Cavity hairpin ThT-light nucleic acid switches: the construction of label- and enzyme-free sensing and imaging platforms.

Thioflavin T (ThT) is a classical fluorescent dye gaining prominence in current research regarding nucleic acid conformations (NACs). However, most NACs with the ability to excite ThT fluorescent are unique or form in demanding conditions, limiting the extensiveness and depth of ThT application in sensing and imaging. Therefore, this study proposed CGG-AAA mismatched cavity hairpin ThT-light nucleic acid switches (CHTLNAS) with excellent fluorescence excitation over 500-fold higher than spontaneous, 17∼20-fold higher than ssDNA and 2.5∼5-fold higher than complementary duplex. Based on the excellent fluorescence excitation, convenient conformation formation, good sequence programmability, and flexible allosteric ability (known as the Worm-crack pod mechanism mediated by the target), it achieved the label- and enzyme-free detection of tetracycline (TET) and berberine (BB) at the pM level within 10 min. Moreover, it was found enable to realize the sensitive tracking of intracellular carriers at the nM level of ThT entry concentration, and prolongated its cell nuclear-entry time of ThT over 8 h, overcoming the non-specific high background signal interference of ThT in the nuclear region, and expanding the diversified application of ThT in cell biology research. Therefore, CHTLNAS is a more universal, practical tool than G-quadruplex or other kinds of NACs for ThT development and utilization in sensing and imaging platforms.

Multidimensional Applications and Challenges of Riboswitches in Biosensing and Biotherapy.

Riboswitches have received significant attention over the last two decades for their multiple functionalities and great potential for applications in various fields. This article highlights and reviews the recent advances in biosensing and biotherapy. These fields involve a wide range of applications, such as food safety detection, environmental monitoring, metabolic engineering, live cell imaging, wearable biosensors, antibacterial drug targets, and gene therapy. The discovery, origin, and optimization of riboswitches are summarized to help readers better understand their multidimensional applications. Finally, this review discusses the multidimensional challenges and development of riboswitches in order to further expand their potential for novel applications.

Structural Antagonism-Aided Conformational Regulation Enables an Aptamer-Loop G-Quadruplex Modular Sensor of ß-Lactoglobulin.

A simple, reliable method for identifying ß-lactoglobulin (ß-LG) in dairy products is needed to protect those with ß-LG allergies. A common, practical strategy for target detection is designing simplified nucleic acid nanodevices by integrating functional components. This work presents a label-free modular ß-LG aptasensor consisting of an aptamer-loop G-quadruplex (G4), the working conformation of which is regulated by conformational antagonism to ensure respective module functionality and the related signal transduction. The polymorphic conformations of the module-fused sequence are systematically characterized, and the cause is revealed as shifting antagonistic equilibrium. Combined with conformational folding dynamics, this helped regulate functional conformations by fine-tuning the sequences. Furthermore, the principle of specific ß-LG detection by parallel G4 topology is examined as binding on the G4 aptamer loop by ß-LG to reinforce the G4 topology and fluorescence. Finally, a label-free, assembly-free, succinct, and turn-on fluorescent aptasensor is established, achieving excellent sensitivity across five orders of magnitude, rapidly detecting ß-LG within 22-min. This study provides a generalizable approach for the conformational regulation of module-fused G4 sequences and a reference model for creating simplified sensing devices for a variety of targets.

Aptamer and Thiol Co-Regulated Color-Shifting Fluorophores via Dynamic Through-Bond/Space Conjugation for Constructing Ratiometric RNA Sensor.

Fluorophores with color-shifting characteristics have attracted enormous research interest in the quantitative application of RNA sensors. It reports here a simple synthesis, luminescent properties, and co-transcription ability of de-conjugated triphenylmethane leucomalachite green (LMG). This novel clusteroluminescence fluorophore is rapidly synthesized from malachite green (MG) in reductive transcription system containing dithiothreitol, emitting fluorescence in the UV region through space conjugation. The co-transcribed MG RNA aptamer (MGA) bound to the ligand, resulting in red fluorescence from the through-bond conjugation. Given the equilibrated color-shifting fluorophores, they are rationally employed in a 3WJ-based rolling circle transcription switch, with the target-aptamer acting as an activator to achieve steric allosterism. This one-pot system allows the target to compete continuously for allosteric sites, and the activated transcription switches continue to amplify MGA forward, achieving accurate Aflatoxin 1 quantification at the picomolar level in 1 h. Due to the programmability of this RNA sensor, the design method of target-competitive aptamers is standardized, making it universally applicable.

