A biosensor for theophylline based on fluorescence detection of ligand-induced hammerhead ribozyme cleavage
A biosensor for theophylline based on fluorescence detection of ligand-induced hammerhead ribozyme cleavage
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DOI：
10.1017/s1355838202028066
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发表时间：
2002-10-01
期刊：
影响因子：
4.5
通讯作者：
Walter, NG
中科院分区：
文献类型：
--
作者：
Sekella, PT;Rueda, D;Walter, NG
最近，Breaker及其同事设计了锤头状核酶，其在与特定配体变构结合时从无催化活性构象重排为活性构象。为了监测裂解活性在真实的时间，我们已经耦合了供体-受体荧光团对的这种锤头状核酶的底物RNA的末端，修改切割在反式存在的支气管扩张剂茶碱。在完整的底物中，荧光团通过荧光共振能量转移（FRET）相互作用。当效应配体结合时，特异性FRET信号分解，底物被切割，产物解离，速率常数取决于配体的浓度。我们的生物传感器在1 mM茶碱中以0.46 min（-1）切割底物，在无效应物的情况下以0.04 min（-1）切割底物，并区分茶碱的结构相对物咖啡因。我们已经测量了这种生物传感器的茶碱依赖性曲线，表明低至1 AM的浓度可以与背景区分开。为了探索变构调节的机制，在催化结构域和配体结合结构域之间的通信结构域中的单个核苷酸被突变以使核酶的非活性构象不稳定。正如预测的那样，该突变体在存在和不存在茶碱的情况下显示出相同的活性（0.3 min（-1））。此外，时间分辨的FRET测量的生物传感器核酶在复杂的不可裂解的基板类似物显示没有显着变化的荧光团的距离分布后，结合效应。
Recently, Breaker and coworkers engineered hammerhead ribozymes that rearrange from a catalytically inactive to an active conformation upon allosteric binding of a specific ligand. To monitor cleavage activity in real time, we have coupled a donor-acceptor fluorophore pair to the termini of the substrate RNA of such a hammerhead ribozyme, modified to cleave in trans in the presence of the bronchodilator theophylline. In the intact substrate, the fluorophores interact by fluorescence resonance energy transfer (FRET). The specific FRET signal breaks down as the effector ligand binds, the substrate is cleaved, and the products dissociate, with a rate constant dependent on the concentration of the ligand. Our biosensor cleaves substrate at 0.46 min(-1) in 1 mM theophylline and 0.04 min(-1) without effector, and discriminates against caffeine, a structural relative of theophylline. We have measured the theophylline-dependence profile of this biosensor, showing that concentrations as low as 1 AM can be distinguished from background. To probe the mechanism of allosteric regulation, a single nucleotide in the communication domain between the catalytic and ligand-binding domains was mutated to destabilize the inactive conformation of the ribozyme. As predicted, this mutant shows the same activity (0.3 min(-1)) in the presence and absence of theophylline. Additionally, time-resolved FRET measurements on the biosensor ribozyme in complex with a noncleavable substrate analog reveal no significant changes in fluorophore distance distribution upon binding of effector.