Dumbbell Dual-Hairpin Triggered DNA Nanonet Assembly for Cascade-Amplified Sensing of Exosomal MicroRNA

Abstract

Exosomal microRNAs (miRNAs) are valuable biomarkers closely associated with cancer progression. Therefore, sensitive and specific exosomal miRNA biosensing has been employed for cancer diagnosis, prognosis, and prediction. In this study, a miRNA-based DNA nanonet assembly strategy is proposed, enabling the biosensing of exosomal miRNAs through dumbbell dual-hairpin under isothermal enzyme-free conditions. This strategy dexterously designs a specific dumbbell dual-hairpin that can selectively recognize exosomal miRNA, inducing conformational changes to cascade-generated X-shaped DNA structures, facilitating the extension of the X-shaped DNA in three-dimensional space, ultimately forming a DNA nanonet assembly. On the basis of the target miRNA, our design enriches the fluorescence signal through the cascade assembly of DNA nanonet and realizes the secondary signal amplification. Using exosomal miR-141 as the target, the resultant fluorescence sensing demonstrates an impressive detection limit of 57.6 pM and could identify miRNA sequences with single-base variants with high specificity. Through the analysis of plasma and urine samples, this method effectively distinguishes between benign prostatic hyperplasia, prostate cancer, and metastatic prostate cancer. Serving as a novel noninvasive and accurate screening and diagnostic tool for prostate cancer, this dumbbell dual-hairpin triggered DNA nanonet assembly strategy is promising for clinical applications.

Scheme 1. Illustration of Our Strategy

(A) The process of our method implementation. (B) Dumbbell dual-hairpin triggered DNA nanonet assembly for cascade-amplified sensing of exosomal microRNA.

Figure 1

Verification of our design. (A) Polyacrylamide gel electrophoresis analysis. Lane 1: 20 bp DNA ladder; lane 2: H1; lane 3: T + H1; lane 4: H1 + H2; lane 5: T + H1 + H2; lane 6: H1 + H2 + H3; lane 7: T + H1 + H2 + H3; lane 8: H1 + H2 + H3 + H4; lane 9: T + H1 + H2 + H3 + H4. (B) Fluorescence spectra in the presence and absence of T. Concentrations: miR-141, 10 nM; H1, H2, H3, H4, 100 nM.

Figure 2

Optimization of important experimental factors. (A) Temperature optimization. (B) Time optimization. (C) Hairpin concentration. (D) pH optimization. Concentrations: miR-141, 10 nM; H1, H2, H3, H4, 100 nM. F and F₀ represent the fluorescence intensities of the probes in the presence and absence of miR-141, respectively.

Figure 3

Analytical performance of the experimental design. (A) Fluorescence spectra of various miR-141 concentrations. (B) The linear relationship between the FL intensity and the concentration of miR-141. (C) Specificity of our design for detection of different miRNA species. (D) Identification capacity of our method to detect miR-141 in a miRNA mixture. Concentrations: miR-141, 0 pM to 1 μM; H1, H2, H3, H4, 100 nM.

Figure 4

Exosomal characterization and quantitation of exosomal miR-141. (A) The workflow for utilizing exosomal miRNAs in our study. (B) Transmission electron microscope. (C) Western blotting. Exosomal concentrations of different cohorts in (D) urine and (E) plasma. Quantitation of exosomal miR-141 of different cohorts in (F) urine and (G) plasma by qRT-qPCR and our method. Concentrations: miR-141, 10 nM; H1, H2, H3, H4, 100 nM.