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DNA nanostructures have shown potential in cancer therapy. However, their clinical application is hampered by the difficulty to deliver them into cancer cells and susceptibility to nuclease degradation. To overcome these limitations, we report herein a periodically ordered nick‐hidden DNA nanowire (NW) with high serum stability and active targeting functionality. The inner core is made of multiple connected DNA double helices, and the outer shell is composed of regularly arranged standing‐up hairpin aptamers. All termini of the components are hidden from nuclease attack, whereas the target‐binding sites are exposed to allow delivery to the cancer target. The DNA NW remained intact during incubation for 24 h in serum solution. Animal imaging and cell apoptosis showed that NWs loaded with an anticancer drug displayed long blood‐circulation time and high specificity in inducing cancer‐cell apoptosis, thus validating this approach for the targeted imaging and therapy of cancers. [ABSTRACT FROM AUTHOR]

Fluorescent, DNA‐stabilized silver nanoclusters (DNA‐AgNCs) are applied in a range of applications within nanoscience and nanotechnology. However, their diverse optical properties, mechanism of formation, and aspects of their composition remain unexplored, making the rational design of nanocluster probes challenging. Herein, a synthetic procedure is described for obtaining a high yield of emissive DNA‐AgNCs with a C‐loop hairpin DNA sequence, with subsequent purification by size‐exclusion chromatography (SEC). Through a combination of optical spectroscopy, gel electrophoresis, inductively coupled plasma mass spectrometry (ICP‐MS), and small‐angle X‐ray scattering (SAXS) in conjunction with the systematic study of various DNA sequences, the low‐resolution structure and mechanism of the formation of AgNCs were investigated. Data indicate that fluorescent DNA‐AgNCs self‐assemble by a head‐to‐head binding of two DNA hairpins, bridged by a silver nanocluster, resulting in the modelling of a dimeric structure harboring an Ag12 cluster. [ABSTRACT FROM AUTHOR]

Toehold‐mediated DNA strand displacement endows DNA nanostructures with dynamic response capability. However, the complexity of sequence design dramatically increases as the size of the DNA network increases. We attribute this problem to the mechanism of toehold‐mediated strand displacement, termed exact strand displacement (ESD), in which one input strand corresponds to one specific substrate. In this work, we propose an alternative to toehold‐mediated DNA strand displacement, termed fuzzy strand displacement (FSD), in which one‐to‐many and many‐to‐one relationships are established between the input strand and the substrate, to reduce the complexity. We have constructed four modules, termed converter, reporter, fuzzy detector, and fuzzy trigger, and demonstrated that a sequence pattern recognition network composed of these modules requires less complex sequence design than an equivalent network based on toehold‐mediated DNA strand displacement. [ABSTRACT FROM AUTHOR]

In recent years, three‐dimensional density maps reconstructed from single particle images obtained by electron cryo‐microscopy (cryo‐EM) have reached unprecedented resolution. However, map interpretation can be challenging, in particular if the constituting structures require de‐novo model building or are very mobile. Herein, we demonstrate the potential of convolutional neural networks for the annotation of cryo‐EM maps: our network Haruspex has been trained on a carefully curated set of 293 experimentally derived reconstruction maps to automatically annotate RNA/DNA as well as protein secondary structure elements. It can be straightforwardly applied to newly reconstructed maps in order to support domain placement or as a starting point for main‐chain placement. Due to its high recall and precision rates of 95.1 % and 80.3 %, respectively, on an independent test set of 122 maps, it can also be used for validation during model building. The trained network will be available as part of the CCP‐EM suite. [ABSTRACT FROM AUTHOR]

G‐quadruplex (G4) DNA structures are linked to fundamental biological processes and human diseases, which has triggered the development of compounds that affect these DNA structures. However, more knowledge is needed about how small molecules interact with G4 DNA structures. This study describes the development of a new class of bis‐indoles (3,3‐diindolyl‐methyl derivatives) and detailed studies of how they interact with G4 DNA using orthogonal assays, biophysical techniques, and computational studies. This revealed compounds that strongly bind and stabilize G4 DNA structures, and detailed binding interactions which for example, show that charge variance can play a key role in G4 DNA binding. Furthermore, the structure–activity relationships generated opened the possibilities to replace or introduce new substituents on the core structure, which is of key importance to optimize compound properties or introduce probes to further expand the possibilities of these compounds as tailored research tools to study G4 biology. [ABSTRACT FROM AUTHOR]

Synthetic DNA has emerged as a powerful self‐assembled material for the engineering of nanoscale supramolecular devices and materials. Recently dissipative self‐assembly of DNA‐based supramolecular structures has emerged as a novel approach providing access to a new class of kinetically controlled DNA materials with unprecedented life‐like properties. So far, dissipative control has been achieved using DNA‐recognizing enzymes as energy dissipating units. Although highly efficient, enzymes pose limits in terms of long‐term stability and inhibition of enzyme activity by waste products. Herein, we provide the first example of kinetically controlled DNA nanostructures in which energy dissipation is achieved through a non‐enzymatic chemical reaction. More specifically, inspired by redox signalling, we employ redox cycles of disulfide‐bond formation/breakage to kinetically control the assembly and disassembly of tubular DNA nanostructures in a highly controllable and reversible fashion. [ABSTRACT FROM AUTHOR]

