Self-assembly of a AuNPs/Ti3C2 MXene hydrogel for cascade amplification of microRNA-122 biosensing.

A three-dimensional (3D) self-assembled AuNPs/Ti3C2 MXene hydrogel (AuNPs/Ti3C2 MXH) nanocomposite was prepared for the fabrication of a novel microRNA-122 electrochemical biosensor. The 3D hydrogel structure was gelated from two-dimensional MXene nanosheets with the assistance of graphite oxide and ethylenediamine. MXene hydrogels supported the in situ formation of Au nanoparticles (AuNPs) that predominantly exploring the (111) facet, and these AuNPs are utilized as carriers for hairpin DNA (hpDNA) probes, facilitating DNA hybridization. MXene acted as both a reductant and stabilizer, significantly improving the electrochemical signal. In addition, the conjugation of PAMAM dendrimer-encapsulated AuNPs and H-DNA worked as an ideal bridge to connect targets and efficient electrochemical tags, providing a high amplification efficiency for the sensing of microRNA-122. A linear relationship between the peak currents and the logarithm of the concentrations of microRNA-122 from 1.0 × 10-2 to 1.0 × 102 fM (I = 1.642 + 0.312 lgc, R2 = 0.9891), is obtained. The detection limit is  0.8 × 10-2 fM (S/N = 3). The average recovery for human serum detection ranged from 97.32 to 101.4% (RSD < 5%).

Plasmon-emitter coupling in cytosine-rich hairpin DNA-templated silver nanoclusters: Thermal reversibility, white light emission, and dynamics inside live cells.

Bio-templated luminescent noble metal nanoclusters (NCs) have attracted great attention for their intriguing physicochemical properties. Continuous efforts are being made to prepare NCs with high fluorescence quantum yield (QY), good biocompatibility, and tunable emission properties for their widespread practical applications as new-generation environment-friendly photoluminescent materials in materials chemistry and biological systems. Herein, we explored the unique photophysical properties of silver nanoclusters (AgNCs) templated by cytosine-rich customized hairpin DNA. Our results indicate that a 36-nucleotide containing hairpin DNA with 20 cytosine (C20) in the loop can encapsulate photostable red-emitting AgNCs with an absolute QY of ∼24%. The luminescent properties in these DNA-templated AgNCs were found to be linked to the coupling between the surface plasmon and the emitter. These AgNCs exhibited excellent thermal sensitivity and were employed to produce high-quality white light emission with an impressive color rendering index of 90 in the presence of dansyl chloride. In addition, the as-prepared luminescent AgNCs possessing excellent biocompatibility can effectively mark the nuclear region of HeLa cells and can be employed as a luminescent probe to monitor the cellular dynamics at a single molecular resolution.

Protein-to-DNA Converter with High Signal Gain.

DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a proximity activation mechanism that converts protein input into DNA barcodes for the DNA exponential amplification reaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.

DNA Framework-Programmed Nanoscale Enzyme Assemblies.

Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in ∼5.9- and ∼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.

DNA liquid crystals with AIE effect toward humidity-indicating biomaterials.

In this study, by fabricating DNA doped with tetraphenylethene-containing ammonium surfactant, the resulting solvent-free DNA ionic complex could undergo a humidity-induced phase change that could be well tracked by the fluorescence signal of the surfactant. Taking advantage of the humidity-induced change in fluorescence, the reported ionic DNA complex could accurately indicate the humidity in real time.

Stable Interstrand Cross-Links Generated from the Repair of 1,N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

Electrical Transport and Dynamics of Confined DNA through Highly Conductive 2D Graphene Nanochannels.

