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US 2026/0092278 A1Published application
Methods to modulate protein translation efficiency
AssigneeWashington University
InventorSergej Djuranovic
Life of this patent
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Abstract
The present disclosure relates to compositions and methods to modulate the level of expression of a protein in a deliberate manner (i.e., tunable regulation of expression) with only a minimal change to the genetic sequence of the gene of interest. The present disclosure therefore also provides compositions and methods to predictably alter protein abundance.
Claims12
A method of modulating the level of expression of a protein, the method comprising:
- Inserting a polynucleotide sequence into a nucleic acid sequence encoding a first protein of interest to produce a modified nucleic acid sequence encoding a second protein, wherein the nucleic acid encoding the first protein of interest comprises a coding region encoding the first protein of interest, wherein the coding region comprises a first, a second, a third, a fourth and a fifth codon in consecutive order, wherein the first codon is located at a 5′-most end of the coding region, and wherein
- (a) the method increases the level of expression of the protein, the polynucleotide sequence is inserted at a position in the coding region selected from between second and third codons, between third and fourth codons, and between fourth and fifth codons, and the polynucleotide sequence is selected from AACATCTAC, AAGTACCCA, CCATTACAT, TACTATGCA, CGAGAAATC, AAATATCGC, AAGTATCAC, AAAATCTTC, AAATACTGT, AAATTCTAT, AAAAATGTC, AACTGTACT, GATACTATT, AAACAAGTT, AAATTCAGT, ACGAATGAC, AAAAAATAT, AATATAACT, and AAAATACAC;
- or
- (b) the method decreases the level of expression of the protein, the polynucleotide sequence is inserted at a position in the coding region selected from between second and third codons, between third and fourth codons, and between fourth and fifth codons, and the polynucleotide sequence is selected from
- GCCCCGGGGG, GAGGTGGAC, GTGGTGGAC, GGGGGCTCC,
- GGGTGGTCC, GGGGGCCCG, GCGGTGGGC, CGGGTGAAC,
- TTGGTGAGC,CGTGTGAAC, CTTGCCGGC, CCGGTGGGC,
- CTCGCGGGG, CTATGGGTA, CCGGTGTTC, GCGGTGGAC,
- CCGGTGAGC, CCCTCGGGG, CTCGCCGGG,
- and
- GAGTCCCCG;
- and expressing the modified nucleic acid sequence in a cell or in a cell-free transcription/translation system thereby increasing or decreasing the amount of second protein translated as compared to the amount of first protein translated, wherein the polynucleotide sequence does not encode a stop codon recognizable by the cell or the cell-free system.
9 more elements
A method of increasing the level of expression of a protein, the method comprising:
- inserting a polynucleotide sequence into a nucleic acid sequence encoding a first protein of interest to produce a modified nucleic acid sequence encoding a second protein, wherein the nucleic acid encoding the first protein of interest comprises a coding region encoding the first protein of interest, wherein the coding region comprises a first, a second, a third, a fourth and a fifth codon in consecutive order, wherein the first codon is located at a 5′-most end of the coding region, and wherein
- the method increases the level of expression of the protein, the polynucleotide sequence is inserted at a position in the coding region selected from between second and third codons, between third and fourth codons, and between fourth and fifth codons, and the polynucleotide sequence encode a peptide selected from KYY, KHY, KYH, KFY, NQY, NHN, KKY, KYC, KMH, KNY, NHY, NYK, KKH, HQH, HMH, KIY, KYQ, NMH, KNF, NIY, KHI, KFC, NYY, NTY, KII, KMY, NNI, NHM, NPI, NKQ, KNN, NNN, KCY, NYH, MHF, NHI, NKI, QIY, NTI, NKH, NCY, MNI, KQY, NCI, KNI, IKH, DYK, KHT, KCI, and KIF;
- and expressing the modified nucleic acid sequence in a cell or in a cell-free transcription/translation system thereby increasing the amount of second protein translated as compared to the amount of first protein translated, wherein the polynucleotide sequence does not encode a stop codon recognizable by the cell or the cell-free system.
A vector comprising a promoter operably linked to a nucleic acid construct R1-R1A-R2-R3-R4-R4A wherein:
- R1 is a start codon;
- R1A is an optional polynucleotide sequence encoding a peptide tag and a cleavage sequence;
- R2 is a polynucleotide sequence 5′-n1n2n3-3′, wherein n1, n2, and n3 are each independently selected from the group consisting of adenine, thymine, uracil, guanine, and cytosine;
- R3 is a polynucleotide sequence selected from Table A, Table B, Table C, or Table D, or a polynucleotide sequence encoding a peptide selected from Table E, Table F, Table G or Table I;
- R4 is a multiple cloning site;
- and
- R4A is an optional polynucleotide sequence encoding a cleavage sequence and a peptide tag;
- wherein R1A, R2, R3, R4, and R4A are joined by phosphodiester bonds.
5 more elements
A method of increasing the level of expression of a protein, the method comprising:
- inserting a polynucleotide sequence into a nucleic acid sequence encoding a first protein of interest to produce a modified nucleic acid sequence encoding a second protein, wherein the nucleic acid encoding the first protein of interest comprises a coding region encoding the first protein of interest, wherein the coding region comprises a first, a second, a third, a fourth and a fifth codon in consecutive order, wherein the first codon is located at a 5′-most end of the coding region, and wherein
- the method increases the level of expression of the protein, the polynucleotide sequence is inserted at a position in the coding region selected from between second and third codons, between third and fourth codons, and between fourth and fifth codons, and the polynucleotide sequence encode a peptide selected from KNM, NMK, NYF, KHH, NNY, NNH, KYM, KCF, NNC, KTI, YNH, NFH, KYS, NNK, KKI, KSY, KYW, NKK, YQH, KFW, KIH, NYT, EYY, IYH, MFK, KYK, DKY, EIY, QYK, DII, KIN, HYF, NIQ, KMQ, KHK, MYY, TKH, KHW, TYH, KYI, HIY, KFI, KTK, KFN, DYH, HMK, IKY, QYI, NOK, and NKN;
- and expressing the modified nucleic acid sequence in a cell or in a cell-free transcription/translation system thereby increasing the amount of second protein translated as compared to the amount of first protein translated, wherein the polynucleotide sequence does not encode a stop codon recognizable by the cell or the cell-free system.
Dependencies are inferred from the claim text.