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DNA circuits are important fundamental tools for performing temporal logic operations with complex structures, but they lack sequence orthogonality. Here, we developed a simple and orthogonal hairpin exonuclease assistance signal (H-EAST) architecture to construct DNA domino circuits with time-delay characteristics and temporal logic operations, which has potential applications in biomolecular computing.

Hydrogels, water-filled networks that can adapt to external stimuli by altering their volume, are known for their high flexibility and biocompatibility. DNA, a critical biomolecule renowned for its exceptional characteristics including information transmission, molecular recognition, and editability, has found widespread applications in the biosensing field as well. The integration of these two biomaterials offers promising opportunities for the development of novel biosensors with enhanced sensitivity, specificity, and adaptability. Therefore, by virtue of the collective features, researchers have recently focused on the construction of responsive DNA hydrogel systems. This feature article describes recent developments in fabricating DNA hydrogels and their applications in the biosensing area. Initially, it focuses on the design strategies employed in preparing DNA hydrogels, encompassing both pure DNA hydrogels and hybridized DNA hydrogels. Subsequently, it summarizes the use of DNA hydrogels in biosensing applications, highlighting their applications in visual detection, electrochemical sensing, and optical biosensing analyses. Furthermore, the underlying responsive mechanisms within these biosensing systems are also described. Lastly, this article presents a comprehensive discussion on the existing challenges and prospects of responsive DNA hydrogels, offering insights into their potential to revolutionize the field of biosensing.

DNA-based nanostructures and nanodevices have recently been employed for a broad range of applications in modulating the assemblies and interaction patterns of different cell membrane receptors. These versatile nanodevices can be rationally designed with modular structures, easily programmed and tweaked such that they may act as smart chemical biology and cell biology tools to reveal insights into complicated cellular signaling processes. Their outstanding in vitro and cellular features have also begun to be further validated for some in vivo applications and demonstrated their great biomedical potential. In this review, we will highlight some key current advances in the molecular engineering and biological applications of DNA-based functional nanodevices, with a focus on how these tools have been used to respond and modulate membrane receptor dimerizations and/or oligomerizations, as a way to control cellular signaling processes. Some current challenges and future directions to further develop and apply these DNA nanodevices will also be discussed.

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.

The microperoxidase-11 hemopeptide exhibits configuration-dependent selectivity for guanine-quadruplexes by specifically uncaging c-MYC guanine-quadruplexes from a duplex DNA.

The versatile reactivity of isothiocyanate intermediates enabled the diversity-oriented synthesis (DOS) of N-heterocycles in a DNA-compatible manner. We first reported a mild in situ conversion of DNA-conjugated amines to isothiocyanates. Subsequently, a set of diverse transformations was successfully developed to construct 2-thioxo-quinazolinones, 1,2,4-thiadiazoles, and 2-imino thiazolines. Finally, the feasibility of these approaches in constructing DELs was further demonstrated through enzymatic ligation and mock pool preparation. This study demonstrated the advantages of combining in situ conversion strategies with DOS, which effectively broadened the chemical and structural diversity of DELs.

We here describe a visible-light photooxidation of sulfinate salts with common alkenes to yield β-hydroxy sulfones on DNA. This process demonstrates a broad substrate compatibility and achieves conversion rates ranging from moderate to excellent. Most importantly, it presents a straightforward, efficient, and metal-free approach for synthesizing Csp 3 -rich DNA-encoded libraries.

Herein, a universal nucleic acid analysis platform was constructed for sensitive and accurate detection of miRNA-155 and ctDNA using isothermal amplification-assisted CRISPR/Cas12a and a tetrahedral DNA nanostructure (TDN) supported sensing interface. Under the optimal experimental conditions, the prepared sensor achieved specific detection of miRNA-155 and ctDNA at as low as aM levels in 2.6 h. Furthermore, the platform was also successfully applied to human serum sample recovery experiments and cancer cell lysates, demonstrating outstanding reliability and accuracy. We firmly believe that this work provides a universal, sensitive, and practical tool for early clinical diagnosis.

