FINDER: A Fluidly Confined CRISPR-Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification.

The accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the naturally confined enzymes on fluid cell membranes, a fluidly confined CRISPR-based DNA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR-Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamer-based DNA logic gate for cell recognition, thus a FINDER system capable of accurate, rapid and sensitive cancer cell identification was constructed. The FINDER rapidly identified target cells in only 20 min, and achieved over 80 % recognition efficiency with only 0.1 % of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.

A Semisynthetic Bioluminescence Sensor for Ratiometric Imaging of Metal Ions In Vivo Using DNAzymes Conjugated to An Engineered Nano-Luciferase.

DNA-based probes have gained significant attention as versatile tools for biochemical analysis, benefiting from their programmability and biocompatibility. However, most existing DNA-based probes rely on fluorescence as the signal output, which can be problematic due to issues like autofluorescence and scattering when applied in complex biological materials such as living cells or tissues. Herein, we report the development of bioluminescent nucleic acid (bioLUNA) sensors that offer laser excitation-independent and ratiometric imaging of the target in vivo. The system is based on computational modelling and mutagenesis investigations of a genetic fusion between circular permutated Nano-luciferase (NLuc) and HaloTag, enabling the conjugation of the protein with a DNAzyme. In the presence of Zn2+ , the DNAzyme sensor releases the fluorophore-labelled strand, leading to a reduction in bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. Consequently, this process induces ratiometric changes in the bioluminescent signal. We demonstrated that this bioLUNA sensor enabled imaging of both exogenous Zn2+ in vivo and endogenous Zn2+ efflux in normal epithelial prostate and prostate tumors. This work expands the DNAzyme sensors to using bioluminescence and thus has enriched the toolbox of nucleic acid sensors for a broad range of biomedical applications.

A Reaction-Based Ratiometric Bioluminescent Platform for Point-of-Care and Quantitative Detection Using a Smartphone.

Fluorescent probes have emerged as powerful tools for the detection of different analytes by virtue of structural tenability. However, the requirement of an excitation source largely hinders their applicability in point-of-care detection, as well as causing autofluorescence interference in complex samples. Herein, based on bioluminescence resonance energy transfer (BRET), we developed a reaction-based ratiometric bioluminescent platform, which allows the excitation-free detection of analytes. The platform has a modular design consisting of a NanoLuc-HaloTag fusion as an energy donor, to which a synthetic fluorescent probe is bioorthogonally labeled as recognition moiety and energy acceptor. Once activated by the target, the fluorescent probe can be excited by NanoLuc to generate a remarkable BRET signal, resulting in obvious color changes of luminescence, which can be easily recorded and quantitatively analyzed by a smartphone. As a proof of concept, a fluorescent probe for HOCl was synthesized to construct the bioluminescent system. Results demonstrated the system showed a constant blue/red emission ratio which is independent to the signal intensity, allowing the quantification of HOCl concentration with high sensitivity (limit of detection (LOD) = 13 nM) and accuracy. Given the universality, this reaction-based bioluminescent platform holds great potential for point-of-care and quantitative detection of reactive species.

DNA-Templated Anchoring of Proteins for Programmable Cell Functionalization and Immunological Response.

Membrane protein engineering exhibits great potential for cell functionalization. Although genetic strategies are sophisticated for membrane protein engineering, there still exist some issues, including transgene insertional mutagenesis, laborious, complicated procedures, and low tunability. Herein, we report a DNA-templated anchoring of exogenous proteins on living cell membranes to realize programmable functionalization of living cells. Using DNA as a scaffold, the model cell membranes are readily modified with proteins, on which the density and ratio of proteins as well as their interactions can be precisely controlled through predictable DNA hybridization. Then, the natural killer (NK) cells were engineered to gain the ability to eliminate the immune checkpoint signaling at the NK-tumor synapse, which remarkably promoted NK cell activation in immunotherapy. Given the versatile functions of exogenous proteins and flexible designs of programmable DNA, this method has the potential to facilitate membrane-protein-based cell engineering and therapy.

Ratiometric Detection of Zn2+ Using DNAzyme-Based Bioluminescence Resonance Energy Transfer Sensors.

