聚集诱导发光型核酸探针的制备及其核酸传感原理研究

Preparation of Aggregation-Induced Emission Nucleic Acid Probes and Study of Their Nucleic Acid Sensing Principles

核酸检测是目前生物诊疗及医学分析领域最为精准的方法之一.其中,广泛应用的聚合酶链式反应(PCR)技术利用TaqMan探针的荧光信号输出,可实现对微量目标序列的高灵敏定量分析.本工作系统性地完成聚集诱导发光(AIE)基元——四苯乙烯(TPE)的亚磷酰胺修饰,并将其设计和制备成一系列单标记特定RNA的核酸探针,为后续高效率利用全自动核酸合成仪制备各类AIE探针奠定基础;利用RNaseA酶特异性切割TPE-RNA1探针中水溶性的RNA部分,通过透射电镜、荧光光谱、质谱等表征证明酶切作用诱发的探针溶解能力变化且伴随着疏水聚集;通过优化探针浓度、反应时间、序列长度等多个参数,实现了AIE效应下荧光增强34.7倍.采用AIE原理取代传统探针中荧光-淬灭基团的双标记策略,扩展了核酸探针的设计策略,并系统表征和探讨AIE型核酸探针的聚集过程,旨在推动更高效、更灵敏的核酸检测探针,拓展其在生物化学检测领域的应用.

Nucleic acid detection is one of the most precise methods in biomedicine and medical diagnostics.Among these techniques,the widely employed polymerase chain reaction(PCR)utilizes the fluorescence signal output of TaqMan probes to achieve highly sensitive quantitative analysis of trace target sequences.This study systematically accomplishes the modification of the aggregation-induced emission(AIE)moiety—tetraphenylethylene(TPE)with phosphoramidite,and designs and synthesizes a series of single-labeled nucleic acid probes specific to particular RNA sequences,laying the foundation for the efficient preparation of various AIE nucleic acid probes using automatic oligonucleotide synthesizer.By specifically cleaving the water-soluble RNA segment within the TPE-RNA1 probe using the RNase A enzyme,various characterization techniques including transmission electron microscopy,fluorescence spectroscopy,mass spectrometry,etc.,demonstrate changes in probe solubility induced by enzymatic cleavage,accompanied by hydrophobic aggregation.Through the optimization of parameters such as probe concentration,reaction time,and sequence length,a 34.7-fold enhancement in fluorescence under the AIE effect was achieved.This study replaces the traditional dual-labeled strategy of fluorescence-quencher moieties in probes with the AIE principle,expanding the design strategy of nucleic acid probes.It systematically characterizes and discusses the aggregation process of AIE-type nucleic acid probes,aiming to promote more efficient and sensitive nucleic acid detection probes and expand their applications in the field of biochemical detection.