聚集诱导发光型核酸探针的制备及其核酸传感原理研究

doi:10.6023/cjoc202403057

有机化学 ›› 2024, Vol. 44 ›› Issue (8): 2554-2562.DOI: 10.6023/cjoc202403057 上一篇下一篇

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聚集诱导发光型核酸探针的制备及其核酸传感原理研究

欧彦^a, 蓝琳^b, 王正雄^b, 王志明^a^,^b^,^*(), 唐本忠^a^,^b

a 华南理工大学广东省分子聚集发光重点实验室发光材料与器件国家重点实验室广州 510640
b 广东省大湾区华南理工大学聚集诱导发光高等研究院广州 510700

收稿日期:2024-03-31 修回日期:2024-05-17 发布日期:2024-06-07
基金资助:
国家自然科学基金(21975077); 广东省自然科学基金(2022B1515020084); 广东省分子聚集发光重点实验室开放基金(South China University of Technology, No. 2019B030301003); 华南理工大学发光材料与器件国家重点实验室自主研发项目(Skllmd-2022-01); 广东省基础与应用基础研究基金(2023B1515040003); 云南省科技厅重点项目(202303AC100021)

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

Yan Ou^a, Lin Lan^b, Zhengxiong Wang^b, Zhiming Wang^a^,^b(), BenZhong Tang^a^,^b

a State Key Laboratory of Luminescent Materials and Devices, Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates, South China University of Technology, Guangzhou 510640
b AIE Institute, Guangzhou 510700

Received:2024-03-31 Revised:2024-05-17 Published:2024-06-07
Contact: *E-mail: wangzhiming@scut.edu.cn
Supported by:
National Natural Science Foundation of China(21975077); Natural Science Foundation of Guangdong Province(2022B1515020084); Open Fund of Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates(South China University of Technology, No. 2019B030301003); Independent Research Project of State Key Lab of Luminescent Materials and Devices (SCUT)(Skllmd-2022-01); Guangdong Basic and Applied Basic Research Foundation(2023B1515040003); Key Project of Yunnan Provincial Department of Science and Technology(202303AC100021)

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摘要/Abstract

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RNase A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.

关键词: 核酸探针, 聚集诱导发光, 亚磷酰胺修饰, 聚合酶链式反应, 核酸检测

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.

Key words: nucleic acid probe, aggregation-induced emission (AIE), modified phosphoramidite, polymerase chain reaction (PCR), nucleic acid detection

引用此文

欧彦, 蓝琳, 王正雄, 王志明, 唐本忠. 聚集诱导发光型核酸探针的制备及其核酸传感原理研究[J]. 有机化学, 2024, 44(8): 2554-2562.

Yan Ou, Lin Lan, Zhengxiong Wang, Zhiming Wang, BenZhong Tang. Preparation of Aggregation-Induced Emission Nucleic Acid Probes and Study of Their Nucleic Acid Sensing Principles[J]. Chinese Journal of Organic Chemistry, 2024, 44(8): 2554-2562.

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图/表 9

图式1. 传统TaqMan探针和AIE型荧光探针水解识别机制

Scheme 1. Recognition mechanism of hydrolysis in traditional TaqMan probe and AIE-based fluorescent probe

图式2. 化合物TPE-P的合成路线

Scheme 2. Synthesis route of compound TPE-P

图1. TPE-OH在不同比例THF/水下的(a)吸收光谱和(b)荧光光谱图; (c) TPE-OH的PL强度随fw的变化; (d) TPE-RNA1在水溶液中测试的归一化UV和PL光谱

Figure 1. (a) Absorption spectra and (b) fluorescence spectra of TPE-OH at different ratios of THF/water; (c) PL intensity of TPE-OH varies with increasing fw; (d) normalized UV and PL spectra of TPE-RNA1 measured in water

图2. (a) TPE-RNA1的荧光强度随RNase A酶水解时间的变化, 以及(b)在482 nm处荧光强度与反应时间的关系

Figure 2. (a) Fluorescence intensity of TPE-RNA1 varies with the hydrolysis time of RNase A enzyme, and (b) fluorescence intensity at 482 nm versus reaction time

图3. 不同浓度(10和50 μmol/L) TPE-RNA1探针被RNase A酶水解前后的透射电镜图

Figure 3. TEM images of the TPE-RNA1 probes before and after hydrolyzed by RNaseA enzyme at different concentrations (10 and 50 μmol/L)

图4. TPE标记的单链核酸(8、15、24和36 nt)在缓冲溶液中的荧光光谱(分子浓度10 μmol/L)

Figure 4. Fluorescence spectra of TPE-labeled single-stranded nucleic acids (8, 15, 24 and 36 nt) in buffer solution (molecular concentration: 10 μmol/L)

图5. TPE-RNA1探针在10 μmol/L到100 μmol/L浓度范围内(a)未加入RNase A酶和(b)加入RNase A酶后的荧光光谱; (c)探针浓度和最强发射波长482 nm下的荧光强度关系图; (d) RNase A酶水解前后的信噪比图

Figure 5. Fluorescence spectra of TPE-RNA1 probe concentration between 10 μmol/L and 100 μmol/L concentration (a) without RNase A enzyme and (b) after RNase A enzyme addition; (c) Curve of fluorescence emission maximum at 482 nm versus TPE-RNA1 concentration; (d) S/N before and after hydrolysis with RNase A enzyme

图6. (a) TPE-RNA1、(b) TPE-RNA1-Eclipse和(c) TPE-RNA1-BHQ-1探针在加入RNase A酶前后的荧光光谱图; (d) RNase A酶水解FAM-RNA和FAM-RNA-Eclipse前后的荧光强度

Figure 6. Fluorescence spectra of (a) TPE-RNA1, (b) TPE-RNA1 Eclipse and (c) TPE-RNA1-BHQ-1 probes with and without RNase A enzyme; (d) Fluorescence intensity of FAM-RNA and FAM-RNA-Eclipse before and after hydrolysis by RNase A enzyme FRNA: FAM-RNA; FRNA＋A: FAM-RNA＋RNase A; FRNAE: FAM-RNA-Eclipse; FRNAE＋A: FAM-RNA-Eclipse＋RNase A.

图7. RNase A酶水解(a) TPE-RNA1、(b) TPE-RNA1-Eclipse和(c) TPE-RNA1-BHQ-1探针的透射电镜图

Figure 7. TEM images of RNase A enzyme hydrolyzed (a) TPE-RAN1, (b) TPE-RNA1-Eclipse and (c) TPE-RNA1-BHQ-1 probes

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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

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