Stimuli-Responsive Autonomous-Motion Molecular Machine for Sensitive Simultaneous Fluorescence Imaging of Intracellular MicroRNAs

Abstract

DNAzymes with enzymatic activity identified from random DNA pools by in vitro selection have recently attracted considerable attention. In this work, a DNAzyme-based autonomous-motion (AM) molecular machine is demonstrated for sensitive simultaneous imaging of different intracellular microRNAs (miRNAs). The AM molecular machine consists of two basic elements, one of which is a target-analogue-embedded double-stem hairpin substrate (TDHS) and the other is a locking-strand-silenced DNAzyme (LSDz). LSDz can be activated by target miRNA and catalytically cleave TDHS, generating Clv-TDHS and releasing free target analogue capable of triggering the next round of cleavage reaction. As such, the molecular machine can exert sustainable autonomous operation, producing an enhanced signal. Because the active target analogue comes from the machine itself and offers cyclical stimulation in a feedback manner, this target-induced autonomous cleavage circuit is termed a self-feedback circuit (SFC). The SFC-based molecular machine can be used to quantify miRNA-21 down to 10 pM without interference from nontarget miRNAs, indicating a substantial improvement in assay performance compared with its counterpart system without an SFC effect. Moreover, due to the enzyme-free process, the AM molecular machine is suitable for miRNA imaging in living cells, and the quantitative results are consistent with the gold standard PCR assay. More interestingly, the AM molecular machine can be used for the simultaneous fluorescence imaging of several intracellular miRNAs, enabling the accurate discrimination of cancerous cells (e.g., HeLa and MCF-7) from healthy cells. The SFC-based autonomous-motion machine is expected to be a promising tool for the research of molecular biology and early diagnosis of human diseases.

Self-Protected DNAzyme Walker with a Circular Bulging DNA Shield for Amplified Imaging of miRNAs in Living Cells and Mice

“…The detection limit was determined to be 10 pM by using 3σ method. Compared to the previous studies (SI Tables S4 and S5), ,− the proposed ES-AuNP system shows a relatively lower detection limit, demonstrating tremendous potential for application in the ultrasensitive detection of miR-21.…”

DNA Hairpins and Dumbbell-Wheel Transitions Amplified Walking Nanomachine for Ultrasensitive Nucleic Acid Detection

“…It is because the DNAzyme sequences are restricted in the loops of Probe D, inhibiting effective hybridization with Probe B. We have then drawn out DNAzyme sequence (Probe C) from Probe D to demonstrate the cleavage activity . After incubating Probe C with Probe B in the presence of Mg 2+ , the band of Probe B disappears, and two new bands with smaller molecule weights are observed, which are ascribed to the cleaved products of Probe B (lane f).…”

Programmable, Universal DNAzyme Amplifier Supporting Pancreatic Cancer-Related miRNAs Detection

“…For example, Lu et al [35] developed a signal amplification strategy based on DNAzyme catalytic cutting and the utilization of CHA to detect low concentrations of endogenous metal ions in cells. Furthermore, they also took advantage of DNAzyme to establish an aptamer sensing platform based on the combination of magnetic beads and DNAzyme for the highly sensitive detection of small molecules such as toxins [36,37]. Based on this, there is the potential to construct a universal DNAzyme amplifier through integration of the programmable nature of DNA nanodevices and the catalytic capability of DNAzyme, which can be conveniently applied for the detection of a wide range of pancreatic cancer-related miRNAs through reasonable sequence design.…”