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

Abstract

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.

Fluorescence Resonance Energy Transfer-Based DNA Tetrahedron Nanotweezer for Highly Reliable Detection of Tumor-Related mRNA in Living Cells

“…20 Nonetheless, stability still remains a problem, and false-positive signals cannot be entirely avoided. 21 In addition, complex steps for the preparation and functionalization of these nanomaterials are commonly required. 22,23 …”

“…29,33,34 Nevertheless, all of them are single-intensity-based sensing probes, which may lead to high false-positive signals from nuclease digestion, protein binding, or thermodynamic fluctuations. 21 Moreover, such sensing is compromised by the local distribution of probes and by drifts of light sources and detectors. These obstacles limit accurate detection and imaging of tumor-related mRNA in living cells.…”

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

“…With the reporter strand designed to be complementary to target RNAs, GNP-Nano-MBs (named sticky flares) can quantify the expression of target RNAs and track the spatial distribution of target RNAs 123 . In addition, with FRET probes instead of single fluorophores conjugated on GNPs, the GNP-Nano-MBs would have less false-positive signals 124 .…”

“…For the detection of single mRNA tumour markers, both MB and Nano-MBs have been successfully applied in detecting survivin mRNA 113, 114, 125, 130 , MnSOD mRNA 103, 115 , c-raf-1 mRNA 119 , TK1 mRNA 124 , STAT5B mRNA 126 and K-ras mRNA 170 in different tumour cells.…”

Photoinduced Electron Transfer-Based Fluorescence Quenching Combined with Rolling Circle Amplification for Sensitive Detection of MicroRNA

“…[7] So, the strategies for specific and sensitive quantitative detection of miRNAs are in urgent need. [8][9][10][11] Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is the gold standard for miRNA quantification. [12,13] The qRT-PCR technique offers good sensitivity and specificity for miRNA detection.…”