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7-Deaza-2'-deoxyisoguanosine forms stable inverse Watson-Crick base pairs with 5-methyl-2'-deoxyisocytidine and purine-purine base pairs with 2'-deoxyguanosine or 5-aza-7-deaza-2'-deoxyguanosine. Both base pairs expand the genetic coding system. The manuscript reports on the functionalization of these base pairs with halogen atoms and clickable side chains introduced at 7-position of the 7-deazapurine base. Oligonucleotides containing the functionalized base pairs were prepared by solid-phase synthesis. To this end, a series of phosphoramidites were synthesized and clickable side chains with short and long linkers were incorporated in oligonucleotides. Fluorescent pyrene conjugates were obtained by postmodification. Functionalization of DNA with a single inverse Watson-Crick base pair by halogens or clickable residues has only a minor impact on duplex stability. Pyrene click adducts increase (long linker) or decrease (short linker) the double helix stability. Stable hybrid duplexes were constructed containing three consecutive purine-purine pairs of 7-functionalized 7-deaza-2'-deoxyisoguanine with guanine or 5-aza-7-deazaguanine in the center and Watson-Crick pairs at both ends. The incorporation of a hybrid base pair tract of 7-deaza-2'-deoxyisoguanosine/5-aza-7-deaza-2'-deoxyguanosine pairs stabilizes the double helix strongly. Fluorescence intensity of pyrene short linker adducts increased when the 7-deazapurine base was positioned opposite to 5-methylisocytosine (inverse base pair) compared to purine-purine base pairs with guanine or 5-aza-7-deazaguanine in opposite positions. For long liker adducts, the situation is more complex. Circular dichroism (CD) spectra of purine DNA differ to those of Watson-Crick double helices and are indicative for the new DNA constructs. The impact of 7-deaza-2'-deoxyisoguanine base pair functionalization is studied for the first time and all experimental details are reported to prepare DNA functionalized at the 7-deazaisoguanine site. The influence of single and multiple incorporations on DNA structure and stability is shown. Clickable residues introduced at the 7-position of the 7-deazaisoguanine base provide handles for Huisgen-Sharpless-Meldal click cycloadditions without harming the stability of purine-pyrimidine and purine-purine base pairs. Other chemistries might be used for bioconjugation. Our investigation paves the way for the functionalization of a new DNA related recognition system expanding the common Watson-Crick regime.

The elasticities of double-stranded (ds) DNA and RNA, which are critical to their biological functions and applications in materials science, can be significantly modulated by solution conditions such as ions and temperature. However, there is still a lack of a comprehensive understanding of the role of solvents in the elasticities of dsRNA and dsDNA in a comparative way. In this work, we explored the effect of ethanol solvent on the elasticities of dsRNA and dsDNA by magnetic tweezers and all-atom molecular dynamics simulations. We found that the bending persistence lengths and contour lengths of dsRNA and dsDNA decrease monotonically with the increase in ethanol concentration. Furthermore, the addition of ethanol weakens the positive twist-stretch coupling of dsRNA, while promotes the negative twist-stretch coupling of dsDNA. Counter-intuitively, the lower dielectric environment of ethanol causes a significant re-distribution of counterions and enhanced ion neutralization, which overwhelms the enhanced repulsion along dsRNA/dsDNA, ultimately leading to the softening in bending for dsRNA and dsDNA. Moreover, for dsRNA, ethanol causes slight ion-clamping across the major groove, which weakens the major groove-mediated twist-stretch coupling, while for dsDNA, ethanol promotes the stretch-radius correlation due to enhanced ion binding and consequently enhances the helical radius-mediated twist-stretch coupling., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

The DNA-encoded library (DEL) is a robust tool for chemical biology and drug discovery. In this study, we developed a DNA-compatible light-promoted reaction that is highly efficient and plate-compatible for DEL construction based on the formation of the indazolone scaffold. Employing this high-efficiency approach, we constructed a DEL featuring an indazolone core, which enabled the identification of a novel series of ligands specifically targeting E1A-binding protein (p300) after DEL selection. Taken together, our findings underscore the feasibility of light-promoted reactions in DEL synthesis and unveil promising avenues for developing p300-targeting inhibitors.

With digital transformation and the general application of new technologies, data storage is facing new challenges with the demand for high-density loading of massive information. In response, DNA storage technology has emerged as a promising research direction. Efficient and reliable data retrieval is critical for DNA storage, and the development of random access technology plays a key role in its practicality and reliability. However, achieving fast and accurate random access functions has proven difficult for existing DNA storage efforts, which limits its practical applications in industry. In this review, we summarize the recent advances in DNA storage technology that enable random access functionality, as well as the challenges that need to be overcome and the current solutions. This review aims to help researchers in the field of DNA storage better understand the importance of the random access step and its impact on the overall development of DNA storage. Furthermore, the remaining challenges and future research trends in random access technology of DNA storage are discussed, with the goal of providing a solid foundation for achieving random access in DNA storage under large-scale data conditions.

The evolution of dynamic DNA nanostructures has propelled DNA nanotechnology into a robust and versatile field, offering groundbreaking applications in nanoscale communication, drug delivery, and molecular computing. Yet, the full potential of this technology awaits further enhancement through optimization of kinetic properties governing conformational changes. In this work, we introduce a mean-field theory to characterize the kinetic behavior of a dynamic DNA origami hinge where each arm bears complementary single-stranded DNA overhangs of different lengths, which can latch the hinge at a closed conformation. This device is currently being investigated for multiple applications, being of particular interest the development of DNA-based rapid diagnostic tests for coronavirus. Drawing from classical statistical mechanics theories, we derive analytical expressions for the mean binding time of these overhangs within a constant hinge. This analysis is then extended to flexible hinges, where the angle diffuses within a predetermined energy landscape. We validate our model by comparing it with experimental measurements of the closing rates of DNA nanocalipers with different energy landscapes and overhang lengths, demonstrating excellent agreement and suggesting fast angular relaxation relative to binding. These findings offer insights that can guide the optimization of devices for specific state lifetimes. Moreover, the framework introduced here lays the groundwork for further advancements in modeling the kinetics of dynamic DNA nanostructures., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Programmed DNA structures and assemblies are readily accessible, but site-specific functionalization is critical to realize applications in various fields such as nanoelectronics, nanomaterials and biomedicine. Besides pre- and post-DNA synthesis conjugation strategies, on-solid support reactions offer advantages in certain circumstances. We describe on-solid support internucleotide coupling reactions, often considered undesirable, and a workaround strategy to overcome them. Palladium coupling reactions enabled on-solid support intra- and interstrand coupling between single-stranded DNAs (ss-DNAs). Dilution with a capping agent suppressed interstrand coupling, maximizing intrastrand coupling. Alternatively, interstrand coupling actually proved advantageous to provide dimeric organic/DNA conjugates that could be conveniently separated from higher oligomers, and was more favorable with longer terphenyl coupling partners.

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (piconewton) integrin-ECM forces have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrin receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop an LR probe composed of an engineered DNA structure that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN (hairpin unfolding) and a high force threshold at 47 pN (duplex shearing). These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live cells. Automated analysis of tens of thousands of events from eight cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 s. A subset of these events mature in magnitude to >47 pN with a median loading rate of 1.1 pN s -1 and primarily localize at the periphery of the cell-substrate junction. The LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of molecular mechanotransduction in living systems.

