DNA Complexes Research Articles

Parallel DNA Synthesis to Produce Multi-Usage Two-Dimensional Barcodes

Data storage on DNA has emerged as a molecular approach to safeguarding digital information. Microarrays are an excellent source of complex DNA sequence libraries and are playing a central role in the development of this technology. However, the amount of DNA recovered from microarrays is often too small, and a PCR amplification step is usually required. Primer information can be conveyed alongside the DNA library itself in the form of readable barcodes made of DNA on the array surface. Here, we present a synthetic method to pattern QR and data matrix barcodes using DNA photolithography, phosphoramidite chemistry and fluorescent labeling. Patterning and DNA library synthesis occur simultaneously and on the same surface. We manipulate the chemical composition of the barcodes to make them indelible, erasable or hidden, and a simple chemical treatment under basic conditions can reveal or degrade the pattern. In doing so, information crucial to retrieval and amplification can be made available by the user at the appropriate stage. The code and its data contained within are intimately linked to the library as they are synthesized simultaneously and on the same surface. This process is, in principle, applicable to any in situ microarray synthesis method, for instance, inkjet or electrochemical DNA synthesis.

Nuclear lipids in chromatin regulation: Biological roles, experimental approaches and existing challenges.

Lipids are crucial for various cellular functions. Besides the storage of energy equivalents, these include forming membrane bilayers and serving as signaling molecules. While significant progress has been made in the comprehension of the molecular and cellular biology of lipids, their functions in the cell nucleus remain poorly understood. The main role of the eukaryotic cell nucleus is to provide an environment for the storage and regulation of chromatin which is a complex of DNA, histones, and associated proteins. Recent studies suggest that nuclear lipids play a role in chromatin regulation and epigenetics. Here, we discuss various experimental methods in lipid-chromatin research, including biophysical, structural, and cell biology approaches, pointing out their strengths and weaknesses. We take the view that nuclear lipids have a far more widespread impact on chromatin than is currently acknowledged. This gap in comprehension is mostly due to existing experimental challenges in the study of lipid-chromatin biology. Several new, interdisciplinary approaches are discussed that could aid in elucidating the roles of nuclear lipids in chromatin regulation and gene expression.

A Comprehensive Review of Pfu DNA Polymerase: Extraction, Production and Applications

Pfu DNA Polymerase, a thermostable enzyme derived from the hyperthermophilic archaeon Pyrococcus furiosus, is widely recognized for its high fidelity and strong processivity. Its 3'-5' exonuclease activity makes it indispensable for the correct amplification of both short and complex DNA strands. These biochemical properties of Pfu DNA Polymerase have encouraged considerable advancements in its extraction and production methods. This review covers some of the traditional approaches for purification, including protein purification and affinity chromatography, and updates on recent advances in recombinant gene expression, automated systems of production, and membrane-based technologies. New ways of engineering enzymes have recently been developed, such as CRISPR-Cas9-mediated gene optimization, which raised the bar on extraction efficiency to meet emerging demand. Once challenging, Pfu DNA Polymerase production has been significantly streamlined through recombinant expression in E. coli both at a laboratory and commercial scale. The optimization techniques that are involved with IPTG concentration and Response Surface Methodology have increased the yield by up to 30%. Auto-induction means even higher biomass outputs are allowed. Today, applications of Pfu DNA Polymerase span from standard PCR up to advanced clinical diagnostics in the fields of molecular biology, forensic analysis, clinical microbiology, and biotechnology.

Reconstitution of SPO11-dependent double-strand break formation.