While fluorescent sensors have been developed for monitoring metal ions in health and diseases, they are limited by the requirement of an excitation light source that can lead to photobleaching and a high autofluorescence background. To address these issues, bioluminescence resonance energy transfer (BRET)-based protein or small molecule sensors have been developed; however, most of them are not highly selective nor generalizable to different metal ions. Taking advantage of the high selectivity and generalizability of DNAzymes, we report herein DNAzyme-based ratiometric sensors for Zn2+ based on BRET. The 8-17 DNAzyme was labeled with luciferase and Cy3. The proximity between luciferase and Cy3 permiQed BRET when coelenterazine, the substrate for luciferase, was introduced. Adding samples containing Zn2+ resulted in a cleavage of the substrate strand, causing dehybridization of the DNAzyme construct, thus increasing the distance between Cy3 and luciferase and changing the BRET signals. Using these sensors, we detected Zn2+ in serum samples and achieved Zn2+ detection with a smartphone camera. Moreover, since the BRET pair is not the component that determines the selectivity of the sensors, this sensing platform has the potential to be adapted for the detection of other metal ions with other metal-dependent DNAzymes.

Precisely controlling the cellular internalization of DNA-decorated semiconductor polymer nanoparticles for drug delivery.

Nonspecific adhesivity of nanoparticles to cells is regarded as a significant issue of nanomedicine, which brings about many serious drawbacks in applications, including low detection sensitivity, non-targeted biotoxicity and poor diagnostic accuracy. Here, we propose for the first time, DNA-decorated semiconductor polymer nanoparticles (SPN-DNAs), whose adhesivity can be significantly alleviated by controlling the density and thickness of DNA layers. This property is demonstrated to be independent of external conditions such as temperature, concentration, incubation time, ionic strength and cell lines. The mechanism of this phenomenon is also discussed. Finally, based on minimized nonspecific adhesivity to cells, a triggered nanoswitch can be constructed to control cellular internalization and drug delivery.

Identification of PKS-NRPS Hybrid Metabolites in Marine-Derived Penicillium oxalicum.

Filamentous fungi are abundant resources of bioactive natural products. Here, 151 marine-derived fungi were collected from the north Yellow Sea and identified by an internal transcribed spacer (ITS) sequence. The crude extracts of all strains were evaluated for their antimicrobial activities and analyzed by HPLC fingerprint. Based on these, strain Penicillium oxalicum MEFC104 was selected for further investigation. Two new polyketide-amino acid hybrid compounds with feature structures of tetramic acid, oxopyrrolidine A and B, were isolated. Their planner structures were assigned by HRESIMS and 1D/2D NMR experiments. The absolute configurations were determined by modified Mosher's method, J-based configuration analysis, and ECD calculations. Furthermore, the biosynthetic pathway was identified by bioinformatic analysis and gene-deletion experiments. This study established a link between oxopyrrolidines and the corresponding biosynthesis genes in P. oxalicum.

A synergistic strategy to develop photostable and bright dyes with long Stokes shift for nanoscopy.

The quality and application of super-resolution fluorescence imaging greatly lie in the dyes' properties, including photostability, brightness, and Stokes shift. Here we report a synergistic strategy to simultaneously improve such properties of regular fluorophores. Introduction of quinoxaline motif with fine-tuned electron density to conventional rhodamines generates new dyes with vibration structure and inhibited twisted-intramolecular-charge-transfer (TICT) formation synchronously, thus increasing the brightness and photostability while enlarging Stokes shift. The new fluorophore YL578 exhibits around twofold greater brightness and Stokes shift than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, YL578 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine based probes (CPY-Halo and 580CP-Halo), known as photostable fluorophores for STED imaging. Furthermore, the strategy is well generalized to offer a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing.

Topological DNA Tetrahedron Encapsulated Gold Nanoparticle Enables Precise Ligand Engineering for Targeted Cell Imaging.

