DNA Secondary Structures Research Articles

A One‐step Fluorescence Sensor Based on Molybdenum Disulfide Nanosheets for the Detection of Potassium Ions

AbstractDetermination of potassium ion concentration in biological samples is of great significance due to its vital role in the maintenance of physiological activities. Single or few‐layered transition metal dichalcogenides (TMD, e. g., MoS2, WS2, TiS2) have recently found wide applications in optoelectronics and biomedical engineering. Herein, for the first time, MoS2 nanosheets were integrated with G‐quadraplex DNA secondary structures to construct a fluorescence sensor for detecting potassium ions. The biosensor showed merits such as ease of preparation, ease of use, low‐cost, broad linear working range, as well as sufficient sensitivity, specificity and practical applicability, demonstrating its promise as an effective potassium ion sensor for practical applications. To the best of our knowledge, this is the first report on the combination of TMD nanomaterials and DNA quadraplex for the development of nanodevices. The proposed TMD nanosheets‐G‐quadraplex sensing formulation could enable the devise of other nanomachines and nanosensors considering the versatility of TMD nanomaterials and G‐quadraplex‐containing aptamers.

Exploring the Relationship between G-Quadruplex Nucleic Acids and Plants: From Plant G-Quadruplex Function to Phytochemical G4 Ligands with Pharmaceutic Potential.

G-quadruplex (G4) oligonucleotides are higher-order DNA and RNA secondary structures of enormous relevance due to their implication in several biological processes and pathological states in different organisms. Strategies aiming at modulating human G4 structures and their interrelated functions are first-line approaches in modern research aiming at finding new potential anticancer treatments or G4-based aptamers for various biomedical and biotechnological applications. Plants offer a cornucopia of phytocompounds that, in many cases, are effective in binding and modulating the thermal stability of G4s and, on the other hand, contain almost unexplored G4 motifs in their genome that could inspire new biotechnological strategies. Herein, we describe some G4 structures found in plants, summarizing the existing knowledge of their functions and biological role. Moreover, we review some of the most promising G4 ligands isolated from vegetal sources and report on the known relationships between such phytochemicals and G4-mediated biological processes that make them potential leads in the pharmaceutical sector.

Mechanistic insights into poly(C)-binding protein hnRNP K resolving i-motif DNA secondary structures

I-motifs are four-strand noncanonical secondary structures formed by cytosine (C)-rich sequences in living cells. The structural dynamics of i-motifs play essential roles in many cellular processes, such as telomerase inhibition, DNA replication, and transcriptional regulation. In cells, the structural dynamics of the i-motif can be modulated by the interaction of poly(C)-binding proteins (PCBPs), and the interaction is closely related to human health, through modulating the transcription of oncogenes and telomere stability. Therefore, the mechanisms of how PCBPs interact with i-motif structures are fundamentally important. However, the underlying mechanisms remain elusive. I-motif structures in the promoter of the c-MYC oncogene can be unfolded by heterogeneous nuclear ribonucleoprotein K (hnRNP K), a PCBP, to activate its transcription. Here, we selected this system as an example to comprehensively study the unfolding mechanisms. We found that the promoter sequence containing 5 C-runs preferred folding into type-1245 to type-1234 i-motif structures based on their folding stability, which was further confirmed by single-molecule FRET. In addition, we first revealed that the c-MYC i-motif structure was discretely resolved by hnRNP K through two intermediate states, which were assigned to the opposite hairpin and neighboring hairpin, as further confirmed by site mutations. Furthermore, we found all three KH (hnRNP K homology) domains of hnRNP K could unfold the c-MYC i-motif structure, and KH2 and KH3 were more active than KH1. In conclusion, this study may deepen our understanding of the interactions between i-motifs and PCBPs and may be helpful for drug development.

Replication Protein A Utilizes Differential Engagement of Its DNA-Binding Domains to Bind Biologically Relevant ssDNAs in Diverse Binding Modes.

Replication protein A (RPA) is a ubiquitous ssDNA-binding protein that functions in many DNA processing pathways to maintain genome integrity. Recent studies suggest that RPA forms a highly dynamic complex with ssDNA that can engage with DNA in many modes that are orchestrated by the differential engagement of the four DNA-binding domains (DBDs) in RPA. To understand how these modes influence RPA interaction with biologically relevant ligands, we performed a comprehensive and systematic evaluation of RPA's binding to a diverse set of ssDNA ligands that varied in sequence, length, and structure. These equilibrium binding data show that WT RPA binds structured ssDNA ligands differently from its engagement with minimal ssDNAs. Next, we investigated each DBD's contributions to RPA's binding modes through mutation of conserved, functionally important aromatic residues. Mutations in DBD-A and -B have a much larger effect on binding when ssDNA is embedded into DNA secondary structures compared to their association with unstructured minimal ssDNA. As our data support a complex interplay of binding modes, it is critical to define the trimer core DBDs' role in binding these biologically relevant ligands. We found that DBD-C is important for engaging DNA with diverse binding modes, including, unexpectedly, at short ssDNAs. Thus, RPA uses its constituent DBDs to bind biologically diverse ligands in unanticipated ways. These findings lead to a better understanding of how RPA carries out its functions at diverse locations of the genome and suggest a mechanism through which dynamic recognition can impact differential downstream outcomes.