Homologous meiotic recombination starts with DNA double-strand breaks (DSBs) generated by SPO11 protein 1 . SPO11 is critical for meiosis in most species but the DSBs it makes are also dangerous because of their mutagenic 2 and gametocidal 3 potential, so cells must foster SPO11's beneficial functions while minimizing its risks 4 . SPO11 mechanism and regulation remain poorly understood. Here we report reconstitution of DNA cleavage in vitro with purified recombinant mouse SPO11 bound to its essential partner TOP6BL. Similar to their yeast orthologs 5,6 , SPO11- TOP6BL complexes are monomeric (1:1) in solution and bind tightly to DNA. Unlike in yeast, however, dimeric (2:2) assemblies of mouse SPO11-TOP6BL cleave DNA to form covalent 5' attachments requiring SPO11 active site residues, divalent metal ions, and SPO11 dimerization. Surprisingly, SPO11 can also manifest topoisomerase activity by relaxing supercoils and resealing DNA that it has nicked. Structure modeling with AlphaFold3 7 illuminates the protein-DNA interface and suggests that DNA is bent prior to cleavage. Deep sequencing of in vitro cleavage products reveals a rotationally symmetric base composition bias that partially explains DSB site preferences in vivo. Cleavage is inefficient on complex DNA substrates, partly because SPO11 is readily trapped in DSB-incompetent (presumably monomeric) binding states that exchange slowly. However, cleavage is improved by using substrates that favor DSB-competent dimer assembly, or by fusing SPO11 to an artificial dimerization module. Our results inform a model in which intrinsically feeble dimerization restrains SPO11 activity in vivo, making it exquisitely dependent on accessory proteins that focus and control DSB formation so that it happens only at the right time and the right places.

Binding investigation of chrysin and flavone with promoter DNA sequence d(CACGTG)2: Multi spectroscopic studies and cytotoxic evaluation

Interactions of chrysin and flavone with d(CACGTG)2 DNA sequence were explored using multi spectroscopic techniques. Absorption and fluorescence spectroscopic results indicated the partial intercalation for chrysin, and flavone interact externally. The binding coefficient (Kb) values of chrysin and flavone are 105 and 104 M-1, respectively. Job plots using fluorescence data confirmed the 1:1 stoichiometry for the chrysin DNA complex, while multiple stoichiometries for the flavone DNA complex in solutions. Fluorescence lifetime analysis data showed that the average lifetime of chrysin increased while the average lifetime of flavone decreased upon complexation with DNA. Competitive displacement studies with EtBr and Hoechst 33258 data suggest partial intercalation. Circular dichroism results showed that DNA structures were perturbed upon chrysin and flavone binding. Circular dichroism and differential scanning calorimetry thermal data showed minute changes in melting temperature at 1:1 complex for chrysin-DNA, while there was no significant change in flavone DNA complex. These slight changes in melting temperatures support the non-intercalative binding of chrysin and flavone. The heat enthalpy changes of the free d(CACGTG)2 DNA increased significantly upon interaction with both chrysin and flavone. Specifically, for chrysin, ΔH1 increased from 88.07 to 196.40 kcal/mol and ΔH2 from 41.14 to 104.00 kcal/mol. In the case of flavone, ΔH1 rose from 88.07 to 127.40 kcal/mol and ΔH2 from 41.14 to 180.70 kcal/mol. The van der Waals forces and hydrophobic interactions are key contributors to the binding between d(CACGTG)2 DNA and both chrysin and flavone. The cytotoxic effect of chrysin and flavone was analyzed using an MTT assay and AO/EtBr staining. MTT data showed that they exhibited a significant antiproliferation effect against MCF-7, DU-145, and HCT-15 cancer cell lines, where flavone is most effective against DU-145 prostate cancer cells, and chrysin has more effects against MCF-7 breast cancer cells. The IC50 values of chrysin and flavone are 74.97 µM, 95.15 µM, 88.66 µM, 78.62 µM and 149.45 µM, 45.17 µM against MCF-7, HCT-15, and DU-145 respectively. Flavone compounds induced apoptosis substantially in all three cancer cell lines compared to the control cells. This study recognizes flavones as potential proapoptotic agents.

Nucleus-Targeting Photosensitizers Enhance Neutrophil Extracellular Traps for Efficient Eradication of Multidrug-Resistant Bacterial Infections.

