Method for introducing site-directed RNA mutation, target editing guide RNA used in the method and target RNA-target editing guide RNA complex
A method for inducing a site-directed RNA mutation is provided. The method includes repairing an RNA mutation by converting target adenosine, which is located at a target editing site of a target RNA, into inosine. The method for inducing a site-directed RNA mutation involves reacting the target RNA having a target adenosine with a target editing guide, which has been constructed so as to form a complementary strand with target RNA, to form a double-stranded complex, and converting the target adenosine to inosine by causing ADAR to act on the double-stranded complex, inducing A-to-I editing capability. The converted inosine is further translated into guanosine.
1. A method for introducing a site-directed RNA mutation comprising:
reacting a 3′-target RNA represented by formula [XXXVIA]:
with a 5′-target editing guide RNA represented by [XXXVIIA], which comprises a base sequence of SEQ ID NO: 99 (GGGUGGAAUA GUAUACCAUU CGUGGUAUAG UAUCCCACCU) between j N n+p and k N 1 :
to obtain a 3′-target RNA-5′-target editing guide RNA complex being represented by formula [XXXVIIIA], which comprises a base sequence of SEQ ID NO: 99 (GGGUGGAAUA GUAUACCAUU CGUGGUAUAG UAUCCCACCU) between j N n+p and k N 1 :
obtaining a 3′-edited target RNA represented by formula [XXXIXA]:
in which a target base adenosine (A*) of the resulting 3′-target RNA-5′-target editing guide RNA complex is converted to inosine (I*) by A-I editing by action of double-stranded specific adenosine deaminase (ADAR); wherein,
the 3′-target RNA comprises a guide-side target complementary region, a target base adenosine and a terminal-side target complementary region, and
in formula [XXXVIA], a symbol of f N1 . . . f Nm corresponds to the terminal-side target complementary region of the 3′-target RNA and indicates a base sequence constructed from the same or different bases, and a symbol of f N is a base selected from adenine, cytosine, guanine and uracil, and a symbol of m indicates a number of bases of the terminal-side target complementary region in a range of 40 to 20,
a symbol of g N1 g N2 . . . g Nn-1 g Nn corresponds to the guide-side target complementary region of the 3′-target RNA and indicates a base from the same or different bases and a symbol of g N is a base selected from adenine, cytosine, guanine and uracil, and a symbol of n indicates a number of bases of the guide-side target complementary region in a range of 1 to 10, and
A* indicates the target base adenine existing at a target editing position; and
the 5′-target editing guide RNA comprises a terminal-side target recognition region, a target editing inducing base, a core-side target recognition region, an ADAR binding core region and a guide-side decoupling region, and
in formula [XXXVIIA], X is cytosine, guanine or adenine and corresponds to the target editing inducing base,
the base sequence of SEO ID NO:99 corresponds to the ADAR binding core region,
a symbol of 3′- k N p k N p−1 . . . k N 2 k N 1 corresponds to the guide-side decoupling region of the 5′-target editing guide RNA and indicates a base sequence constructed from the same or different bases, and a symbol of k N is a base elected from adenine, cytosine, guanine and uracil, and a symbol of p indicates a number of bases of the guide-side decoupling region in a range of 10 or less,
a symbol of h N1 . . . h Nm corresponds to the terminal-side target recognition region of the 5′-target editing guide RNA and indicates a base sequence constructed from the same or different bases, and a symbol of h N is a base selected from adenine, cytosine, guanine and uracil, and a symbol of m indicates the same meaning as described above, and a number of bases in the terminal-side target recognition region is the same number of bases in the base sequence of the terminal-side target complementary region of the 3′-target RNA, and each base in the terminal-side target recognition region forms a base-pair with the corresponding base of the terminal-side target complementary region of the 3′-target RNA,
a symbol of j N1 j N2 . . . j Nn+p corresponds to a base sequence of a X adjacent partial region and an ADAR adjacent partial region in the core-side target recognition region of the 5′-target editing guide RNA and indicates a base sequence constructed from the same or different bases, and a symbol of j N is a base selected from adenine, cytosine, guanine and uracil, a symbol of n indicates the same meaning as described above and is the same number of bases in a base sequence of the X adjacent partial region of the core-side target recognition region, a symbol of p indicates the same meaning as described above, and is the same number of bases in a base sequence of the ADAR adjacent partial region of the core-side target recognition region, and a base sequence of the X adjacent partial region forms a base-pair with a base sequence of the guide-side target complementary region of the 3′-target RNA, and a base sequence of the guide-side decoupling region forms a base-pair with a base sequence of the ADAR adjacent partial region in the core-side target recognition region as forming the 3′-target RNA-5′-target editing guide RNA complex.
2. The method according to claim 1 , wherein n is 3 and p is 0.
3. The method according to claim 1 , wherein, the action of ADAR is derived from intravital ADAR mechanism.