The famous ''light-switch'' ruthenium complex [Ru(bpy) 2 (dppz)](PF 6 ) 2 (1) has been long known for its DNA binding properties in vitro . However, the biological utility of this compound has been hampered by its poor cellular uptake in living cells. Here we report a bioimaging application of 1 as cell viability probe in both 2D cells monolayer and 3D multi-cellular tumor spheroids of various human cancer cell lines (U87, HepG2, A549). When compared to propidium iodide, a routinely used cell viability probe, 1 was found to enhance the staining of dead cells in particular in tumor spheroids. 1 has high photostability, longer Stokes shift, and displays lower cytotoxicity compared to propidium iodide, which is a known carcinogenic. Finally, 1 was also found to displace the classical DNA binding dye Hoechst in dead cells, which makes it a promising dye for time-dependent imaging of dead cells in cell cultures, including multi-cellular tumor spheroids.

A programmably engineered stochastic RNA nanowalker powered by duplex-specific nuclease (DSN) is developed. By utilizing poly-adenine-based spherical nucleic acids (polyA-SNA) to accurately regulate the densities of DNA tracks, the nanowalker showcases its capability to identify miRNA-21, miRNA-486, and miRNA-155 with quick kinetics and attomolar sensitivity, positioning it as a promising option for cancer clinical surveillance.

We developed a system to detect multiple target biomolecules through sensing motif-tethered oligodeoxynucleotides. DNA-based molecular probes gave the primary amine motif upon reaction with the target biomolecules, glutathione (GSH) and H 2 O 2 . After labelling with biotin, the product DNAs were selectively collected to be quantified by qPCR.

The coupling of structural transitions to heat capacity changes leads to destabilization of macromolecules at both elevated and lowered temperatures. DNA origami not only exhibit this property but also provide a nanoscopic observable of cold denaturation processes by directing intramolecular strain to the most sensitive elements within their hierarchical architecture.

We present a triple-mode nanosensor platform for nucleic acid detection utilizing fluorescence anisotropy and Förster resonance energy transfer (FRET) strategies. The self-assembled nanoprobes serve as mass amplifiers, nanoquenchers, or nanodonors, exhibiting high FRET efficiencies (64.4-86.5%) and demonstrating excellent detection capabilities in DNA and microRNA analysis.

To avoid the unexpected aggregation and reduce the cytotoxicity of nanomaterials as optical probes in cell imaging applications, we propose a programmed DNA-cube as a carrier for silver nanoparticles (Ag NPs) to construct a specific hydrogen sulfide (H 2 S) responsive platform (Ag NP@DNA-cube) for diagnosing colorectal cancer (CRC) in this study. The DNA-cube maintains good dispersion of Ag NPs while providing excellent biocompatibility. Based on the characteristic overexpression of endogenous H 2 S in CRC cells, the Ag NPs are etched by H 2 S within target cells into silver sulfide quantum dots, thereby selectively illuminating the target cells. The Ag NP@DNA-cube exhibits a specific fluorescence response to CRC cells and achieves satisfactory imaging.

Chemically conjugated branched DNA was successfully synthesized by a copper-free click reaction to construct sophisticated and higher-order polyhedral DNA nanostructures with pre-defined units in one pot, which can be used as an efficient nanoplatform to precisely organize multiple gold nanoparticles in predesigned patterns.

The structure of the complex formed by naphthyridine carbamate dimer (NCD) binding to CGG repeat sequences in DNA, associated with fragile X syndrome, has been elucidated using 15 N-labeled NCD and 1 H- 15 N HSQC. In a fully saturated state, two NCD molecules consistently bind to each CGG/CGG unit, maintaining a 1 : 2 binding stoichiometry.

DNA-stabilized silver nanoclusters with 10 to 30 silver atoms are interesting biocompatible nanomaterials with intriguing fluorescence properties. However, they are not well understood, since atom-scale high level theoretical calculations have not been possible due to a lack of firm experimental structural information. Here, by using density functional theory (DFT), we study the recently atomically resolved (DNA) 2 -Ag 16 Cl 2 nanocluster in solvent under the lowest-lying singlet (S 1 ) and triplet (T 1 ) excited states, estimate the relative emission maxima for the allowed (S 1 → S 0 ) and dark (T 1 → S 0 ) transitions, and evaluate the transient absorption spectra. Our results offer a potential interpretation of the recently reported transient absorption and dual emission of similar DNA-stabilized silver nanoclusters, providing a mechanistic view on their photophysical properties that are attractive for applications in biomedical imaging and biophotonics.