The lipopolyplex, a multicomponent nonviral gene carrier, generally demonstrates superior colloidal stability, reduced cytotoxicity, and high transfection efficiency. In this study, a new concept, photochemical reaction-induced transfection, using photosensitizer (PS)-loaded lipopolyplexes was applied, which led to enhanced transfection and cytotoxic effects by photoexcitation of the photosensitizer. Hypericin, a hydrophobic photosensitizer, was encapsulated in the lipid bilayer of liposomes. The preformed nanosized hypericin liposomes enclosed the linear polyethylenimine (lPEI)/pDNA polyplexes, resulting in the formation of hypericin lipopolyplexes (Hy-LPP). The diameters of Hy-LPP containing 50 nM hypericin and 0.25 μg of pDNA were 185.6 ± 7.74 nm and 230.2 ± 4.60 nm, respectively, measured by dynamic light scattering (DLS) and atomic force microscopy (AFM). Gel electrophoresis confirmed the encapsulation of hypericin and pDNA in lipopolyplexes. Furthermore, in vitro irradiation of intracellular Hy-LPP at radiant exposures of 200, 600, and 1000 mJ/cm 2 was evaluated. It demonstrated 60- to 75-fold higher in vitro luciferase expression than that in nonirradiated cells. The lactate dehydrogenase (LDH) assay supported that reduced transfection was a consequence of photocytotoxicity. The developed photosensitizer-loaded lipopolyplexes improved the transfection efficiency of an exogenous gene or induced photocytotoxicity; however, the frontier lies in the applied photochemical dose. The light-triggered photoexcitation of intracellular hypericin resulted in the generation of reactive oxygen species (ROS), leading to photoselective transfection in HepG2 cells. It was concluded that the two codelivered therapeutics resulted in enhanced transfection and a photodynamic effect by tuning the applied photochemical dose.

Nucleic Acid Nanocapsules (NANs) are nucleic acid nanostructures that radially display oligonucleotides on the surface of cross-linked surfactant micelles. Their chemical makeup affords the stimuli-responsive release of therapeutically active DNA-surfactant conjugates into the cells. While NANs have so far demonstrated the effective cytosolic delivery of their nucleic acid cargo, as seen indirectly by their gene regulation capabilities, there remain gaps in the molecular understanding of how this process happens. Herein, we examine the enzymatic degradation of NANs and confirm the identity of the DNA-surfactant conjugates formed by using mass spectrometry (MS). With surface enhanced (resonance) Raman spectroscopy (SE(R)RS), we also provide evidence that the energy-independent translocation of such DNA-surfactant conjugates is contingent upon their release from the NAN structure, which, when intact, otherwise buries the hydrophobic surfactant tail in its interior. Such information suggests a critical role of the surfactant in the lipid disruption capability of the DNA surfactant conjugates generated from degradation of the NANs. Using NANs made with different tail lengths (C 12 and C 10 ), we show that this mechanism likely holds true despite significant differences in the physical properties (i.e., critical micelle concentration (CMC), surfactants per micelle, N agg ) of the resultant particles (C 12 and C 10 NANs). While the total cellular uptake efficiencies of C 12 and C 10 NANs are similar, there were differences observed in their cellular distribution and localized trafficking, even after ensuring that the total concentration of DNA was the same for both particles. Ultimately, C 10 NANs appeared less diffuse within cells and colocalized less with lysosomes over time, achieving more significant knockdown of the target gene investigated, suggesting more effective endosomal escape. These differences indicate that the surfactant assembly and disassembly properties, including the number of surfactants per particle and the CMC can have important implications for the cellular delivery efficacy of DNA micelles and surfactant conjugates.

The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA., (© 2024. The Author(s).)

In this work, an ultrasensitive electrochemiluminescence (ECL) biosensor was constructed based on DNA-stabilized Au Ag nanoclusters (DNA-Au Ag NCs) as the efficient luminophore and Au NPs@Ti 3 C 2 as a new coreaction accelerator for determining microRNA-221 (miRNA-221) related to liver cancer. Impressively, DNA-Au Ag NCs were stabilized by the high affinity of the periodic 3C sequence, exhibiting an excellent ECL efficiency of 27% compared with classical BSA-Au Ag NCs (16%). Moreover, the Au NPs@Ti 3 C 2 nanocomposites, as a new coreaction accelerator, were first introduced to accelerate the production of abundant sulfate free radicals (SO 4 •- ) for promoting the ECL efficiency of DNA-Au Ag NCs in the DNA-Au Ag NCs/Au NPs@Ti 3 C 2 /S 2 O 8 2- ternary system due to the energy band of Au NPs@Ti 3 C 2 being well-matched with the frontier orbital of S 2 O 8 2- . Furthermore, the trace target (miRNA-221) could drive the rolling circle amplification to generate an amount of output DNA with periodic 3C and 10A sequences. Through covalent bonds on the surface of poly A and Au NPs, the distance between the luminophor and the coreaction accelerator could be narrowed to further enhance the detection sensitivity. As a result, the constructed sensor has been applied for the ultrasensitive detection of miRNA-221 with a low detection limit of 50 aM and successfully monitored miRNA-221 in MHCC-97L and HeLa cell lysates. This strategy could be utilized for guiding the synthesis of light-emitting DNA-metal NCs, which has great potential in the construction of ultrasensitive biosensors for the early diagnosis of diseases.

Homogeneous electrochemiluminescence (ECL) has gained attention for its simplicity and stability. However, false positives due to solution background interference pose a challenge. To address this, magnetic ECL nanoparticles (Fe 3 O 4 @Ru@SiO 2 NPs) were synthesized, offering easy modification, magnetic separation, and stable luminescence. These were utilized in an ECL sensor for miRNA-155 (miR-155) detection, with locked DNAzyme and substrate chain (mDNA) modified on their surface. The poor conductivity of long-chain DNA significantly impacts the conductivity and electron transfer capability of Fe 3 O 4 @Ru@SiO 2 NPs, resulting in weaker ECL signals. Upon target presence, unlocked DNAzyme catalyzes mDNA cleavage, leading to shortened DNA chains and reduced density. In contrast, the presence of short-chain DNA has minimal impact on the conductivity and electron transfer capability of Fe 3 O 4 @Ru@SiO 2 NPs. Simultaneously, the material surface's electronegativity decreases, weakening the electrostatic repulsion with the negatively charged electrode, resulting in the system detecting stronger ECL signals. This sensor enables homogeneous ECL detection while mitigating solution background interference through magnetic separation. Within a range of 100 fM to 10 nM, the sensor exhibits a linear relationship between ECL intensity and target concentration, with a 26.91 fM detection limit. It demonstrates high accuracy in clinical sample detection, holding significant potential for clinical diagnostics. Future integration with innovative detection strategies may further enhance sensitivity and specificity in biosensing applications.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

Monotherapy, especially the use of antibodies targeting vascular endothelial growth factor (VEGF), has shown limitations in treating choroidal neovascularization (CNV) since reactive oxygen species (ROS) also exacerbate CNV formation. Herein, we developed a combination therapy based on a DNA origami platform targeting multiple components of ocular neovascularization. Our study demonstrated that ocular neovascularization was markedly suppressed by intravitreal injection of a rectangular DNA origami sheet modified with VEGF aptamers (Ap) conjugated to an anti-VEGF antibody (aV) via matrix metalloproteinase (MMP)-cleavable peptide linkers in a mouse model of CNV. Typically, the DNA origami-based therapeutic platform selectively accumulates in neovascularization lesions owing to the dual-targeting ability of the aV and Ap, followed by the cleavage of the peptide linker by MMPs to release the antibody. Together, the released antibody and Ap inhibited VEGF activity. Moreover, the residual bare DNA origami could effectively scavenge ROS, reducing oxidative stress at CNV sites and thus maximizing the synergistic effects of inhibiting neovascularization.