Construction Strategy of Functionalized Liposomes and Multidimensional Application.

Liposomes are widely used in the biological field due to their good biocompatibility and surface modification properties. With the development of biochemistry and material science, many liposome structures and their surface functional components have been modified and optimized one by one, pushing the liposome platform from traditional to functionalized and intelligent, which will better satisfy and expand the needs of scientific research. However, a main limiting factor effecting the efficiency of liposomes is the complicated environmental conditions in the living body. Currently, in order to overcome the above problem, functionalized liposomes have become a very promising strategy. In this paper, binding strategies of liposomes with four main functional elements, namely nucleic acids, antibodies, peptides, and stimuli-responsive motif have been summarized for the first time. In addition, based on the construction characteristics of functionalized liposomes, such as drug-carrying, targeting, long-circulating, and stimulus-responsive properties, a comprehensive overview of their features and respective research progress are presented. Finally, the paper critically presents the limitations of these functionalized liposomes in the current applications and also prospectively suggests the future development directions, aiming to accelerate realization of their industrialization.

An Effective Docking-Guided Strategy for Rational Tailoring of Fluorescent Aptamer Switches of Dimethylindole Red Analogue.

The light-up aptamer-dimethylindole red (DIR) complexes have been applied in biochemistry analysis as promising signal transduction tools. However, the unfavorable repulsions between DIR and the long-sequence aptamer switch hinder the complex's further development, and it is urgent to engineer a feasible and efficient strategy for synchronously and rationally adjusting the DIR chemical structure and the DIR aptamer performance. Herein, we communicate a versatile docking-guided rational tailoring strategy to effectively upgrade a DNA aptamer which specifically turns on the fluorescence of a synthesized amino-functionalized DIR analogue (NH2-DIR). After optimizing with three-level tailoring strategies including molecule docking-guided tailoring, coarse tailoring, and fine tailoring, the NH2-DIR aptamer switch with higher binding affinity and specificity, considerable fluorescence-activation ability, and 40% shortened length was obtained. Integrating the experimental and docking results, the binding mechanism between NH2-DIR and the tailored aptamer was deciphered via three types of interactions.

Synthesis of Amorphous/Crystalline Hetero-Phase Nanozymes With Peroxidase-Like Activity by Coordination-Driven Self-Assembly for Biosensors.

Nanozymes and amorphous nanomaterials attract great attention owing to their extraordinary properties. However, the requirements for special synthesis conditions become the bottleneck of their development. Herein, a new strategy involving the DNA-based coordination-driven self-assembly is reported for the synthesis of a novel amorphous/crystalline hetero-phase nanozyme (Fe-DNA). For the synthesis of both nanozymes and amorphous materials, this strategy is simple and controllable, avoiding the traditionally employed harsh conditions. Benefitting from the amorphous structure and the superior physicochemical properties, the synthesized Fe-DNA nanozyme is subsequently found to exhibit a smaller Michaelis constant value for hydrogen peroxide (H2 O2 ) (0.81 mm) than that of horseradish peroxidase (HRP) (3.70 mm), demonstrating the stronger affinity of the Fe-DNA nanozyme toward H2 O2 . The Fe-DNA nanozyme also shows significant peroxidase-like activity but only negligible oxidase-like activity, a characteristic which releases the corresponding assay system from oxygen interference, thereby improving the performance of the nanozyme-based sensing platform. In addition, compared with other nanozymes, the novel Fe-DNA nanozyme is degradable via phosphate; thus, mitigating potential environmental threat. This work provides novel amorphous/crystalline hetero-phase nanozymes and opens a new avenue for the design of amorphous nanomaterials and nanozymes.

Decellularized Extracellular Matrix for Remodeling Bioengineering Organoid's Microenvironment.

