Electrochemical cytosensor utilizing tetrahedral DNA/bimetallic AuPd holothurian-shaped nanoparticles for ultrasensitive non-destructive detection of circulating tumor cells.

As a real-time fluid biopsy method, the detection of circulating tumor cells (CTCs) provides important information for the early diagnosis, precise treatment, and prognosis of cancer. However, the low density of CTCs in the peripheral blood hampers their capture and detection with high sensitivity and selectivity using currently available methods. Hence, we designed a sandwich-type electrochemical aptasensor that utilizes holothurian-shaped AuPd nanoparticles (AuPd HSs), tetrahedral DNA nanostructures (TDNs), and CuPdPt nanowire networks (NWs) interwoven with a graphdiyne (GDY) sheet for ultrasensitive non-destructive detection of MCF-7 breast cancer cells. CuPdPt NW-GDY effectively enhanced the electron transfer rate and coupled with the loaded TDNs. The TDNs could capture MCF-7 cells with precision and firmness, and the resulting composite complex was combined with AuPd HSs to form a sandwich-type structure. This novel aptasensor showed a linear range between 10 and 106 cells mL-1 and an ultralow detection limit of 7 cells mL-1. The specificity, stability, and repeatability of the measurements were successfully verified. Moreover, we used benzonase nuclease to achieve non-destructive recovery of cells for further clinical studies. According to the results, our aptasensor was more sensitive measuring the number of CTCs than other approaches because of the employment of TDNs, CuPdPt NW-GDY, and AuPd HSs. We designed a reliable sensor system for the detection of CTCs in the peripheral blood, which could serve as a new approach for cancer diagnosis at an early stage.

DNA origami scaffold promoting nerve guidance and regeneration.

Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.

Construction of lymph nodes-targeting tumor vaccines by using the principle of DNA base complementary pairing to enhance anti-tumor cellular immune response.

Tumor vaccines, a crucial immunotherapy, have gained growing interest because of their unique capability to initiate precise anti-tumor immune responses and establish enduring immune memory. Injected tumor vaccines passively diffuse to the adjacent draining lymph nodes, where the residing antigen-presenting cells capture and present tumor antigens to T cells. This process represents the initial phase of the immune response to the tumor vaccines and constitutes a pivotal determinant of their effectiveness. Nevertheless, the granularity paradox, arising from the different requirements between the passive targeting delivery of tumor vaccines to lymph nodes and the uptake by antigen-presenting cells, diminishes the efficacy of lymph node-targeting tumor vaccines. This study addressed this challenge by employing a vaccine formulation with a tunable, controlled particle size. Manganese dioxide (MnO2) nanoparticles were synthesized, loaded with ovalbumin (OVA), and modified with A50 or T20 DNA single strands to obtain MnO2/OVA/A50 and MnO2/OVA/T20, respectively. Administering the vaccines sequentially, upon reaching the lymph nodes, the two vaccines converge and simultaneously aggregate into MnO2/OVA/A50-T20 particles through base pairing. This process enhances both vaccine uptake and antigen delivery. In vitro and in vivo studies demonstrated that, the combined vaccine, comprising MnO2/OVA/A50 and MnO2/OVA/T20, exhibited robust immunization effects and remarkable anti-tumor efficacy in the melanoma animal models. The strategy of controlling tumor vaccine size and consequently improving tumor antigen presentation efficiency and vaccine efficacy via the DNA base-pairing principle, provides novel concepts for the development of efficient tumor vaccines.

Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides.

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction. This study explores simple, new approaches to extraction using engineered smart nanomaterials, namely NA-binding, intrinsically disordered proteins (IDPs), that undergo triggered liquid-liquid phase separation (LLPS). Two types of NA-binding IDPs are studied, both based on genetically engineered elastin-like polypeptides (ELPs), model IDPs that exhibit a lower critical solution temperature in water and can be designed to exhibit LLPS at desired temperatures in a variety of biological solutions. We show that ELP fusion proteins with natural NA-binding domains can be used to extract DNA and RNA from physiologically relevant solutions. We further show that LLPS of pH responsive ELPs that incorporate histidine in their sequences can be used for both binding, extraction and release of NAs from biological solutions, and can be used to detect SARS-CoV-2 RNA in samples from COVID-positive patients.

Synthetic molecular switches driven by DNA-modifying enzymes.