Histidine and arginine are two amino acids that exhibit beneficial properties for gene delivery. In particular, the imidazole group of histidine facilitates endosomal release, while the guanidinium group of arginine promotes cellular entry. Consequently, a dual-charged copolymer library based on these amino acids was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The content of the N -acryloyl-l-histidine (His) monomer was systematically increased, while maintaining consistent levels of methyl N -acryloyl-l-argininate hydrochloride (ArgOMe) or N -(4-guanidinobutyl)acrylamide hydrochloride (GBAm). The resulting polymers formed stable, nanosized polyplexes when complexed with nucleic acids. Remarkably, candidates with increased His content exhibited reduced cytotoxicity profiles and enhanced transfection efficiency, particularly retaining this performance level at lower pDNA concentrations. Furthermore, endosomal release studies revealed that increased His content improved endosomal release, while ArgOMe improved cellular entry. These findings underscore the potential of customized dual-charged copolymers and the synergistic effects of His and ArgOMe/GBAm in enhancing gene delivery.

DNA-based Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is an effective super resolution microscopy technique, and its optimization is key to improve nanoscale detection. The state-of-the-art improvements that are at the base of this optimization have been first routinely validated on DNA nanostructure devices before being tested on biological samples. This allows researchers to finely tune DNA-PAINT imaging features in a more controllable in vitro environment. Dye-labeled oligonucleotide probes with short hybridization domains can expand DNA-PAINT's detection by targeting short nucleotide sequences and improving resolution, speed, and multiplexing. However, developing these probes is challenging as their brief bound state makes them difficult to capture under routine imaging conditions. To extend dwell binding times and promote duplex stability, we introduced structural and chemical modifications to our imager probes. The modifications included mini-hairpins and/or Bridged Nucleic Acids (BNA); both of which increase the thermomechanical stability of a DNA duplex. Using this approach we demonstrate DNA-PAINT imaging with approximately 5 nm resolution using a 4-nucleotide hybridization domain that is 43% shorter than previously reported probes. Imager probes with such short hybridization domains are key for improving detection on DNA nanostructure devices because they have the capability to target a larger number of binding domains per localization unit. This is essential for metrology applications such as Nucleic Acid Memory (NAM) where the information density is dependent on the binding site length. The selected imager probes reported here present imaging resolution equivalent to current state-of-the-art DNA-PAINT probes, creating a strategy to image shorter DNA domains for nanoscience and nanotechnology alike.

Periodontitis, with its persistent nature, causes significant distress for most sufferers. Current treatments, such as mechanical cleaning and surgery, often fail to fully address the underlying overactivation of fibroblasts that drives this degradation. Targeting the post-transcriptional regulation of fibroblasts, particularly at the 3'-untranslated regions (3'UTR) of pathogenic genes, offers a therapeutic strategy for periodontitis. Herein, we developed a DNA nanorobot for this purpose. This system uses a dynamic DNA nanoframework to incorporate therapeutic microRNAs through molecular recognition and covalent bonds, facilitated by DNA monomers modified with disulfide bonds. The assembled-DNA nanoframework is encapsulated in a cell membrane embedded with a fibroblast-targeting peptide. By analyzing the 3'UTR regions of pathogenic fibroblast genes FOSB and JUND , we identified the therapeutic microRNA as miR-1-3p and integrated it into this system. As expected, the DNA nanorobot delivered the internal components to fibroblasts by the targeting peptide and outer membrane that responsively releases miR-1-3p under intracellular glutathione. It resulted in a precise reduction of mRNA and suppression of protein function in pathogenic genes, effectively reprogramming fibroblast behavior. Our results confirm that this approach not only mitigates the inflammation but also promotes tissue regeneration in periodontal models, offering a promising therapeutic avenue for periodontitis.

BRCA1 gene and carcinoembryonic antigen (CEA) are important markers of breast cancer, so accurate detection of them is significant for early detection and diagnosis of breast cancer. In this study, a potential-resolved ratio electrochemiluminescence (ECL) biosensor using perylene diimide (PDI)-metal-organic framework and DNA nanoflowers (NFs)-CdS quantum dots (QDs) was constructed for detection of BRCA1 and CEA. Specifically, PDI-MOF and CdS QDs can generate potential-resolved intense ECL signals only using one coreactant, so the detection procedure can be effectively simplified. PDI-MOF was first attached to the electrode by graphene oxide, and the dopamine (DA) probe was linked to quench the ECL signal by DNA hybridization. In the presence of target BRCA1, it can form a bipedal DNA walker, so the quenching molecules (DA) were detached from the electrode via the walker amplification process aided by Mg 2+ , so that the PDI signal at -0.25 V was restored for the BRCA1 assay. Moreover, CdS QDs@DNA NFs as amplified signal probes were formed by self-assembly, and the target CEA-amplified product introduced the CdS QDs@DNA NFs to the electrode, so the QD ECL signal at -1.42 V was enhanced, while the ECL signal of PDI is unchanged; thus, CEA detection was achieved by the ratio value between them. Therefore, the detection accuracy is guaranteed by detection of two cancer markers and a ratio value. This biosensor has a great contribution to the development of new ECL materials and a novel ECL technique for fast and efficient multitarget assays, showing great significance for the early monitoring and diagnosis of breast cancer.

Our study focuses on synthesizing and exploring the potential of three N-(4) substituted thiosemicarbazones derived from cinnamic aldehyde, alongside their Ru(II)-(η 6 -p-cymene)/(η 6 -benzene) complexes. The synthesized compounds were comprehensively characterized using a range of analytical techniques, including FT-IR, UV-visible spectroscopy, NMR ( 1 H, 13 C), and HRMS. We investigated their electronic and physicochemical properties via density functional theory (DFT). X-ray crystal structures validated structural differences identified by DFT. Molecular docking predicted promising bioactivities, supported by experimental observations. Notably, docking with EGFR suggested an inhibitory potential against this cancer-related protein. Spectroscopic titrations revealed significant DNA/BSA binding affinities, particularly with DNA intercalation and BSA hydrophobic interactions. RuPCAM displayed the strongest binding affinity with DNA (K b = 6.23 × 10 7 M -1 ) and BSA (K b = 9.75 × 10 5 M -1 ). Assessed the cytotoxicity of the complexes on cervical cancer cells (HeLa), and breast cancer cells (MCF-7 and MDA-MB 231), revealing remarkable potency. Additionally, selectivity was assessed by examining MCF-10a normal cell lines. The active complexes were found to trigger apoptosis, a vital cellular process crucial for evaluating their potential as anticancer agents utilizing staining assays and flow cytometry analysis. Intriguingly, complexation with Ru(II)-arene precursors significantly amplified the bioactivity of thiosemicarbazones, unveiling promising avenues toward the creation of powerful anticancer agents.