Over the past decade, stem cell- and tumor-derived organoids are the most promising models in developmental biology and disease modeling, respectively. The matrix is one of three main elements in the construction of an organoid and the most important module of its extracellular microenvironment. However, the source of the currently available commercial matrix, Matrigel, limits the application of organoids in clinical medicine. It is worth investigating whether the original decellularized extracellular matrix (dECM) can be exploited as the matrix of organoids and improving organoid construction are very important. In this review, tissue decellularization protocols and the characteristics of decellularization methods, the mechanical support and biological cues of extraccellular matrix (ECM), methods for construction of multifunctional dECM and responsive dECM hydrogel, and the potential applications of functional dECM are summarized. In addition, some expectations are provided for dECM as the matrix of organoids in clinical applications.

Coordination-Driven One-Step Rapid Self-Assembly Synthesis of Dual-Functional Ag@Pt Nanozyme.

Realizing high-precise and adjustable regulation of engineering nanozyme is important in nanotechnology. Here, Ag@Pt nanozymes with excellent peroxidase-like and antibacterial effects are designed and synthesized by nucleic acid and metal ions coordination-driven one-step rapid self-assembly. The adjustable NA-Ag@Pt nanozyme is synthesized within 4 min using single-stranded nucleic acid as templates, and peroxidase-like enhancing FNA-Ag@Pt nanozyme is received by regulating functional nucleic acids (FNA) based on NA-Ag@Pt nanozyme. Both Ag@Pt nanozymes that are developed not only has simple and general synthesis approaches, but also can produce artificial precise adjustment and possess dual-functional. Moreover, when lead ion-specific aptamers as FNA are introduced to NA-Ag@Pt nanozyme, the Pb2+ aptasensor is successfully constructed by increasing electron conversion efficiency and improving the specificity of nanozyme. In addition, both nanozyme has good antibacterial properties, with ~100% and ~85% antibacterial efficiency against Escherichia coli and Staphylococcus aureus, respectively. This work provides a synthesis method of novelty dual-functional Ag@Pt nanozymes and successful application in metal ions detection and antibacterial agents.

One-Pot Controllable Assembly of a Baicalin-Condensed Aptamer Nanodrug for Synergistic Anti-Obesity.

The rapid, simple and low-cost preparation of DNA micro-nano-architectures remain challenging in biosensing and therapy. Polymerase chain reaction (PCR)-driven DNA micro-nano-flowers are used to construct a nanosized baicalin-compressed-aptamer-nanodrug (bcaND) via one-pot assembly for targeted and synergistic anti-obesity. In the design, the tailored Adipo-8 (tAdi-8) overhang in the PCR amplicon displays anti-obesity targeting activity, while the baicalin loaded in the bcaND by embedding the amplicon plays a three-fold role as a lipid-lowering factor, bcaND size compressor, and uncoupling protein-1 (UCP1)-raised thermogenic activator. The ingenious bcaND represents an advanced multifunctional nanomaterial capable of adjusting the morphology at an optimal 400/1 molar ratio of Mg2+ to phosphate groups, compressing the size from 2.699 µm to 214.76 nm using 1 mg/mL baicalin at a temperature of 70 °C, an effective payload with amplicons of up to 98.94%, and a maximum baicalin load of 86.21 g/g DNA. Responsive release in acidic conditions (pH 5.0) occurs within 72 h, accelerating thermogenesis via UCP1 up-regulation by 2.5-fold in 3T3-L1-preadipocytes and 13.7-fold in the white-adipose-tissue (WAT) of mice, targeting adipocytes and visceral white adipose tissue. It plays an efficient synergistic role in obesity therapy in vitro and in vivo, providing a new direction for DNA self-assembly nanotechnology.

Extension characteristics of TdT and its application in biosensors.

The advantages of rapid amplification of nucleic acid without a template based on terminal deoxyribonucleotidyl transferase (TdT) have been widely used in the field of biosensors. However, the catalytic efficiency of TdT is affected by extension conditions. The sensitivity of TdT- mediated biosensors can be improved only under appropriate conditions. Therefore, in this review, we provide a comprehensive overview of TdT extension characteristics and its applications in biosensors. We focus on the relationship between TdT extension conditions and extension efficiency. Furthermore, the construction strategy of TdT-mediated biosensors according to five different recognition types and their applications in targets are discussed and, finally, several current challenges and prospects in the field are taken into consideration.