Alkylation at the O 6 position of guanine is a common and highly mutagenic form of DNA damage. Direct repair of O 6 -alkylguanines by the "suicide" enzyme O 6 -methylguanine DNA methyltransferase (MGMT, AGT, AGAT) maintains genome stability and inhibits carcinogenesis. In this study, a fluorescent analogue of thymidine containing trans -stilbene ( ts T) is quenched by O 6 -methylguanine residues in the opposite strand of DNA by molecular dynamics that propagate through the duplex with as much as ∼9 Å of separation. Increased fluorescence of ts T or the cytosine analogue ts C resulting from MGMT-mediated DNA repair were distinguishable from non-covalent DNA-protein binding following protease digest. To our knowledge, this is the first study utilizing molecular rotor base analogues to detect DNA damage and repair activities in duplex DNA.

DNA nanostructures that respond to external stimuli have found applications in several areas such as biosensing, drug delivery and molecular computation. The use of different types of stimuli in a single operation provides another layer of control for the reconfiguration of nucleic acid nanostructures. This work demonstrates the use of a ribonuclease to "unset" a nucleic acid nanodevice based on the paranemic crossover (PX) DNA and specific DNA inputs to "reset" the structure into a juxtaposed DNA (JX 2 ) configuration, resulting in a 180° rotation of the helical domains. Such operations would be useful in translational applications where DNA nanostructures can be designed to reconfigure on the basis of more than one stimulus.

A compact 8-17 DNAzyme was modified with a Cu II -meditated artificial base pair to develop a metal-responsive allosteric DNAzyme. The base sequence was rationally designed based on the reported three-dimensional structure. The activity of the modified DNAzyme was enhanced 5.1-fold by the addition of one equivalent of Cu II ions, showing good metal responsiveness. Since it has been challenging to modify compactly folded DNAzymes without losing their activity, this study demonstrates the utility of the metal-mediated artificial base pairing to create stimuli-responsive functional DNAs.

Here, we develop an all-in-one strategy for efficient assembly of an electrochemical aptasensor. A multifunctional structure based on a tetrahedral DNA nanostructure (TDN) was synthesized via a one-step annealing process, providing DNA fixation, target recognition, signal amplification and space regulation. Based on the integration of this multifunctional structure, the sensing interface was assembled in one step. A ratiometric aptasensor was constructed by anchoring methylene blue (MB) to the TDN and ferrocene (Fc) on the cDNA. Using the ratio of the currents obtained from Fc and MB as a measure, the developed aptasensor shows excellent analytical performance for fumonisin B1 detection. This strategy is universal and could simplify the fabrication of aptasensors.

DNA methylation is a major epigenetic modification that is closely related to human health. Many experimental techniques as well as theoretical methods have been used to detect the modified nucleotides and identify their effects on molecular binding. It remains challenging to resolve the effect of few methylations of nucleic acids. Using super-resolution force spectroscopy, we firstly revealed that single cytosine methylation increases the mechanical stability of the DNA duplex by 1.9 ± 0.3 pN. Methylation also induces significant chiral selectivity towards drug molecules such as d , l -tetrahydropalmatine. Our results precisely quantify the mechanical effect of methylation and suggest that drug design should take methylation into consideration for enhanced selectivity.

The co-assembly of lipids and other compounds has recently gained increasing interest. Here, we report the formation of stimuli-responsive lipid-DNA origami fibers through the electrostatic co-assembly of cationic lipids and 6-helix bundle (6HB) DNA origami. The photosensitive lipid degrades when exposed to UV-A light, which allows a photoinduced, controlled release of the 6HBs from the fibers. The presented complexation strategy may find uses in developing responsive nanomaterials e.g. for therapeutics.

Combining the Hg 2+ -induced oxidization of silver nanoparticles with the cation exchange reaction between Ag + and CuS nanoparticles for cascade signal amplification, a sensitive, universal and label-free ICP-MS bioassay for nucleic acids and proteins was developed. By replacing the loop sequence of the T-Hg-T hairpin structure with specific sequences or aptamers to different biomarkers, it has great promise for the early detection of biomarkers potentially for diagnosis of cancerous diseases.

DNA is self-assembled with Fmoc-amino acids and Cu 2+ to construct a supramolecular catechol oxidase-mimetic catalyst, which exhibits remarkable activity in catalyzing colorimetric reactions. This catalytic system is used for the detection of DNA hybridization with a high selectivity and a low detection limit.