Confining DNA in nanochannels is an important approach to studying its structure and transportation dynamics. Graphene nanochannels are particularly attractive for studying DNA confinement due to their atomic flatness, precise height control, and excellent mechanical strength. Here, using femtosecond laser etching and wetting transfer, we fabricate graphene nanochannels down to less than 4.3 nm in height, with the length-to-height ratios up to 103. These channels exhibit high stability, low noise, and self-cleaning ability during the long-term ionic current recording. We report a clear linear relationship between DNA length and the residence time in the channel and further utilize this relationship to differentiate DNA fragments based on their lengths, ranging widely from 200 bps to 48.5 kbps. The graphene nanochannel presented here provides a potential platform for label-free analyses and reveals fundamental insights into the conformational dynamics of DNA and proteins in confined space.

Real-time MBDi-RPA using methyl-CpG binding protein 2: A real-time detection method for simple and rapid estimation of CpG methylation status.

BACKGROUND: Analysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent. RESULTS: MBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging. SIGNIFICANCE AND NOVELTY: Real-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.

Mechanism of DNA origami folding elucidated by mesoscopic simulations.

Many experimental and computational efforts have sought to understand DNA origami folding, but the time and length scales of this process pose significant challenges. Here, we present a mesoscopic model that uses a switchable force field to capture the behavior of single- and double-stranded DNA motifs and transitions between them, allowing us to simulate the folding of DNA origami up to several kilobases in size. Brownian dynamics simulations of small structures reveal a hierarchical folding process involving zipping into a partially folded precursor followed by crystallization into the final structure. We elucidate the effects of various design choices on folding order and kinetics. Larger structures are found to exhibit heterogeneous staple incorporation kinetics and frequent trapping in metastable states, as opposed to more accessible structures which exhibit first-order kinetics and virtually defect-free folding. This model opens an avenue to better understand and design DNA nanostructures for improved yield and folding performance.

In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

DNA Packaging and Polycation Length Determine DNA Susceptibility to Free Radical Damage in Condensed DNA.

In nature, DNA exists primarily in a highly compacted form. The compaction of DNA in vivo is mediated by cationic proteins: histones in somatic nuclei and protamines in sperm chromatin. The extreme, nearly crystalline packaging of DNA by protamines in spermatozoa is thought to be essential for both efficient genetic delivery as well as DNA protection against damage by mutagens and oxidative species. The protective role of protamines is required in sperm, as they are sensitive to ROS damage due to the progressive loss of DNA repair mechanisms during maturation. The degree to which DNA packaging directly relates to DNA protection in the condensed state, however, is poorly understood. Here, we utilized different polycation condensing agents to achieve varying DNA packaging densities and quantify DNA damage by free radical oxidation within the condensates. Although we see that tighter DNA packaging generally leads to better protection, the length of the polycation also plays a significant role. Molecular dynamics simulations suggest that longer polyarginine chains offer increased protection by occupying more space on the DNA surface and forming more stable interactions. Taken together, our results suggest a complex interplay among polycation properties, DNA packaging density, and DNA protection against free radical damage within condensed states.

Structural basis of DNA crossover capture by Escherichia coli DNA gyrase.

DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of Escherichia coli DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.

Deconvoluting the Effect of Cell-Penetrating Peptides for Enhanced and Controlled Insertion of Large-Scale DNA Nanopores.

DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay. The results demonstrated that incorporating an increasing number of SynB1 or GALA peptides into the DNA nanopore significantly enhanced the membrane perforation. Kinetic analysis revealed that the DNA nanopore scaffold played a role in prearranging the CPPs, which facilitated membrane interaction and pore formation. Notably, the use of pH-responsive GALA peptides allowed highly efficient and pH-controlled insertion of large DNA pores. Furthermore, single-channel recording elucidated that the insertion process of single GALA-modified nanopores into planar lipid bilayers was dynamic, likely forming transient large toroidal pores. Overall, our study highlights the potential of CPPs as insertion enhancers for DNA nanopores, which opens avenues for improved molecule sensing and the controlled release of cargo molecules.

Dissecting the CRISPR Cas1-Cas2 Protospacer Binding and Selection Mechanism by Using Molecular Dynamics Simulations.