The incorporation of a methyl group to DNA nucleotides (methylation) is a well known phenomenon in epigenetic regulation. Methylation can result in gene silencing. Here we investigate the electronic properties of cytosine methylation in different DNA systems using first‐principles density functional theory calculations. Specifically, four systems are investigated consisting of two stacked base pairs, including the deoxyribose‐phosphate backbone. These four systems are the unmethylated GpC‐CpG, the single methylated GpC‐mCpG, the double symmetrical methylated GpCm‐mCpG, and the quadruple symmetrical GpCmm‐mmCpG. Single base‐pair cases are also considered with and without the deoxyribose‐phosphate backbone (with: pG‐pC, pG‐pCm, and pG‐pCmm; without: G−C, G−Cm, and G‐Cmm). These structures are relaxed using the conjugate gradient method. Nanoscale properties, such as the electronic density of states, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) wave functions, charge Mulliken population, hydrogen bond energy, binding energy, and dipole moments between unmethylated and methylated relaxed structures are obtained and compared. These results demonstrate that the deoxyribose‐phosphate backbone plays a crucial role in the spatial distribution of HOMO and LUMO wave functions in the stacked systems modulating reactivity and stability. Cytosine methylation on the GpC‐CpG system results in a stabilization effect. Dipole moment modification in the studied systems could favor specific protein‐DNA interactions due to the methyl group non‐polarity, producing hydrophobic DNA regions. These findings suggest specific rules of interaction between methylated regions and gene silencing elements at the nanoscale level. [ABSTRACT FROM AUTHOR]

Biomolecules undergo motions on the micro‐to‐millisecond timescale to adopt low‐populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr–Purcell–Meiboom–Gill (CPMG), chemical exchange saturation transfer (CEST), and R1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R1ρ experiment are more broadly sensitive to motions on the micro‐to‐millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high‐power radio‐frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro‐to‐millisecond timescale. Given the ease of implementing high‐power fields in CEST, this should make it easier to characterize micro‐to‐millisecond dynamics in biomolecules. [ABSTRACT FROM AUTHOR]

Programmable assembly of nanoparticles (NPs) into well‐defined architectures has attracted attention because of tailored properties resulting from coupling effects. However, general and precise approaches to control binding modes between NPs remain a challenge owing to the difficulty in manipulating the accurate positions of the functional patches on the surface of NPs. Here, a strategy is developed to encage spherical NPs into pre‐designed octahedral DNA origami frames (DOFs) through DNA base‐pairings. The DOFs logically define the arrangements of functional patches in three dimensions, owing to the programmability of DNA hybridization, and thus control the binding modes of the caged nanoparticle with designed anisotropy. Applying the node‐and‐spacer approach that was widely used in crystal engineering to design coordination polymers, patchy NPs could be rationally designed with lower symmetry encoded to assemble a series of nano‐architectures with high‐order geometries. [ABSTRACT FROM AUTHOR]

Programming intelligent DNA nanocarriers for the targeted transport of molecular payloads in living cells has attracted extensive attention. In vivo activation of these nanocarriers usually relies on external light irradiation. An interest is emerging in the automatic recognition of intracellular surroundings by nanocarriers and their in situ activation under the control of programmed DNA‐computation circuits. Herein, we report the integration of DNA circuits with framework nucleic acid (FNA) nanocarriers that consist of a truncated square pyramid (TSP) cage and a built‐in duplex cargo containing an antisense strand of the target mRNA. An i‐motif and ATP aptamer embedded in the TSP are employed as logic‐controlling units to respond to H+ and ATP inside cellular compartments, triggering the release of the sensing element for fluorescent mRNA imaging. Logic‐controlled FNA devices could be used to target drug delivery, enabling precise disease treatment. [ABSTRACT FROM AUTHOR]

Numerous applications of metal‐mediated base pairs (metallo‐base‐pairs) to nucleic acid based nanodevices and genetic code expansion have been extensively studied. Many of these metallo‐base‐pairs are formed in DNA and RNA duplexes containing Watson–Crick base pairs. Recently, a crystal structure of a metal–DNA nanowire with an uninterrupted one‐dimensional silver array was reported. We now report the crystal structure of a novel DNA helical wire containing HgII‐mediated T:T and T:G base pairs and water‐mediated C:C base pairs. The Hg‐DNA wire does not contain any Watson–Crick base pairs. Crystals of the Hg‐DNA wire, which is the first DNA wire structure driven by HgII ions, were obtained by mixing the short oligonucleotide d(TTTGC) and HgII ions. This study demonstrates the potential of metallo‐DNA to form various structural components that can be used for functional nanodevices. [ABSTRACT FROM AUTHOR]

Guanine quadruplexes, recently reported to form in vivo, represent a broad spectrum of non‐canonical conformations of nucleic acids. The actual conformation might differ between water solutions and crowding or dehydrating solutions that better reflect the conditions in the cell. Here we show, using spectroscopic techniques, that most guanine substitutions prevent the conformational switch from antiparallel or hybrid forms to parallel ones when induced by dehydrating agents. The inhibitory effect does not depend on the position of the substitution, but, interestingly, on the type of substitution and, to some extent, on its destabilising potential. A parallel form might be induced in some cases by ligands such as N‐methyl mesoporphyrin IX and even this ligand‐induced switch is inhibited by guanine substitution. The ability or inability to have a conformation switch, based on actual conditions, might significantly influence potential conformation‐dependent quadruplex interactions. [ABSTRACT FROM AUTHOR]

The synthesis and characterisation of three novel iridium(III) bis‐cyclometallated complexes is reported. Their photophysics have been fully characterised by classical methods and revealed charge‐transfer (CT) and ligand‐centred (LC) transitions. Their ability to selectively interact with G‐quadruplex telomeric DNA over duplex DNA has been studied by circular dichroism (CD), bio‐layer interferometry (BLI) and surface plasmon resonance (SPR) analyses. Interestingly, one of the complexes was able to promote photoinduced electron transfer (PET) with the guanine DNA base, which in turn led to oxidative damage (such as the formation of 8‐oxoguanine) to the telomeric sequence. To the best of our knowledge, this is the first study of highly photo‐oxidising bis‐cyclometallated iridium(III) complexes with G‐quadruplex telomeric DNA. [ABSTRACT FROM AUTHOR]

