed in specific period of a cell's life cycle wherein said reference set is a subset of said cell's total genome or is a set of viral genes coordinately expressed in said specific period of a cell's life cycle and which are a subset of a virus's genome, wherein said set of genes have a common temporal expression during a virus's life cycle,
c. for each codon of said coding sequence, computing a parameter that effects translation rate for said codon and its synonymous codons over all sequences in the reference set, wherein said parameter is relative synonymous codons frequencies (RSCF), and
d. substituting at least one codon of said coding sequence with a synonymous codon with a faster rate of translation in said specific period of a cell's life cycle, in order to optimize said coding sequence or substituting at least one codon of said coding sequence with a synonymous codon with a slower rate of translation in said specific period of a cell's life cycle, in order to deoptimize said coding sequence,
thereby producing a nucleic acid molecule optimized or deoptimized for expression in a specific period of a cell's life cycle.
12. A method for producing a modified virus, the method comprising:
a. selecting an endogenous viral sequence,
b. performing steps b-d of the method of claim 11 to optimize said endogenous viral sequence, thereby producing a reengineered viral sequence, wherein said selected coding sequence of steps b-d of the method of claim 11 is the selected endogenous viral sequence of step a, and
c. replacing the endogenous viral sequence with the reengineered viral sequence, thereby producing a modified virus.
13. A method for producing an attenuated virus, the method comprising the method of claim 12 wherein,
a. said viral sequence is an essential viral sequence, and
b. said synonymous codon with a different translation rate has a slower translation rate, thereby producing an attenuated virus.
14. The method of claim 11 , wherein said temporal expression during a virus's life cycle is selected from the group consisting of: early expression in the virus life cycle, intermediate expression in the virus life cycle, and late expression in the virus life cycle.
15. The method of claim 11 , wherein said common temporal expression during a virus's life cycle of said set of genes is mRNA from said genes being translated at a common time in said virus's life cycle.
16. A method for producing a nucleic acid molecule optimized for expression in a specific period of a cell's life cycle, the method comprising:
a. selecting a coding sequence,
b. selecting a reference set of genes expressed in a specific period of a cell's life cycle wherein said reference set is a subset of said cell's total genome or is a set of viral genes coordinately expressed in said cellular context and which are a subset of a virus's genome, wherein said set of genes have a common temporal expression during a virus's life cycle,
c. for each codon of said coding sequence, computing a parameter that effects translation rate for said codon and its synonymous codons over all sequences in the reference set, wherein said parameter is relative synonymous codons frequencies (RSCF), and
d. substituting at least one codon of said coding sequence with a synonymous codon with a faster rate of translation in said specific period of a cell's life cycle, in order to optimize said coding sequence,
thereby producing a nucleic acid molecule optimized for expression in specific period of a cell's life cycle.
17. The method of claim 16 , wherein said temporal expression during a virus's life cycle is selected from the group consisting of: early expression in the virus life cycle, intermediate expression in the virus life cycle, and late expression in the virus life cycle.
18. The method of claim 16 , wherein said common temporal expression during a virus's life cycle of said set of genes is mRNA from said genes being translated at a common time in said virus's life cycle.