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Mapping the endocytic vesicular acidification process is of prior importance to better understand the health and pathological processes of cells. Herein, by integrating a pH-sensitive i-motif and a pair of fluorescence resonance energy transfer (FRET) into a tetrahedral DNA framework (TDF), we develop a pH-responsive DNA nanomachine, allowing for efficient sensing of pH from 7.0 to 5.5 via the pH-triggered spatial proximity modulation of FRET. The inheriting endo-lysosome-targeting ability of TDF enables spatiotemporal tracking of endocytic vesicle acidification during the endosomal maturation process. Analysis of pH-dependent FRET response at single fluorescent spot level reveals the significant difference of endocytic vesicular acidification between normal and cancer cells. The performance of pH-responsive DNA nanomachine underlines its potential for studies on vesicle acidification-related pathologies as a universal platform.

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DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles.

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In conventional ratiometric photoelectrochemical (PEC) sensors, the detection and reference signals are output sequentially from two independent photosensitive materials. In such a "two-to-two" ratiometric mode, unavoidable difference during dual-interface modification exists, resulting in questionable ratiometric signals and detection results. To address this issue, we propose a novel "one-to-two" ratiometric PEC sensor on a single electrode interface through pH-modulated band alignment engineering. The double ratiometric signals are generated by the synergistic action of a pH-responsive CuTCPP/WS2 photoelectric substrate material and the i-motif sensing tool. Specifically, a ternary heterostructure to generate a photoanodic detection signal is formed under alkaline conditions between CuTCPP/WS2 and signal label CdS QDs binding to the i-motif. While under acidic conditions, a photocurrent polarity conversion and signaling labels detachment, induced by the band realignment of CuTCPP/WS2 and the i-motif conformational switching, produce a reliable internal reference photocathodic signal. The feasibility of this two-wing signal generation strategy is validated by detecting mycotoxin ochratoxin A, which achieves accurate and reliable ratio detection results. Overall, this work provides guidance for the design of a PEC ratiometric determination system and exhibits great potential to be applied in practical analysis research.

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Although more than 10% of the human genome has the potential to fold into non-B DNA, the formation of non-canonical structural motifs as part of long dsDNA chains are usually considered as unfavorable from a thermodynamic point of view. However, recent experiments have confirmed that non-canonical motifs do exist and are non-randomly distributed in genomic DNA. This distribution is highly dependent not only on the DNA sequence but also on various other factors such as environmental conditions, DNA topology and the expression of specific cellular factors in different cell types. In this study, we describe a new strategy used in the preparation of DNA minicircles containing different non-canonical motifs which arise as a result of imperfect base pairing between complementary strands. The approach exploits the fact that imperfections in the pairing of complementary strands thermodynamically weaken the dsDNA structure at the expense of enhancing the formation of non-canonical motifs. In this study, a completely different concept of stable integration of a non-canonical motif into dsDNA is presented. Our approach allows the integration of various types of non-canonical motifs into the dsDNA structure such as hairpin, cruciform, G-quadruplex and i-motif forms but also combinations of these forms. Small DNA minicircles have recently become the subject of considerable interest in both fundamental research and in terms of their potential therapeutic applications.

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BACKGROUND: Vascular endothelial growth factor (VEGF) is overexpressed in most malignant tumors, which has important impact on tumor angiogenesis and development. Its gene promoter i-motif structure formed by C-rich sequence can regulate gene expression, which is a promising new target for anti-tumor therapy. METHODS: We screened various compounds and studied their effects on VEGF through extensive experiments, including SPR, MST, TO displacement, FRET, CD, ESI-MS, NMR, MTT, clone formation, qPCR, Western blot, dual-luciferase reporter assay, immunofluorescence, cell scrape, apoptosis, transwell assay, and animal model. RESULTS: After extensive screening, bisacridine derivative B09 was found to have selective binding and stabilization to VEGF promoter i-motif, which could down-regulate VEGF gene expression. B09 showed potent inhibition on MCF-7 and HGC-27 cell proliferation and metastasis. B09 significantly inhibited tumor growth in xenograft mice model with HGC-27 cells, showing decreased VEGF expression analyzed through immunohistochemistry. CONCLUSION: B09 could specifically regulate VEGF gene expression, possibly through interacting with promoter i-motif structure. As a lead compound, B09 could be further developed for innovative anti-cancer agent targeting VEGF.