Modulating molecular structure and function at the nanoscale drives innovation across wide-ranging technologies. Electrical control of the bonding of individual DNA base pairs endows DNA with precise nanoscale structural reconfigurability, benefiting efforts in DNA origami and actuation. Here, alloxazine DNA base surrogates were synthesized and incorporated into DNA duplexes to function as a redox-active switch of hydrogen bonding. Circular dichroism (CD) revealed that 24-mer DNA duplexes containing one or two alloxazines exhibited CD spectra and melting transitions similar to DNA with only canonical bases, indicating that the constructs adopt a B-form conformation. However, duplexes were not formed when four or more alloxazines were incorporated into a 24-mer strand. Thiolated duplexes incorporating alloxazines were self-assembled onto multiplexed gold electrodes and probed electrochemically. Square-wave voltammetry (SWV) revealed a substantial reduction peak centered at -0.272 V vs Ag/AgCl reference. Alternating between alloxazine oxidizing and reducing conditions modulated the SWV peak in a manner consistent with the formation and loss of hydrogen bonding, which disrupts the base pair stacking and redox efficiency of the DNA construct. These alternating signals support the assertion that alloxazine can function as a redox-active switch of hydrogen bonding, useful in controlling DNA and bioinspired assemblies.

Dynamic DNA-based nanodevices offer versatile molecular-level operations, but the majority of them suffer from sluggish kinetics, impeding the advancement of device complexity. In this work, we present the self-assembly of a cationic peptide with DNA to expedite toehold-mediated DNA strand displacement (TMSD) reactions, a fundamental mechanism enabling the dynamic control and actuation of DNA nanostructures. The target DNA is modified with a fluorophore and a quencher, so that the TMSD process can be monitored by recording the time-dependent fluorescence changes. The boosting effect of the peptides is found to be dependent on the peptide/DNA N/P ratio, the toehold/invader binding affinity, and the ionic strength with stronger effects observed at lower ionic strengths, suggesting that electrostatic interactions play a key role. Furthermore, we demonstrate that the cationic peptide enhances the responsiveness and robustness of DNA machinery tweezers or logic circuits (AND and OR) involving multiple strand displacement reactions in parallel and cascade, highlighting its broad utility across DNA-based systems of varying complexity. This work offers a versatile approach to enhance the efficiency of toehold-mediated DNA nanodevices, facilitating flexible design and broader applications.

Toehold-mediated strand displacement (TMSD) reaction, one of the DNA nanotechnologies, has great potential as s biological programmable platform in the cellular environment. Various artificial nucleic acids have been developed to improve stability and affinity for biological applications. However, the lack of understanding of the kinetics of TMSD reaction among artificial nucleic acids has limited their applications. We herein systematically characterized the kinetics of TMSD reactions with acyclic xeno nucleic acids (XNAs): serinol nucleic acid (SNA), acyclic D-threoninol nucleic acid (D-aTNA), and acyclic L-threoninol nucleic acid (L-aTNA). We found that the strand displacement reactions by D-aTNA and by L-aTNA were highly dependent on temperature. D-aTNA and L-aTNA systems were orthogonal to each other, and chirality of the input can be switched by using SNA as an interface. We also applied TMSD reactions of XNAs to a seesaw gate amplification system which utilizes the orthogonality. This work will contribute to the developments of thermoresponsive and bioorthogonal nucleic acid circuits., (© 2024 Wiley-VCH GmbH.)

The inherent chiral structures of DNA serve as attractive scaffolds to construct DNA hybrid catalysts for valuable enantioselective transformations. Duplex and G-quadruplex DNA-based enantioselective catalysis has made great progress, yet novel design strategies of DNA hybrid catalysts are highly demanding and atomistic analysis of active centers is still challenging. DNA i-motif structures could be finely tuned by different cytosine-cytosine base pairs, providing a new platform to design DNA catalysts. Herein, we found that a human telomeric i-motif DNA containing cytosine-silver(I)-cytosine (C-Ag + -C) base pairs interacting with Cu(II) ions (i-motif DNA(Ag + )/Cu 2+ ) could catalyze Diels-Alder reactions with full conversions and up to 95 % enantiomeric excess. As characterized by various physicochemical techniques, the presence of Ag + is proved to replace the protons in hemiprotonated cytosine-cytosine (C : C + ) base pairs and stabilize the DNA i-motif to allow the acceptance of Cu(II) ions. The i-motif DNA(Ag + )/Cu 2+ catalyst shows about 8-fold rate acceleration compared with DNA and Cu 2+ . Based on DNA mutation experiments, thermodynamic studies and density function theory calculations, the catalytic center of Cu(II) ion is proposed to be located in a specific loop region via binding to one nitrogen-7 atom of an unpaired adenine and two phosphate-oxygen atoms from nearby deoxythymidine monophosphate and deoxyadenosine monophosphate, respectively., (© 2024 Wiley-VCH GmbH.)

The importance of avian influenza virus (AIV) detection in clinical diagnosis and prognosis has been deeply recognized. In this study, the ultrasensitive detection of AIV subtype H5N1 was achieved by ICP-MS combined with DNA dendrimer-carried silver nanoparticle (AgNP) labeling. First, a magnetic control system was constructed by anchoring double-strand DNAs (dsDNAs) which contained a complementary sequence of H5N1 and two locked triggers on the surface of magnetic beads (MBs). When H5N1 was present, the two triggers were released and initiated dendrimer hybridization chain reactions which led to the generation of DNA dendrimer-carried AgNPs on the surface of the MBs. Finally, the AgNPs were collected via magnetic separation, digested by nitric acid, and tested using ICP-MS. The signal intensities of 107 Ag were positively correlated with the concentrations of H5N1. Notably, the DNA dendrimer assembly contributed to significant signal amplification and good sensitivity with the limit of detection as low as 2.0 × 10 -11 mol L -1 . Moreover, the method displayed favorable selectivity against mismatched H5N1 and good recoveries in human serum samples. It is a promising analytical tool for the H5N1 virus and other subtypes of AIV, and has potential value in clinical diagnosis and prognosis of infectious diseases.

