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US 6,309,830 B1Granted
Human suppressor tRNA oligonucleotides and methods of use for same
AssigneeHuman Gene Therapy Research Institute
InventorsRekha G. Panchal, Charles J. Link, Jr.
Life of this patent
Estimating the term…
Abstract
Novel synthetic suppressor tRNA have been provided which provide read-through of internal nonsense mutations, or which can site-specifically alter translation of transcribed sequences. Uses of the same are also provided in genetic engineering protocols including gene therapy treatment of diseases such as Xeroderma pigmentosum.
Claims29
An oligonucleotide sequence which encodes a synthetic suppressor tRNA consisting of:
- A) a structural gene sequence from a known human tRNA gene, comprising no more than twenty 3′ flanking residues and no 5′ flanking residues of the human tRNA gene sequence, said sequence comprising an anticodon region for pairing with mRNA;
- B) a total length of less than 150 nucleotides;
- C) and wherein said anticodon region has been modified to recognize a codon different from that which is recognized by the anticodon of the known human tRNA gene.
An oligonucleotid e which encodes a synthetic suppressor tRNA consisting of:
- A) a structural gene sequence from a known human tRNA gene, comprising no more than twenty 3′ flanking residues and no 5′ flanking residues of the human tRNA gene sequence, said sequence comprising an anticodon region for pairing with mRNA;
- B) a total length of less than 150 nucleotides;
- C) wherein said anticodon sequence has been modified to recognize a codon different from that which is recognized by the anticodon of the known human tRNA gene, wherein said oligonucleotide has a sequence selected from the group consisting of SEQ ID NOS:
- 1-10, and their complements.
1 more element
A method of restoring translation to a nucleotide sequence which includes a nonsense mutation in a cell comprising:
- introducing to said cell in vitro a synthetic suppressor tRNA oligonucleotide, said oligonucleotide consisting of
- A) a structural gene sequence from a known human tRNA gene, comprising no more than twenty 3′ flanking residues and no 5′ flanking residues of the human tRNA gene sequence, said sequence comprising an anticodon region for pairing with mRNA;
- B) a total length of less than 150 nucleotides;
- C) wherein said anticodon region has been modified to recognize a codon different from that which is recognized by the anticodon of the known human tRNA structural gene, wherein said modified anticodon is complementary to said nonsense mutation, such that said synthetic suppressor tRNA oligonucleotide encodes an amino acid which is normally deleted by said nonsense mutation in a nucleotide sequence;
- wherein said synthetic suppressor tRNA oligonucleotide restores translation to said nucleotide sequence, which includes a nonsense mutation.
2 more elements
A method of restoring translation to a nucleotide sequence which includes a nonsense mutation in a cell comprising:
- introducing to said cell in vitro a synthetic suppressor tRNA oligonucleotide, said oligonucleotide consisting of:
- A) a structural gene sequence derived from a known human tRNA gene, comprising no more than twenty 3′ flanking residues and no 5′ flanking residues of the human tRNA gene sequence, said sequence comprising an anticodon region for pairing with mRNA;
- B) a total length of less than 150 nucleotides;
- C) wherein said anticodon sequence has been modified to recognize a codon different from that which is recognized by the anticodon of the known human tRNA gene, wherein said modified anticodon is complementary to said nonsense mutation, such that said synthetic suppressor tRNA oligonucleotide encodes an amino acid which is normally deleted by said nonsense mutation in a nucleotide sequence, wherein said oligonucleotide has a sequence selected from the group consisting of SEQ ID NOS:
- 1-10, and their complements;
- wherein said synthetic suppressor tRNA oligonucleotide restores translation to said nucleotide sequence, which includes a nonsense mutation.
3 more elements
A method for introducing a site-specific mutation in a protein containing a nonsense mutation comprising:
- introducing to a cell in vitro a synthetic suppressor tRNA oligonucleotide, said oligonucleotide consisting of:
- A) a structural gene sequence derived from a known human tRNA gene, comprising no more than twenty 3′ flanking residues and no 5′ flanking residues of the human tRNA gene sequence, said sequence comprising an anticodon region for pairing with mRNA;
- B) a total length of less than 150 nucleotides;
- C) and wherein said anticodon sequence has been modified to recognize a codon different from that which is recognized by the anticodon of the known human tRNA gene, wherein said anticodon is one which will pair with said nonsense mutation and said tRNA structural gene sequence encodes an amino acid which is deleted by said nonsense mutation;
- wherein said synthetic suppressor tRNA oligonucleotide introduces a site-specific mutation in a protein containing a nonsense mutation.
2 more elements
A method for introducing a site-specific mutation in a protein containing a nonsense mutation comprising:
- introducing to a cell in vitro a synthetic suppressor tRNA oligonucleotide, said oligonucleotide consisting of:
- A) a structural gene sequence derived from a known human tRNA gene, comprising no more than twenty 3′ flanking residues and no 5′ flanking residues of the human tRNA gene sequence, said sequence comprising an anticodon region for pairing with mRNA;
- B) a total length of less than 150 nucleotides;
- C) and wherein said anticodon sequence has been modified to recognize a codon different from that which is recognized by the anticodon of the known human tRNA gene, wherein said anticodon is one which will pair with said nonsense mutation and said tRNA structural gene sequence encodes an amino acid which is deleted by said nonsense mutation, wherein said oligonucleotide has a sequence selected from the group consisting of SEQ ID NOS:
- 1-10, and their complements;
- wherein said synthetic suppressor tRNA oligonucleotide introduces a site-specific mutation in a protein containing a nonsense mutation.
3 more elements
A method for designing a synthetic suppressor tRNA comprising:
- A) identifying a tRNA sequence of interest;
- B) identifying the anticodon of said tRNA sequence;
- C) designing an alternate anticodon sequence such that a different amino acid is translated in relation to the said anticodon than would normally be;
- (D) synthesizing an oligonucleotide comprising:
- a) a human tRNA structural gene sequence comprising no more than twenty 3′ flanking residues and no 5′ flanking residues, said sequence encoding an anticodon region for pairing with mRNA;
- b) a total length of less than 150 nucleotides;
- c) an anticodon sequence which has been modified to recognize a codon different from that which is originally recognized, wherein said anticodon is one which will pair with a nonsense mutation and said tRNA structural gene sequence encodes an amino acid which is deleted by said nonsense mutation.
4 more elements
Dependencies are inferred from the claim text.