Nucleic acids can be damaged by ultraviolet (UV) irradiation, forming structural photolesions such as cyclobutane-pyrimidine-dimers (CPD). In modern organisms, sophisticated enzymes repair CPD lesions in DNA, but to our knowledge, no RNA-specific enzymes exist for CPD repair. Here, we show for the first time that RNA can protect itself from photolesions by an intrinsic UV-induced self-repair mechanism. This mechanism, prior to this study, has exclusively been observed in DNA and is based on charge transfer from CPD-adjacent bases. In a comparative study, we determined the quantum yields of the self-repair of the CPD-containing RNA sequence, GAU = U to GAUU (0.23%), and DNA sequence, d(GAT = T) to d(GATT) (0.44%), upon 285 nm irradiation via UV/Vis spectroscopy and HPLC analysis. After several hours of irradiation, a maximum conversion yield of ∼16% for GAU = U and ∼33% for d(GAT = T) was reached. We examined the dynamics of the intermediate charge transfer (CT) state responsible for the self-repair with ultrafast UV pump - IR probe spectroscopy. In the dinucleotides GA and d(GA), we found comparable quantum yields of the CT state of ∼50% and lifetimes on the order of several hundred picoseconds. Charge transfer in RNA strands might lead to reactions currently not considered in RNA photochemistry and may help understanding RNA damage formation and repair in modern organisms and viruses. On the UV-rich surface of the early Earth, these self-stabilizing mechanisms likely affected the selection of the earliest nucleotide sequences from which the first organisms may have developed.

Sequence-encoded biomolecules such as DNA and peptides are powerful programmable building blocks for nanomaterials. This paradigm is enabled by decades of prior research into how nucleic acid and amino acid sequences dictate biomolecular interactions. The properties of biomolecular materials can be significantly expanded with non-natural interactions, including metal ion coordination of nucleic acids and amino acids. However, these approaches present design challenges because it is often not well-understood how biomolecular sequence dictates such non-natural interactions. This Feature Article presents a case study in overcoming challenges in biomolecular materials with emerging approaches in data mining and machine learning for chemical design. We review progress in this area for a specific class of DNA-templated metal nanomaterials with complex sequence-to-property relationships: DNA-stabilized silver nanoclusters (Ag N -DNAs) with bright, sequence-tuned fluorescence colors and promise for biophotonics applications. A brief overview of machine learning concepts is presented, and high-throughput experimental synthesis and characterization of Ag N -DNAs are discussed. Then, recent progress in machine learning-guided design of DNA sequences that select for specific Ag N -DNA fluorescence properties is reviewed. We conclude with emerging opportunities in machine learning-guided design and discovery of Ag N -DNAs and other sequence-encoded biomolecular nanomaterials.

2-Thiobenzazole is among the privileged heterocyclic scaffolds in medicinal chemistry. Constructing such structural components in DNA-encoded libraries (DELs) may promote related bioactive hit discovery in a high-throughput fashion. Herein, we reported a DNA-compatible mild-condition synthetic methodology to efficiently forge functionalized 2-thiobenzazole scaffolds, realizing on-DNA sulfhydryl incorporation with broad substrate scope, thereby expanding the scope of 2-thiobenzazole-focused DNA-encoded chemical libraries.

Nanopore sequencing maps biochemical processes on DNA by detecting negative peaks in the sequence alignment profile. Protein-bound DNA and single-strand broken DNA cannot pass through nanopores, resulting in unaligned regions in the genome MAP. This novel approach provides a clear representation of genomic biochemical events.

We herein report a convenient and effective method for the purification of DNA-conjugated materials with a benchtop minicentrifuge. We demonstrate the fast isolation of DNA-modified small gold nanoparticles (5 nm), liposomes, and DNA nanostructures using fluorescent methods and gel electrophoresis. Our method is cost-effective and efficient and would accelerate the development of DNA nanotechnology.

In the last decade, point accumulation for imaging in nanoscale topography (PAINT) has emerged as a versatile tool for single-molecule localization microscopy (SMLM). Currently, DNA-PAINT is the most widely used, in which a transient stochastically binding DNA docking-imaging pair is used to reconstruct specific characteristics of biological or synthetic materials on a single-molecule level. Slowly, the need for PAINT probes that are not dependent on DNA has emerged. These probes can be based on (i) endogenous interactions, (ii) engineered binders, (iii) fusion proteins, or (iv) synthetic molecules and provide complementary applications for SMLM. Therefore, researchers have been expanding the PAINT toolbox with new probes. In this review, we provide an overview of the currently existing probes that go beyond DNA and their applications and challenges.