Brief introduction to terminal deoxyribonucleotidyl transferase (TdT) characteristics.Provided a systematic and comprehensive summary of TdT extension conditions.Summarized the four effect factors of catalytic efficiency based on extension conditions and enzyme conformation.Sensing strategies of TdT-mediated biosensors for five different recognitions were summarized in detail.The applications of TdT-mediated biosensors in six targets were introduced in detail.

Applications and Challenges of Bacteriostatic Aptamers in the Treatment of Common Pathogenic Bacteria Infections.

The continuous evolution and spread of common pathogenic bacteria is a major challenge in diagnosis and treatment with current biotechnology and modern molecular medicine. To confront this challenge, scientists urgently need to find alternatives for traditional antimicrobial agents. Various bacteriostatic aptamers obtained through SELEX screening are one of the most promising strategies. These bacteriostatic aptamers can reduce bacterial infection by blocking bacterial toxin infiltration, inhibiting biofilm formation, preventing bacterial invasion of immune cells, interfering with essential biochemical processes, and other mechanisms. In addition, aptamers may also help enhance the function of other antibacterial materials/drugs when used in combination. This paper has reviewed the bacteriostatic aptamers in the treatment of common pathogenic bacteria infections. For this aspect, first, bacteriostatic aptamers and their screening strategies are summarized. Then, the effect of molecular tailoring and modification on the performance of the bacteriostatic aptamer is analyzed, and the antibacterial mechanism and antibacterial strategy based on aptamers are introduced. Finally, the key technical challenges and their development prospects in clinical treatment are also carefully discussed.

Advanced screening and tailoring strategies of pesticide aptamer for constructing biosensor.

The rapid development of aptamers has helped address the challenges presented by the wide existed pesticides contaminations. Screening of aptamers with excellent performance is a prerequisite for successfully constructing biosensors, while further tailoring of aptamers with enhanced activity greatly improved the assay performance. Firstly, this paper reviewed the advanced screening strategies for pesticides aptamers, including immobilization screening that preserves the native structures of targets, non-immobilized screening based on nanomaterials, capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX), virtual screening in silico, high-throughput selection, and rational secondary library generation methods, which contributed significantly to improve the success rate of screening, reduce the screening time, and ensure aptamer binding affinity. Secondly, the precise tailoring strategies for pesticides aptamers were modularly elaborated, containing deletion, splitting, elongation, and fusion, which provided various advantages like cost-efficiency, enhanced binding affinity, and new derived functional motifs. Thirdly, the developed aptamer-based biosensors (aptasensors) for pesticide detection were systematically reviewed according to the different signal output modes. Finally, the challenges and future perspectives of pesticide detection are discussed comprehensively.

Aptamer functionalized nucleic acid nano drug for targeted synergistic therapy for colon cancer.

Due to its complicated pathophysiology, propensity for metastasis, and poor prognosis, colon cancer is challenging to treat and must be managed with a combination of therapy. Using rolling circle transcription (RCT), this work created a nanosponge therapeutic medication system (AS1411@antimiR-21@Dox). Using the AS1411 aptamer, this approach accomplished targeted delivery to cancer cells. Furthermore, analysis of cell viability, cell apoptosis, cell cycle arrest, reactive oxygen species (ROS) content, and mitochondrial membrane potential (MMP) levels revealed that functional nucleic acid nanosponge drug (FND) can kill cancer cells. Moreover, transcriptomics uncovered a putative mechanism for the FND anti-tumor effect. These pathways, which included mitotic metaphase and anaphase as well as the SMAC-mediated dissociation of the IAP: caspase complexes, were principally linked to the cell cycle and cell death. In conclusion, by triggering cell cycle arrest and apoptosis, the nano-synergistic therapeutic system allowed for the intelligent and effective targeted administration of RNA and chemotherapeutic medicines for colon cancer treatment. The system allowed for payload efficiency while being customizable, targeted, reliable, stable, and affordable.

