A triplex ribozyme expression system based on a single hairpin ribozyme.
Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.
Aquino-Jarquin
Guillermo
G
Laboratorio de Terapia Génica, Departamento de Genética y Biología Molecular, CINVESTAV, México D.F., México.
Benítez-Hess
María Luisa
ML
DiPaolo
Joseph A
JA
Alvarez-Salas
Luis M
LM
D045744:Cell Line, Tumor; D005260:Female; D015870:Gene Expression; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D009690:Nucleic Acid Conformation; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000378:metabolism*
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; D012097:Repressor Proteins; C418481:hairpin ribozyme; C059731:oncogene protein E7, Human papillomavirus type 16