Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 129-139 |
| Number of pages | 11 |
| Journal | Molecular Therapy |
| Volume | 7 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1 2003 |
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All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Molecular Biology
- Genetics
- Pharmacology
- Drug Discovery
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Rapid identification of efficient target cleavage sites using a hammerhead ribozyme library in an iterative manner. / Pan, Wei Hua; Xin, Ping; Bui, Vuong; Clawson, Gary.
In: Molecular Therapy, Vol. 7, No. 1, 01.01.2003, p. 129-139.Research output: Contribution to journal › Article
TY - JOUR
T1 - Rapid identification of efficient target cleavage sites using a hammerhead ribozyme library in an iterative manner
AU - Pan, Wei Hua
AU - Xin, Ping
AU - Bui, Vuong
AU - Clawson, Gary
PY - 2003/1/1
Y1 - 2003/1/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0037219310&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037219310&partnerID=8YFLogxK
U2 - 10.1016/S0000-000X(00)00000-0
DO - 10.1016/S0000-000X(00)00000-0
M3 - Article
C2 - 12573626
AN - SCOPUS:0037219310
VL - 7
SP - 129
EP - 139
JO - Molecular Therapy
JF - Molecular Therapy
SN - 1525-0016
IS - 1
ER -