DNA origami nanostructures (DONs) are able to scavenge reactive oxygen species (ROS) and their scavenging efficiency toward ROS radicals was shown to be comparable to that of genomic DNA. Herein, we demonstrate that DONs are highly efficient singlet oxygen quenchers outperforming double-stranded (ds) DNA by several orders of magnitude. To this end, a ROS mixture rich in singlet oxygen is generated by light irradiation of the photosensitizer methylene blue and its cytotoxic effect on Escherichia coli cells is quantified in the presence and absence of DONs. DONs are found to be vastly superior to dsDNA in protecting the bacteria from ROS-induced damage and even surpass established ROS scavengers. At a concentration of 15 nM, DONs are about 50 000 times more efficient ROS scavengers than dsDNA at an equivalent concentration. This is attributed to the dominant role of singlet oxygen, which has a long diffusion length and reacts specifically with guanine. The dense packing of the available guanines into the small volume of the DON increases the overall quenching probability compared to a linear dsDNA with the same number of base pairs. DONs thus have great potential to alleviate oxidative stress caused by singlet oxygen in diverse therapeutic settings., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Parallel-stranded G-quadruplex structures are found to be common in the human promoter sequences. We tested highly fluorescent 9-methoxyluminarine ligand (9-MeLM) binding interactions with different parallel G-quadruplexes DNA by spectroscopic methods such as fluorescence and circular dichroism (CD) titration as well as UV melting profiles. The results showed that the studied 9-MeLM ligand interacted with the intramolecular parallel G-quadruplexes (G4s) with similar affinity. The binding constants of 9-methoxyluminarine with different parallel G4s were determined. The studies upon oligonucleotides with different flanking sequences on c- MYC G-quadruplex suggest that 9-methoxyluminarine may preferentially interact with 3'end of the c- MYC promoter. The high decrease in 9-MeLM ligand fluorescence upon binding to all tested G4s indicates that 9-methoxyluminarine molecule can be used as a selective fluorescence turn-off probe for parallel G-quadruplexes.

Proteins self-assemble to function in living cells. They may execute essential tasks in the form of monomers, complexes, or supramolecular cages via oligomerization, achieving a sophisticated balance between structural topology and functional dynamics. The modularity and programmability make DNA origami unique in mimicking these key features. Here, we demonstrate three-dimensional reconfigurable DNA origami pincers (DOPs) that multitask on giant unilamellar vesicles (GUVs). By programmably adjusting their pinching angle, the DOPs can dynamically control the degree of GUV remodeling. When oligomerized on the GUV to form origami cages, the DOP units interact with one another and undergo reorganization, resulting in the capture, compartmentalization, and detachment of lipid fragments. This oligomerization process is accompanied with membrane disruptions, enabling the passage of cargo across the membrane. We envisage that interfacing synthetic cells with engineered, multifunctional DNA nanostructures may help to confer customized cellular properties, unleashing the potential of both fields.

Fluorescence-based oligonucleotide probes have a great importance in research of molecular interactions. Molecular beacons (MBs) are special case of fluorescent probes that form a stem-loop shape, bringing together a fluorophore and quencher, thus emitting fluorescence only when hybridized to a complementary target. Here we describe a new method for the quantitation of MB hybridization based on the measurement of changes in free energy instead of the fluorescence intensity. The MB energy state can be measured by micro-fluorescence detection. The approach allowed to determine hybridization energy of the MB with target nucleotide directly from fluorescence spectra and distinguish the MB in unfolded and hybridized states. Moreover, the method enabled us to discriminate between DNA duplexes with perfect complementarity or a single-nucleotide mismatch, based on the first direct experimental prove of enthalpy-entropy compensation., Competing Interests: Oleh Fedorych is founder, shareholder and holds patent covering the method. Ruben R. Rosencrantz is a shareholder. Sophia Rosencrantz and Vitali Matyash have no competing interests to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Rosencrantz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The manuscript reports on 7-deazapurine and pyrimidine nucleoside and oligonucleotide cycloadducts formed by the inverse electron demand Diels-Alder (iEDDA) reaction with 3,6-di(pyrid-2-yl)-1,2,4,5-tetrazine. Cycloadducts were constructed from ethynylated and vinylated nucleobases. Oligonucleotides were synthesized containing iEDDA modifications, and the impact on duplex stability was investigated. iEDDA reactions were performed on nucleoside triple bond side chains. Oxidation was not required in these cases as dihydropyridazine intermediates are not formed. In contrast, oxidation is necessary for reactions performed on alkenyl compounds. This was verified on 5-vinyl-2'-deoxyuridine. A diastereomeric mixture of 1,2-dihydropyridazine cycloadduct intermediates was isolated, characterized, and later oxidized. 12-mer oligonucleotides containing 1,2-pyridazine inverse Diels-Alder cycloadducts and their precursors were hybridized to short DNA duplexes. For that, a series of phosphoramidites was prepared. DNA duplexes with 7-functionalized 7-deazaadenines and 5-functionalized pyrimidines display high duplex stability when spacer units are present between nucleobases and pyridazine cycloadducts. A direct connectivity of the pyridazine moiety to nucleobases as reported for metabolic labeling of vinyl nucleosides reduced duplex stability strongly. Oligonucleotides bearing linkers with and without pyridazine cycloadducts attached to the 7-deazaadenine nucleobase significantly reduced mismatch formation with dC and dG.

Slalom chromatography (SC) was discovered in 1988 for analyzing double-stranded (ds) DNA. However, its progress was impeded by practical issues such as low-purity particles, sample loss, and lack of a clear retention mechanism. With the rise of cell and gene therapies and the availability today of bio-inert ultra-high-pressure liquid chromatography (UHPLC) columns and systems, SC has regained interest. In SC, the elution order is opposite to that observed in hydrodynamic chromatography (HDC): larger DNA molecules are more retained than small ones. Yet, the underlying SC retention mechanism remains elusive. We provide the physicochemical background necessary to explain, at a microscopic scale, the full transition from a HDC to a SC retention mechanism. This includes the persistence length of the DNA macromolecule (representing DNA stiffness), their relaxation time (τ R ) from the non-equilibrium contour length to the equilibrium entropic configuration, and the relationship between the mobile phase shear rate (〈γ̇〉) in packed columns and the DNA extended length. We propose a relevant retention model to account for the simultaneous impact of hydrodynamic chromatography (HDC) and SC on the retention factors of a series of large and linear dsDNAs (ranging from 2 to 48 kbp). SC data were acquired using bio-inert MaxPeak TM Columns packed with 1.7μm BEH TM 45 Å, 1.8μm BEH 125 Å, 2.4μm BEH 125 Å, 5.3μm BEH 125 Å, and 11.3μm BEH 125 Å Particles, an ACQUITY TM UPLC TM I-class PLUS System, and either 1 × PBS (pH 7.4) or 100 mM phosphate buffer (pH 8) as the mobile phase. SC is a non-equilibrium retention mode that is dominant when the Weissenberg number (Wi=〈γ̇〉τ R ) is much larger than 10 and the average extended length of DNA exceeds the particle diameter. HDC, on the other hand, is an equilibrium retention mode that dominates when Wi<1 (DNA chains remaining in their non-extended configuration). Maximum dsDNA resolution is observed in a mixed HDC-SC retention mode when the extended length of the DNA is approximately half the particle diameter. This work facilitates the development of methods for characterizing various plasmid DNA mixtures, containing linear, supercoiled, and relaxed circular dsDNAs which all have different degree of molecular stiffness., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The engineering of enzymatic activity generally involves alteration of the protein primary sequences, which introduce structural changes that give rise to functional improvements. Mechanical forces have been used to interrogate protein biophysics, leading to deep mechanistic insights in single-molecule studies. Here, we use simple DNA springs to apply small pulling forces to perturb the active site of a thermostable alcohol dehydrogenase. Methods were developed to enable the study of different spring lengths and spring orientations under bulk catalysis conditions. Tension applied across the active site expanded the binding pocket volume and shifted the preference of the enzyme for longer chain-length substrates, which could be tuned by altering the spring length and the resultant applied force. The substrate specificity changes did not occur when the DNA spring was either severed or rotated by ∼90°. These findings demonstrate an alternative approach in protein engineering, where active site architectures can be dynamically and reversibly remodeled using applied mechanical forces.