Neutrophil extracellular traps (NETs) are web-like complexes of DNA and proteins that are extruded by activated neutrophils and play critical roles as major components of the innate immune response against pathogen invasion. However, some microbes have developed strategies to evade NET attacks, leading to impaired immune defenses and persistent infections. In this study, an engineered neutrophil strategy for enhancing the antibacterial activity of NETs is developed. A nucleus-targeting photosensitizer (NCP) with strong reactive oxygen species production and a strong DNA-binding capacity is synthesized. NCP-loaded neutrophils are subsequently constructed via direct incubation of NCP with neutrophils, and the NCP is closely inserted into the nucleus DNA. Upon activation by bacteria-related toxins, NCP-coupled NETs can be released rapidly, actively trapping bacteria and providing a high local concentration of NCP around them. Both in vitro and in vivo results revealed that NCP-coupled NETs can effectively eradicate various multidrug-resistant bacteria and biofilms through photodynamic therapy, overcome bacterial immune evasion, and promote tissue recovery from severe wound infections. This design can significantly strengthen NET function, providing a non-antibiotic alternative platform for treating bacterial infectious diseases.

The Imbalance of Homeostasis in Neutrophil Extracellular Traps is Associated with Portal Vein Thrombosis in Patients with Decompensated Cirrhosis.

Portal vein thrombosis (PVT) is a challenging complication in liver cirrhosis, with no currently available sensitive diagnostic markers. This study aimed to investigate the potential of neutrophil extracellular traps (NETs) and Deoxyribonuclease (DNase) as diagnostic indicators for PVT in chronic hepatitis B (CHB)-related decompensated cirrhosis. We analyzed 145 CHB-related decompensated cirrhosis patients from the Ditan study and 33 from the Changgung validation study, categorizing them based on PVT occurrence. Plasma samples were assessed for NET markers, including cell-free DNA (cfDNA) and histone-DNA complexes, along with DNase activity. PVT patients exhibited elevated levels of cfDNA and histone-DNA complexes, and reduced DNase activity. This pattern persisted regardless of hepatocellular carcinoma (HCC) status. Histone-DNA levels, DNase activity, and hemoglobin were identified as independent risk factors for PVT. Receiver operating characteristic curve analysis revealed that high histone-DNA levels may serve as a potential diagnostic marker for PVT, with an area under the curve of 0.8628 in the Ditan study and 0.7521 in the Changgung study. When combined with cfDNA and DNase activity, the area under the curve improved to 0.8774 in the Ditan study and 0.7975 in the Changgung study. Imbalances in NET homeostasis are associated with PVT in CHB-related decompensated cirrhosis, including cases involving HCC. Histone-DNA complexes, a significant risk factor for PVT, show potential as a diagnostic marker for PVT in decompensated cirrhosis, particularly in HBV-related HCC.

Allosteric stem-loop multicomponent DNAzyme: A versatile stimuli-responsive switch for nanotweezer operations and multifunctional biosensing

DNAzyme, possessing the capabilities of both encoding information and catalyzing chemical reactions, holds significant value in advancing the widespread application of dynamic nanotechnology. Particularly, multicomponent DNAzyme (MNAzyme) has attracted considerable attention due to its remarkable versatility and practicality. However, the inevitable signal leakage of MNAzyme constrains the applications in complex nucleic acid reaction networks. Herein, we present an allosteric stem-loop multicomponent DNAzyme (ASLM) that incorporates a blocking domain to mitigate leakage resulting from secondary structures of MNAzyme while simultaneously stabilizing binding affinity during complex formation. As the sequence of blocking domain serves as an extension independent of the functional region in MNAzyme, this modulation structure can be flexibly assembled with conformation-switchable nanotweezer to form a time-responsive molecular device, enabling dynamic and dissipative operations through nucleic acid and RNase H. More importantly, the ASLM structure can be directly incorporated into DNA catalytic circuits for detecting multiple biomolecules (nucleic acid, protease, and small molecule). With its scalability and versatility, this effector-activated allosteric regulator will serve as a universal stimuli-responsive switch in complex and hierarchical DNA networks, expanding the scope of DNA nanotechnology in molecular device construction and diagnostic biosensing.

