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US 2020/0048634 A1Published application
Methods to modulate protein translation efficiency
AssigneeWashington University
InventorSergej Djuranovic
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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.
Claims19
A method for 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
- (a) the polynucleotide sequence is inserted between the second and third codons of the nucleic acid sequence encoding the first protein of interest, and the polynucleotide sequence is selected from (i) 5′-n1n2n3-3′, (ii) 5′-n1n2n3n4n5n6-3′, and (iii) 5′-n1n2n3n4n5n6n7n8n9-3′, wherein n1, n2, n3, n4, n5, n6, n7, n8, and n9 are each independently selected from the group consisting of adenine, thymine, uracil, guanine, and cytosine;
- (b) the polynucleotide sequence is inserted between the third and fourth codons of the nucleic acid sequence encoding the first protein of interest, and the polynucleotide sequence is selected from (i) 5′-n1n2n3-3′, and (ii) 5′-n1n2n3n4n5n6-3′, wherein n1, n2, n3, n4, n5, and n6 are each independently selected from the group consisting of adenine, thymine, uracil, guanine, and cytosine;
- or
- (c) the polynucleotide sequence is inserted between the fourth and fifth codons of the nucleic acid sequence encoding the first protein of interest, and the polynucleotide sequence is 5′-n1n2n3-3′, wherein n1, n2, and n3 are each independently selected from the group consisting of adenine, thymine, uracil, guanine, and cytosine;
- and
- expressing the modified nucleic acid sequence in a cell or in a cell-free transcription/translation system, wherein the inserted polynucleotide sequence does not encode a stop codon recognizable by the cell or the cell-free system, thereby increasing or decreasing the amount of the second protein as compared to the first protein of interest.
4 more elements
A method to modulate the level of expression of a protein, the method comprising:
- changing one or more nucleotides of a nucleic acid sequence encoding a first protein of interest, wherein the one or more changes are at codon 3, the one or more changes are at codon 4, the one or more changes are at codon 5, or any combination thereof, to produce a modified nucleic acid sequence comprising a polynucleotide sequence of Table A, Table B or Table C, at codons 3, 4 and 5;
- and the modified nucleic acid sequence encodes a second protein;
- 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.
1 more element
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
Dependencies are inferred from the claim text.