Sandwich capture ultrasensitive sensor based on biohybrid interface for the detection of Cronobacter sakazakii.

A simple, rapid and ultrasensitive visual sensing method for the detection of Cronobacter sakazakii (C. sakazakii) based on a biohybrid interface was established. During the entire sensing process, quadruple-cascade amplification showed its superior sensing performance. First, the prepared immunomagnetic beads (IMB) were used to isolate and enrich specific targets from the food matrix. After adding the fusion aptamer, the aptamer sequence specifically recognized the target and formed the immune sandwich structure of antibody-target-fusion aptamer. In addition, the fusion aptamer also included the template sequence of exponential amplification reaction (EXPAR), which contained the antisense sequence of the G-rich sequence. Therefore, a large number of G-rich sequences can be generated after EXPAR can be triggered in the presence of Bst. DNA polymerase, nicking endonuclease, cDNA, and dNTP. They were self-assembled into G-quadruplex structures and then combined with hemin to form G4/hemin DNAzyme, resulting in visible coloration and measuring absorbance at 450 nm for quantitative detection. The assay showed a limit of detection (LOD) of 2 CFU/mL in pure culture and 12 CFU/g in milk powder in optimal conditions. This method provides a promising strategy for rapid and point-of-care testing (POCT) since it does not require DNA extraction, medium culturing, and expensive instrumentation. KEY POINTS: •Single-cell level detection of C. sakazakii with ultrasensitive and rapidness •The fusion aptamer integrated recognition and amplification •Sensing analysis of C. sakazakii based on cascade amplification of biohybrid interface.

Smart Nucleic Acid Hydrogels with High Stimuli-Responsiveness in Biomedical Fields.

Due to their hydrophilic, biocompatible and adjustability properties, hydrogels have received a lot of attention. The introduction of nucleic acids has made hydrogels highly stimuli-responsiveness and they have become a new generation of intelligent biomaterials. In this review, the development and utilization of smart nucleic acid hydrogels (NAHs) with a high stimulation responsiveness were elaborated systematically. We discussed NAHs with a high stimuli-responsiveness, including pure NAHs and hybrid NAHs. In particular, four stimulation factors of NAHs were described in details, including pH, ions, small molecular substances, and temperature. The research progress of nucleic acid hydrogels in biomedical applications in recent years is comprehensively discussed. Finally, the opportunities and challenges facing the future development of nucleic acid hydrogels are also discussed.

Recent Developments in Delivery of MicroRNAs Utilizing Nanosystems for Metabolic Syndrome Therapy.

Metabolic syndrome (MetS) is a set of complex, chronic inflammatory conditions that are characterized by central obesity and associated with an increased risk of cardiovascular diseases. In recent years, microRNAs (miRNAs) have become an important type of endocrine factors, which play crucial roles in maintaining energy balance and metabolic homeostasis. However, its unfavorable properties such as easy degradation in blood and off-target effect are still a barrier for clinical application. Nanosystem based delivery possess strong protection, high bioavailability and control release rate, which is beneficial for success of gene therapy. This review first describes the current progress and advances on miRNAs associated with MetS, then provides a summary of the therapeutic potential and targets of miRNAs in metabolic organs. Next, it discusses recent advances in the functionalized development of classic delivery systems (exosomes, liposomes and polymers), including their structures, properties, functions and applications. Furthermore, this work briefly discusses the intelligent strategies used in emerging novel delivery systems (selenium nanoparticles, DNA origami, microneedles and magnetosomes). Finally, challenges and future directions in this field are discussed provide a comprehensive overview of the future development of targeted miRNAs delivery for MetS treatment. With these contributions, it is expected to address and accelerate the development of effective NA delivery systems for the treatment of MetS.

Fluorescent detection of Cu (II) ions based on DNAzymatic cascaded cyclic amplification.