Several challenges need to be overcome when applying nucleic acids as therapeutic agents. We developed a new way to control the onset of the release of cholesterol-conjugated oligonucleotides with a simple, versatile, and cheap platform. Moreover, we combine the platform into a dual-release system that can release a hydrophobic drug with zero-order kinetics, followed by a rapid release of cholesterol-conjugated DNA.

The use of terminal deoxynucleotidyl transferase for the first time in a mechanistic exploration-through colorimetric sensing and isothermal titration calorimetric studies-has provided direct experimental evidence of a boronic acid moiety bridging two DNA duplexes via the 3' hydroxyl groups, offering new opportunities and insights into the domain of DNA (nano)biotechnology.

A novel and reusable electrochemiluminescence biosensor was developed based on tetrahedral DNA (TDN) signal amplification for ultrasensitive detection of miRNA-27a. The flowered nickel-iron layered double hydroxide@AuNPs (NiFe-LDH@AuNPs) composites increase the amount of hairpin DNA fixed on the electrode. When miRNA is present, TDN-Ru(bpy) 3 2+ acts as an ECL probe, forming a stable sandwich structure with miRNA-27a and hairpin DNA through base complementation pairing, thus achieving miRNA detection. This biosensor has the characteristics of high sensitivity, excellent selectivity, and good reproducibility.

Dimethyllumazine (DML)-thiazole orange (TO) conjugates were developed for fluorescence sensing of thymine glycol (Tg)-containing DNAs based on the selective recognition of the A nucleobase opposite the Tg residue. Additionally, this conjugate has demonstrated an inhibitory activity towards endonuclease III, a DNA repair enzyme, through its competitive binding to Tg-containing DNAs.

An amplified DNA logic sensor was constructed for the identification of multiple biomarkers, in which the inputs of targets triggered the disassembly of a V-shaped probe (VSP) structure by a strand displacement reaction, leading to the synthesis of silver nanoclusters (AgNCs) for electrocatalytic reduction of H 2 O 2 . The sensing platform achieved sensitive detection of methylated DNA and microRNA 122 with detection limits down to 3.4 and 4.1 fM, respectively, and can be used for the assay of clinical serum samples from healthy volunteers and liver injury patients with satisfactory results. The DNA logic sensor exhibited the advantages of convenience, low cost, and versatility without the involvement of electroactive label modification, which is helpful for disease diagnosis as well as the fundamental investigation of interfacial electrochemistry and molecular biology.

The potential for using DNA nanostructures for drug delivery applications requires understanding and ideally tuning their biostability. Here we investigate how biological degradation varies with size of a DNA nanostructure. We designed DNA tetrahedra of three edge lengths ranging from 13 to 20 bp and analyzed nuclease resistance for two nucleases and biostability in fetal bovine serum. We found that DNase I had similar digestion rates across sizes but appeared to incompletely digest the smallest tetrahedron, while T5 exonuclease was notably slower to digest the largest tetrahedron. In fetal bovine serum, the 20 bp tetrahedron was degraded four times faster than the 13 bp. These results show that DNA nanostructure size can influence nuclease degradation, but suggest a complex relationship that is nuclease specific.

Here we report the development of an equimolar conjugate of a metal-organic cage (MOC) and DNA (MOC-DNA). Several MOC-DNA conjugates were assembled into a programmed structure by coordinating with a template DNA having a complementary base sequence. Moreover, conjugation with the MOC drastically enhanced the permeability of DNA through the lipid bilayer, presenting great potential as a drug delivery system.

Structural selectivity of G-quadruplex ligands is essential for cellular applications since there is an excess of nucleic acids forming duplex structures compared to G-quadruplex structures in living cells. In this study, we developed new structure-selective G-quadruplex ligands utilizing a simple and fast screening system. The affinity, selectivity, enzymatic inhibitory activity and cytotoxicity of the structure-selective G-quadruplex ligands were demonstrated along with a structural selectivity-cytotoxicity relationship of G-quadruplex ligands.