A novel electrochemical biosensor was developed for the detection of epinephrine (EP) by immobilizing double-strand DNA (dsDNA) bound with copper ions on a gold electrode (Cu 2+ /dsDNA/MCH/AuE). The electrochemical behavior of EP at Cu 2+ /dsDNA/MCH/AuE was examined, and the results demonstrated a significant enhancement in the electrocatalytic oxidation peak current of EP due to the formation of a stable G-Cu(II)-G sandwich structure between Cu 2+ and guanine at the modified electrode. The modification process of the electrode was characterized by scanning electron microscopy, infrared spectroscopy, electrochemical impedance spectroscopy, and differential pulse voltammetry. A study on the effect of pH in phosphate buffer solution on the electrochemical oxidation of EP indicated that the catalytic oxidation process was pH-dependent. A plot of catalytic current versus EP concentration exhibited a dual-linear relationship within two ranges: 1.0-12.5 μM and 12.5-1000.0 μM, with correlation coefficients of 0.995 and 0.997, respectively. The limit of detection was determined to be 47 nM (S/N = 3). According to the calculated Hill coefficient (0.99), it can be concluded that the electrocatalytic process followed the Michaelis-Menten kinetic mechanism. The maximum catalytic current I m was 25 μA, while the apparent Michaelis-Menten constant K m was 1.425 mM. These findings indicated excellent electrocatalytic activity of the modified electrode towards oxidation of EP. The developed biosensor successfully detected EP in spiked mouse serum as well as epinephrine hydrochloride injection with high selectivity, sensitivity, stability, and accuracy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Food adulteration presents a significant challenge due to the evasion of legal oversight and the difficulty of identification. Addressing this issue, there is an urgent need for on-site, rapid, visually based small-scale equipment, along with large-scale screening technology, to enable prompt results without providing opportunities for dishonest traders to react. Colorimetric reactions offer advantages in terms of speed, visualization, and miniaturization. However, there is a scarcity of suitable colorimetric reactions for food adulteration detection, and interference from colored food impurities and easily comparable color results affects accuracy. To overcome limitations, this study introduces a novel approach utilizing polydopamine magnetic nanoparticles to enrich DNA in food samples, effectively eliminating interfering components. By employing gold nanoparticles to generate magnetic-gold nanoparticles, a single magnetic bead achieves simultaneous enrichment, impurity removal, and detection. The use of paper-based biosensors and visualization equipment allows for the visualization and digital analysis of results, achieving a low detection limit of 4.59 nmol mL -1 . The method also exhibits high accuracy and repeatability, with a RSD ranging from 1.6 % to 4.0 %. This innovative colorimetric method addresses the need for rapid, miniaturized, and large-scale detection, thus providing a solution for food adulteration challenges., Competing Interests: Declaration of Competing Interest The author declare that there are no conflicts of interest associated with this manuscript. This research was conducted with utmost integrity and impartiality, adhering to the highest ethical standards. We have no financial or personal relationships that could inappropriately influence the content or interpretation of this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Performing spectroscopic measurements on biomolecules labeled with fluorescent probes is a powerful approach to locating the molecular behavior and dynamics of large systems at specific sites within their local environments. The indocarbocyanine dye Cy3 has emerged as one of the most commonly used chromophores. The incorporation of Cy3 dimers into DNA enhances experimental resolution owing to the spectral characteristics influenced by the geometric orientation of excitonically coupled monomeric units. Various theoretical models and simulations have been utilized to aid in the interpretation of the experimental spectra. In this study, we employ all-atom molecular dynamics simulations to study the structural dynamics of Cy3 dimers internally linked to the dsDNA backbone. We used quantum mechanical calculations to derive insights from both the linear absorption spectra and the circular dichroism data. Furthermore, we explore potential limitations within a commonly used force field for cyanine dyes. The molecular dynamics simulations suggest the presence of four possible Cy3 dimeric populations. The spectral simulations on the four populations show one of them to agree better with the experimental signatures, suggesting it to be the dominant population. The relative orientation of Cy3 in this population compares very well with previous predictions from the Holstein-Frenkel Hamiltonian model.

Nucleosomes are the basic compaction unit of chromatin and nucleosome structure and their higher-order assemblies regulate genome accessibility. Many post-translational modifications alter nucleosome dynamics, nucleosome-nucleosome interactions, and ultimately chromatin structure and gene expression. Here, we investigate the role of two post-translational modifications associated with actively transcribed regions, H3K36me3 and H4K5/8/12/16ac, in the contexts of tri-nucleosome arrays that provide a tractable model system for quantitative single-molecule analysis, while enabling us to probe nucleosome-nucleosome interactions. Direct visualization by AFM imaging reveals that H3K36me3 and H4K5/8/12/16ac nucleosomes adopt significantly more open and loose conformations than unmodified nucleosomes. Similarly, magnetic tweezers force spectroscopy shows a reduction in DNA outer turn wrapping and nucleosome-nucleosome interactions for the modified nucleosomes. The results suggest that for H3K36me3 the increased breathing and outer DNA turn unwrapping seen in mononucleosomes propagates to more open conformations in nucleosome arrays. In contrast, the even more open structures of H4K5/8/12/16ac nucleosome arrays do not appear to derive from the dynamics of the constituent mononucleosomes, but are driven by reduced nucleosome-nucleosome interactions, suggesting that stacking interactions can overrule DNA breathing of individual nucleosomes. We anticipate that our methodology will be broadly applicable to reveal the influence of other post-translational modifications and to observe the activity of nucleosome remodelers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dopamine is a neurotransmitter in the central nervous system that is essential for many bodily and mental processes, and a lack of it can cause Parkinson's disease. DNA tetrahedral (TD) nanocages are promising in bio-nanotechnology, especially as a nanocarrier. TD is highly programmable, biocompatible, and capable of cell differentiation and proliferation. It also has tissue and blood-brain barrier permeability, making it a powerful tool that could overcome potential barriers in treating neurological disorders. In this study, we used DNA TD as a carrier for dopamine to cells and zebrafish embryos. We investigated the mechanism of complexation between TD and dopamine hydrochloride using gel electrophoresis, fluorescence and circular dichroism (CD) spectroscopy, atomic force microscopy (AFM), and molecular dynamic (MD) simulation tools. Further, we demonstrate that these dopamine-loaded DNA TD nanostructures enhanced cellular uptake and differentiation ability in SH-SY5Y neuroblastoma cells. Furthermore, we extended the study to zebrafish embryos as a model organism to examine survival and uptake. The research provides valuable insights into the complexation mechanism and cellular uptake of dopamine-loaded DNA tetrahedral nanostructures, paving the way for further advancements in nanomedicine for Parkinson's disease and other neurological disorders.