Aqueous hybrid soft nanomaterials consisting of plural supramolecular architectures with a high degree of segregation (orthogonal coexistence) and precise hierarchy at the nano‐ and microscales, which are reminiscent of complex biomolecular systems, have attracted increasing attention. Remarkable progress has been witnessed in the construction of DNA nanostructures obtained by rational sequence design and supramolecular nanostructures of peptide derivatives through self‐assembly under aqueous conditions. However, orthogonal self‐assembly of DNA nanostructures and supramolecular nanostructures of peptide derivatives in a single medium has not yet been explored in detail. In this study, DNA microspheres, which can be obtained from three single‐stranded DNAs, and three different supramolecular nanostructures (helical nanofibers, straight nanoribbons, and flowerlike microaggregates) of semi‐artificial glycopeptides were simultaneously constructed in a single medium by a simple thermal annealing process, which gives rise to hybrid soft nanomaterials. Fluorescence imaging with selective staining of each supramolecular nanostructure uncovered the orthogonal coexistence of these structures with only marginal impact on their morphology. Additionally, the biostimuli‐responsive degradation propensity of each supramolecular architecture is retained, and this may allow the construction of active soft nanomaterials exhibiting intelligent biofunctions. [ABSTRACT FROM AUTHOR]

By using X‐ray crystallography, we show that the complexes Λ/Δ‐[Ru(TAP)2(11‐CN‐dppz)]2+ (TAP=1,4,5,8‐tetraazaphenanthrene, dppz=dipyridophenazine) bind DNA G‐quadruplex in an enantiospecific manner that parallels the specificity of these complexes with duplex DNA. The Λ complex crystallises with the normally parallel stranded d(TAGGGTTA) tetraplex to give the first such antiparallel strand assembly in which syn‐guanosine is adjacent to the complex at the 5′ end of the quadruplex core. SRCD measurements confirm that the same conformational switch occurs in solution. The Δ enantiomer, by contrast, is present in the structure but stacked at the ends of the assembly. In addition, we report the structure of Λ‐[Ru(phen)2(11‐CN‐dppz)]2+ bound to d(TCGGCGCCGA), a duplex‐forming sequence, and use both structural models to provide insight into the motif‐specific luminescence response of the isostructural phen analogue enantiomers. [ABSTRACT FROM AUTHOR]

Herein, the direct visualization of the dynamic interaction between a photoresponsive transcription factor fusion, GAL4–VVD, and DNA using high‐speed atomic force microscopy (HS‐AFM) is reported. A series of different GAL4–VVD movements, such as binding, sliding, stalling, and dissociation, was observed. Inter‐strand jumping on two double‐stranded (ds) DNAs was also observed. Detailed analysis using a long substrate DNA strand containing five GAL4‐binding sites revealed that GAL4–VVD randomly moved on the dsDNA using sliding and hopping to rapidly find specific binding sites, and then stalled to the specific sites to form a stable complex formation. These results suggest the existence of different conformations of the protein to enable sliding and stalling. This single‐molecule imaging system with nanoscale resolution provides an insight into the searching mechanism used by DNA‐binding proteins. [ABSTRACT FROM AUTHOR]

DNA sequences rich in cytosine have the propensity, under acidic pH, to fold into four‐stranded intercalated DNA structures called i‐motifs. Recent studies have provided significant breakthroughs that demonstrate how chemists can manipulate these structures for nanobiotechnology and therapeutics. The first section of this Minireview discusses the development of advanced functional nanostructures by synthetic conjugation of i‐motifs with organic scaffolds and metal nanoparticles and their role in therapeutics. The second section highlights the therapeutic targeting of i‐motifs with chemical scaffolds and their significance in biology. For this, first we shed light on the long‐lasting debate regarding the stability of i‐motifs under physiological conditions. Next, we present a comparative analysis of recently reported small molecules for specifically targeting i‐motifs over other abundant DNA structures and modulating their function in cellular systems. These advances provide new insights into i‐motif‐targeted regulation of gene expression, telomere maintenance, and therapeutic applications. Non‐canonical DNA structures: i‐Motifs are four‐stranded DNA structures formed by the folding of cytosine‐rich sequences present in telomeres and gene promoters. Compared to complementary G‐quadruplexes, i‐motifs are less explored. This Minireview describes significant advances in chemically regulated i‐motif‐based functional nanostructures and small‐molecule targeting of i‐motifs for therapeutics. [ABSTRACT FROM AUTHOR]

Quantum mechanical (QM) and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations of a recently reported dinuclear mercury(II)-mediated base pair were performed aiming to analyse its intramolecular bonding pattern, its stability, and to obtain clues on the mechanism of the incorporation of mercury(II) into the DNA. The dynamic distance constraint was employed to find initial structures, control the dissociation process in an unbiased fashion and to determine the free energy required. A strong influence of the exocyclic carbonyl or amino groups of neighbouring base pairs on both the bonding pattern and the mechanism of incorporation was observed. During the dissociation simulation, an amino group of an adenine moiety of the adjacent base pair acts as a turnstile to rotate the mercury(II) ion out of the DNA core region. The calculations provide an important insight into the mechanism of formation of this dinuclear metal-mediated base pair and indicate that the exact location of a transition metal ion in a metal-mediated base pair may be more ambiguous than derived from simple model building.

