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

doi:10.6023/cjoc202403057

Chinese Journal of Organic Chemistry ›› 2024, Vol. 44 ›› Issue (8): 2554-2562.DOI: 10.6023/cjoc202403057 Previous Articles Next Articles

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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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Abstract

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

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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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Fig. & Tab. 9

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

Scheme 2. Synthesis route of compound TPE-P

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

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

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

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

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.

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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