Patent ID: 9546364
Date: 2017-01-17
CPC Classifications: C12N

Claim:
1. A solid-phase method of preparing an miRNA guide strand precursor having a lariat structure, comprising preparing on a solid support a lariat structure having a miRNA sequence that is at least partially complementary to a target mRNA: a. adding at least two nucleotide residues to the solid support in a 3′ to 5′ direction; b. adding residues to the 5′ and 2′ positions of the 5′ terminal residue, producing a 5′ strand and a 2′ strand; c. terminating the 5′ strand with a terminal residue having either a 5′ alkyne group or a 5′ azide group; d. terminating the 2′ strand with a terminal residue having a 3′ terminal alkyne when the terminal residue of the 5′ strand has a 5′ azide group or a 3′ azide group when the terminal residue of the 5′ strand has a 5′ alkyne group; e. ligating the 5′ terminal alkyne or azide of the terminal residue of the 5′ strand with the 3′-terminal azide or alkyne, respectively, of the terminal residue of the 2′ strand to produce a triazole linkage between the terminal residue of the 5′ strand and the terminal residue of the 2′ strand; and f. deprotecting the lariat structure and cleaving the structure from the solid support, wherein the total number of nucleotide residues in the miRNA guide strand precursor is between 20 and 25 residues and has a sequence that is at least partially complementary to an mRNA, thereby producing a miRNA guide strand precursor.