A modular strategy for tailoring fluorescent biosensors from ribonucleopeptide complexes
A modular strategy for tailoring fluorescent biosensors from ribonucleopeptide complexes
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DOI：
10.1021/ja063965c
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发表时间：
2006-10-04
影响因子：
15
通讯作者：
Morii, Takashi
中科院分区：
文献类型：
--
作者：
Hagihara, Masaki;Fukuda, Masatora;Morii, Takashi
荧光生物传感器，促进试剂敏感检测的小分子是至关重要的工具，在治疗和诊断领域。然而，从大分子受体构建具有所需特性（即，用于配体检测的检测波长和浓度范围）的荧光生物传感器不是一个简单的任务。ATP结合核糖核酸肽（RNP）受体转化为荧光ATP传感器，而不进行化学修饰的ATP结合RNA中的核苷酸。ATP结合RNP的RNA亚基和用芘基修饰的肽形成稳定的荧光RNP复合物，该复合物在与ATP结合时显示荧光强度增加。将ATP结合RNP受体转化为荧光ATP传感器的策略应用于通过使用从体外选择ATP结合RNP和一系列荧光团修饰的肽亚基获得的RNA亚基池来产生荧光ATP结合RNP文库。基于在配体存在和不存在下的荧光发射强度变化的荧光RNP文库的简单筛选提供了发射波长从390至670 nm变化的荧光ATP或GTP传感器。在增加浓度的ATP存在下筛选荧光发射强度变化允许荧光RNP文库的滴定分析，其提供在宽浓度范围的ATP下响应的ATP传感器。使用模块化RNP受体的组合策略，在这里报告，使定制的荧光传感器的特定配体的大分子受体的详细结构信息的知识。
Fluorescent biosensors that facilitate reagentless sensitive detection of small molecules are crucial tools in the areas of therapeutics and diagnostics. However, construction of fluorescent biosensors with desired characteristics, that is, detection wavelengths and concentration ranges for ligand detection, from macromolecular receptors is not a straightforward task. An ATP-binding ribonucleopeptide (RNP) receptor was converted to a fluorescent ATP sensor without chemically modifying the nucleotide in the ATP-binding RNA. The RNA subunit of the ATP-binding RNP and a peptide modified with a pyrenyl group formed a stable fluorescent RNP complex that showed an increase in the fluorescence intensity upon binding to ATP. The strategy to convert the ATP-binding RNP receptor to a fluorescent ATP sensor was applied to generate fluorescent ATP-binding RNP libraries by using a pool of RNA subunits obtained from the in vitro selection of ATP-binding RNPs and a series of fluorophore-modified peptide subunits. Simple screening of the fluorescent RNP library based on the fluorescence emission intensity changes in the absence and presence of the ligand afforded fluorescent ATP or GTP sensors with emission wavelengths varying from 390 to 670 nm. Screening of the fluorescence emission intensity changes in the presence of increasing concentrations of ATP allowed titration analysis of the fluorescent RNP library, which provided ATP sensors responding at wide concentration ranges of ATP. The combinatorial strategy using the modular RNP receptor reported here enables tailoring of a fluorescent sensor for a specific ligand without knowledge of detailed structural information for the macromolecular receptor.