Taking inspiration from natural systems, in which molecular switches are ubiquitous in the biochemistry regulatory network, we aim to design and construct synthetic molecular switches driven by DNA-modifying enzymes, such as DNA polymerase and nicking endonuclease. The enzymatic treatments on our synthetic DNA constructs controllably switch ON or OFF the sticky end cohesion and in turn cascade to the structural association or disassociation. Here we showcase the concept in multiple DNA nanostructure systems with robust assembly/disassembly performance. The switch mechanisms are first illustrated in minimalist systems with a few DNA strands. Then the ON/OFF switches are realized in complex DNA lattice and origami systems with designated morphological changes responsive to the specific enzymatic treatments.

Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure.

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

Nucleosomal DNA has topological memory.

One elusive aspect of the chromosome architecture is how it constrains the DNA topology. Nucleosomes stabilise negative DNA supercoils by restraining a DNA linking number difference (∆Lk) of about -1.26. However, whether this capacity is uniform across the genome is unknown. Here, we calculate the ∆Lk restrained by over 4000 nucleosomes in yeast cells. To achieve this, we insert each nucleosome in a circular minichromosome and perform Topo-seq, a high-throughput procedure to inspect the topology of circular DNA libraries in one gel electrophoresis. We show that nucleosomes inherently restrain distinct ∆Lk values depending on their genomic origin. Nucleosome DNA topologies differ at gene bodies (∆Lk = -1.29), intergenic regions (∆Lk = -1.23), rDNA genes (∆Lk = -1.24) and telomeric regions (∆Lk = -1.07). Nucleosomes near the transcription start and termination sites also exhibit singular DNA topologies. Our findings demonstrate that nucleosome DNA topology is imprinted by its native chromatin context and persists when the nucleosome is relocated.

A target-triggered strand displacement-assisted target recycling based on carbon dots-based fluorescent probe and MSNs@PDA nanoparticles for miRNA amplified detection and fluorescence imaging.

A target-triggered strand displacement-assisted target recycling based on carbon dots-based fluorescent probe and mesoporous silica nanoparticles@polydopamine (MSNs@PDA) was established to detect miRNA. The surface of MSNs rich in mesopores was coated with a layer of PDA, which can adsorb and quench the fluorescence of single-stranded Fuel DNA with fluorescent carbon dots (CDs) modified at the end through fluorescence resonance energy transfer (FRET). After adding double-stranded DNA-gold nanoparticles (dsDNA-AuNPs) and target let-7a, it will trigger two toehold-mediated strand displacement reactions (TSDR), leading to the recovery of fluorescence and the recycling of target let-7a (excitation wavelength: 380 nm; emission wavelength: 458 nm). The recovery value of fluorescence is proportional to the logarithm of the target microRNA let-7a concentration, thus realizing the sensitivity amplification detection of disease markers. The MSNs@PDA@Fuel DNA-CDs/dsDNA-AuNPs nanoplatform based on the strategy of "on-off-on" and TSDR cyclic amplification may hold great potential as an effective and safe nanoprobe for accurate fluorescence imaging of diseases related to miRNA with low abundances.

Prediction of Transcription Factor Binding Sites on Cell-Free DNA Based on Deep Learning.

Transcription factors (TFs) are important regulatory elements for vital cellular activities, and the identification of transcription factor binding sites (TFBS) can help to explore gene regulatory mechanisms. Research studies have proved that cfDNA (cell-free DNA) shows relatively higher coverage at TFBS due to the protection by TF from degradation by nucleases and short fragments of cfDNA are enriched in TFBS. However, there are still great difficulties in the noninvasive identification of TFBSs from experimental techniques. In this study, we propose a deep learning-based approach that can noninvasively predict TFBSs of cfDNA by learning sequence information from known TFBSs through convolutional neural networks. Under the addition of long short-term memory, our model achieved an area under the curve of 84%. Based on this model to predict cfDNA, we found consistent motifs in cfDNA fragments and lower coverage occurred upstream and downstream of these cfDNA fragments, which is consistent with a previous study. We also found that the binding sites of the same TF differ in different cell lines. TF-specific target genes were detected from cfDNA and were enriched in cancer-related pathways. In summary, our method of locating TFBSs from plasma has the potential to reflect the intrinsic regulatory mechanism from a noninvasive perspective and provide technical guidance for dynamic monitoring of disease in clinical practice.

Binding energies and hydrogen bonds effects on DNA-cisplatin interactions: a DFT-xTB study.

