Detection of miRNA Using a Double-Strand Displacement Biosensor with a Self-Complementary Fluorescent Reporter

Abstract

Design of rapid, selective, and sensitive DNA and ribonucleic acid (RNA) biosensors capable of minimizing false positives from nuclease degradation is crucial for translational research and clinical diagnostics. We present proof-of-principle studies of an innovative micro-ribonucleic acid (miRNA) reporter-probe biosensor that displaces a self-complementary reporter, while target miRNA binds to the probe. The freed reporter folds into a hairpin structure to induce a decrease in the fluorescent signal. The self-complementarity of the reporter facilitates the reduction of false positives from nuclease degradation. Nanomolar limits of detection and quantitation were capable with this proof-of-principle design. Detection of miRNA occurs within 10 min and does not require any additional hybridization, labeling, or rinsing steps. The potential for medical applications of the reporter-probe biosensor is demonstrated by selective detection of a cancer regulating microRNA, Lethal-7 (Let-7a). Mechanisms for transporting the biosensor across the cell membrane will be the focus of future work.

Toehold-mediated DNA displacement-based surface-enhanced Raman scattering DNA sensor utilizing an Au–Ag bimetallic nanodendrite substrate

“…The intensity decrease continued up to 60 min and no meaningful intensity change occurred afterward. This observation is analogous with those reported in previous publications [12,19] in which a double-strand was also pre-immobilized on a substrate surface before measurements; while, the displacement was completed approximately in 10 min when a double-strand was in a solution without immobilization on the surface [7,11,28]. The Raman spectrum acquired at 60 min of the displacement from the 200 pM sample (black) is also shown in Fig.…”

“…Due to these advantages, several biosensors based on toehold-mediated DNA displacement have been recently reported. Larkey et al [11] demonstrated a miRNA fluorescence sensor utilizing fluorescence quenching that occurs when two different dyes tagged at both ends of a freed reporter DNA became close by forming a hairpin structure (limit of detection (LOD): 10 nM). A quartz crystal microbalance (QCM)-based sensing scheme was also studied [12].…”

A DNA Circuit for IsomiR Detection

“…[1] miRNAs are often annotated as single sequences,a lthough many length and nucleotide variants of the same miRNA have been identified. [5] Numerous isothermal miRNA amplification methods have been developedt hat utilise the hybridisation chain reaction, [6] DNAzymes and enzymes. [3] The biologicals ignificance of miRNAs has stimulated the development of techniques in which thermally stable polymerases are used to replicate reverse-transcribedm iRNA by thermalc ycling.…”

Enhanced fluorescence detection of miRNA-16 on a photonic crystal

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We report a novel sensing method for fluorescence-labelled microRNAs (miRNAs) spotted on an all-dielectric photonic structure. [11][12][13][14][15] Regardless of the strategies adopted for sample preparation, it is nevertheless beneficial to have sensing transduction means capable of enhancing the measured signal at the detection stage. As a result, the Limit of Detection (LoD) is demonstrated to decrease by a factor of about 50.

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“…5 Detecting miRNAs is one of the new challenges of analytical technologies. [11][12][13][14][15] Regardless of the strategies adopted for sample preparation, it is nevertheless beneficial to have sensing transduction means capable of enhancing the measured signal at the detection stage. In an attempt to increase the specificity of recognition while decreasing the minimum detectable amount of miRNA (Limit of Detection, LoD), several detection approaches have been proposed in the past.…”