i-Motifs are non-canonical secondary structures of DNA formed by mutual intercalation of hemi-protonated cytosine-cytosine base pairs, most typically in slightly acidic conditions (pH<7.0). These structures are well-studied in vitro and have recently been suggested to exist in cells. Despite nearly a decade of active research, the quest for small-molecule ligands that could selectively bind to and stabilize i-motifs continues, and no reference, bona fide i-motif ligand is currently available. This is, at least in part, due to the lack of robust methods to assess the interaction of ligands with i-motifs, since many techniques well-established for studies of other secondary structures (such as CD-, UV-, and FRET-melting) may generate artifacts when applied to i-motifs. Here, we describe an implementation of automated, potentiometric (pH) titrations as a robust isothermal method to assess the impact of ligands or cosolutes on thermodynamic stability of i-motifs. This approach is validated through the use of a cosolute previously known to stabilize i-motifs (PEG2000) and three small-molecule ligands that are able to stabilize, destabilize, or have no effect on the stability of i-motifs, respectively., (Copyright © 2024. Published by Elsevier Inc.)

Recently, we observed the first example of a left‐handed G‐quadruplex structure formed by natural DNA, named Z‐G4. We analysed the Z‐G4 structure and inspected its primary 28‐nt sequence in order to identify motifs that convey the unique left‐handed twist. Using circular dichroism spectroscopy, NMR spectroscopy, and X‐ray crystallography, we revealed a minimal sequence motif of 12 nt (GTGGTGGTGGTG) for formation of the left‐handed DNA G‐quadruplex, which is found to be highly abundant in the human genome. A systematic analysis of thymine loop mutations revealed a moderate sequence tolerance, which would further broaden the space of sequences prone to left‐handed G‐quadruplex formation. It's just a jump to the left: Based on analysis and modification of the two sequence blocks of the natural left‐handed DNA G‐quadruplex Z‐G4, the 12‐nt sequence GTGGTGGTGGTG was identified as a minimal sequence motif for left‐handed G‐quadruplex formation. This motif is abundant in the human genome. [ABSTRACT FROM AUTHOR]

The high‐throughput assessment of the secondary structures adopted by DNA oligonucleotides is currently hampered by the lack of suitable biophysical methods. Fluorescent sensors hold great potential in this respect; however, the moderate selectivity that they display for one DNA conformation over the others constitutes a major drawback to the development of accurate assays. Moreover, the use of single sensors impedes a comprehensive classification of the tested sequences. Herein, we propose to overcome these limitations through the development of a fluorescence sensor array constituted by easily accessible, commercial dyes. Multivariate analysis of the emission data matrix produced by the array allows the conformational preferences of DNA sequences of interest to be explored, either by calculating the probability of group membership in the six predefined structural categories (three G‐quadruplex groups, double‐stranded, and two groups of single‐stranded forms) or by revealing their particular structural features. The assay enables rapid screening of synthetic DNA oligonucleotides in a 96‐well plate format. Shining the light on DNA. A fluorescence sensor array constituted by easily accessible, commercial dyes has been developed to investigate the secondary structures of DNA. Multivariate analysis of the emission data produced by the array permits high‐throughput assessment of secondary structures adopted by DNA oligonucleotides (see figure). [ABSTRACT FROM AUTHOR]

The formation of growth‐mediated structures from gold nanoparticle seeds was studied in the presence of amine‐modified single‐strand DNA sequences and reducing agents such as hydroxylamine and hydroquinone. In the case of hydroxylamine, spherical gold nanoparticle seeds (0.45 nM) were incubated with amine‐modified single‐strand DNA probes PMR (amine‐5′‐ACATCAGT‐3′) and PML (amine‐5′‐GATAAGCT‐3′), which resulted in gold nanoflowers and nanospheres, respectively. When the concentration of the nanoparticle seeds was varied (0.15–0.45 nM), only the PMR sequence showed growth‐mediated development of gold nanoflowers. The size of the gold nanoparticles obtained is independent of the seed concentration for both PMR and PML sequences. In contrast, in the presence of the reducing agent hydroquinone, the growth processes are identical in for both the sequences. At a lower seed concentrations (0.15 nM), gold nanoflowers of larger size were observed for both sequences, whereas at higher seed concentrations (0.45 nM), much smaller gold nanospheres resulted. The formation and stability of nanoflowers and nanospheres for PMR and PML with hydroxylamine‐based reduction were further studied in detail with diverse controlled amine‐modified (5′‐, 3′‐ and both end‐modified) and non‐modified DNA sequences with other mutants of these two sequences. All grown up! The formation of growth‐mediated structures from gold nanoparticle seeds was studied in the presence of amine‐modified single‐strand DNA sequences and reducing agents such as hydroxylamine and hydroquinone. Both the reducing agent and the DNA sequences were critical in determining the size and shape of the gold structures formed. The resulting nanoflowers and nanospheres were used to detect the DNA sequence for miR‐21. [ABSTRACT FROM AUTHOR]

