Exonuclease III-aided recycling amplification of proximity ligation assay using thymine-melamine-thymine triplex structure for ultrasensitive fluorometric determination of melamine

A fluorescence sensing strategy was described for the determination of melamine with ultrasensitivity and high specificity. This strategy relies on the combination of proximity ligation assay (PLA)-modulated thymine-melamine-thymine (T-melamine-T) triplex structure along with exonuclease III (Exo III)-aided recycling amplification. The reaction system involves three DNA probes (poly A, tDNA and molecular beacon (MB)). Probe ploy A is an adenine enriched sequence containing an apurinic/apyrimidinic site (AP site). It can provide a hydrophobic vacancy for specially recognizing melamine to bind the nucleobase through pseudo-base pairing and participate in PLA modulated T-melamine-T triplex structure. Probe tDNA can form T-melamine-T with probe poly A via PLA only in the presence of melamine. Probe MB is modified with a fluorophore at its 5′-terminus and a quencher at an internal position, which can self-hybridize to form a hairpin structure, positioning the fluorophore in close proximity to the quencher. Therefore, the binding of MB did not yield any fluorescence signal. However, in the presence of melamine, a T-melamine-T triplex structure is formed through PLA. Probe MB can hybridize with t T-melamine-T triplex structure to yield a substrate for interaction with Exo III. As a result, the T-melamine-T triplex structure is released. Next, the released T-melamine-T triplex structure will hybridize with another MB probe to achieve the Exo III-aided recycle amplification. This is the first example of PLA-modulated T-melamine-T triplex structure coupled with Exo III-aided recycling amplification. Under optimal conditions, the assay has a detection range over 4 orders of magnitude and a limit of detection of 4.4 nM. Moreover, the assay is rapid, simple and inexpensive. Hence, the strategy may open new avenues for ultrasensitive and highly specific detection of melamine encounted in food analysis.