In situ monitoring of intracellular microRNAs (miRNAs) often encounters the challenges of surrounding complexity, coexistence of precursor miRNAs (pre-miRNAs) and the degradation of biological enzyme in living cells. Here, we designed a novel probe encapsulated DNA tetrahedral molecular sieve (DTMS) to realize the size-selective detection of intracellular miRNA 21 that can avoid the interference of pre-miRNAs. In such strategy, quencher (BHQ-1) labeled probe DNA (S6-BHQ 1) was introduced into the inner cavity of fluorophore (FAM) labeled DNA tetrahedral scaffolds (DTS) to prepare DTMS, making the FAM and BHQ-1 closely proximate, and resulting the sensor in a "signal-off" state. In the presence of miRNA 21, strand displacement reaction happened to form more stable DNA double-stranded structure, accompanied by the release of S6-BHQ 1 from the inner cavity of DTMS, making the sensor in a "signal-on" state. The DTMS based sensing platform can then realized the size-selective detection of miRNA 21 with a detection limit of 3.6 pM. Relying on the mechanical rigidity of DTS and the encapsulation of DNA probe using DTMS, such proposed method achieved preferable reproducibility and storage stability. Moreover, this sensing system exhibited good performance for monitoring the change of intracellular miRNA 21 level during the treatment with miRNA-related drugs, demonstrating great potential for biological studies and accurate disease diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The convergence of DNA origami and surface-enhanced Raman spectroscopy (SERS) has opened a new avenue in bioanalytical sciences, particularly in the detection of single-molecule proteins. This breakthrough has enabled the development of advanced sensor technologies for diagnostics. DNA origami offers a highly controllable framework for the precise positioning of nanostructures, resulting in superior SERS signal amplification. In our investigation, we have successfully designed and synthesized DNA origami-based gold nanorod monomer and dimer assemblies. Moreover, we have evaluated the potential of dimer assemblies for label-free detection of a single biomolecule, namely epidermal growth factor receptor (EGFR), a crucial biomarker in cancer research. Our findings have revealed that the significant Raman amplification generated by DNA origami-assembled gold nanorod dimer nanoantennas facilitates the label-free identification of Raman peaks of single proteins, which is a prime aim in biomedical diagnostics. The present work represents a significant advancement in leveraging plasmonic nanoantennas to realize single protein SERS for the detection of various cancer biomarkers with single-molecule sensitivity.

T-2 toxin, a hazardous mycotoxin often present in cereals and products based on cereals, poses a substantial risk to humans and animals due to its high toxicity. The development of uncomplicated, quick and highly sensitive methods for detecting T-2 toxin is imperative. In this work, a portable sensing system was constructed using water column height as a readout device in combination with a controlled release system, which allows for an accurate quantitative analysis of T-2 toxin without the need for expensive instrumentation or skilled technicians. Hyaluronic acid (HA) hydrogel was constructed by double cross-linked DNA/aptamer hybrids with polyethyleneimine (PEI) and embedded with platinum nanoparticles (Pt NPs). The aptamer specifically bound to T-2 toxin in its presence, resulting in the disruption of the hydrogel and subsequent release of the Pt NPs. These Pt NPs were later mixed with a solution of H 2 O 2 in a confined reaction flask, leading to the decomposition of H 2 O 2 into O 2 . A glass capillary tube containing a column of red water had been inserted into the cap of the reaction flask, and the low solubility of O 2 led to an increase in pressure within the reaction unit, causing the red water column to rise. There is a good linear correlation between the height of the capillary liquid level and the T-2 toxin concentration in the range of 20 ng/mL to 6 μg/mL. The system has been successfully used to detect T-2 toxin in samples of barley tea and corn., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The DNA binding and cellular uptake of the lambda enantiomer of two bis-tetraazaphenanthrene (TAP) Ru(II) polypyridyl complexes containing either a linear dppn ( 1 ) or a hooked bdppz ( 2 ) benzodipyridophenazine ligand are reported, and the role of different charge-transfer states of the structural isomers in the photo-oxidation of guanine is explored. Both complexes possess characteristic metal-to-ligand charge-transfer (MLCT) bands between 400 and 500 nm and emission at ca. 630 nm in an aerated aqueous solution. Transient visible absorption (TrA) spectroscopy reveals that 400 nm excitation of 1 yields a dppn-based metal-to-ligand charge-transfer (MLCT) state, which in turn populates a dppn intraligand ( 3 IL) state. In contrast, photoexcitation of 2 results in an MLCT state on the TAP ligand and not the intercalating bdppz ligand. Both 1 and 2 bind strongly to double-stranded guanine-rich DNA with a loss of emission. Combined TrA and time-resolved infrared (TRIR) spectroscopy confirms formation of the guanine radical cation when 2 is bound to the d(G 5 C 5 ) 2 duplex, which is not the case when 1 is bound to the same duplex and indicates a different mechanism of action in DNA. Utilizing the long-lived triplet excited lifetime, we show good uptake and localization of 2 in live cells as well as isolated chromosomes. The observed shortening of the excited-state lifetime of 2 when internalized in cell chromosomes is consistent with DNA binding and luminescent quenching due to guanine photo-oxidation.

Soon after its introduction in 1987, polymerase chain reaction (PCR) has become a technique widely employed in diagnostic medical devices and forensic science with the intention of amplifying genetic information. PCR prescribes that each of its cycles must include a heating subprocess at 95 °C or more (denominated DNA denaturation and provided for allowing a claimed orderly separation of the two complementary nucleotides strands), which can produce significant damage to DNA, caused by high-speed collisions with surrounding molecules. Since such disruption should be prevented in order to reliably employ PCR, a study of the mechanics of such loss of structural integrity is herein presented, preceded by a review of the fundamental literature which has elucidated the effects of molecular agitation on DNA fragmentation. The main conclusion of this retrospective survey is that the body of examined theoretical and experimental evidence consistently and redundantly confirms scarce resilience and significant loss of structural integrity when DNA is heated at temperatures above 90 °C, even for 1 minute. Such conclusion contradicts the claimed paradigm of PCR fidelity and raises the concern that, at least for long sequences, if PCR can amplify some information, such amplified information may be unreliable for diagnostic or forensic applications, since it originates from sequences of nucleotides subjected to random fragmentation and reaggregation. Such a low-reliability scenario should be preventively considered in the various fields where DNA amplification methodologies are employed which provide for high-temperature heating under conditions equal to or similar to those prescribed by the PCR protocols reviewed in this study.

