具有折叠构象的多肽/蛋白基荧光分子信标

We propose to develop peptide and protein based fluorescent molecular beacons. For this purpose, we propose to employ α-/β-turn, β-sheet and β-hairpin structures as the basic structural framework, which will be elaborately equipped with fluorophore and quencher at the two terminuses of the folded structures, that affords fluorescent response following bimolecular processes such as formation of excimer and exciplex, photo-induced electron transfer, proton transfer and resonant energy transfer. The binding groups are designed to locate at amino acid residues that are either separated or linked as a segment in the peptide chain within the folded structure. The analyte binding simultaneously to several binding groups would hence lead initially to local structural changes which are transmitted and amplified among the noncovalent interaction network of the folded structure, that finally results in a substantial change in the beacon structure in terms of the altered distance and orientation of the fluorophore against the quencher, giving amplified fluorescent signaling output with higher selectivity. The peptide and protein based fluorescent molecular beacons are expected to be applicable to a variety of analytes, ranging from proteins and peptides to small molecules, with potential applications of fluorescent sensing and imaging in abiotic samples and biological systems.

本项目拟发展多肽/蛋白基荧光分子信标。提出以天然多肽或人工拟肽、蛋白质高级结构中的转角、β-折叠和β-发夹为信标的基本结构框架，将荧光团和猝灭剂连接于折叠结构两个末端，组成以激基缔合物、电子转移/质子转移和共振能量转移等双分子作用过程为荧光响应机制的荧光信号报告基团。巧妙地依据蛋白质高级结构中多重次级键的网络结构特征和肽链中氨基酸残基或氨基酸残基序列的选择性结合能力，将目标物结合基团分别修饰至不同的氨基酸残基上，多个结合基团共同参与目标物结合，引起相关氨基酸残基处局部结构变化，后者经肽链或蛋白高级结构中的次级键网络传递并放大，诱发信标分子的整体结构变化，给出放大的荧光响应，实现高选择性高灵敏荧光传感。信标的荧光响应启动自目标物结合导致的信标分子的局部结构变化，不仅适用于蛋白质、多肽，亦适于短肽和小分子目标物，是一类广谱的荧光传感探针分子，更可用于生命体系的动态荧光响应和荧光成像。

传统的荧光分子信标一般包含发夹形状的核酸片段并依据“核酸-核酸相互作用”进行目标识别。本项目发展了基于折叠构象的多肽/蛋白基荧光分子信标，以折叠构象的天然多肽或人工拟肽为基本结构框架，两末端引入荧光团和猝灭剂，藉由目标物诱发的整体结构变化给出放大的荧光响应，达至高灵敏度荧光传感。据此构筑了系列肽基酰胺基硫脲之β-转角折叠短肽分子，于其两末端引入适宜的荧光基团，发展了比率荧光响应的肽基荧光分子信标；并以折叠短肽为螺旋构筑基元构造了系列新型超分子螺旋结构，继而应用于超分子手性研究和阴离子跨膜转运；以脯氨酸和喹啉折叠体之折叠构象为结构骨架，藉由动态化学键发展了葡萄糖、多巴、唾液酸、手性胺等重要生物小分子的选择性传感识别体系，包括手性识别；以双硫键或螺吡喃为骨架结构开发了基于仿生变构效应的光学传感体系。作为拓展研究，构建了多类光化学传感体系，尤其发展了基于动态聚集体的化学传感策略。研究工作不仅有力推动了荧光分子信标和光学传感研究的发展，更发展了新一代仿生超分子螺旋的构建策略和应用研究。.在项目执行期间内，共发表SCI收录期刊论文26篇，其中JCR一区和二区期刊论文分别为15篇和9篇；IF > 5.5的期刊论文16篇；有3篇论文被国际权威刊物选为封面论文。“基于动态超分子聚集体的化学传感”获教育部自然科学二等奖。项目负责人组织了2次国际学术会议，并14次应邀参加国际学术会议。培养博士生8人、硕士毕业生12人。.本项目基本按任务书既定计划完成研究任务，并有所拓展；青年教师的整体研究水平提升明显，为学科发展注入新的动力。