Removal of the guanine cap on the 5′ terminus of RNA
View Patent ↗Methods and compositions are provided for efficiently removing a guanine cap from the 5′ end of an RNA using enzymes. Decapped RNA can be used for cloning, sequencing or other RNA manipulations.
1. An in vitro method for removing guanosine monophosphate (GMP) or methyl GMP from the 5′ end of a capped RNA, the capped RNA comprising GpppN or methylated GpppN, the method comprising
(a) combining an isolated 5′ deadenylase with the capped RNA; and
(b) removing GMP or methyl GMP from the capped RNA.
2. A method according to claim 1 , wherein the guanine cap comprises a 7-methylated guanine.
3. A method according to claim 1 , wherein the guanine cap is unmethylated.
4. A method according to any of claims 1 through 3 , wherein one or more of the 2′ hydroxy groups of the first 2 ribose sugars of the 5′ end of the RNA are methylated.
5. An in vitro artificial mixture comprising an isolated 5′ deadenylase and a guanine capped RNA.
6. The mixture according to claim 5 , wherein the RNA is as large as 1 kb or greater or as small as 2 nucleotides.
7. The mixture according to claim 5 wherein the isolated 5′ deadenylase is a recombinant enzyme.