Cas1 and Cas2 are highly conserved proteins among the clustered regularly interspaced short palindromic repeat Cas (CRISPR-Cas) systems and play a crucial role in protospacer selection and integration. According to the double-forked CRISPR Cas1-Cas2 complex, we conducted extensive all-atom molecular dynamics simulations to investigate the protospacer DNA binding and recognition. Our findings revealed that single-point amino acid mutations in Cas1 or in Cas2 had little impact on the binding of the protospacer, both in the binding and precatalytic states. In contrast, multiple-point amino acid mutations, particularly G74A, P80L, and V89A mutations on Cas2 and Cas2' proteins (m-multiple1 system), significantly affected the protospacer binding and selection. Notably, mutations on Cas2 and Cas2' led to an increased number of hydrogen bonds (#HBs) between Cas2&Cas2' and the dsDNA in the m-multiple1 system compared with the wild-type system. And the strong electrostatic interactions between Cas1-Cas2 and the protospacer DNA (psDNA) in the m-multiple1 system again suggested the increase in the binding affinity of protospacer acquisition. Specifically, mutations in Cas2 and Cas2' can remotely make the protospacer adjacent motif complementary (PAMc) sequences better in recognition by the two active sites, while multiple mutations K211E, P202Q, P212L, R138L, V134A, A286T, P282H, and P294H on Cas1a/Cas1b&Cas1a'/Cas1b' (m-multiple2 system) decrease the #HBs and the electrostatic interactions and make the PAMc worse in recognition compared with the wild-type system.

Linear dichroism reveals the perpendicular orientation of DNA bases in the RecA and Rad51 recombinase filaments: A possible mechanism for the strand exchange reaction.

Linear dichroism spectroscopy is used to investigate the structure of RecA family recombinase filaments (RecA and Rad51 proteins) with DNA for clarifying the molecular mechanism of DNA strand exchange promoted by these proteins and its activation. The measurements show that the recombinases promote the perpendicular base orientation of single-stranded DNA only in the presence of activators, indicating the importance of base orientation in the reaction. We summarize the results and discuss the role of DNA base orientation.

Building a stable and robust anti-interference DNA dissipation system by eliminating the accumulation of systemic specified errors.

BACKGROUND: The emergence of DNA nanotechnology has enabled the systematic design of diverse bionic dissipative behaviors under the precise control of nucleic acid nanodevices. Nevertheless, when compared to the dissipation observed in robust living systems, it is highly desirable to enhance the anti-interference for artificial DNA dissipation to withstand perturbations and facilitate repairs within the complex biological environments. RESULTS: In this study, we introduce strategically designed "trash cans" to facilitate kinetic control over interferences, transforming the stochastic binding of individual components within a homogeneous solution into a competitive binding process. This approach effectively eliminates incorrect binding and the accumulation of systemic interferences while ensuring a consistent pattern of energy fluctuation from response to silence. Remarkably, even in the presence of numerous interferences differing by only one base, we successfully achieve complete system reset through multiple cycles, effectively restoring the energy level to a minimum. SIGNIFICANCE: The system was able to operate stably without any adverse effect under conditions of irregular interference, high-abundance interference, and even multiplex interferences including DNA and RNA crosstalk. This work not only provides an effective paradigm for constructing robust DNA dissipation systems but also greatly broadens the potential of DNA dissipation for applications in high-precision molecular recognition and complex biological reaction networks.

Efficient DNA walker guided by ordered cruciform-shaped DNA track for ultrasensitive and rapid electrochemical detection of lead ion.

