Site-Specific Scissors Based on Myeloperoxidase for Phosphorothioate DNA

Methods to detect and quantify disease biomarkers with high specificity and sensitivity in biological fluids play a key role in enabling clinical diagnosis, including point-of-care testing. Myeloperoxidase (MPO) is an emerging biomarker for the detection of inflammation, neurodegenerative diseases, and cardiovascular disease, where excess MPO can lead to oxidative damage to biomolecules in homeostatic systems. While numerous methods have been developed for MPO analysis, most techniques are challenging in clinical applications due to the lack of amplification methods, high cost, or other practical drawbacks. Enzyme-linked immunosorbent assays are currently used for the quantification of MPO in clinical practice, which is often limited by the availability of antibodies with high affinity and specificity and the significant nonspecific binding of antibodies to the analytical surface. In contrast, nucleic acid-based biosensors are of interest because of their simplicity, fast response time, low cost, high sensitivity, and low background signal, but detection targets are limited to nucleic acids and non-nucleic acid biomarkers are rare. Recent studies reveal that the modification of a genome in the form of phosphorothioate is specifically sensitive to the oxidative effects of the MPO/H 2 O 2 /Cl − system. We developed an oxidative cleavage-based threedimensional DNA biosensor for rapid, ratiometric detection of HOCl and MPO in a "one-pot" method, which is simple, stable, sensitive, specific, and time-saving and does not require a complex reaction process, such as PCR and enzyme involvement. The constructed biosensor has also been successfully used for MPO detection in complex samples. This strategy is therefore of great value in disease diagnosis and biomedical research.

mentioning

confidence: 99%

Oxidative Cleavage-Based Three-Dimensional DNA Biosensor for Ratiometric Detection of Hypochlorous Acid and Myeloperoxidase

Wang

et al. 2021

“…With the successful evaluation of sequence-mimic switches, we then moved to the optimization of the actual enzyme reaction-assisted programmable transcriptional switch module (Figures S2 and S3). The site-specific cleavage reaction for PT modification includes three components: MPO, Cl − , and 28 and according to these optimization results, we could confirm that the MPO−H 2 O 2 enzymatic cleavage system could successfully be implemented and further used for transcriptional switches. We designed a set of transcriptional switches that share the same loop structure with a hidden T7-RNAP promoter sequence and the same stem structure with a PTmodified triplex sequence (Figure 2e).…”

Section: ■ Experimental Sectionmentioning

confidence: 59%

“…27 Meanwhile, MPO, a mammalian enzyme known to oxidize Cl − to HClO at cytoplasm conditions, enables a site-specific cleaved phosphorothioate (PT)-modified substrate under certain conditions in the cell. 28 The enzymatic system would provide an endogenous tool box for the orthogonally activatable DNA switches to directly study external stimulations and the cell signal network. As shown in Figure 1a (left), these strands are rationally designed so that the structure of triplex DNA with PT modification prevents efficient transcription of FLAP due to the incomplete formation of the promoter sequence.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Enzyme Reaction-Assisted Programmable Transcriptional Switches for Bioactive Molecule Detection

Tang,

Chen,

et al. 2023

Bioactive molecules are highly worthwhile to recognize and explore the latent pathogenic mechanism. Conventional methods for bioactive molecule detection, including mass spectrometry and fluorescent probe imaging, are limited due to the complex processing and signal interference. Here, we designed enzyme-reaction-assisted programmable transcriptional switches for the detection of bioactive molecules. The approach is based on the use of programmable enzyme site-specific cleavage-assisted DNA triplex-based conformational switches that, upon responding to bioactive molecules, can trigger the transcription of fluorescent light-up aptamers. Thanks to the programmable nature of the sensing platform, the method can be adapted to different bioactive molecules, and we demonstrated the enzyme-small molecule catalytic reaction combination of myeloperoxidase (MPO)–hydrogen peroxide (H2O2) as a model that transcriptional switches was capable of detecting H2O2 and possessed the specificity and anti-interference ability in vitro. Furthermore, we successfully applied the switches into cells to observe the detection feasibility in vivo, and dynamically monitored changes of H2O2 in cellular oxidative stress levels. Therefore, we attempt to amalgamate the advantages of enzyme reaction with the pluripotency of programmable transcriptional switches, which can take both fields a step further, which may promote the research of biostimuli and the construction of DNA molecular devices.

“…To maintain more general bioanalytical applicability, the bio-friendly luminol/reactive oxygen species (ROS) system based on dissolved oxygen with better stability would provide a promising strategy. ,− ROS is generated from the electrochemical reduction reaction of dissolved oxygen and water. These ROS species promote the generation of the excited state of luminol by accelerating the formation of endoperoxides, which emit photons with an enhancement of ECL intensity. ,, Previously, ultrasound (US), as a nondestructive and biologically friendly technology, can split water into ROS in situ .…”

Section: Introductionmentioning

confidence: 99%

Self-Enhanced Electrochemiluminescence Imaging System Based on the Accelerated Generation of ROS under Ultrasound

Zhu

Jialin

et al. 2023

Electrochemiluminescence (ECL) imaging, as an optical technology, has been developed at full tilt in the field of life science and nanomaterials. However, the relatively low ECL intensity or the high co-reactant concentration needed in the electrochemical reaction blocks its practical application. Here, we developed an ECL imaging system based on the rGO-TiO2–x composite material, where the co-reactant, reactive oxygen species (ROS), is generated in situ under the synergetic effect of of ultrasound (US) and electric irradiation. The rGO-TiO2–x composites facilitate the separation of electron (e–) and hole (h+) pairs and inhibit recombination triggered by external US irradiation due to the high electroconductivity of rGO and oxygen-deficient structures of TiO2, thus significantly boosting ROS generation. Furthermore, the increased defects on rGO accelerate the electron transfer rate, improving the electrocatalytic performance of the composite and forming more ROS. This high ultrasonic–electric synergistic efficacy is demonstrated through the enhancement of photon emission. Compared with the luminescence intensity triggered by US irradiation and electric field, an enhancement of ∼20-fold and 10-fold of the US combined with electric field-triggered emission is observed from this composite. Under the optimized conditions, using dopamine (DA) as a model target, the sensitivity of the US combined ECL strategy for detection of DA is two orders of magnitude higher than that of the ECL method. The successful detection of DA at low concentrations makes us believe that this strategy provides the possibility of applying ECL imaging for cellular single-molecule analysis and cancer therapy.