The protein-induced fluorescence change technique was employed to investigate the interactions between proteins and their DNA substrates modified with the Cy3 fluorophore. It has been reported that the human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal proline-tryptophan-tryptophan-proline (PWWP) domain (the N-terminal 100 amino acids of HDGF) capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancer. This project investigated the specific binding behavior of HDGF, the PWWP domain, and the C140 domain (the C-terminal 140 amino acids of HDGF) sequentially using protein-induced fluorescence change. We found that the binding of HDGF and its related proteins on Cy3-labeled 15 bp SMYD1 dsDNA will cause a significant decrease in the recorded Cy3 fluorophore intensity, indicating the occurrence of protein-induced fluorescence quenching. The dissociation equilibrium constant was determined by fitting the bound fraction curve to a binding model. An approximate 10-time weaker SMYD1 binding affinity of the PWWP domain was found in comparison to HDGF. Moreover, the PWWP domain is required for DNA binding, and the C140 domain can enhance the DNA binding affinity. Furthermore, we found that the C140 domain can regulate the sequence-specific binding capability of HDGF on SMYD1 .

DNA methylation is an epigenetic modification that plays a crucial role in various biological processes. Aberrant DNA methylation is closely associated with the onset of diseases, and the specific localization of methylation sites in the genome offers further insight into the connection between methylation and diseases. Currently, there are numerous methods available for site-specific methylation detection. Electrochemical biosensors have garnered significant attention due to their distinct advantages, such as rapidity, simplicity, high sensitivity, low cost, and the potential for miniaturization. In this paper, we present a systematic review of the primary sensing strategies utilized in the past decade for analyzing site-specific methylation and their applications in electrochemical sensors, from a novel perspective focusing on the localization analysis of site-specific methylation. These strategies include bisulfite treatment, restriction endonuclease treatment, other sensing strategies, and deamination without direct bisulfite treatment. We hope that this paper can offer ideas and references for establishing site-specific methylation electrochemical analysis in clinical practice.

The "AT-hook" is a peculiar DNA-binding domain that interacts with DNA in the minor groove in correspondence to AT-rich sequences. This domain has been first described in the HMGA protein family of architectural factors and later in various transcription factors and chromatin proteins, often in association with major groove DNA-binding domains. In this review, using a literature search, we identified about one hundred AT-hook-containing proteins, mainly chromatin proteins and transcription factors. After considering the prototypes of AT-hook-containing proteins, the HMGA family, we review those that have been studied in more detail and that have been involved in various pathologies with a particular focus on cancer. This review shows that the AT-hook is a domain that gives proteins not only the ability to interact with DNA but also with RNA and proteins. This domain can have enzymatic activity and can influence the activity of the major groove DNA-binding domain and chromatin docking modules when present, and its activity can be modulated by post-translational modifications. Future research on the function of AT-hook-containing proteins will allow us to better decipher their function and contribution to the different pathologies and to eventually uncover their mutual influences.

Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The histidine-metal (His-Me) superfamily nucleases are renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how the single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases., Competing Interests: CC, GZ, YG No competing interests declared, (© 2024, Chang et al.)

Transcription factors specifically bind to their consensus sequence motifs and regulate transcription efficiency. Transcription factors are also able to non-specifically contact the phosphate backbone of DNA through electrostatic interaction. The homeodomain of Meis1 TALE human transcription factor (Meis1-HD) recognizes its target DNA sequences via two DNA contact regions, the L1-α1 region and the α3 helix (specific binding mode). This study demonstrates that the non-specific binding mode of Meis1-HD is the energetically favored process during DNA binding, achieved by the interaction of the L1-α1 region with the phosphate backbone. An NMR dynamics study suggests that non-specific binding might set up an intermediate structure which can then rapidly and easily find the consensus region on a long section of genomic DNA in a facilitated binding process. Structural analysis using NMR and molecular dynamics shows that key structural distortions in the Meis1-HD-DNA complex are induced by various single nucleotide mutations in the consensus sequence, resulting in decreased DNA binding affinity. Collectively, our results elucidate the detailed molecular mechanism of how Meis1-HD recognizes single nucleotide mutations within its consensus sequence: (i) through the conformational features of the α3 helix; and (ii) by the dynamic features (rigid or flexible) of the L1 loop and the α3 helix. These findings enhance our understanding of how single nucleotide mutations in transcription factor consensus sequences lead to dysfunctional transcription and, ultimately, human disease., (© 2024. The Author(s).)

Motile droplets using Marangoni convection are attracting attention for their potential as cell-mimicking small robots. However, the motion of droplets relative to the internal and external environments that generate Marangoni convection has not been quantitatively described. In this study, we used an aqueous two-phase system [poly(ethylene glycol) (PEG) and dextran] in an elongated chamber to generate motile dextran droplets in a constant PEG concentration gradient. We demonstrated that dextran droplets move by Marangoni convection, resulting from the PEG concentration gradient and the active transport of PEG and dextran into and out of the motile dextran droplet. Furthermore, by spontaneously incorporating long DNA into the dextran droplets, we achieved cell-like motility changes controlled by coexisting environment-sensing molecules. The DNA changes its position within the droplet and motile speed in response to external conditions. In the presence of Mg 2+ , the coil-globule transition of DNA inside the droplet accelerates the motile speed due to the decrease in the droplet's dynamic viscosity. Globule DNA condenses at the rear part of the droplet along the convection, while coil DNA moves away from the droplet's central axis, separating the dipole convections. These results provide a blueprint for designing autonomous small robots using phase-separated droplets, which change the mobility and molecular distribution within the droplet in reaction with the environment. It will also open unexplored areas of self-assembly mechanisms through phase separation under convections, such as intracellular phase separation.

N 2 -Alkyl-2'-deoxyguanosine ( N 2 -alkyl-dG) is a major type of minor-groove DNA lesions arising from endogenous metabolic processes and exogenous exposure to environmental contaminants. The N 2 -alkyl-dG lesions, if left unrepaired, can block DNA replication and transcription and induce mutations in these processes. Nevertheless, the repair pathways for N 2 -alkyl-dG lesions remain incompletely elucidated. By utilizing a photo-cross-linking coupled with mass spectrometry-based quantitative proteomic analysis, we identified a series of candidate N 2 -alkyl-dG-binding proteins. We found that two of these proteins, i.e., high-mobility group protein B3 (HMGB3) and SUB1, could bind directly to N 2 - n Bu-dG-containing duplex DNA in vitro and promote the repair of this lesion in cultured human cells. In addition, HMGB3 and SUB1 protected cells against benzo[ a ]pyrene-7,8-diol-9,10-epoxide (BPDE). SUB1 exhibits preferential binding to both the cis and trans diastereomers of N 2 -BPDE-dG over unmodified dG. On the other hand, HMGB3 binds favorably to trans - N 2 -BPDE-dG; the protein, however, does not distinguish cis - N 2 -BPDE-dG from unmodified dG. Consistently, genetic ablation of HMGB3 conferred diminished repair of trans - N 2 -BPDE-dG, but not its cis counterpart, whereas loss of SUB1 conferred attenuated repair of both diastereomers. Together, we identified proteins involved in the cellular sensing and repair of minor-groove N 2 -alkyl-dG lesions and documented a unique role of HMGB3 in the stereospecific recognition and repair of N 2 -BPDE-dG.

Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 μL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.

G-quadruplex (G4) structures play roles in various biological processes, but the challenge lies in specific targeting. To address this, we synthesized a conjugate capable of recognizing the G4 structure and its proximal duplex. Our conjugate can enable recognition of specific G4s in the human genome to understand and target those structures.