Rapid identification of efficient target cleavage sites using a hammerhead ribozyme library in an iterative manner.
A major limitation to the effectiveness of ribozymes is definition of accessible sites in targeted RNAs. Although library selection procedures have been developed, they are generally difficult to perform and have not been widely employed. Here we describe a selection technology that utilizes a randomized, active hammerhead ribozyme (Rz) library in an iterative manner. After two rounds of binding under inactive conditions, the selected, active Rz library is incubated with target RNA, and the sites of cleavage are identified on sequencing gels. We performed this library-selection protocol using human papillomavirus type 16 E6/E7 mRNA as target and constructed Rz targeted to the identified sites. Rz targeted to sites identified with this procedure were generally highly active in vitro and, more importantly, they were highly active in cell culture, whereas their catalytically inactive counterparts were not. This protocol can be used to identify a set of potential target sites within a relatively short time.
Pan
Wei-Hua
WH
Department of Pathology, Gittlen Cancer Research Institute, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033, USA.
Xin
Ping
P
Bui
Vuong
V
Clawson
Gary A
GA
D001483:Base Sequence; D017931:DNA Primers; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; C059731:oncogene protein E7, Human papillomavirus type 16