Research

Neutrophils release neutrophil extracellular traps (NETs) to neutralize infections, a process that also contributes to immunothrombosis. While beneficial in localized infections, excessive NET formation can lead to widespread coagulopathy and organ failure. While the roles of NET-associated proteins such as histones in immunothrombosis are well characterized, NET-derived DNAs are much less known. To address this issue, we report herein the direct interaction between thrombin and DNA scaffolds and further, the discovery of short tandem repeats of single-stranded (ATTCC)n in NETs that selectively bind thrombin, a crucial enzyme involved in both blood clot formation and immune response. We have also developed a strategy of selective targeting ss(ATTCC)n using antisense locked nucleic acids (LNAs), effectively disrupting NET-thrombin interactions. This discovery reveals an unexplored role of ssDNA within NETs and provides a novel avenue for developing targeted therapeutic interventions for immunothrombosis-related disorders.

Potassium ions (K+) within the tumor microenvironment, along with dysregulation of K+ channels, play critical roles in supporting cancer cell survival and preventing their elimination. Directly monitoring changes in K+ homeostasis within cancer cells is invaluable for understanding these processes. However, achieving high selectivity over other biological metal ions, a detection dynamic range that aligns with intracellular K+ levels, and broad accessibility to research laboratories remain technically challenging for current K+ imaging probes. In this study, we report the in vitro selection of the first K+-specific RNA-cleaving DNAzyme and the development of a K+-specific DNAzyme fluorescent sensor with exceptional selectivity, achieving over 1000-fold selectivity against Na+ and more than 100-fold selectivity over other major biologically relevant metal ions. This sensor has an apparent dissociation constant (105 mM) that is close to the intracellular level of K+, and it has a broad detection range from 21 to 200 mM K+. Using this tool, we reveal a progressive decline in intracellular K+ levels in breast cancer cells with more advanced progression states. Moreover, we demonstrate that elevated extracellular K+ levels interfere with the efficacy of anticancer compounds like ML133 and Amiodarone, suggesting an underappreciated role of microenvironmental K+ in chemoresistance. Notably, blocking the Kir2.1 channel activity restored treatment sensitivity, presenting a potential strategy to overcome chemoresistance in aggressive cancers. These findings underscore the role of K+ homeostasis in tumor progression and support further exploration of ion-channel-targeted cancer therapies.

Detecting Coenzyme A (CoA) in cells is vital for understanding its role in metabolism. DNA aptamers, though widely used for monitoring many other molecules, have not been effective for CoA detection, as previous attempts at obtaining DNA aptamers for CoA using SELEX resulted in aptamers that only recognize the adenine moiety of CoA. This “tyranny” of adenine dominating in SELEX has, therefore, hampered the SELEX of aptamers specific for CoA. To meet this challenge, we employed a capture SELEX method by incorporating rigorous counter selections against adenine, adenosine, ATP, pantetheine, and pantothenic acid, resulting in a highly specific DNA aptamer for CoA over adenosine, ATP and other related metabolites such as NADH, with a dissociation constant of 48.9 μM. This aptamer was then converted to a fluorescent sensor for CoA across pH 6.4-8.0. Confocal microscopy showed its ability to visualize CoA in living cells, with fluorescence changes observed upon manipulating CoA levels. This method broadens SELEX's application and presents a promising approach for studying and understanding CoA dynamics.

In living organisms, cells synergistically couple cascade reaction pathways to achieve inter- and intracellular signal transduction by transmembrane protein receptors. The construction and assembly of synthetic receptor analogs that can mimic such biological processes is a central goal of synthetic biochemistry and bionanotechnology to endow receptors with user-defined signal transduction effects. However, designing artificial transmembrane receptors with the desired input, output, and performance parameters are challenging. Here we show that the dimerization of synthetic transmembrane DNA receptors executes a systematically engineered sensing and actuation cascade in response to external molecular signals. The synthetic DNA receptors are composed of three parts, including an extracellular signal reception part, a lipophilic transmembrane anchoring part, and an intracellular signal output part. Upon the input of external signals, the DNA receptors can form dimers on the cell surface triggered by configuration changes, leading to a series of downstream cascade events including communication between donor and recipient cells, gene transcription regulation, protein level control, and cell apoptosis. We believe this work establishes a flexible cell surface engineering strategy that is broadly applicable to implement sophisticated biological functions.

