ABSTRACT
DNAzymes have emerged as an important class of sensors for a wide variety of metal ions, with florescence DNAzyme sensors as the most widely used in different sensing and imaging applications because of their fast response time, high signal intensity, and high sensitivity. However, the requirements of an external excitation light source and its associated power increase the cost and size of the fluorometer, making it difficult to be used for portable detections. To overcome these limitations, we report herein a DNAzyme sensor that relies on chemiluminescence resonance energy transfer (CRET) without the need for external light. The sensor is constructed by combining the functional motifs from both Pb2+-dependent 8-17 DNAzyme conjugated to fluorescein (FAM) and hemin/G-quadruplex that mimics horseradish peroxidase to catalyze the oxidation of luminol by H2O2 to yield chemiluminescence. In the absence of Pb2+, the hybridization between the enzyme and substrate strands bring the FAM and hemin/G-quadruplex in close proximity, resulting in CRET. The presence of Pb2+ ions can drive the cleavage on the substrate strand, resulting in a sharp decrease in the melting temperature of hybridization and thus separation of the FAM from hemin/G-quadruplex. The liberated CRET pair causes a ratiometric increase in the donor's fluorescent signal and a decrease in the acceptor signal. Using this method, Pb2+ ions have been measured rapidly (<15 min) with a low limit of detection at 5 nM. By removing the requirement of exogenous light excitation, we have demonstrated a simple and portable detection using a smartphone, making the DNAzyme-CRET system suitable for field tests of lake water. Since DNAzymes selective for other metal ions or targets, such as bacteria, can be obtained using in vitro selection, the method reported here opens a new avenue for rapid, portable, and ratiometric detection of many targets in environmental monitoring, food safety, and medical diagnostics.
Subject(s)
Biosensing Techniques , DNA, Catalytic , G-Quadruplexes , DNA, Catalytic/metabolism , Energy Transfer , Hemin , Hydrogen Peroxide , Ions , LuminescenceABSTRACT
Metal ions can be beneficial or toxic depending on their identity, oxidation state, and concentration. Therefore, the ability to detect and quantify different types of metal ions using portable sensors or in situ imaging agents is important for better environmental monitoring, in vitro medical diagnostics, and imaging of biological systems. While numerous metal ions in different oxidation states are present in the environment and biological systems, only a limited number of them can be detected effectively using current methods. In this Account, we summarize research results from our group that overcome this limitation by the development of a novel class of activity-based sensors based on metal-dependent DNAzymes, which are DNA molecules with enzymatic activity. First, we have developed an in vitro selection method to obtain DNAzymes from a large DNA library of up to 1015 sequences that can carry out cleavage of an oligonucleotide substrate only in the presence of a specific metal ion with high selectivity. Negative selection steps can further be used to improve the selectivity against potentially competing targets by removing sequences that recognize the competing metal ions. Second, we have developed a patented catalytic beacon method to transform the metal-dependent DNAzyme cleavage reaction into a turn-on fluorescent signal by attaching a fluorophore and quenchers to the DNAzyme complex. Because of the difference in the melting temperatures of DNA hybridization before and after metal-ion-dependent cleavage of the DNAzyme substrate, the fluorophore on the DNA cleavage product can be released from its quenchers to create a turn-on fluorescent signal. Because DNAzymes are easy to conjugate with other signaling moieties, such as gold nanoparticles, lanthanide-doped upconversion nanoparticles, electrochemical agents, and gadolinium complexes, these DNAzymes can also readily be converted into colorimetric sensors, upconversion luminescence sensors, electrochemical sensors, or magnetic resonance contrast agents. In addition to describing recent progress in developing and applying these metal ion sensors for environmental monitoring, point-of-care diagnostics, cellular imaging, and in vivo imaging in zebrafish, we summarize major advantages of this class of activity-based sensors. In addition to advantages common to most activity-based sensors, such as enzymatic turnovers that allow for signal amplification and the use of initial rates instead of absolute signals for quantification to avoid interferences from sample matrices, the DNAzyme-based sensors allow for in vitro selection to expand the method to almost any metal ion under a variety of conditions, negative selection to improve the selectivity against competing targets, and reselection of DNAzymes and combination of active and inactive variants to fine-tune the dynamic range of detection. The use of melting temperature differences to separate target binding from signaling moieties in the catalytic beacon method allows the use of different fluorophores and nanomaterials to extend the versatility and modularity of this sensing platform. Furthermore, sensing and imaging artifacts can be minimized by using an inactive mutant DNAzyme as a negative control, while spatiotemporal control of sensing/imaging can be achieved using optical, photothermal, and endogenous orthogonal caging methods. Finally, current challenges, opportunities, and future perspectives for DNAzymes as activity-based sensors are also discussed.