The design and characterization of new ruthenium(II) complexes aimed at targeting G‐quadruplex DNA is reported. Importantly, these complexes are based on oxidizing 1,4,5,8‐tetraazaphenanthrene (TAP) ancillary ligands known to favour photo‐induced electron transfer (PET) with DNA. The photochemistry of complexes 1–4 has been studied by classical methods, which revealed two of them to be capable of photo‐abstracting an electron from guanine. From studies of the interactions with DNA through luminescence, circular dichroism, bio‐layer interferometry, and surface plasmon resonance experiments, we have demonstrated the selectivity of these complexes for telomeric G‐quadruplex DNA over duplex DNA. Preliminary biological studies of these complexes have been performed: two of them showed remarkable photo‐cytotoxicity towards telomerase‐negative U2OS osteosarcoma cells, whereas very low mortality was observed in the dark at the same photo‐drug concentration. G‐quadruplex‐targeting Ru complexes: Three new photo‐oxidizing ruthenium(II) complexes based on the 2‐(4‐chlorophenyl)‐1H‐imidazo[4,5‐f][1,10]phenanthroline (CPIP) ligand have been synthesized. These complexes selectively target G‐quadruplex DNA over duplex DNA. [ABSTRACT FROM AUTHOR]

i‐Motifs are tetraplex DNAs known to be stable at acidic pH. The structure of i‐motifs is important in DNA nanotechnology; i‐motif‐forming sequences with consecutive cytosine (C) molecules are abundant throughout the human genome. There is, however, little information on the structure of C‐rich DNAs under physiologically relevant neutral conditions. The electron dynamics of i‐motifs, crucial to both biology and materials applications, also remains largely unexplored. In this work, we report a combined femtosecond and nanosecond broadband time‐resolved fluorescence (TRF) and steady‐state fluorescence investigation on homo‐oligomer dC20, a human telomeric sequence (HTS) 5′‐dC3(TA2C3)3, and its analogue performed with different excitation at both acidic and neutral pH. Our study provides direct observation of intrinsic fluorescence and the first full probe of the real‐time dynamics of the intrinsic fluorescence from i‐motifs formed from varied sequences and pH conditions. The results obtained demonstrate concrete evidence for the existence at neutral pH of i‐motifs from both dC20 and the HTS. It also identifies that, under neutral conditions, the i‐motif from dC20 adopting the bimolecular folding structure is significantly more stable than the HTS i‐motif featuring the unimolecular topology. Our femtosecond and nanosecond TRF study unveils excitation dynamics distinctive of the interdigitated architecture of i‐motifs with the excited states involved exhibiting deactivation over a remarkably broad timescale through multiple channels involving proton‐coupled electron transfer lasting tens of picoseconds, as signified by the solvent kinetic isotope effect, and structure‐dependent charge recombination in the hundreds of picoseconds to tens of nanoseconds time regime. Return to the fold: Intrinsic fluorescence measurements revealed formation of i‐motifs at neutral pH from both the homo‐cytosine oligomer and C‐rich human telomeric DNA that possess largely varied structural stability owing to the difference in folding molecularity (see figure). Studying the intrinsic fluorescence from these i‐motifs uncovered multiple timescale electron dynamics distinctive of the interdigitated architecture of i‐motifs. [ABSTRACT FROM AUTHOR]

DNA quadruplexes have been the subject of investigation because of their biological relevance and because of their potential application in supramolecular chemistry. Similarly, RNA quadruplexes are now gaining increasing attention. Although DNA and RNA quadruplexes are structurally very similar, the latter show higher stability. In this study we report dispersion‐corrected density functional theory (DFT‐D) quantum chemical calculations that were undertaken to understand the difference in stabilities of RNA and DNA quadruplexes. The smallest meaningful model of a stack of quartets, interacting with alkali metal cations, was simulated in an aqueous environment. The energy decomposition analysis allows for in‐depth examination of the interaction energies, emphasising the role of noncovalent interactions and better electrostatics in determining RNA‐GQs higher stabilities, particularly pinpointing the role of the extra 2′‐OH groups. Furthermore, our computations present new insights on why the cation is required for self‐assembly: unexpectedly the cation is not necessary to relieve the repulsion between the oxygen atoms in the central cavity, but it is needed to overcome the entropic penalty. Why so stable? Dispersion‐corrected (DFT‐D) quantum chemical calculations on the stabilities of RNA and DNA quadruplexes show electrostatic interactions and hydrogen bonds make the decisive difference (see figure). [ABSTRACT FROM AUTHOR]

Despite evidence that histone H3 and H4 proteins may act as the precursor for orientating the DNA sequence to form nucleosome structures, there is no direct evidence of the formed compact structure. Here, it is demonstrated that a histone H3–H4 octasome could be constructed without the involvement of histone H2A–H2B under in vitro reconstitution conditions. Atomic force microscopy was used to obtain the first direct observation of the octasome structure, which exhibited a similar core‐protein size as that of a nucleosome but with a shorter core histone‐binding DNA region. The octasome also displayed a one‐step histone‐dissociation pattern under heat treatment, distinct micrococcal nuclease and peplomycin accessibility, which suggests a different wrapping pattern to that in nucleosomes. It′s a wrap! Here it is demonstrated that a histone H3–H4 octasome can be constructed without involving histone H2A–H2B under in vitro reconstitution conditions. The first direct observation of the octasome structure was achieved by high speed atomic force microscopy, and it was found to exhibit a similar core‐protein size to a nucleosome but with a shorter core histone‐binding DNA region. [ABSTRACT FROM AUTHOR]

Abstract: Small molecules that target G‐quadruplex (G4) DNA structures are not only valuable to study G4 biology but also for their potential as therapeutics. This work centers around how different design features of small molecules can affect the interactions with G4 DNA structures, exemplified by the development of synthetic methods to bis‐indole scaffolds. Our synthesized series of bis‐indole scaffolds are structurally very similar but differ greatly in the flexibility of their core structures. The flexibility of the molecules proved to be an advantage compared to locking the compounds in the presumed bioactive G4 conformation. The flexible derivatives demonstrated similar or even improved G4 binding and stabilization in several orthogonal assays even though their entropic penalty of binding is higher. In addition, molecular dynamics simulations with the c‐MYC G4 structure showed that the flexible compounds adapt better to the surrounding. This was reflected by an increased number of both stacking and polar interactions with both the residues in the G4 DNA structure and the DNA residues just upstream of the G4 structure. [ABSTRACT FROM AUTHOR]