A fluorescent biosensor based on the cascaded cyclic amplification-lighted copper nanoparticles has been developed, optimized, and validated. In the double-modular cascaded cyclic amplification, a DNAzymatic cyclic amplification unit transforms metal ion signal to specific DNA sequences, and a linear/exponential integrated amplification unit converts as-prepared DNA codes to identical thymine (T)-rich DNA templates. T-rich scaffolds can induce the generation of red fluorescent copper nanoparticles, with fluorescence emission at 625 nm upon the excitation at 340 nm, as signal vehicles for quantitative detection of metal ions. Copper ions, selected as the model target, could be detected in a wide linear range from 10 to 104 nM depending on the increased fluorescent intensity, and the detection limit is 5.6 ± 0.52 nM (n = 3) within 40 min, which is 4 orders of magnitude lower than the limits set in drinking water. In the detection of Cu2+ in real tap and lake water, the results between inductively coupled plasma mass spectrometry (ICP-MS) and our proposed biosensor were consistent, illustrating the practicability of the fabricated method. In summary, the established fluorescent biosensor compensates the deficiency of immunoassays failing to analyze metal ions, broadens ranges of biomarkers responding to cleaved DNAzymes, provides an open platform sensing different metal ions, and meets the increasing need for the ultrasensitive detection in the field of food safety, environmental monitoring, and medical diagnosis.

A Universal Electrochemical Biosensor Using Nick-HCR Nanostructure as Molecular Gate of Nanochannel for Detecting Chromium(III) Ions and MicroRNA.

Solid-state nanochannels demonstrating excellent mechanical properties and chemical stability combined with programmable DNA provide an opportunity to control on-demand ion transport. However, poor functionalization of the nanochannels limits the types of detected targets, as well as its universality in the sensing field. To solve these issues, a universal nanochannel sensing platform was developed by employing a nick hybridization chain reaction (nHCR) nanostructure as a molecular gate, which could generally respond to the universal sequence Y. Metal ion-dependent DNAzyme cleavage was used to transfer the chromium(III) (Cr3+) ions into nucleic acid X, which was further amplified and converted into universal sequence Y. Upon adding sequence Y into the nHCR nanostructure-functionalized nanochannel, the disassembly of the nHCR molecular gate turned on the ionic current signal inside the nanochannel. The ON-OFF ratio displayed a linear relationship with the Cr3+ concentration in the range from 200 fM to 20 nM. In less than 66 min, the nanochannel-based biosensing platform successfully detected Cr3+ ions as low as 200 fM. In addition, the detection of microRNA with a concentration as low as 1 pM was achieved by only regulating the sequence of template X'-Y'.

A colorimetric zinc(II) assay based on the use of hairpin DNAzyme recycling and a hemin/G-quadruplex lighted DNA nanoladder.

A method is described for sensitive colorimetric determination of zinc(II) ions that uses (a) a Zn(II)-responsive hairpin DNAzyme that assists target recycling, (b) hybridization chain reaction, and (c) hemin/G-quadruplex DNA nanoladder. The Zn(II)-responsive split of the hairpin DNAzyme (HD) acts as the recognition and transformation probe. Upon addition of Zn (II) and enzyme strand, a duplex is formed in the loop region of hairpin. The caged initiator sequence is subsequently liberated from the HD by the Zn(II)-selective split of the substrate strand. This cleavage induces an enzyme strand recycling for the next round of cleavage. As a result, the initiator DNA is accumulated and cross-opened H1 and H2 to start a hybridization chain reaction (HCR). The caged G-quadruplex is released after the HCR to recruit hemin to form the hemin/G-quadruplex that is inserted into the DNA nanoladder. Once formed in the DNA nanoladder, these act as catalytic labels for the ABTS, resulting a green color change. This cascade amplification strategy allows 10 nM to 100 µM of Zn(II) to be linearly quantified by colorimetry at 415 nm with a detection limit of 3.5 nM. The recoveries ranged from 97.7 to 108.3% were obtained, confirming high reliability of the method for Zn2+ analysis in lake water samples. Graphical abstractA colorimetric assay for Zn2+ using hairpin DNAzyme (HD) assistant cycle and hemin/G-quadruplex lighted nanoladders is designed. A Zn2+-responsive split of HD is designed as the recognition and transformation probe. The hemin/G-Quadruplex inserted nanoladder act as reporter for signal readout.