Method for making a 21 nucleotide double stranded RNA chemically linked at one end
View Patent ↗The present invention provides methods and compositions for inhibiting gene expression using double stranded RNA molecules that are between 15 and 21 nucleotides in length and are complementary to a target gene sequence.
1. A method for making an oligoribonucleotide having a double stranded structure (dsRNA) consisting of two RNA strands that are chemically linked at one end by a carbon-carbon covalent linker,
wherein a first strand of the dsRNA is fully complementary to a segment of an RNA transcript of a target gene and a second strand of the dsRNA is complementary to the first strand,
wherein the dsRNA is 21 nucleotides in length, and wherein said oligoribonucleotide specifically inhibits the expression of a target gene, comprising the steps of:
a. synthesizing a first oligoribonucleotide strand (S1) which comprises a nucleic acid sequence, wherein said nucleic acid sequence is fully complementary to an mRNA transcript of the target gene and is 21 nucleotides in length,
b. synthesizing a second oligoribonucleotide strand (S2) which consists of a nucleic acid sequence complementary to the oligoribonucleotide strand S1,
c. hybridizing said S1 strand to said S2 strand under conditions that allow the formation of a double stranded structure, thereby making an oligoribonucleotide having a double stranded structure consisting of two RNA strands, and
d. chemically linking the 3′ end of one of the strands to the 5′ end of the other strand with said carbon-carbon covalent linker.
2. The method of claim 1 , wherein said S1 and S2 strands are synthesized chemically.
3. The method of claim 1 , wherein said S1 and S2 strands are synthesized in a transcription reaction.
4. The method of claim 3 , wherein said transcription reaction occurs extracellularly.
5. The method of claim 1 , wherein the target gene is a mammalian gene.
6. The method of claim 1 , wherein at least one of said oligoribonucleotide strands comprises at least one chemically modified nucleotide.
7. The method of claim 6 , wherein said chemically modified nucleotide is chosen from the group of: a 2′-amino or a 2′-methyl substituted nucleotide, or a locked nucleotide.
8. The method of claim 1 , wherein said carbon-carbon covalent linker is a C18 linker.