The rational design of DNA tracks is an effective pathway to guide the autonomous movement and high-efficiency recognition in DNA walkers, showing outstanding advantages for the cascade signal amplification of electrochemical biosensors. However, the uncontrolled distance between two adjacent tracks on the electrode could increase the risk of derailment and interruption of the reaction. Hence, a novel four-way balanced cruciform-shaped DNA track (C-DNT) was designed as a structured pathway to improve the effectiveness and stability of the reaction in DNA walkers. In this work, two kinds of cruciform-shaped DNA were interconnected as a robust structure that could avoid the invalid movement of the designed DNA walker on the electrode. When hairpin H2 was introduced onto the electrode, the strand displacement reaction (SDR) effectively triggered movements of the DNA walker along the cruciform-shaped track while leaving ferrocene (Fc) on the electrode, leading to a significant enhancement of the electrochemical signal. This design enabled the walker to move in an excellent organized and controllable manner, thus enhancing the reaction speed and walking efficiency. Compared to other walkers moving on random tracks, the reaction time of the C-DNT-based DNA walker could be reduced to 20 min. Lead ion (Pb2+) was used as a model target to evaluate the analytical performance of this biosensor, which exhibited a low detection limit of 0.033 pM along with a wide detection ranging from 0.1 pM to 500 nM. This strategy presented a novel concept for designing a high-performance DNA walker-based sensing platform for the detection of contaminants.

An electrochemical biosensor for the amplification of thrombin activity by perylene-mediated photoinitiated polymerization.

BACKGROUND: Thrombin, a coagulation system protease, is a key enzyme involved in the coagulation cascade and has been developed as a marker for coagulation disorders. However, the methods developed in recent years have the disadvantages of complex operation, long reaction time, low specificity and sensitivity. Meanwhile, thrombin is at a lower level in the pre-disease period. Therefore, to accurately diagnose the disease, it is necessary to develop a fast, simple, highly sensitive and specific method using signal amplification technology. RESULTS: We designed an electrochemical biosensor based on photocatalytic atom transfer radical polymerization (photo-ATRP) signal amplification for the detection of thrombin. Sulfhydryl substrate peptides (without carboxyl groups) are self-assembled to the gold electrode surface via Au-S bond and serve as thrombin recognition probes. The substrate peptide is cleaved in the presence of thrombin to generate -COOH, which can form a carboxylate-Zr(IV)-carboxylate complex via Zr(IV) and initiator (α-bromophenylacetic acid, BPAA). Subsequently, an electrochemical biosensor was prepared by introducing polymer chains with electrochemical signaling molecules (ferrocene, Fc) onto the electrode surface by photocatalytic (perylene, Py) mediated ATRP using ferrocenylmethyl methacrylate (FMMA) as a monomer. The concentration of thrombin was evaluated by the voltammetric signal generated by square wave voltammetry (SWV), and the result showed that the biosensor was linear between 1.0 ng/mL âˆ¼ 10 fg/mL, with a lower detection limit of 4.0 fg/mL (∼0.1 fM). Moreover, it was shown to be highly selective for thrombin activity in complex serum samples and for thrombin inhibition screening. SIGNIFICANCE: The biosensor is an environmentally friendly and economically efficient strategy while maintaining the advantages of high sensitivity, anti-interference, good stability and simplicity of operation, which has great potential for application in the analysis of complex samples.

G4-Ligand-Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

Use of DNA forceps to measure receptor-ligand dissociation equilibrium constants in a single-molecule competition assay.

The ability of biophysicists to decipher the behavior of individual biomolecules has steadily improved over the past thirty years. However, it still remains unclear how an ensemble of data acquired at the single-molecule level compares with the data acquired on an ensemble of the same molecules. We here propose an assay to tackle this question in the context of dissociation equilibrium constant measurements. A sensor is built by engrafting a receptor and a ligand onto a flexible dsDNA scaffold and mounting this assembly on magnetic tweezers. This way, looking at the position of the magnetic bead enables one to determine in real-time if the two molecular partners are associated or not. Next, to quantify the affinity of the scrutinized single-receptor for a given competitor, various amounts of the latter molecule are introduced in solution and the equilibrium response of the sensor is monitored throughout the titration protocol. Proofs of concept are established for the binding of three rapamycin analogs to the FKBP12 cis-trans prolyl isomerase. For each of these drugs the mean affinity constant obtained on a ten of individual receptors agrees with the one previously determined in a bulk assay. Furthermore, experimental contingencies are sufficient to explain the dispersion observed over the single-molecule values.