Ligand-functionalized plasmonic nanoparticles have been widely used for targeted imaging in living systems. However, ligand presentation and encoding on the nanoparticle's surface in a stoichiometrically controllable manner remains a great challenge. Herein, we propose a method to construct ligand-engineered plasmonic nanoprobes by using nanoparticle encapsulation with topological DNA tetrahedrons, which enables the programmed ligand loading for precise regulation of targeting efficiency of nanoprobes in biorelated applications. With this method, we demonstrated the preparation of functionalized plasmonic nanoprobes by programmed loading of RGD peptides and aptamers onto the DNA tetrahedron encapsulated gold nanoparticles with controllable stoichiometric ratios. The cell imaging and particle counting assays suggested that the targeting efficiency of the nanoprobes could be readily modulated by tailoring the number and stoichiometric ratios of the loaded ligands, respectively. It can be anticipated that this robust strategy could provide new opportunities for the construction of efficacious nanoprobes and delivery systems for versatile bioapplications.

Molecular engineering of organic-based agents for in situ bioimaging and phototherapeutics.

In situ monitoring of the location and transportation of bioactive molecules is essential for deciphering diverse biological events in the field of biomedicine. In addition, obtaining the in situ information of lesions will provide a clear perspective for surgeons to perform precise resection in clinical surgery. Notably, delivering drugs or operating photodynamic therapy/photothermal therapy in situ by labeling the lesion regions of interest can improve treatment and reduce side effects in vivo. In various advanced imaging and therapy modalities, optical theranostic agents based on organic small molecules can be conveniently modified as needed and can be easily internalized into cells/lesions in a non-invasive manner, which are prerequisites for in situ bioimaging and precision treatment. In this tutorial review, we first summarize the in situ molecular immobilization strategies to retain small-molecule agents inside cells/lesions to prevent their diffusion in living organisms. Emphasis will be focused on introducing the application of these strategies for in situ imaging of biomolecules and precision treatment, particularly pertaining to why targeting therapy in situ is required.

DNAzyme Amplified Aptasensing Platform for Ochratoxin A Detection Using a Personal Glucose Meter.

Aptamer-based sensors have emerged as a major platform for detecting small-molecular targets, because aptamers can be selected to bind these small molecules with higher affinity and selectivity than other receptors such as antibodies. However, portable, accurate, sensitive, and affordable detection of these targets remains a challenge. In this work, we developed an aptasensing platform incorporating magnetic beads and a DNAzyme for signal amplification, resulting in high sensitivity. The biosensing platform was constructed by conjugating a biotin-labeled aptamer probe of small-molecular targets such as toxins and a biotin-labeled substrate strand on magnetic beads, and the DNAzyme strand hybridized with the aptamer probe to block the substrate cleavage activity. The specific binding of the small-molecular target by the aptamer probe can replace the DNAzyme strand and then induce the hybridization between the DNAzyme strand and substrate strand, and the iterative signal amplification reaction of hydrolysis and cleavage of the substrate chain occurs in the presence of a metal ion cofactor. Using invertase to label the substrate strand, the detection of small molecules of the toxin is successfully transformed into the measurement of glucose, and the sensitive analysis of small molecules such as toxins can be realized by using the household portable glucose meter as a readout. This platform is shown to detect ochratoxin, a common toxin in food, with a linear detection range of 5 orders of magnitude, a low detection limit of 0.88 pg/mL, and good selectivity. The platform is easy to operate and can be used as a potential choice for quantitative analysis of small molecules, at home or under point-of-care settings. Moreover, by changing and designing the aptamer probe and the arm of DNAzyme strand, it can be used for the analysis of other analytes.

A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging.

Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

DNA origami-based protein networks: from basic construction to emerging applications.

Natural living systems are driven by delicate protein networks whose functions are precisely controlled by many parameters, such as number, distance, orientation, and position. Focusing on regulation rather than just imitation, the construction of artificial protein networks is important in many research areas, including biomedicine, synthetic biology and chemical biology. DNA origami, sophisticated nanostructures with rational design, can offer predictable, programmable, and addressable scaffolds for protein assembly with nanometer precision. Recently, many interdisciplinary efforts have achieved the precise construction of DNA origami-based protein networks, and their emerging application in many areas. To inspire more fantastic research and applications, herein we highlight the applicability and potentiality of DNA origami-based protein networks. After a brief introduction to the development and features of DNA origami, some important factors for the precise construction of DNA origami-based protein networks are discussed, including protein-DNA conjugation methods, networks with different patterns and the controllable parameters in the networks. The discussion then focuses on the emerging application of DNA origami-based protein networks in several areas, including enzymatic reaction regulation, sensing, bionics, biophysics, and biomedicine. Finally, current challenges and opportunities in this research field are discussed.