Subject(s)
Biosensing Techniques/methods , DNA, Catalytic/metabolism , Metals/analysis , Animals , Base Sequence , DNA, Catalytic/genetics , Environmental Monitoring , Humans , Metals/chemistryABSTRACT
Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal-binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg2+ -specific 10-23 or Zn2+ -specific 8-17 RNA-cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg2+ or Zn2+ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein-based sensors.
Subject(s)
Biosensing Techniques/methods , DNA, Catalytic/metabolism , Diagnostic Imaging/methods , Ions/chemistry , Metals/chemistry , HumansABSTRACT
Many different metal ions are involved in various biological functions including metallomics and trafficking, and yet there are currently effective sensors for only a few metal ions, despite the first report of metal sensors for calcium more than 40 years ago. To expand upon the number of metal ions that can be probed in biological systems, we and other laboratories employ the in vitro selection method to obtain metal-specific DNAzymes with high specificity for a metal ion and then convert these DNAzymes into fluorescent sensors for these metal ions using a catalytic beacon approach. In this Forum Article, we summarize recent progress made in developing these DNAzyme sensors to probe metal ions in living cells and in vivo, including several challenges that we were able to overcome for this application, such as DNAzyme delivery, spatiotemporal control, and signal amplification. Furthermore, we have identified a key remaining challenge for the quantitative detection of metal ions in living cells and present a new design and the results of a Förster resonance energy transfer (FRET)-based DNAzyme sensor for the ratiometric quantification of Zn2+ in HeLa cells. By converting existing DNAzyme sensors into a ratiometric readout without compromising the fundamental catalytic function of the DNAzymes, this FRET-based ratiometric DNAzyme design can readily be applied to other DNAzyme sensors as a major advance in the field to develop much more quantitative metal-ion probes for biological systems.
Subject(s)
DNA, Catalytic/chemistry , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Metals/analysis , DNA, Catalytic/metabolism , HeLa Cells , Humans , Ions/analysis , Ions/metabolism , Metals/metabolismABSTRACT
Bioorthogonal control of metal-ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18-base pair recognition site of a homing endonuclease (I-SceI), which is found by chance only once in 7×1010 â bp of genomic sequences, and can thus form a near bioorthogonal pair with I-SceI for DNAzyme activation with minimal effect on living cells. Once I-SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg2+ to release the fluorophore-labeled DNA fragment and produce a fluorescent turn-on signal for Mg2+ . Thus I-SceI bioorthogonally activates the 10-23 DNAzyme for imaging of Mg2+ in HeLa cells.
Subject(s)
Biosensing Techniques/methods , DNA, Catalytic/metabolism , Fluorescent Dyes/chemistry , Magnesium/analysis , Magnesium/chemistry , Molecular Imaging/methods , Catalysis , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/metabolism , HeLa Cells , Humans , Spectrometry, FluorescenceABSTRACT
Manganese is an essential trace element in the human body that acts as a cofactor in many enzymes and metabolisms. It is important to develop methods to detect Mn2+ in living cells. While fluorescent sensors have been very effective in detecting other metal ions, Mn2+-specific fluorescent sensors are rarely reported due to nonspecific fluorescence quenching by the paramagnetism of Mn2+ and poor selectivity against other metal ions such as Ca2+ and Mg2+. To address these issues, we herein report in vitro selection of an RNA-cleaving DNAzyme with exceptionally high selectivity for Mn2+. Through converting it into a fluorescent sensor using a catalytic beacon approach, Mn2+ sensing in immune cells and tumor cells has been achieved. The sensor is also used to monitor degradation of manganese-based nanomaterials such as MnOx in tumor cells. Therefore, this work provides an excellent tool to detect Mn2+ in biological systems and monitor the Mn2+-involved immune response and antitumor therapy.
