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US 6,057,153 AGranted
Stabilized external guide sequences
AssigneeYale University
InventorsShaji T. George, Michael Ma, Martina Werner, Umberto Pace, Allan R. Goldberg
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Abstract
Modified external guide sequence (EGS) molecules that mediate cleavage of specific target RNAs have been constructed. The modified molecules are external guide sequence molecules for RNAse P which are designed to specifically bind to and promote RNAse P-mediated cleavage of target RNA molecules and to have enhanced nuclease resistance. Specific regions are modified to achieve enhanced stability while maintaining RNAse P activity. Modified external guide sequence molecules suitable for use in the treatment of hepatitis B viral infections have been constructed.
Claims18
An external guide sequence comprising an isolated oligonucleotide molecule comprising
- a RNAse P cleavage targeting sequence, and
- a recognition sequence complementary to a targeted sequence in a target RNA molecule,
- wherein the recognition sequence comprises an A recognition arm and a D recognition arm, wherein the A recognition arm is located at the 3' end of the external guide sequence and the D recognition arm is located at the 5' end of the external guide sequence,
- wherein the RNAse P cleavage targeting sequence forms a structure corresponding to the T stem and T loop of precursor tRNA,
- wherein the A recognition arm, the D recognition arm, and the structure corresponding to the T stem consist of nucleotides selected from the group consisting of ribonucleotides with a 5'-phosphate, ribonucleotides with a 5'-phosphorothioate, 2'-modified ribonucleotides with a 5'-phosphate, 2'-modified ribonucleotides with a 5'-phosphorothioate, deoxyribonucleotides with a 5'-phosphate, and deoxyribonucleotides with a 5'-phosphorothioate,
- wherein the structure corresponding to the T loop consists of nucleotides selected from the group consisting of ribonucleotides with a 5'-phosphate, ribonucleotides with a 5'-phosphorothioate, 2'-modified ribonucleotides with a 5'-phosphate, 2'-modified ribonucleotides with a 5'-phosphorothioate, deoxyribonucleotides with a 5'-phosphate, and deoxyribonucleotides with a 5'-phosphorothioate,
- wherein the external guide sequence promotes RNAse P-mediated cleavage of the target RNA molecule, and
- wherein at least one nucleotide in the external guide sequence is selected from the group consisting of modified nucleotides and unmodified deoxyribonucleotides.
5 more elements
A method for cleaving a target RNA molecule comprising
- bringing into contact, under conditions that promote RNAse P cleavage, RNAse P, the target RNA molecule, and an external guide sequence which comprises an isolated oligonucleotide molecule comprising
- a RNAse P cleavage targeting sequence, and
- a recognition sequence complementary to a targeted sequence in a target RNA molecule,
- wherein the recognition sequence comprises an A recognition arm and a D recognition arm, wherein the A recognition arm is located at the 3' end of the external guide sequence and the D recognition arm is located at the 5' end of the external guide sequence,
- wherein the RNAse P cleavage targeting sequence forms a structure corresponding to the T stem and T loop of precursor tRNA,
- wherein the A recognition arm, the D recognition arm, and the structure corresponding to the T stem consist of nucleotides selected from the group consisting of ribonucleotides with a 5'-phosphate, ribonucleotides with a 5'-phosphorothioate, 2'-modified ribonucleotides with a 5'-phosphate, 2'-modified ribonucleotides with a 5'-phosphorothioate, deoxyribonucleotides with a 5'-phosphate, and deoxyribonucleotides with a 5'-phosphorothioate,
- wherein the structure corresponding to the T loop consists of nucleotides selected from the group consisting of ribonucleotides with a 5'-phosphate, ribonucleotides with a 5'-phosphorothioate, 2'-modified ribonucleotides with a 5'-phosphate, 2'-modified ribonucleotides with a 5'-phosphorothioate, deoxyribonucleotides with a 5'-phosphate, and deoxyribonucleotides with a 5'-phosphorothioate,
- wherein the external guide sequence promotes RNAse P-mediated cleavage of the target RNA molecule, and
- wherein at least one nucleotide in the external guide sequence is selected from the group consisting of modified nucleotides and unmodified deoxyribonucleotides.
6 more elements
Dependencies are inferred from the claim text.