Abstract: Photoactivated DNA interstrand cross‐linking agents have a wide range of biological applications. Recently, several aryl boronates have been reported to induce DNA interstrand cross‐link (ICL) formation via carbocations upon photoirradiation. Herein, we synthesized a series of new bifunctional phenyl compounds to test the generality of such a mechanism, and to understand how the chemical structure influences carbocation formation and the DNA cross‐linking process. These compounds efficiently form DNA ICLs via generated benzyl cations upon 350 nm irradiation. The DNA cross‐linking efficiency and the pathway for carbocation generation depend on both the aromatic substituents and the leaving groups. Bromine as a leaving group facilitates the DNA cross‐linking process in comparison with trimethyl ammonium salt. Both electron‐donating and ‐withdrawing substituents induce bathochromic shifts, which favor photoinduced DNA ICL formation. For the bromides, the benzyl cation intermediates were generated through oxidation of the corresponding benzyl radicals. However, for the ammonia salts, the benzyl cations were formed through two pathways: either through oxidation of the benzyl radicals or by direct heterolysis of the C−N bond. Photoinduced C−N homolysis to form benzyl radicals occurred with compounds having donating substituents, whereas direct heterolysis of the C−N bond occurred with those bearing withdrawing substituents. The adducts formed between 1 a and four natural nucleosides were characterized, indicating that the alkylation sites for the photogenerated benzyl cations are dG, dA, and dC. [ABSTRACT FROM AUTHOR]

Abstract: DNA nanostructure‐based mechanical systems that control the distance between elements of interest have demonstrated great potential for various applications, including nanoplasmonic systems, molecular reactors, and other nanotechnology platforms. However, previously reported systems could not collectively manipulate a 2D or 3D nanoscale network of elements to various forms in multiple stages. A reconfigurable DNA accordion rack structure is introduced that is a DNA beam lattice that changes its conformation with a small amount of short‐length DNA locks as the controlling input. The lattice shape of the 2D DNA accordion rack and the diameter and the height of the 3D DNA nanotubular structure made of the DNA accordion rack could be controlled. Furthermore, by sequentially repeating the detachment and the attachment of the different DNA locks using strand displacement, the shape reconfiguration was repeatedly carried out. [ABSTRACT FROM AUTHOR]

Abstract: In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K+ channel proteins are assembled through zinc‐finger protein (ZFP)‐adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP‐fused Kir3 channels and ZFP‐based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K+ channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells. [ABSTRACT FROM AUTHOR]

The activation of stimulator of interferon genes (STING) plays a key role for provoking effective innate and adaptive immunity, yet is constrained by the limited clinical translation of STING agonists. Herein, we construct a smart DNA sponge to deliver STING agonists for robust cancer immunotherapy via the synergistic activation of innate immune pathways. The compact DNA sponge (Mn-CpG), assembling through a simple rolling circle amplification reaction, is encoded with numerous STING agonist (Mn2+) and toll-like receptor 9 (TLR9) agonist (CpG oligodeoxynucleotide) and allows their on-demand cellular delivery We show that Mn-CpG sponge can trigger the synergistic type I interferon response and robust T cell priming through a convenient coordination of different signaling pathways. In a poorly immunogenic breast cancer model, the method achieves the boosted immune responses with significantly inhibited tumor growth and metastasis. [Display omitted] • The compact DNA sponge realizes the on-demand cellular delivery of STING and TLR9 agonists into antigen-presenting cells. • This system actualizes two innate immune stimuli to achieve effective antitumour immunity with decreased autoimmune toxicity. • It provides a simple yet powerful tactic that renders DNA-based STING activation with synergistic innate immunomodulation. [ABSTRACT FROM AUTHOR]

Abstract: A new type of DNA ligand that contains a phosphate‐binding group and a photoresponsive azobenzene moiety is reported. When the azobenzene is in trans configuration, the ligand binds to the minor groove of a double‐stranded DNA, whereas it partially desorbs upon trans–cis isomerisation with light. The ability to photoswitch the ligand upon interaction with DNA is evidenced by (chir)optical signatures, and deciphered by the differences of binding geometry, stability, and dynamics of the DNA/ligand complexes for the two isomers. We exploit these properties to photomodulate DNA‐templated self‐assembly, through the incorporation of another π‐stacking DNA ligand, which together with the photoresponsive ligand form mixed supramolecular complexes along DNA. Our study demonstrates that well‐designed photoresponsive DNA binders can be used to modulate multicomponent supramolecular DNA assemblies. [ABSTRACT FROM AUTHOR]

There has been increasing interest in the development of small molecules that can selectively bind to G-quadruplex DNA structures. The latter have been associated with a number of key biological processes and therefore are proposed to be potential targets for drug development. Herein, we report the first example of a reduction-activated G-quadruplex DNA binder. We show that a new octahedral platinum(IV)-salphen complex does not interact with DNA in aqueous media at pH 7.4; however, upon addition of bioreductants such as ascorbic acid or glutathione, the compound is readily reduced to the corresponding square planar platinum(II) complex. In contrast to the parent platinum(IV) complex, the in situ generated platinum(II) complex has good affinity for G-quadruplex DNA. [ABSTRACT FROM AUTHOR]

