Terminal protection of small-molecule-linked DNA for sensitive fluorescence detection of protein binding based on nucleic acid amplification

A novel fluorescence biosensing strategy based on terminal protection triggered nucleic acid amplification reaction upon target binding was developed for the sensitive detection of small molecule–protein interactions. This method relies on the specific interactions between target proteins and their small molecule-linked DNA to produce a ligation probe. Introduction of a target protein into the assay system protected the small molecule-linked DNA from Exonuclease I (Exo I)-mediated digestion and served as the circular template to trigger rolling circle amplification (RCA). The RCA product containing thousands of repeated sequences could hybridize with the TaqMan probes (reporter probes) and were then detected by Exonuclease III (Exo III)-aided recycling amplification reaction, generating remarkable fluorescence signals. This developed strategy transduced small molecule–protein interactions into DNA amplification and detection techniques. Due to the primary RCA followed by the secondary Exo III-aided recycling amplification, the developed method was demonstrated to have very high sensitivity for the detection of a tumor biomarker of folate receptor with a limit of detection of 0.8 pM. The novel strategy holds great promise for the study of small molecule–protein interactions with desirable specificity and sensitivity achieved as a result of terminal protection and nucleic acid amplification.