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Cytosine-rich DNA sequences can fold into intercalated motifs known as i-motifs, through noncanonical hydrogen bonding interactions. Molecular probes can provide valuable insights into the conformational stability and potential cellular functions of i-motifs. W5K5, a decapeptide composed of alternating tryptophan (W) and lysine (K) units, has been identified as a lead candidate to modulate the structural dynamics of the hypoxia-inducible factor 1-alpha (HIF-1α) DNA i-motif. This finding is expected to facilitate the rational design of peptide-based probes for studying the structure and functional dynamics of i-motifs.

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BACKGROUND/AIM: Dynamic DNA sequences (i.e. sequences capable of forming hairpins, G-quadruplexes, i-motifs, and triple helices) can cause replication stress and associated mutations. One example of such a sequence occurs in the RACK7 gene in human DNA. Since this sequence forms i-motif structures at neutral pH that cause replication stress and result in spontaneous deletions in prostate cancer cells, our initial aim was to determine its potential utility as a biomarker of prostate cancer. MATERIALS AND METHODS: We cloned and sequenced the region in RACK7 where i-motif deletions often occur in DNA obtained from eight individuals. Expressed prostatic secretions were obtained from three individuals with a positive biopsy for prostate cancer and two with individuals with a negative biopsy for prostate cancer. Peripheral blood specimens were obtained from two control healthy bone marrow donors and a marrow specimen was obtained from a third healthy marrow donor. Follow-up computer searches of the genomes of 74 mammalian species available at the NCBI ftp site or frequencies of 6 dynamic sequences known to produce mutations or replication stress using a program written in Mathematica were subsequently performed. RESULTS: Deletions were found in RACK7 in specimens from both older normal adults, as well as specimens from older patients with cancer, but not in the youngest normal adult. The deletions appeared to show a weak trend to increasing frequency with patient age. This suggested that endogenous mutations associated with dynamic sequences might accumulate during aging and might serve as biomarkers of biological age rather than direct biomarkers of cancer. To test that hypothesis, we asked whether or not the genomic frequencies of several dynamic sequences known to produce replication stress or mutations in human DNA were inversely correlated with maximum lifespan in mammals. CONCLUSION: Our results confirm this correlation for six dynamic sequences in 74 mammalian genomes studied, thereby suggesting that spontaneously induced replication stress and mutations linked to dynamic sequence frequency may limit lifespan by limiting genome stability.

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Cytosine rich sequences can form intercalated, i-motif DNA structures stabilized by hemi-protonated cytosine:cytosine base pairing. These sequences are often located in regulatory regions of genes such as promoters. Ligands targeting i-motif structures may provide potential leads for treatments for genetic disease. The focus on ligands interacting with i-motif DNA has been increasing in recent years. Here, we describe the fluorescent intercalator displacement (FID) assay using thiazole orange binding i-motif DNA and assess the binding affinity of a ligand to the i-motif DNA by displacing thiazole orange. This provides a time and cost-effective high throughput screening of ligands against secondary DNA structures for hit identification.

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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.

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Endocytosis is a major bottleneck toward cytosolic delivery of nucleic acids, as the vast majority of nucleic acid drugs remain trapped within endosomes. Current trends to overcome endosomal entrapment and subsequent degradation provide varied success; however, active delivery agents such as cell-penetrating peptides have emerged as a prominent strategy to improve cytosolic delivery. Yet, these membrane-active agents have poor selectivity for endosomal membranes, leading to toxicity. A hallmark of endosomes is their acidic environment, which aids in degradation of foreign materials. Here, we develop a pH-triggered spherical nucleic acid that provides smart antisense oligonucleotide (ASO) release upon endosomal acidification and selective membrane disruption, termed DNA EndosomaL Escape Vehicle Response (DELVR). We anchor i-Motif DNA to a nanoparticle (AuNP), where the complement strand contains both an ASO sequence and a functionalized endosomal escape peptide (EEP). By orienting the EEP toward the AuNP core, the EEP is inactive until it is released through acidification-induced i-Motif folding. In this study, we characterize a small library of i-Motif duplexes to develop a structure-switching nucleic acid sequence triggered by endosomal acidification. We evaluate antisense efficacy using HIF1a, a hypoxic indicator upregulated in many cancers, and demonstrate dose-dependent activity through RT-qPCR. We show that DELVR significantly improves ASO efficacy in vitro. Finally, we use fluorescence lifetime imaging and activity measurement to show that DELVR benefits synergistically from nuclease- and pH-driven release strategies with increased ASO endosomal escape efficiency. Overall, this study develops a modular platform that improves the cytosolic delivery of nucleic acid therapeutics and offers key insights for overcoming intracellular barriers.

