Aptazyme-based biosensors using a eukaryotic cell-free translation system.
I have constructed a novel aptazyme-based biosensor system for detecting cofactors of the aptazymes using a cell-free luciferase synthesis in wheat germ extract. In this system, the activity of the aptazyme that is fused to a 5'-untranslated region of a luciferase gene can be detected as luciferase expression. In translating the aptazyme-fused mRNA as-is using a wheat germ cell-free translation system, the luciferase is almost not expressed because of the following triple suppression effects: (1) 5'-terminal three bases and (2) 5'-terminal duplex prevent the ribosome from binding to own mRNA; (3) if the ribosome binds, translation of a mimic gene in the aptazyme inhibits that of the downstream luciferase gene (OFF state). In contrast, in the presence of the aptazyme cofactor, the aptazyme in mRNA is self-cleaved to produce an aptazyme-free luciferase gene, which is translated efficiently (ON state). The ON/OFF efficiency and the detection limit of the aptazyme-based biosensor for theophylline are much higher and lower, respectively, compared to those of previously-reported one that utilizes a prokaryotic translation system.
Ogawa
Atsushi
A
Senior Research Fellow Center, Ehime University, Matsuyama, Ehime 790-8577, Japan.
D015374:Biosensing Techniques; D002474:Cell-Free System; D005057:Eukaryotic Cells / Q000378:metabolism; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis; D016337:RNA, Catalytic / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry; D013806:Theophylline / Q000032:analysis
D016337:RNA, Catalytic; D012333:RNA, Messenger; D013806:Theophylline; D008156:Luciferases