Charge Transfer Interaction Dynamics between 2-Methyl-8-hydroxyquinoline and 2,4-Dinitrophenol: Synthesis, Spectroscopic Characterization, DNA Binding and DFT Studies

A novel charge transfer (CT) complex was formed by combining the electron-acceptor 2,4-dinitrophenol (DNP) with the electron-donor 2-methyl-8-hydroxyquinoline (MHQ). The complex obtained was further characterized using both experimental and theoretical methods. The Benesi-Hildebrand equation can be utilized to determine various spectroscopic physical measurements such as the molar absorptivity (εCT) and formation constant (KCT). The CT complex has a stoichiometry of 1:1. Multiple spectroscopic methods were employed to investigate the resultant solid compound. The existence of charge and proton transfer in the resultant complex was confirmed by FT-IR, 1H NMR, SEM-EDX and powder-XRD studies. An electron absorption spectroscopy examination was done to analyze the complex DNA binding capability. The resulting complex was determined to have an intercalative binding mechanism, with an intrinsic binding constant (Kb) value of 4.2 × 106 M-1. The experimental results were corroborated by performing theoretical calculations using DFT using a CAM-B3LYP/6-31G(d,p) basis set. The geometrical parameters, electrostatic potential maps (MEPs) and Mullikan charges were computed and examined in agreement with the experimental observations. In addition to electron transmission, the stability of the complex is influenced by the presence of a hydrogen bond.

Ultra-sensitive immobilization-free homogeneous electrochemiluminescence biosensor for thrombin detection via electrostatic interaction and Exo I-powered signal amplification

Herein, we present the development of an ultra-sensitive immobilization-free homogeneous electrochemiluminescence (ECL) biosensor, leveraging the electrostatic repulsion between a negatively charged indium tin oxide (ITO) electrode and DNA and exonuclease I (Exo I)-powered signal amplification, to achieve highly efficient detection of thrombin. Specifically, the aptamer and the complementary DNA engage in the formation of a double-stranded DNA (dsDNA) complex. This negatively charged dsDNA structure subsequently associates with a positively charged ECL indicator, namely ruthenium phenanthroline (Ru(phen)32+), resulting in the generation of the dsDNA-Ru(phen)32+ ECL probe. The negatively charged dsDNA-Ru(phen)32+ experiences electrostatic repulsion from the negatively charged ITO electrode, resulting in a low ECL signal. Nonetheless, upon the addition of thrombin, the aptamer preferentially binds to thrombin, triggering the releases of the embedded Ru(phen)32+ facilitated by Exo I and hence resulting in a robust and enhanced ECL signal. The amplified ECL signal is linearly correlated with the logarithm of thrombin concentration within a detection range spanning from 10 fmol/mL to 50 pmol/mL, with a remarkable detection limit of 3.21 fmol/mL. This strategy eliminates the need for cumbersome labeling steps, avoids the electrode modification process, overcoming the low immobilization efficiency of aptamers and poor signal transduction of indicators labeled at the end of DNA.

Design of Experiments to Tailor the Potential of BSA-Coated Peptide Nanocomplexes for Temozolomide/p53 Gene Co-Delivery.

Background: Gene therapy can be viewed as a promising/valuable therapeutic approach directed to cancer treatment, including glioblastoma. Concretely, the combination of gene therapy with chemotherapy could increase its therapeutic index due to a synergistic effect. In this context, bovine serum albumin (BSA)-coated temozolomide (TMZ)-peptide (WRAP5)/p53 gene-based plasmid DNA complexes were developed to promote payload co-delivery. Methods: Design of experiments (DoE) was employed to unravel the BSA-coated TMZ-WRAP5/p53 nanocomplexes with the highest potential by considering the nitrogen to phosphate groups ratio (N/P), and the BSA concentration as inputs and the size, polydispersity index, surface charge and p53-based plasmid complexation capacity (CC) as DoE outputs. Results: The obtained quadratic models were statistically significant (p-value < 0.05) with an adequate coefficient of determination, and the correspondent optimal points were successfully validated. The optimal complex formulation had N/P of 1.03, a BSA concentration of 0.08%, a size of approximately 182 nm, a zeta potential of +9.8 mV, and a pDNA CC of 96.5%. The optimal nanocomplexes are approximately spherical. A cytotoxicity assay showed that these BSA-coated TMZ-WRAP5/p53 complexes did not elicit toxicity in normal brain cells, and a hemolysis study demonstrated the hemocompatibility of the complexes. The complexes were stable in cell culture medium and fetal bovine serum and assured pDNA protection and release. Moreover, the optimal BSA-coated complexes were able of gene transcription and promoted a significant inhibition of glioblastoma cell viability. Conclusions: The reported findings instigate the development of future research to evaluate their potential utility to TMZ/p53 co-delivery. The DoE tool proved to be a powerful approach to explore and tailor the composition of BSA-coated TMZ-WRAP5/p53 complexes, which are expected to contribute to the progress toward a more efficient therapy against cancer and, more specifically, against glioblastoma.