DNA origami nanostructures are self-assembled into almost arbitrary two- and three-dimensional shapes from a long, single-stranded viral scaffold strand and a set of short artificial oligonucleotides. Each DNA strand can be functionalized individually using well-established DNA chemistry, representing addressable sites that allow for the nanometre precise placement of various chemical entities such as proteins, molecular chromophores, nanoparticles, or simply DNA motifs. By means of microscopic and spectroscopic techniques, these entities can be visualized or detected, and either their mutual interaction or their interaction with external stimuli such as radiation can be studied. This gives rise to the Lab-on-a-DNA origami approach, which is introduced in this Feature Article, and the state-of-the-art is summarized with a focus on light-harvesting nanoantennas and DNA platforms for single-molecule analysis either by optical spectroscopy or atomic force microscopy (AFM). Light-harvesting antennas can be generated by the precise arrangement of chromophores to channel and direct excitation energy. At the same time, plasmonic nanoparticles represent a complementary approach to focus light on the nanoscale. Plasmonic nanoantennas also allow for the observation of single molecules either by Raman scattering or fluorescence spectroscopy and DNA origami platforms provide unique opportunities to arrange nanoparticles and molecules to be studied. Finally, the analysis of single DNA motifs by AFM allows for an investigation of radiation-induced processes in DNA with unprecedented detail and accuracy.

Herein, we report two newly synthesized silver cluster-assembled materials (SCAMs), [Ag 14 (S t Bu) 10 (CF 3 COO) 4 (bpa) 2 ] n (bpa = 1,2-bis(4-pyridyl)acetylene) and [Ag 12 (S t Bu) 6 (CF 3 COO) 6 (bpeb) 3 ] n (bpeb = 1,4-bis(pyridin-4-ylethynyl)benzene) composed of Ag 14 and Ag 12 chalcogenolate cluster cores, respectively, bridged by acetylenic bispyridine linkers. The linker structures and electrostatic interaction between positively charged SCAMs and negatively charged DNA confer the SCAMs with the ability to suppress the high background fluorescence of single-stranded (ss) DNA probes with SYBR Green I nucleic acid stain, leading to high signal-to-noise ratio for label-free target DNA detection.

We designed a series of DNA sequences comprising a trinucleotide repeat segment and a small molecule-binding aptamer. Optimization of the DNA sequences and reaction conditions enabled chemical control of phase separation of DNA condensates. Our results demonstrate a new strategy to regulate biomolecular phase transition.

Control over synthetic DNA-based nanodevices can be achieved with a variety of physical and chemical stimuli. Actuation with light, however, is as advantageous as difficult to implement without modifying DNA strands with photo-switchable groups. Herein, we show that DNA nanodevices can be controlled using visible light in photo-switchable aqueous buffer solutions in a reversible and highly programmable fashion. The strategy presented here is non-invasive and allows the remote control with visible light of complex operations of DNA-based nanodevices such as the reversible release/loading of cargo molecules.

The formation of G-quadruplex structures (G4s) in vitro from guanine (G)-rich nucleic acid sequences of DNA and RNA stabilized with monovalent cations, typically K + and Na + , under physiological conditions, has been verified experimentally and some of them have high-resolution NMR or X -ray crystal structures; however, the biofunction of these special noncanonical secondary structures of nucleic acids has not been fully understood and their existence in vivo is still controversial at present. It is generally believed that the folding and unfolding of G4s in vivo is a transient process. Accumulating evidence has shown that G4s may play a role in the regulation of certain important cellular functions including telomere maintenance, replication, transcription and translation. Therefore, both DNA and RNA G4s of human cancer hallmark genes are recognized as the potential anticancer drug target for the investigation in cancer biology, chemical biology and drug discovery. The relationship between the sequence, structure and stability of G4s, the interaction of G4s with small molecules, and insights into the rational design of G4-selective binding ligands have been intensively studied over the decade. At present, some G4-ligands have achieved a new milestone and successfully entered the human clinical trials for anticancer therapy. Over the past few decades, numerous efforts have been devoted to anticancer therapy; however, G4s for molecular recognition and live cell imaging and for application as antibacterial agents and antibiofilms against antibiotic resistance have been obviously underexplored. The recent advances in G4-ligands in these areas are thus selected and discussed concentratedly in this article in order to shed light on the emerging role of G4s in chemical biology and therapeutic prospects against bacterial infections. In addition, the recently published molecular scaffolds for designing small ligands selectively targeting G4s in live cell imaging, bacterial biofilm imaging, and antibacterial studies are discussed. Furthermore, a number of underexplored G4-targets from the cytoplasmic membrane-associated DNA, the conserved promoter region of K. pneumoniae genomes, the RNA G4-sites in the transcriptome of E. coli and P. aeruginosa , and the mRNA G4-sites in the sequence for coding the vital bacterial FtsZ protein are highlighted to further explore in G4-drug development against human diseases.