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We have elucidated the hnRNP K promoter as a hotspot for tetraplex-based molecular switches receptive to micro-environmental stimuli. We have characterised the structural features of four tetraplex-forming loci and identified them as binding sites of transcription factors. These segments form either G-quadruplex or i-motif structures, the structural dynamicity of which has been studied in depth via several biophysical techniques. The tetraplexes display high dynamicity and are influenced by both pH and KCl concentrations in vitro. The loci complementary to these sequences form additional non-canonical secondary structures. In the cellular context, the most eminent observation of this study is the binding of hnRNP K to the i-motif forming sequences in its own promoter. We are the first to report a probable transcriptional autoregulatory function of hnRNP K in coordination with higher-order DNA structures. Herein, we also report the positive interaction of the endogenous tetraplexes with Sp1, a well-known transcriptional regulator. Treatment with tetraplex-specific small molecule ligands further uncovered G-quadruplexes' functioning as repressors and i-motifs as activators in this context. Together, our findings strongly indicate the critical regulatory role of the identified tetraplex elements in the hnRNP K promoter.Communicated by Ramaswamy H. Sarma.

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Sophisticated dynamic molecular systems with diverse functions have been fabricated by using the fundamental tool of toehold-mediated strand displacement (TMSD) in the field of dynamic DNA nanotechnology. However, simple approaches to reset these TMSD-based dynamic systems are lacking due to the difficulty in creating kinetically favored pathways to implement the backward resetting reactions. Here, we develop a facile proton-driven strategy to achieve complete resetting of a modular DNA circuit by integrating a pH-responsive intermolecular CG-C+ triplex DNA and an i-motif DNA into the conventional DNA substrate. The pH-programmed strategy allows modular DNA components to specifically associate/dissociate to promote the forward/backward TMSD reactions, thereby enabling the modular DNA circuit to be repeatedly operated at a constant temperature without generating any DNA waste products. Leveraging this tractable approach, we further constructed two resettable DNA logic gates used for logical computation and two resettable catalytic DNA systems with good performance in signal transduction and amplification.

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Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress.

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The development of precisely engineered vehicles for intracellular delivery and the controlled release of payloads remains a challenge. DNA-based nanomaterials offer a promising solution based on the A-T-G-C alphabet-dictated predictable assembly and high programmability. Herein, we present a self-immolative DNA nanogel vaccine, which can be tracelessly released in the intracellular compartments and activate the immune response. Three building blocks with cytosine-rich overhang domains are designed to self-assemble into a DNA nanogel framework with a controlled size. Two oligo agonists and one antigen peptide are conjugated to the building blocks via an acid-labile chemical linker. Upon internalization into acidic endosomes, the formation of i-motif configurations leads to dissociation of the DNA nanogel vaccine. The acid-labile chemical linker is cleaved, releasing the agonists and antigen in their traceless original form to activate antigen-presenting cells and an immune response. This study presents a novel strategy for constructing delivery platforms for intracellularly stimuli-triggered traceless release of therapeutics.

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Unmethylated cytosine-phosphate-guanine (CpG) DNA is recognized by Toll-like receptor 9, expressed in the endosomes of immune cells, and induces the secretion of proinflammatory cytokines. CpG DNA is, therefore, expected to be used as vaccine adjuvants, but there are many obstacles for its therapeutic application, such as poor cellular uptake and biostability. Long single-stranded DNA (lssDNA) synthesized by rolling circle amplification can be a useful delivery carrier for CpG DNA because of its cellular uptake efficiency, but the immunostimulatory effect is transient because it is easily degraded in endosomes. To improve its stability, we constructed lssDNA which forms hydrogel by i-motifs in an acidic environment mimicking endosome, and incorporated CpG DNA into lssDNA (i-CpG-lssDNA). We synthesized lssDNA containing the optimized i-motif sequence, and confirmed the formation of a DNA hydrogel in an acidic environment. The i-CpG-lssDNA elicited a potent proinflammatory cytokine production in murine macrophages, compared to CpG DNA-containing lssDNA without i-motifs. Consistently, its intradermal administration induced potent inflammatory cytokines at the regional lymph nodes. These results suggested that i-CpG-lssDNA could serve as a novel type of adjuvant for the induction of a potent immune response.

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Due to the advantages of its numerous modification sites, predictable structure, high thermal stability, and excellent biocompatibility, DNA is the ideal choice as a key component of biosensors. DNA biosensors offer significant advantages over existing bioanalytical techniques, addressing limitations in sensitivity, selectivity, and limit of detection. Consequently, they have attracted significant attention from researchers worldwide. Here, we exemplify four foundational categories of functional nucleic acids: aptamers, DNAzymes, i-motifs, and G-quadruplexes, from the perspective of the structure-driven functionality in constructing DNA biosensors. Furthermore, we provide a concise overview of the design and detection mechanisms employed in these DNA biosensors. Noteworthy advantages of DNA as a sensor component, including its programmable structure, reaction predictility, exceptional specificity, excellent sensitivity, and thermal stability, are highlighted. These characteristics contribute to the efficacy and reliability of DNA biosensors. Despite their great potential, challenges remain for the successful application of DNA biosensors, spanning storage and detection conditions, as well as associated costs. To overcome these limitations, we propose potential strategies that can be implemented to solve these issues. By offering these insights, we aim to inspire subsequent researchers in related fields.