Biomineralized nanoparticles enable an enzyme-assisted DNA signal amplification in living cells.

The enzymatic-assisted signal amplification of DNA sensors is rarely applied in living cells due to the difficulties in protein delivery. In this study, we have proposed a biomineralization-based DNA nanoprobe to transport nucleases and DNA sensors for enzyme-assisted imaging of microRNA in living cells.

Aptamer-Functionalized DNA Nanostructures for Biological Applications.

DNA nanostructures hold great promise for various applications due to their remarkable properties, including programmable assembly, nanometric positional precision, and dynamic structural control. The past few decades have seen the development of various kinds of DNA nanostructures that can be employed as useful tools in fields such as chemistry, materials, biology, and medicine. Aptamers are short single-stranded nucleic acids that bind to specific targets with excellent selectivity and high affinity and play critical roles in molecular recognition. Recently, many attempts have been made to integrate aptamers with DNA nanostructures for a range of biological applications. This review starts with an introduction to the features of aptamer-functionalized DNA nanostructures. The discussion then focuses on recent progress (particularly during the last five years) in the applications of these nanostructures in areas such as biosensing, bioimaging, cancer therapy, and biophysics. Finally, challenges involved in the practical application of aptamer-functionalized DNA nanostructures are discussed, and perspectives on future directions for research into and applications of aptamer-functionalized DNA nanostructures are provided.

DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells.

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.

"Apollo Program" in Nanoscale: Landing and Exploring Cell-Surface with DNA Nanotechnology.

Plasma membranes are the fundamental mediators through which cells communicate with their surrounding environment. The techniques to monitor or synthetically manipulate the cell membranes are attractive tools to engineer the functions of cells as well as their local microenvironment. Current advances of biomolecular science enable the insertion of functional compounds onto cell-surface via external integration or genetic engineering to manipulate cell membrane function. Recently, the DNA nanotechnology made it possible to use synthetic DNA as an emerging and promising molecular toolkit for anchoring and exploring cell-surface. In this review, the latest advances of DNA nanotechnology on cell-surface are summarized. We first give an overview of commonly used strategies for installing DNA nanodevices onto cell-surface including amphiphilic interaction, covalent modification, and affinity labeling. Then the biological applications of DNA nanodevices on cell membranes are reviewed. By integrating functional nucleic acids as recognition elements, DNA sensors are fabricated to monitor the cellular microenvironment and membrane activities. In addition, the programmable behaviors of DNA on cell-surface are also discussed, which include biomimicry and the regulation of membrane functions. Finally, we analyze the current challenges in the development of DNA nanotechnology on cell-surface as well as their prospects in bioimaging and cancer therapy.

DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells.

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.

Valency-Controlled Molecular Spherical Nucleic Acids with Tunable Biosensing Performances.

Spherical nucleic acids (SNAs) play critical roles in many fields, such as molecular diagnostics, disease therapeutics, and materials application. Due to the important role of DNA density on the properties of SNAs, the controlled synthesis of monodisperse SNAs with precise DNA density is an important approach for the structure-function relationship study and finite functions regulation of SNAs. In particular, the construction of monodisperse SNAs in a valency-tunable and site-specific manner is highly important; however, it is still challenging. Herein, on the basis of the high controllability, nanometer precision, and addressable modification ability of framework nucleic acid (FNA), we develop the concept of valency-controlled framework nucleic acid core-based molecular spherical nucleic acids (FNA-mSNAs) with tunable biosensing performances. The FNA-mSNAs consist of a valency-tunable FNA-based DNA nanocube as the core and a controlled, precise number of DNA strands per core. By simply alternating the binding site number for shell DNA strands on the DNA nanocube, homogeneous FNA-mSNAs with different valencies were easily designed, which enabled the molecular level study of the effect of valency on their properties, such as nuclease stability and cellular uptake. Furthermore, taking advantage of the addressable modification ability of FNA, the first heterogeneous molecular SNAs with tunable valency were demonstrated. Importantly, the valency of heterogeneous FNA-mSNAs was able to tune their biosensing performance, such as response dynamics, detection sensitivity, and response range. With these remarkable features, FNA-mSNAs provide new research methods for the development of functional SNAs at the molecular level for a wide range of biological applications.