Glycosylated RNAs (glycoRNAs) have recently emerged as a new class of molecules of substantial interest owing to their potential roles in cellular processes and diseases. However, studying glycoRNAs is challenging owing to the lack of effective research tools including, but not limited to, imaging techniques to study the spatial distribution of glycoRNAs. Recently, we reported the development of a glycoRNA imaging technique, called sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA), to visualize sialic acid-containing glycoRNAs with high sensitivity and specificity. Here we describe the experimental design principles and detailed step-by-step procedures for ARPLA-assisted glycoRNA imaging across multiple cell types. The procedure includes details for target selection, oligo design and preparation, optimized steps for RNA in situ hybridization, glycan recognition, proximity ligation, rolling circle amplification and a guideline for image acquisition and analysis. With properly designed probe sets and cells prepared, ARPLA-based glycoRNA imaging can typically be completed within 1 d by users with expertise in biochemistry and fluorescence microscopy. The ARPLA approach enables researchers to explore the spatial distribution, trafficking and functional contributions of glycoRNAs in various cellular processes.

The global demand for lithium has soared in recent years due to the wide use of lithium batteries. To meet this demand, we herein report developing novel on-site sample preparation methods for the extraction of Li+ from relevant materials, including brine water, spodumene rock, as well as lithium-ion battery electrodes, and a DNAzyme-based fluorescent sensor for sensitive and robust detection of Li+ in these samples down to 1.4 mM (10 ppm) using a portable fluorometer. The system can distinguish key threshold lithium levels that indicate economic value across several industries, including 200 ppm Li+ for brine mining, 6 % Li2O or SC6 for rock mining, and Li+-specific aging in LIBs. The methods developed and demonstrated in this work will allow highly selective, on-site, portable detection of lithium in both environmental samples to identify new lithium resources and in battery electrodes to guide recycling strategies in order to meet the global demand for lithium. 

Transmembrane channels play a vital role in regulating the permeation process, and have inspired recent development of biomimetic channels. Herein, we report a class of artificial biomimetic nanochannels based on DNAzyme-functionalized glass nanopipettes to realize delicate control of channel permeability, whereby the surface wettability and charge can be tuned by metal ions and DNAzyme-substrates, allowing reversible conversion between different permeability states. We demonstrate that the nanochannels can be reversibly switched between four different permeability states showing distinct permeability to various functional molecules. By embedding the artificial nanochannels into the plasma membrane of single living cells, we achieve selective transport of dye molecules across the cell membrane. Finally, we report on the advanced functions including gene silencing of miR-21 in single cancer cells and selective transport of Ca2+ into single PC-12 cells. In this work, we provide a versatile tool for the design of rectifying artificial nanochannels with on-demand functions.

Technologies that may visualize and control cell responses are in increasing demand, among which cell surface engineering and intracellular bioimaging are the most impactful directions. Both intracellular and extracellular microenvironments are highly heterogeneous, in which different types of proteins, glycans, lipid molecules, genetic molecules, inorganic molecules, and metal ions are present. Similar to protein enzymes, most DNAzymes exhibit enzymatic activity in the presence of specific metal ions. DNAzymes appear to be a good choice for the manipulation and bioimaging of living cells. By modifying cell surfaces and intracellular mitochondria with engineered DNAzyme molecular machines, both inter- and intra-cellular regulations with programmable selectivity can be achieved. Amplification techniques, such as catalytic and molecular beacons or DNA walkers, which utilize the enzymatic turn-over property of DNAzyme, and catalytic hairpin assembly were used for amplifying the signals and lowering the detection limit of DNAzyme sensors.