Development of a novel computational technique to create DNA and cell geometrical models for Geant4-DNA

BackgroundThis study aimed to develop a novel human cell geometry for the Geant4-DNA simulation toolkit that explicitly incorporates all 23 chromosome pairs of the human cell. This approach contrasts with the existing, default human cell, geometrical model, which utilizes a continuous Hilbert curve. MethodsA Python-based tool named “complexDNA” was developed to facilitate the design of both simple and complex DNA geometries. This tool was employed to construct a human cell geometry with individual pairs of chromosomes. Subsequently, the performance of this chromosomal model was compared to the standard human cell model provided in the “molecularDNA” Geant4-DNA example. ResultsSimulations using the new chromosomal model revealed minimal discrepancies in DNA damage yield and fragment size distribution compared to the default human cell model. Notably, the chromosomal model demonstrated significant computational efficiency, requiring approximately three times less simulation time to achieve equivalent results. ConclusionsThis work highlights the importance of incorporating chromosomal structure into human cell models for radiation biology research. The “complexDNA” tool offers a valuable resource for creating intricate DNA structures for future studies. Further refinements, such as implementing smaller voxels for euchromatin regions, are proposed to enhance the model’s accuracy.

SETD3-mediated histidine methylation of MCM7 regulates DNA replication by facilitating chromatin loading of MCM.

The minichromosome maintenance complex (MCM) DNA helicase is an important replicative factor during DNA replication. The proper chromatin loading of MCM is a key step to ensure replication initiation during S phase. Because replication initiation is regulated by multiple biological cues, additional changes to MCM may provide better understanding towards this event. Here, we report that histidine methyltransferase SETD3 promotes DNA replication in a manner dependent on enzymatic activity. Nascent-strand sequencing (NS-seq) shows that SETD3 regulates replication initiation, as depletion of SETD3 attenuates early replication origins firing. Biochemical studies reveal that SETD3 binds MCM mainly during S phase, which is required for the CDT1-mediated chromatin loading of MCM. This MCM loading relies on histidine-459 methylation (H459me) on MCM7 which is catalyzed by SETD3. Impairment of H459 methylation attenuates DNA synthesis and chromatin loading of MCM. Furthermore, we show that CDK2 phosphorylates SETD3 at Serine-21 during the G1/S phase, which is required for DNA replication and cell cycle progression. These findings demonstrate a novel mechanism by which SETD3 methylates MCM to regulate replication initiation.

Convolutional network learning of self-consistent electron density via grid-projected atomic fingerprints

The self-consistent field (SCF) generation of the three-dimensional (3D) electron density distribution (ρ) represents a fundamental aspect of density functional theory (DFT) and related first-principles calculations, and how one can shorten or bypass the SCF loop represents a critical question in electronic structure theory from both practical and fundamental standpoints. Herein, a machine learning strategy, DeepSCF, is presented in which the map between the SCF ρ and the initial guess density (ρ0) constructed by the summation of neutral atomic densities is learned using 3D convolutional neural networks (CNNs). High accuracy and transferability of DeepSCF are achieved by first encoding ρ0 on a 3D grid and then expanding the input features to include atomic fingerprints beyond ρ0. The prediction of the residual density (δρ) rather than ρ itself is targeted, and given that δρ is indicative of chemical bonding information, a dataset of small-sized organic molecules featuring diverse bonding characters is adopted. The fidelity of DeepSCF is finally enhanced by subjecting the atomic geometries of the dataset to random rotations and strains. The effectiveness of DeepSCF is demonstrated using a complex carbon nanotube-based DNA sequencer model. This work evidences that the nearsightedness in electronic structure can be optimally represented via the spatial locality in CNNs, offering insight into the success of various machine learning-based atomistic materials simulations.

