Colorimetric and fluorescent dual-mode detection of microRNA based on duplex-specific nuclease assisted gold nanoparticle amplification.

The efficient and precise delivery of antisense oligonucleotides (ASOs) to target cells is of great value in gene silencing. However, the specificity and packaging capacity of delivery system still remains challenging. Here, we designed an i-motif forming-initiated in situ bipedal hybridization chain reaction (pH-Apt-BiHCR) amplification strategy for specific target cells imaging and enhanced gene delivery of ASOs. As a proof of concept, an 8-nt ASO modified with locked nucleic acid (LNA) which is complementary to the seed region of microRNA21 (miR-21) was used for gene silencing studies. Benefiting from the design of hairpin-contained i-motif, the stimuli-responsive assembly of pH-Apt-BiHCR was successfully achieved on MCF-7 cells surface based on the specific recognition of aptamer. Using this strategy, the pH-Apt-BiHCR not only contains repeated fluorescence resonance energy transfer (FRET) units for activatable tumor imaging with high contrast but also arrays with plenty of LNA ASOs as interference molecules for cancer cells inhibition. An in vitro assay showed that this strategy presented an excellent response ability in buffer within a narrow pH range (6.0–7.0) with a transition midpoint (pHT) of 6.44 ± 0.06. Moreover, live cell studies revealed that it realized a specific activatable imaging of target cells, while the ASOs arrayed pH-Apt-BiHCR exhibited improved internalization via an endocytosis pathway and enhanced gene silencing to MCF-7 cells compared to single ASO alone. We believe that this design will inspire the development of novel probes for early diagnosis and therapy of cancer cells

It has been reported that PIWI-interacting RNAs (piRNAs) play critical roles in activating invasion and metastasis, evading growth suppressors, and sustaining proliferative signaling of cancer and can be regarded as a novel biomarker candidate. Thus, it is necessary to develop an effective method for imaging and regulating cancer-related piRNAs to diagnose and treat cancers. Herein, we designed aptamer-functionalized activatable DNA tetrahedron nanoprobes (apt-ADTNs) to image and regulate endogenous piRNAs in cancer cells. As proof of concept, overexpressed piRNA-36026 in MCF-7 cells was used for this study. In brief, aptamer AS1411 and piRNA-36026 antisequence with Cy5 fluorescent dye are appended from the DNA tetrahedron; then, a short oligonucleotide with black hole quencher 2 (Q-oligo) is complementary with piRNA-36026 antisequence to quench the fluorescence of Cy5. The apt-ADTNs can recognize the MCF-7 cells through aptamer AS1411, and then enter the cells. Q-oligo is detached from the apt-ADTNs because of the binding between apt-ADTNs and piRNA-36026, leading to the recovery of the Cy5 fluorescence signal. Meanwhile, the hybridization of apt-ADTNs and piRNA-36026 results in down-regulating of dissociative piRNA-36026 in cytoplasm and the subsequent apoptosis of MCF-7 cells. As the achievement of synchronously imaging and regulating piRNA-36026 in MCF-7 cells, we believe that this design holds great promise in application of diagnosis and therapy for cancer.

The exhaustive investigating interactions between recognition probes and amyloid aggregates, especially simultaneous recognition events, are challenging and crucial for the design of biosensing probes and further diagnosis of amyloid diseases. In the present work, the interactions of aptamers (Apts) with β-amyloid (Aβ) aggregates were explored thoroughly by single-molecule force spectroscopy (SMFS). Indeed, it was found that the interaction of aptamer1 (Apt1)−amyloid aggregates was different from that of aptamer2 ( Apt2)−Aβ 40 aggregates at the single-molecule level. Especially, the interaction force of Apt1−Aβ 40 fibril showed a double distinguishing Gaussian fitting. The only unimodal distribution of the force histogram was displayed for the interactions of Apt2−Aβ 40 oligomer, Apt2−Aβ 40 fibril, and Apt1−Aβ 40 oligomer. More intriguingly, two Apts could bind to amyloid aggregates simultaneously.With the assistance of two Apts recognition, a novel sensitive dual Apt-based surface plasmon resonance (SPR) sensor using Au nanoparticles (AuNPs) was developed for quantifying Aβ 40 aggregates. The dual Apt-based SPR sensor not only avoided the limitation of steric hindrance and epitope but also employed simple operation as well as inexpensive recognition probes. A detection limit as low as 0.2 pM for Aβ 40 oligomer and 0.05 pM for Aβ 40 fibril could be achieved. Moreover, the established sensor could be successfully applied to detect Aβ 40 aggregates in artificial cerebrospinal fluid (CSF) and undiluted real CSF. This work could provide a strategy to monitor a simultaneous recognition event using SMFS and broaden the application of Apts in the diagnosis of neurodegenerative diseases.