CONTEXT: A systematic study of hydrogen bonds in base pairs and the interaction of cisplatin with DNA fragments was carried out. Structure, binding energies, and electron density were analyzed. xTB has proven to be an accurate method for obtaining structures and binding energies in DNA structures. Our xTB values for DNA base binding energy were in the same order and in some cases better than CAM-B3LYP values compared to experimental values. Double-stranded DNA-cisplatin structures have been calculated and the hydrogen bonds of water molecules are a decisive factor contributing to the preference for the cisplatin-Guanine interaction. Higher values of the water hydrogen bonding energies were obtained in cisplatin-Guanine structures. Furthermore, the electrostatic potential was used to investigate and improve the analysis of DNA-cisplatin structures. METHODS: We applied the xTB method and the CAM-B3LYP functional combined with def2-SVP basis set to perform and analyze of the bonding energies of the cisplatin interaction and the effects of the hydrogen bonds. Results were calculated employing the xTB and the ORCA software.

The RNA-DNA world and the emergence of DNA-encoded heritable traits.

The RNA world hypothesis confers a central role to RNA molecules in information encoding and catalysis. Even though evidence in support of this hypothesis has accumulated from both experiments and computational modelling, the transition from an RNA world to a world where heritable genetic information is encoded in DNA remains an open question. Recent experiments show that both RNA and DNA templates can extend complementary primers using free RNA/DNA nucleotides, either non-enzymatically or in the presence of a replicase ribozyme. Guided by these experiments, we analyse protocellular evolution with an expanded set of reaction pathways made possible through the presence of DNA nucleotides. By encapsulating these reactions inside three different types of protocellular compartments, each subject to distinct modes of selection, we show how protocells containing DNA-encoded replicases in low copy numbers and replicases in high copy numbers can dominate the population. This is facilitated by a reaction that leads to auto-catalytic synthesis of replicase ribozymes from DNA templates encoding the replicase after the chance emergence of a replicase through non-enzymatic reactions. Our work unveils a pathway for the transition from an RNA world to a mixed RNA-DNA world characterized by Darwinian evolution, where DNA sequences encode heritable phenotypes.

Multiarmed DNA jumper and metal-organic frameworks-functionalized paper-based bioplatform for small extracellular vesicle-derived miRNAs assay.

Small extracellular vesicle-derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na+, combined with a disposable paper-based electrode modified with a Zr-MOF-rGO-Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing.

Ligandability Assessment of IL-1ß by Integrated Hit Identification Approaches.

Human interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that plays a critical role in the regulation of the immune response and the development of various inflammatory diseases. In this publication, we disclose our efforts toward the discovery of IL-1ß binders that interfere with IL-1ß signaling. To this end, several technologies were used in parallel, including fragment-based screening (FBS), DNA-encoded library (DEL) technology, peptide discovery platform (PDP), and virtual screening. The utilization of distinct technologies resulted in the identification of new chemical entities exploiting three different sites on IL-1ß, all of them also inhibiting the interaction with the IL-1R1 receptor. Moreover, we identified lysine 103 of IL-1ß as a target residue suitable for the development of covalent, low-molecular-weight IL-1ß antagonists.

Immobilization-free and label-free electrochemical DNA biosensing based on target-stimulated release of redox reporter and its catalytic redox recycling.

Herein, we demonstrate a simple, homogenous and label-free electrochemical biosensing system for sensitive nucleic acid detection based on target-responsive porous materials and nuclease-triggered target recycling amplification. The Fe(CN)63- reporter was firstly sealed into the pores of Fe3O4 nanoparticles by probe DNA. Target DNA recognition triggered the controllable release of Fe(CN)63- for the redox reaction with the electron mediator of methylene blue enriched in the dodecanethiol assembled electrode and thereby generating electrochemical signal. The exonuclease III (Exo III)-assisted target recycling and the catalytic redox recycling between Fe(CN)63- and methylene blue contributed for the enhanced signal response toward target recognition. The low detection limit toward target was obtained as 478 fM and 1.6 pM, respectively, by square wave voltammetry and cyclic voltammetry methods. It also possessed a well-discrimination ability toward mismatched strands and high tolerance to complex sample matrix. The coupling of bio-gated porous nanoparticles, nuclease-assisted target amplification and catalytic redox recycling afforded the sensing system with well-controllable signal responses, sensitive and selective DNA detection, and good stability, reusability and reproducibility. It thus opens a new avenue toward the development of simple but sensitive electrochemical biosensing platform.

Hydrophobic Surfactant-DNA Complex (Surf-DNA) Enables DNA-Encoded-Library-Compatible Decarboxylative Arylation under Anhydrous Conditions.