DNA Catalysis: Design, Function, and Optimization.

Catalytic DNA has gained significant attention in recent decades as a highly efficient and tunable catalyst, thanks to its flexible structures, exceptional specificity, and ease of optimization. Despite being composed of just four monomers, DNA's complex conformational intricacies enable a wide range of nuanced functions, including scaffolding, electrocatalysis, enantioselectivity, and mechano-electro spin coupling. DNA catalysts, ranging from traditional DNAzymes to innovative DNAzyme hybrids, highlight the remarkable potential of DNA in catalysis. Recent advancements in spectroscopic techniques have deepened our mechanistic understanding of catalytic DNA, paving the way for rational structural optimization. This review will summarize the latest studies on the performance and optimization of traditional DNAzymes and provide an in-depth analysis of DNAzyme hybrid catalysts and their unique and promising properties.

ROS‐Activatable Prodrug of Doxazolidine as Novel Cancer Therapy Paradigm

AbstractOvercoming severe side effects from anticancer agents without decreasing their effects on tumor growth is a major challenge. A prodrug technology is reported using agents that are spatiotemporally activated primarily in tumors while the extratumoral toxicity to healthy cells is minimized. A ROS‐activatable prodrug of a strong anticancer agent, doxazolidine (doxaz), is developed. Doxaz is a DNA alkylating agent with a half‐life of 3 min and significantly higher cytotoxicity than the clinically used parental compound doxorubicin (dox). Importantly, doxaz is not affected by p‐glycoprotein expression since it irreversibly alkylates DNA while dox inhibits the topoisomerase II DNA complex. As drug activators, reactive oxygen species (ROS) are already produced inside cancer cells in higher abundance than in normal cells but additionally generated by external stimuli such as radionuclides (via radiolysis of water) and/or ROS‐inducing drugs. We synthesized the prodrug, Doxaz‐BA, and evaluated its efficacy in vitro in cell cultures and then in vivo in xenograft mouse models. Doxaz‐BA is effective in a broad range of cancer cells since most cancer cells produce higher levels of ROS. Combining with clinically relevant radiotracers such as 18F‐FDG or other tumor‐tropic agents / ROS inducing drugs results in a tumor‐specific and enhanced localized therapy paradigm.

The N-terminal activation function AF-1 domain of ERα interacts directly with the C-terminal AF-2-holding ligand-binding domain to recruit the coactivator proteins.

Cryoelectron microscopy (cryo-EM) clarified the quaternary structure of the DNA complex of coactivator-bound estrogen receptor alpha (ERα), revealing the adjacency of the N-terminal domain (NTD) and C-terminal ligand-binding domain (LBD). ERα-NTD and LBD constitute activation function 1 (AF-1) and activation function 2 (AF-2), respectively. These domains are essential for transcription activation. Their spatial proximity was judged to be essential for ERα to recruit the SRC coactivator proteins. In the present study, we first evaluated untethered free ERα-NTD(AF-1) [residues 1-180] and its-truncated desNTD(AF-1)-ERα [residues 181-595] in a luciferase reporter gene assay. ERα-NTD(AF-1) was completely inactive, whereas desNTD(AF-1)-ERα exhibited 66% activity of wild-type ERα. Surprisingly, ERα-NTD(AF-1) was found to inhibit desNTD(AF-1)-ERα markedly. Therefore, assuming that ERα-NTD(AF-1) must also inhibit wild-type full-length ERα, we co-expressed ERα-NTD(AF-1) and full-length ERα. As expected, ERα-NTD(AF-1) inhibited ERα in a dose-dependent manner, but non-competitively for 17β-estradiol. When their intracellular transport was examined immunocytochemically, ERα-NTD(AF-1) showed a distinct translocation from the cytoplasm to the nucleus, despite being expressed solely in the cytoplasm without full-length ERα. This nuclear translocation was attributable to a direct interaction between ERα-NTD(AF-1) and full-length ERα consisting of the nuclear localization signal. The present results demonstrated that, in full-length ERα, the N-terminally tethered NTD(AF-1) domain collaborates with the C-terminal LBD(AF-2) for coactivator recruitment.