Recently, smart DNA hydrogels, which are generally formed by the self-assembly of oligonucleotides or through the cross-linking of oligonucleotide–polymer hybrids, have attracted tremendous attention. However, the difficulties of fabricating DNA hydrogels limit their practical applications. We report herein a novel method for producing pH-responsive hydrogels by rolling circle amplification (RCA). In this method, pH-sensitive cross-linking sites were introduced into the polymeric DNA chains during DNA synthesis. As the DNA sequence can be precisely defined by its template, the properties of such hydrogels can be finely tuned in a very facile way through template design. We have investigated the process of hydrogel formation and pH-responsiveness to provide rationales for functional hydrogel design based on the RCA reaction. [ABSTRACT FROM AUTHOR]

Physicochemical methods have been used to investigate interactions occurring in solution between the dicarbene gold(I) complex [Au(9-methylcaffein-8-ylidene)2]BF4 (AuNHC) and a human telomeric DNA sequence, namely Tel23. Circular dichroism measurements allow identification of the conformational changes experienced by Tel23 upon interaction with AuNHC, and the respective binding stoichiometries and constants were determined. Computational studies provide a good link between previous crystallographic results of the same system and the present solution data, offering an exhaustive description of the inherent noncovalent metallodrug-DNA interactions. Remarkably, we found that a preformed AuNHC/Tel23 adduct is capable of producing strong and selective inhibition of the enzyme telomerase. The latter feature is mechanistically relevant and might account for the conspicuous in vitro anticancer properties of the investigated dicarbene gold(I) complex. [ABSTRACT FROM AUTHOR]

Metal-mediated base pairs have been extensively utilized in many research fields, including genetic-code extension, novel therapeutics development, and nanodevice design. Compared to other cations, AgI is more flexible in pairing with natural base pairs. Herein, we present a DNA structure containing two C-AgI-C pairs and the first reported G-AgI-G pair in a short 8mer DNA strand. This structure not only provides detailed insight into these AgI-mediated base-pairing patterns in DNA, but also represents the first nonhelical DNA structure driven by heavy-metal ions, thus further contributing to the structural diversity of DNA. This unique complex structure is highly sequence-dependent, thus implying functional potentials as a new DNA aptamer that can bind and recognize silver ions. These results not only advance our understanding of the interactions between AgI and nucleobases, but also provide a unique structural component for the rational design of new DNA nanodevices. [ABSTRACT FROM AUTHOR]

G-rich and C-rich DNA can fold into the tetrastranded helical structures G quadruplex or C quadruplex (i-motif), which are considered to be specific drug targets for cancer therapy. A large number of small molecules (so-called ligands), which can bind and modulate the stability of G quadruplex structures, have been widely examined. Much less is known, however, about the ligand binding interactions with the C quadruplex (i-motif). By combining steady-state measurements (UV/Vis, fluorescence, and induced circular dichroism (ICD)) with time-resolved laser flash photolysis spectroscopy, we have studied the binding interactions of cationic porphyrin (5,10,15,20-tetrakis( N-methylpyridinium-4-yl)-21 H,23 H-porphyrin, abbreviated as TMPyP4) with i-motifs (C3TA2)3C3T and (C4A4C4)2. The intercalation binding mode through π-π stacking of the porphyrin macrocycle and the C:C+ hemiprotonated base pair has been identified for the first time. The coexistent binding modes of intercalation (≈80 %) versus external major-groove binding (≈20 %) have been determined quantitatively, thereby allowing a fuller understanding of the porphyrin-i-motif interactions. The ionic strength was found to play an important role in affecting affects the binding modes, with the progressive increase in the ionic strength resulting in the gradual decrease in the intercalation percentage and an increase in the groove-binding percentage. Furthermore, an extended study of the porphyrin derivative with four bulky side-arm substituents (T4) suggests a complete prohibition of the intercalation mode owing to large steric hindrance, thereby providing a novel groove-binding ligand with site selectivity. These results provide in-depth mechanistic insights to better understand the ligand interactions with i-motifs and guidance for related applications in anticancer drug design. [ABSTRACT FROM AUTHOR]

We describe the synthesis and pairing properties of the novel DNA analogue 7′,5′-bicyclo(bc)-DNA. In this analogue, the point of attachment of the connecting phosphodiester group is switched from the 3′ to the 7′ position of the underlying bicyclic sugar unit and is thus in a topological position that is inaccessible in natural DNA. The corresponding phosphoramidite building blocks carrying all natural nucleobases were synthesized and incorporated into oligonucleotides. From Tm experiments of duplexes with complementary DNA and RNA we find that single modifications are generally well tolerated with some variability as to the nature of the nucleobase. Fully modified oligonucleotides show low affinity for RNA and DNA complements. However, they form antiparallel homo-duplexes with similar thermal stability as DNA. CD spectra of the homo-duplexes show distinct changes in the helix conformation compared to natural DNA. A conformational analysis at the ab initio level of the mononucleosides revealed two minimal energy structures which primarily deviate in the conformation of the cyclopentane ring. Molecular dynamics simulation of a 7′,5′-bc-DNA homo-duplex revealed a right-handed structure with a smaller helical rise and a significantly wider minor groove compared to DNA. Interestingly, this duplex is characterized by an atypical, alternating 6′- endo/6′- exo conformational pattern of consecutive nucleotides which seems to be responsible for the poor binding to natural nucleic acids. [ABSTRACT FROM AUTHOR]