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DNA sequences containing at least four runs of repetitive cytosines can fold into tetra-helical structures called i-Motifs (iMs). The interest in these DNA secondary structures is increasing due to their therapeutical and technological applications. Still, limited knowledge of their folding requirements is currently available. We developed a novel step-by-step pipeline for the systematic screening of putative iM-forming model sequences. Focusing on structures comprising only three cytosine-cytosine+ base pairs, we investigated what the minimal lengths of the loops required for formation of an intra-molecular iM are. Our data indicate that two and three nucleotides are required to connect the strands through the minor and majorgrooves of the iM, respectively. Additionally, they highlight an asymmetric behavior according to the distribution of the cytosines. Specifically, no sequence containing a single cytosine in the first and third run was able to fold into intra-molecular iMs with the same stability of those formed when the first and the third run comprise two cytosines. This knowledge represents a step forward toward the development of prediction tools for the proper identification of biologically functional iMs, as well as for the rational design of these secondary structures as technological devices.

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I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2'-deoxyribose, arabinose, and 2'-deoxy-2'-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3'-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

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Development of probe systems that provide unique spectral signatures for duplex, G-quadruplex (GQ) and i-motif (iM) structures is very important to understand the relative propensity of a G-rich-C-rich promoter region to form these structures. Here, we devise a platform using a combination of two environment-sensitive nucleoside analogs namely, 5-fluorobenzofuran-modified 2'-deoxyuridine (FBF-dU) and 5-fluoro-2'-deoxyuridine (F-dU) to study the structures adopted by a promoter region of the c-Myc oncogene. FBF-dU serves as a dual-purpose probe containing a fluorescent and 19 F NMR label. When incorporated into the C-rich sequence, it reports the formation of different iMs via changes in its fluorescence properties and 19 F signal. F-dU incorporated into the G-rich ON reports the formation of a GQ structure whose 19 F signal is clearly different from the signals obtained for iMs. Rewardingly, the labeled ONs when mixed with respective complementary strands allows us to determine the relative population of different structures formed by the c-Myc promoter by the virtue of the probe's ability to produce distinct and resolved 19 F signatures for different structures. Our results indicate that at physiological pH and temperature the c-Myc promoter forms duplex, random coil and GQ structures, and does not form an iM. Whereas at acidic pH, the mixture largely forms iM and GQ structures. Taken together, our system will complement existing tools and provide unprecedented insights on the population equilibrium and dynamics of nucleic acid structures under different conditions.

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The assembly of enzyme [glucose oxidase (GOx)]-loaded stimuli-responsive DNA-based hydrogels on electrode surfaces, and the triggered control over the stiffness of the hydrogels, provides a means to switch the bioelectrocatalytic functions of the hydrogels. One system includes the assembly of GOx-loaded, pH-responsive, hydrogel matrices cross-linked by two cooperative nucleic acid motives comprising permanent duplex nucleic acids and "caged" i-motif pH-responsive duplexes. Bioelectrocatalyzed oxidation of glucose leads to the formation of gluconic acid that acidifies the hydrogel resulting in the separation of the i-motif constituents and lowering the hydrogel stiffness. Loading of the hydrogel matrices with insulin results in the potential-triggered, glucose concentration-controlled, switchable release of insulin from the hydrogel-modified electrodes. The switchable bioelectrocatalyzed release of insulin is demonstrated in the presence of ferrocenemethanol as a diffusional electron mediator or by applying an electrically wired integrated matrix that includes ferrocenyl-modified GOx embedded in the hydrogel. The second GOx-loaded, stimuli-responsive, DNA-based hydrogel matrix associated with the electrode includes a polyacrylamide hydrogel cooperatively cross-linked by duplex nucleic acids and "caged" G-quadruplex-responsive duplexes. The hydrogel matrix undergoes K+-ions/crown ether-triggered stiffness changes by the cyclic K+-ion-stimulated formation of G-quadruplexes (lower stiffness) and the crown ether-induced separation of the G-quadruplexes (higher stiffness). The hydrogel matrices demonstrate switchable bioelectrocatalytic functions guided by the stiffness properties of the hydrogels.

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