Detection of small organic analytes by fluorescing molecular switches.
A sensor system was developed for the determination of theophylline concentrations based on a theophylline-dependent allosteric ribozyme (Soukup, G. A.; Breaker, R. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3584) in combination with an RNA substrate which is double-labeled with a fluorophore and a quencher dye. In the presence of theophylline, a hammerhead ribozyme domain is switched into an active conformation by the action of a theophylline-binding aptamer domain. Upon substrate cleavage, the quencher is removed from the vicinity of the fluorophore, causing an increased fluorescence signal. Real-time analysis of the cleavage reactions both under single and multiple turnover conditions revealed a dependence on the cleavage rate within a range from 0.01 to 2mM theophylline. The structurally similar molecule caffeine, however, had no detectable influence on the fluorescence signal.
Frauendorf
C
C
Freie Universität Berlin, FB Biologie, Chemie & Pharmazie, Institut für Chemie, Thielallee 63, 14195, Berlin, Germany.
Jäschke
A
A
D000495:Allosteric Site; D001483:Base Sequence; D001665:Binding Sites; D001993:Bronchodilator Agents / Q000032:analysis*; D002110:Caffeine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D007202:Indicators and Reagents; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D017421:Sequence Analysis; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013329:Structure-Activity Relationship; D013806:Theophylline / Q000032:analysis* / Q000378:metabolism; D013997:Time Factors
D001993:Bronchodilator Agents; D007202:Indicators and Reagents; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D002110:Caffeine; D012313:RNA; D013806:Theophylline