Background G-rich sequences undergo unique structural equilibria to form G-quadruplexes (G4) both in vitro and in cell systems. Several pathologies emerged to be directly related to G4 occurrence at defined genomic portions. Additionally, G-rich sequences are significantly represented around transcription start sites (TSS) thus leading to the hypothesis of a gene regulatory function for G4. Thus, the tuning of G4 formation has been proposed as a new powerful tool to regulate gene expression to treat related pathologies. However, up-to date this approach did not provide any new really efficient treatment. Scope of review Here, we summarize the most recent advances on the correlation between the structural features of G4 in human promoters and the role these systems physiologically exert. In particular we focus on the effect of G4 localization among cell compartments and along the promoters in correlation with protein interaction networks and epigenetic state. Finally the intrinsic structural features of G4 at promoters are discussed to unveil the contribution of different G4 structural modules in this complex architecture. Major conclusions It emerges that G4s play several roles in the intriguing and complex mechanism of gene expression, being able to produce opposite effects on the same target. This reflects the occurrence of a highly variegate network of several components working simultaneously. General significance The resulting picture is still fuzzy but some points of strength are definitely emerging, which prompts all of us to strengthen our efforts in view of a selective control of gene expression through G4 modulation. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. [ABSTRACT FROM AUTHOR]

X-ray crystal structures of three Λ-[Ru(L)2dppz]2+ complexes (dppz=dipyridophenazine; L=1,10-phenanthroline (phen), 2,2′-bipyridine (bpy)) bound to d((5BrC)GGC/GCCG) showed the compounds intercalated at a 5′-CG-3′ step. The compounds bind through canted intercalation, with the binding angle determined by the guanine NH2 group, in contrast to symmetrical intercalation previously observed at 5′-TA-3′ sites. This result suggests that canted intercalation is preferred at 5′-CG-3′ sites even though the site itself is symmetrical, and we hypothesise that symmetrical intercalation in a 5′-CG-3′ step could give rise to a longer luminescence lifetime than canted intercalation. [ABSTRACT FROM AUTHOR]

The catalytic activity of G-quadruplex/hemin is much lower than that of proteinous enzymes, so it is very important to increase its activity. Very recently, flanking sequences, which can be regarded as an external part of G-quadruplexes, were found to enhance the activity of G-quadruplex/hemin DNAzyme. However, little is known about the effect of internal parts, such as loop sequences and linkers, on the activity. In the present study, adenine repeats were incorporated into several designed G-quadruplex structures either in the loops, bulges, or linkers, and the constructed G-quadruplex/hemin DNAzyme exhibit about fivefold improvement in peroxidase-mimicking activity in some cases. The enhancement effect may result from the formation of compound I, protoporphyrin⋅FeIV=O.+, accelerated by dA repeats, which was demonstrated by H2O2 decay kinetics and pH dependency analysis. The novel enhancement methods described here may help in the development of high-activity DNAzymes, illustrated by a dimer G-quadruplex with flanking adenine at one end, a relatively long adenine run in one loop, and another adenine run in the linker. [ABSTRACT FROM AUTHOR]

Developing multifunctional nanozymes with photothermal-augmented enzyme-like reaction dynamics in the second near-infrared (NIR-II) biowindow is of significance for nanocatalytic therapy (NCT). Herein, DNA-templated Ag@Pd alloy nanoclusters (DNA-Ag@Pd NCs) are prepared as a kind of novel noble-metal alloy nanozymes by using cytosine-rich hairpin-shaped DNA structures as growth templates. DNA-Ag@Pd NCs exhibit high photothermal conversion efficiency (59.32%) under 1270 nm laser and photothermally augmented peroxidase-mimicking activity with synergetic enhancement between Ag and Pd. In addition, hairpin-shaped DNA structures on the surface of DNA-Ag@Pd NCs endow them with good stability and biocompatibility in vitro and in vivo, and enhanced permeability and retention effect at tumor sites. Upon intravenous injection, DNA-Ag@Pd NCs demonstrate high-contrast NIR-II photoacoustic imaging-guided efficient photothermal-augmented NCT of gastric cancer. This work provides a strategy to synthesize versatile noble-metal alloy nanozymes in a bioinspired way for highly efficient therapy of tumors., (© 2023 Wiley-VCH GmbH.)

Efficient incorporation of modified nucleotides by DNA polymerases is essential for many cutting-edge biomolecular technologies. The present study compares the acceptance of either alkene- or alkyne-modified nucleotides by KlenTaq DNA polymerase and provides structural insights into how 7-deaza-adenosine and deoxyuridine with attached alkene-modifications are incorporated into the growing DNA strand. Thereby, we identified modified nucleotides that prove to be superior substrates for KlenTaq DNA polymerase compared with their natural analogues. The knowledge can be used to guide future design of functionalized nucleotide building blocks. [ABSTRACT FROM AUTHOR]

Small molecules are used in the G-quadruplex (G4) research field in vivo and in vitro, and there are increasing demands for ligands that selectively stabilize different G4 structures. Thioflavin T (ThT) emits an enhanced fluorescence signal when binding to G4 structures. Herein, we show that ThT can be competitively displaced by the binding of small molecules to G4 structures and develop a ThT-displacement high-throughput screening assay to find novel and selective G4-binding compounds. We screened approximately 28 000 compounds by using three different G4 structures and identified eight novel G4 binders. Analysis of the structural conformation and stability of the G4 structures in presence of these compounds demonstrated that the four compounds enhance the thermal stabilization of the structures without affecting their structural conformation. In addition, all four compounds also increased the G4-structure block of DNA synthesis by Taq DNA polymerase. Also, two of these compounds showed selectivity between certain Schizosaccharomyces pombe G4 structures, thus suggesting that these compounds or their analogues can be used as selective tools for G4 DNA studies. [ABSTRACT FROM AUTHOR]