Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction

Abstract

In this work, we developed a novel, label-free, and enzyme-free strategy for the colorimetric detection of microRNA (miRNA), which relies on a target-catalyzed toehold-mediated strand displacement (TMSD) reaction. The system employs a detection probe that specifically binds to the target miRNA and sequentially releases a catalyst strand (CS) intended to trigger the subsequent TMSD reaction. Thus, the presence of target miRNA releases the CS that mediates the formation of an active G-quadruplex DNAzyme which is initially caged and inactivated by a blocker strand. In addition, a fuel strand that is supplemented for the recycling of the CS promotes another TMSD reaction, consequently generating a large number of active G-quadruplex DNAzymes. As a result, a distinct colorimetric signal is produced by the ABTS oxidation promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Based on this novel strategy, we successfully detected miR-141, a promising biomarker for human prostate cancer, with high selectivity. The diagnostic capability of this system was also demonstrated by reliably determining target miR-141 in human serum, showing its great potential towards real clinical applications. Importantly, the proposed approach is composed of separate target recognition and signal transduction modules. Thus, it could be extended to analyze different target miRNAs by simply redesigning the detection probe while keeping the same signal transduction module as a universal signal amplification unit, which was successfully demonstrated by analyzing another target miRNA, let-7d.

Three-Way Junction-Induced Isothermal Amplification with High Signal-to-Background Ratio for Detection of Pathogenic Bacteria

“…The limit of detection (LOD) was estimated to be 37.6 pM based on the definition of LOD = 3σ/S, where σ and S are the standard deviation of the blank and the slope of the linear relationship, respectively. This is comparable to those from previous strategies for the detection of target nucleic acids [ 5 , 41 , 42 , 43 , 44 , 45 , 46 ].…”

Principles and Applications of Nucleic Acid Strand Displacement Reactions

“…In another approach, RNA target molecules were used to trigger the formation of a Au-NP containing DNA network for colorimetric readout, which facilitated differentation between the 16S rRNAs of closely related bacteria with a detection limit of ≥5 × 10 5 CFU . Similar as for the HCR and CHA discussed above, also a large number of miRNA sensors have been developed (Figure ), which utilize strand displacement reactions in their sensing scheme, achieving detection of miRNA from the nanomolar down to the femtomolar range. ,− Of note, Zhang et al achieved detection of miRNA at a concentration of 67 aM using a DNA walker scheme, and Shen et al also used strand displacement techniques for sensitive miRNA imaging …”

The use of a 2-aminopurine-containing split G-quadruplex for sequence-specific DNA detection

“…The longer ones generated the slightly, reduced fluorescence signal, which is attributed to the protruding regions that might interfere with the effective formation of active G-quadruplex structure. That these observations are a consequence of target DNA interactions with the probe was evidenced by the results of polyacrylamide gel electrophoresis analysis [28,29]. As shown in Figure 3(b), the split G-rich segments in the presence of target DNA produce new electrophoretic bands, whose positions match those of the hybridization products of target DNA with the split segments (S1 and S2) of ASG.…”