FRET Nanoflares for Intracellular mRNA Detection: Avoiding False Positive Signals and Minimizing Effects of System Fluctuations

A new class of intracellular nanoprobe, termed fluorescence resonance energy transfer (FRET) nanoflares, was developed to sense mRNA in living cells. It consists of a gold nanoparticle (AuNP), recognition sequences, and flares. Briefly, the AuNP functionalized with recognition sequences hybridized to flares, which are designed as hairpin structures and fluorescently labeled donors and acceptors at two ends, respectively. In the absence of targets, the flares are captured by binding with the recognition sequences, separating of the donor and acceptor, and inducing low FRET efficiency. However, in the presence of targets, the flares are gradually displaced from the recognition sequences by the targets, subsequently forming hairpin structures that bring the donor and acceptor into close proximity and result in high FRET efficiency. Compared to the conventional single-dye nanoflares, the upgraded FRET nanoflares can avoid false positive signals by chemical interferences (such as nuclease and GSH) and thermodynamic fluctuations. Moreover, the signal generation in FRET nanoflares can be easily made with ratiometric measurement, minimizing the effect of system fluctuations.