Integrating Single-Walled Carbon Nanotubes into Supramolecular Assemblies: From Basic Interactions to Emerging Applications.

Integrating single-walled carbon nanotubes (SWCNTs) into supramolecular self-assemblies harnesses the distinctive mechanical, optical, and electronic properties of the nanoparticles alongside the structural and chemical properties of the assemblies. Organic molecules capable of forming supramolecular assemblies through hydrophobic, van der Waals, and π-π interactions have been demonstrated to be particularly effective in dispersing and functionalizing SWCNTs, as these same interactions facilitate the binding to the hydrophobic graphene-like surface of the SWCNTs. This review discusses a variety of self-assembling structures that were shown to integrate SWCNTs, ranging from simple micelles and ring structures to complex DNA origami and three-dimensional hydrogels formed by low-molecular-weight gelators. We explore the integration of SWCNTs into various supramolecular assemblies and highlight emerging applications of these composite materials, such as the mechanical enforcement of self-assembling hydrogels and leveraging the near-infrared (NIR) fluorescence properties of SWCNTs for monitoring the molecular self-assembly process. Notably, the distinctive NIR fluorescence of SWCNTs, which overlaps with the biological transparency window, offers significant opportunities for noninvasive sensing applications within the supramolecular platforms. Future research into a deeper understanding of the interactions between SWCNTs and different supramolecular frameworks will expand the potential applications of SWCNT-integrated supramolecular assemblies in fields like biomedical engineering, electronic devices, and environmental sensing.

Ultrasensitive Amplification-Free Quantification of a Methyl CpG-Rich Cancer Biomarker by Single-Molecule Kinetic Fingerprinting.

The most well-studied epigenetic marker in humans is the 5-methyl modification of cytosine in DNA, which has great potential as a disease biomarker. Currently, quantification of DNA methylation relies heavily on bisulfite conversion followed by PCR amplification and NGS or microarray analysis. PCR is subject to potential bias in differential amplification of bisulfite-converted methylated versus unmethylated sequences. Here, we combine bisulfite conversion with single-molecule kinetic fingerprinting to develop an amplification-free assay for DNA methylation at the branched-chain amino acid transaminase 1 (BCAT1) promoter. Our assay selectively responds to methylated sequences with a limit of detection below 1 fM and a specificity of 99.9999%. Evaluating complex genomic DNA matrices, we reliably distinguish <5% DNA methylation at the BCAT1 promoter in whole blood DNA from completely unmethylated whole-genome amplified DNA. Taken together, these results demonstrate the feasibility and sensitivity of our amplification-free, single-molecule quantification approach to improve the early detection of methylated cancer DNA biomarkers.

FastAd: A versatile toolkit for rapid generation of single adenoviruses or diverse adenoviral vector libraries

Adenoviruses (Ads) are potent gene delivery vectors for in vitro and in vivo applications. However, current methods for their construction are time-consuming and inefficient, limiting their rapid production and utility in generating complex genetic libraries. Here, we introduce FastAd, a rapid and easy-to-use technology for inserting recombinant “donor” DNA directly into infectious “receiver” Ads in mammalian cells by the concerted action of two efficient recombinases: Cre and Bxb1. Subsequently, the resulting mixed recombinant Ad population is subjected to negative selections by flippase (FLP) recombinase to remove viruses that missed the initial recombination. With this approach, recombinant Ad production time is reduced from two months to 10 days or less. FastAd can be applied for inserting complex genetic DNA libraries into Ad genomes, as evidenced by the generation of barcode libraries with over 3 million unique clones from a T25 flask-scale of transfection. Furthermore, we leveraged FastAd to construct an Ad library containing a comprehensive genome-wide CRISPR/Cas9 guide RNA (gRNA) library and demonstrated its effectiveness in uncovering novel virus-host interactions. In summary, FastAd enables the rapid generation of single Ad vectors or complex genetic libraries, facilitating not only novel applications of Ad vectors but also research in foundational Ad biology.