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

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.