DNA-encoded libraries (DELs) are a key technology for identifying small-molecule hits in both the pharmaceutical industry and academia, but their chemical diversity is largely limited to water-compatible reactions to aid in the solubility and integrity of encoding DNA tags. To broaden the DEL chemical space, we present a workflow utilizing DNA-cationic surfactant complexation that enables dissolution and reactions on-DNA in anhydrous organic solvents. We demonstrate its utility by developing DEL-compatible photoredox decarboxylative C(sp2)-C(sp3) coupling under water-free conditions. The workflow is optimized for the 96-well format necessary for large-scale DEL productions, and it enables screening and optimization of DEL-compatible reactions in organic solvents.

Digital Sort-Enabled Counting Allows Absolute Electrical Quantification of Target Nucleic Acid.

Quantitative nucleic acid amplification tests are of great importance for diagnostics, but current approaches require complex and costly optical setups that limit their nonlaboratory applications. Herein we describe the implementation of a microfluidics platform that can perform binary DNA-amplification-activated droplet sorting. The digital sort-enabled counting (DISCO) platform enables label-free absolute quantification of the nucleic acid. This is achieved by provoking a pH change in droplets through a loop-mediated isothermal amplification (LAMP) reaction, followed by using sorting by interfacial tension (SIFT) to direct positive and negative droplets to different outlets. With the use of on-chip electrodes at both outlets, we demonstrate that the digital electrical counting of target DNA and RNA can be realized. DISCO is a promising approach for realizing sensitive nucleic acid quantification in point-of-care settings.

Innovative On-DNA Synthesis of Sulfides and Sulfoximines: Enriching the DEL Synthesis Toolbox.

DNA-encoded library (DEL) technologies enable the fast exploration of gigantic chemical space to identify ligands for the target protein of interest and have become a powerful hit finding tool for drug discovery projects. However, amenable DEL chemistry is restricted to a handful of reactions, limiting the creativity of drug hunters. Here, we describe a new on-DNA synthetic pathway to access sulfides and sulfoximines. These moieties, usually contemplated as challenging to achieve through alkylation and oxidation, can now be leveraged in routine DEL selection campaigns.

Accelerated diagnosis: a crosslinking catalytic hairpin assembly system for rapid and sensitive SARS-CoV-2 RNA detection.

The COVID-19 pandemic has underscored the urgent need for rapid and reliable strategies for early detection of SARS-CoV-2. In this study, we propose a DNA nanosphere-based crosslinking catalytic hairpin assembly (CCHA) system for the rapid and sensitive SARS-CoV-2 RNA detection. The CCHA system employs two DNA nanospheres functionalized with catalytic hairpin assembly (CHA) hairpins. The presence of target SARS-CoV-2 RNA initiated the crosslinking of DNA nanospheres via CHA process, leading to the amplification of fluorescence signals. As a result, the speed of SARS-CoV-2 diagnosis was enhanced by significantly increasing the local concentration of the reagents in a crosslinked DNA product, leading to a detection limit of 363 fM within 5 min. The robustness of this system has been validated in complex environments, such as fetal bovine serum and saliva. Hence, the proposed CCHA system offers an efficient and simple approach for rapid detection of SARS-CoV-2 RNA, holding substantial promise for enhancing COVID-19 diagnosis.

Inverse design of a pyrochlore lattice of DNA origami through model-driven experiments.

Sophisticated statistical mechanics approaches and human intuition have demonstrated the possibility of self-assembling complex lattices or finite-size constructs. However, attempts so far have mostly only been successful in silico and often fail in experiment because of unpredicted traps associated with kinetic slowing down (gelation, glass transition) and competing ordered structures. Theoretical predictions also face the difficulty of encoding the desired interparticle interaction potential with the experimentally available nano- and micrometer-sized particles. To overcome these issues, we combine SAT assembly (a patchy-particle interaction design algorithm based on constrained optimization) with coarse-grained simulations of DNA nanotechnology to experimentally realize trap-free self-assembly pathways. We use this approach to assemble a pyrochlore three-dimensional lattice, coveted for its promise in the construction of optical metamaterials, and characterize it with small-angle x-ray scattering and scanning electron microscopy visualization.

Diamond-lattice photonic crystals assembled from DNA origami.

Colloidal self-assembly allows rational design of structures on the micrometer and submicrometer scale. One architecture that can generate complete three-dimensional photonic bandgaps is the diamond cubic lattice, which has remained difficult to realize at length scales comparable with the wavelength of visible or ultraviolet light. In this work, we demonstrate three-dimensional photonic crystals self-assembled from DNA origami that act as precisely programmable patchy colloids. Our DNA-based nanoscale tetrapods crystallize into a rod-connected diamond cubic lattice with a periodicity of 170 nanometers. This structure serves as a scaffold for atomic-layer deposition of high-refractive index materials such as titanium dioxide, yielding a tunable photonic bandgap in the near-ultraviolet.