Metal-mediated nucleobase pairing can play a central role in the expression of metal-responsive DNA functions. We report the Cu II -mediated stabilisation of DNA duplexes bearing damaged nucleobases, 1, N 6 -ethenoadenine (εA), as metal-binding sites, which was utilised to construct a metal-responsive DNAzyme. Consecutive incorporation of three or more εA-εA mismatch pairs allowed for Cu II -dependent significant duplex stabilisation through metal-mediated εA-Cu II -εA base pairing. Subsequently, a split DNAzyme with three εA-Cu II -εA base pairs was strategically designed. The activity of the εA-modified DNAzyme was enhanced by 5.3-fold upon addition of Cu II ions. This study demonstrates the utility of εA lesions for building metal-responsive DNA architectures.

Artificial metal-mediated DNA base pairing is a promising strategy for creating highly functionalized DNA supramolecules. Here we report a novel ligand-type triazole-4-carboxylate (TazC) nucleoside that is readily prepared by the click reaction. TazC nucleosides were found to form a stable TazC-Cu II -TazC base pair inside DNA duplexes, resulting in Cu II -specific duplex stabilization (Δ T m = +7.7 °C). This study demonstrates that the triazole derivatives are useful in the development of metal-mediated base pairing.

A rectangle DNA origami nanostructure equipped with doxorubicin-derived prodrugs targeting a tumor cell-specific enzyme (NQO1) is constructed. Combining the high prodrug payload of DNA origami and NQO1-activated chemotherapy, this nanosystem presents therapeutic selectivity for NQO1-overexpressing MCF-7 cells over healthy L02 cells, offering a potent strategy for precision cancer therapy.

We present an efficient and readily applicable strategy for the covalent ligation of proteins to DNA origami by using the SpyCatcher-SpyTag (SC-ST) connector system. This approach showed orthogonality with other covalent connectors and has been used exemplarily for the immobilization and study of stereoselective ketoreductases to gain insight into the spatial arrangement of enzymes on DNA nanostructures.

Guanine-rich DNA sequences are known to fold into secondary structures called G-quadruplexes (G4s), which can form from either individual DNA strands (intra-molecular) or multiple DNA strands (inter-molecular, iG4s). Intra-molecular G4s have been the object of extensive biological investigation due to their enrichment in gene-promoters and telomers. On the other hand, iG4s have never been considered in biological contexts, as the interaction between distal sequences of DNA to form an iG4 in cells was always deemed as highly unlikely. In this feature article, we challenge this dogma by presenting our recent discovery of the first human protein (CSB) displaying astonishing picomolar affinity and binding selectivity for iG4s. These findings suggest potential for iG4 structures to form in cells and highlight the need of further studies to unravel the fundamental biological roles of these inter-molecular DNA structures. Furthermore, we discuss how the potential for formation of iG4s in neuronal cells, triggered by repeat expansions in the C9orf72 gene, can lead to the formation of nucleic-acids based pathological aggregates in neurodegenerative diseases like ALS and FTD. Finally, based on our recent work on short LNA-modified probes, we provide a prespective on how the rational design of G4-selective chemical tools can be leveraged to further elucidate the biological relevance of iG4 structures in the context of ageing-related diseases.

Dynamic regulation of DNA origami nanostructures is important for the fabrication of intelligent DNA nanodevices. Toehold-mediated strand displacement is a common regulation strategy, which utilizes trigger strands to assemble and disassemble nanostructures. Such trigger strands are required to be completely complementary to the corresponding substrate strands, which strictly demands orthogonality and accuracy of the sequence design. Herein, we present a disassembly strategy of DNA origami dimers based on polymerase-triggered strand displacement, where the polymerase primers, as the trigger strands, were only partially complementary to the toehold region of the substrate strands. To demonstrate the programmability of trigger strands, we utilized primers with different sequence combination patterns to disassemble DNA origami dimers. The statistical summary of AFM images and fluorescence curves proved the feasibility of the new strategy. The utilization of polymerase-triggered strand displacement on the disassembly of DNA origami structures enriches the toolbox for the dynamic regulation of DNA nanostructures.