Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In Vivo Using High-Intensity Focused Ultrasound

Abstract

Detecting metal ions in vivo with a high spatiotemporal resolution is critical to understanding the roles of the metal ions in both healthy and disease states. Although spatiotemporal controls of metal-ion sensors using light have been demonstrated, the lack of penetration depth in tissue and in vivo has limited their application. To overcome this limitation, we herein report the use of highintensity focused ultrasound (HIFU) to remotely deliver on-demand, spatiotemporally resolved thermal energy to activate the DNAzyme sensors at the targeted region both in vitro and in vivo. A Zn 2+selective DNAzyme probe is inactivated by a protector strand to block the formation of catalytic enzyme structure, which can then be activated by an HIFU-induced increase in the local temperature. With this design, Zn 2+ -specific fluorescent resonance energy transfer (FRET) imaging has been demonstrated by the new DNAzyme-HIFU probes in both HeLa cells and mice. The current method can be applied to monitor many other metal ions for in vivo imaging and medical diagnosis using metal-specific DNAzymes that have either been obtained or can be selected using in vitro selection.

Modular Engineering of DNAzyme-Based Sensors for Spatioselective Imaging of Metal Ions in Mitochondria

“…Of note, the fluorescence intensity of activated L-DZ gradually enhanced with increasing Zn 2+ concentration (Figure c and Figure S3). The limit of detection is calculated to be 0.2 μM (Figure S4), which is comparable to that of reported 17E DNAzyme sensors . As expected, unactivated L-DZ showed no obvious signal changes with the increasing Zn 2+ concentration (Figure S3).…”

“…The limit of detection is calculated to be 0.2 μM (Figure S4), which is comparable to that of reported 17E DNAzyme sensors. 27 As expected, unactivated L-DZ showed no obvious signal changes with the increasing Zn 2+ concentration (Figure S3). In addition, activated L-DZ showed good selectivity toward Zn 2+ with a negligible response to other metal ions (Figure 2d).…”

“…DNAzymes, a class of functional DNA with enzymatic activities, provide intriguing opportunities for detection of metal ions. − Recently, benefiting from the convenient synthesis and modification, flexible designability, and excellent biocompatibility, a variety of DNAzyme sensors have been developed for metal-ion imaging in living cells. − Nevertheless, such biosensors may nonspecifically respond to surrounding metal ions encountered in transit, leading to irreversible signal leakage. To address this limitation, several stimuli-responsive strategies have been developed for conditional control over the sensing function of DNAzyme sensors. − For example, we reported an endogenous enzyme-activated DNAzyme sensor to achieve cell-selective imaging of metal ions . In particular, photoactivation strategies with high spatiotemporal precision have been developed to control the imaging activity of DNAzyme sensors.…”

Near-Infrared-Light-Mediated DNA-Logic Nanomachine for Bioorthogonal Cascade Imaging of Endogenous Interconnected MicroRNAs and Metal Ions

“…Recent advances in DNA nanotechnology provide an unprecedented opportunity to construct DNA nanoprobes for the fluorescence imaging of miRNAs or metal ions individually. − Generally, miRNA imaging has predominantly relied on hybridization of the target miRNA to complementary DNA sequences embedded in the nanoprobes. On the other hand, DNAzymes, a class of functional DNA that possess metal-ion-dependent catalytic activity, have been extensively investigated as building units of DNA nanoprobes for metal ion imaging. − In addition, numerous signal amplification technologies, , including rolling circle amplification, , catalytic DNA hairpin assembly, − and hybridization chain reaction (HCR), − have been introduced for designing DNA nanoprobes for the amplified imaging of low abundance of endogenous miRNAs or metal ions in vitro and in vivo . Also, several approaches for controlling the intracellular activation of DNA nanoprobes by various internal or external stimuli have been reported for spatiotemporally controlled purposes. − More recently, multiplexed molecular imaging in vivo has been realized by integration with DNA logic gate systems. − Despite these successes, to our best knowledge, a multiplexed DNA nanoprobe for bioorthogonal controlled visualization of miRNAs and metal ions expressions within the context of in situ tumors is currently lacking.…”

Robust DNA Cross-Linked Polymeric Lighting-Up Nanogel Facilitates Sensitive Live Cell MicroRNA Imaging

“…In recent years, with the fast development of nucleic acid amplification technologies, they were extensively used to achieve low levels of biomarker detection in live cells. − Unlike enzymatic signal amplification methods that require the addition of exogenous enzymes and complex steps, , nonenzyme amplification approaches including DNAzyme, , catalytic hairpin assembly (CHA), , and hybridization chain reaction (HCR), , have evolved as efficient strategies for the visualization of intracellular RNAs. For instance, a branched HCR circuit pathway for mRNA signal amplification and imaging and different sensitive RNA imaging approaches in living cells based on enzyme-free amplification reactions , were reported recently.…”