ABSTRACT
Visualizing redox-active metal ions, such as Fe2+ and Fe3+ ions, are essential for understanding their roles in biological processes and human diseases. Despite the development of imaging probes and techniques, imaging both Fe2+ and Fe3+ simultaneously in living cells with high selectivity and sensitivity has not been reported. Here, we selected and developed DNAzyme-based fluorescent turn-on sensors that are selective for either Fe2+ or Fe3+, revealing a decreased Fe3+/Fe2+ ratio during ferroptosis and an increased Fe3+/Fe2+ ratio in Alzheimer's disease mouse brain. The elevated Fe3+/Fe2+ ratio was mainly observed in amyloid plaque regions, suggesting a correlation between amyloid plaques and the accumulation of Fe3+ and/or conversion of Fe2+ to Fe3+. Our sensors can provide deep insights into the biological roles of labile iron redox cycling.
Subject(s)
Alzheimer Disease , Mice , Animals , Humans , Alzheimer Disease/diagnostic imaging , Iron , Metals , Brain/diagnostic imaging , Brain/metabolism , Plaque, Amyloid , Amyloid beta-Peptides/metabolismABSTRACT
Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, causing delays in treatment and further spread of the virus. Developing new sensors that can inform on the infectability of clinical or environmental samples will meet this unmet challenge. However, very few methods can obtain sensing molecules that can recognize an intact infectious virus and differentiate it from the same virus that has been rendered non-infectious by disinfection methods. Here, we describe a protocol to select aptamers that can distinguish infectious viruses vs non-infectious viruses using systematic evolution of ligands by exponential enrichment (SELEX). We take advantage of two features of SELEX. First, SELEX can be tailor-made to remove competing targets, such as non-infectious viruses or other similar viruses, using counter selection. Additionally, the whole virus can be used as the target for SELEX, instead of, for example, a viral surface protein. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need to disrupt of the virus. This method thus allows recognition agents to be obtained based on functional differences in the surface of pathogens, which do not need to be known in advance.
Subject(s)
Aptamers, Nucleotide , Virus Diseases , Viruses , Aptamers, Nucleotide/metabolism , Humans , Ligands , Membrane Proteins , SELEX Aptamer Technique/methods , Viruses/metabolismABSTRACT
Viral infections are a major global health issue, but no current method allows rapid, direct, and ultrasensitive quantification of intact viruses with the ability to inform infectivity, causing misdiagnoses and spread of the viruses. Here, we report a method for direct detection and differentiation of infectious from noninfectious human adenovirus and SARS-CoV-2, as well as from other virus types, without any sample pretreatment. DNA aptamers are selected from a DNA library to bind intact infectious, but not noninfectious, virus and then incorporated into a solid-state nanopore, which allows strong confinement of the virus to enhance sensitivity down to 1 pfu/ml for human adenovirus and 1 × 104 copies/ml for SARS-CoV-2. Applications of the aptamer-nanopore sensors in different types of water samples, saliva, and serum are demonstrated for both enveloped and nonenveloped viruses, making the sensor generally applicable for detecting these and other emerging viruses of environmental and public health concern.
ABSTRACT
Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal-binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg2+-specific 10-23 or Zn2+-specific 8-17 RNA-cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg2+ or Zn2+ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein-based sensors.
ABSTRACT
Fluorescent aptamer sensors have shown enormous potential for intracellular imaging of small molecule metabolites. Since metabolites distribute differently at different subcellular locations and their concentrations and locations fluctuate with time, methods are needed for spatiotemporally controlled monitoring of these metabolites. Built upon previous success in temporal control of aptamer-based sensors, we herein report an aptamer sensor containing a photocleavable linker and using DQAsomes to target mitochondria for spatiotemporally controlled monitoring of ATP in the mitochondria of living cells. The photocleavable modification on the DNA ATP aptamer sensor can prevent sensor activation before reaching mitochondria and the sensor can then be activated upon light irradiation. The sensor has a detection limit of 3.7 µM and high selectivity against other nucleotides, allowing detection of ATP concentration fluctuations in mitochondria induced by Ca2+ or oligomycin. This work represents the first successful delivery of a DNA aptamer sensor to mitochondria, providing a new platform for targeted delivery to subcellular organelles for monitoring energy producing processes, as well as mitochondrial dysfunction-related diseases in different cells.