4′‐SCF3‐Labeling Constitutes a Sensitive 19F NMR Probe for Characterization of Interactions in the Minor Groove of DNA

AbstractFluorinated nucleotides are invaluable for 19F NMR studies of nucleic acid structure and function. Here, we synthesized 4′‐SCF3‐thymidine (T ) and incorporated it into DNA by means of solid‐phase DNA synthesis. NMR studies showed that the 4′‐SCF3 group exhibited a flexible orientation in the minor groove of DNA duplexes and was well accommodated by various higher order DNA structures. The three magnetically equivalent fluorine atoms in 4′‐SCF3‐DNA constitute an isolated spin system, offering high 19F NMR sensitivity and excellent resolution of the positioning of T within various secondary and tertiary DNA structures. The high structural adaptability and high sensitivity of T make it a valuable 19F NMR probe for quantitatively distinguishing diverse DNA structures with single‐nucleotide resolution and for monitoring the dynamics of interactions in the minor groove of double‐stranded DNA.

4'-SCF3 -Labeling Constitutes a Sensitive 19 F NMR Probe for Characterization of Interactions in the Minor Groove of DNA.

Fluorinated nucleotides are invaluable for 19 F NMR studies of nucleic acid structure and function. Here, we synthesized 4'-SCF3 -thymidine (T ) and incorporated it into DNA by means of solid-phase DNA synthesis. NMR studies showed that the 4'-SCF3 group exhibited a flexible orientation in the minor groove of DNA duplexes and was well accommodated by various higher order DNA structures. The three magnetically equivalent fluorine atoms in 4'-SCF3 -DNA constitute an isolated spin system, offering high 19 F NMR sensitivity and excellent resolution of the positioning of T within various secondary and tertiary DNA structures. The high structural adaptability and high sensitivity of T make it a valuable 19 F NMR probe for quantitatively distinguishing diverse DNA structures with single-nucleotide resolution and for monitoring the dynamics of interactions in the minor groove of double-stranded DNA.

DNA‐Encoded Nanomaterials with Controllable Properties

AbstractDNA as an attractive capping ligand or template has been extensively utilized for synthesizing nanomaterials, and fabricating nano‐systems with tuneable properties. This review aims to summarize related significant progress on DNA‐encoded nanomaterials and their controllable properties including catalysis, surface plasmon resonance, chirality, surface‐enhanced Raman scattering and fluorescence, moreover, to present the underlying clues to engineer nanomaterials with DNA on the basis of DNA‐metal ion and DNA‐nanomaterial interactions. The apparent utility of diverse DNA modules including DNA homopolymers (e. g., poly adenine), secondary structures (e. g., triplex) and DNA nanostructures (e. g., DNA origami), etc., is revealed, which is derived from their strong specific interactions, stimulus‐responsive features, or excellent addressable functions, etc. Accompanied by insight into the underlying mechanism of DNA‐participated interactions, it is anticipated that, new DNA‐encoded nanomaterials will be reported and contribute to exploration of intelligent nanocatalysts, nanoprobes and nanomedicines.

Photo-Oxidizing Ruthenium(II) Complexes with Enhanced Visible-Light Absorption and G-quadruplex DNA Binding Abilities.

Photosensitizers that gather high photo-oxidizing power and strong visible-light absorption are of great interest in the development of new photo-chemotherapeutics. Indeed, such compounds constitute attractive candidates for the design of type I photosensitizers that are not dependent on the presence of oxygen. In this paper, we report on the synthesis and studies of new ruthenium(II) complexes that display strong visible-light absorption and can oxidize guanine residues under visible-light irradiation, as evidenced by nanosecond transient absorption spectroscopy. The reported compounds also tightly bind to G-quadruplex DNA structures from the human telomeric sequence (TTAGGG repeat). The kinetic and thermodynamic parameters of the interaction of these Ru(II) complexes with G-quadruplex and duplex DNA were studied thanks to luminescence titrations and bio-layer interferometry measurements, which revealed higher affinities towards the non-canonical G-quadruplex architecture. Docking experiments and non-covalent ionic analysis allowed us to gain information on the mode and the strength of the interaction of the compounds towards G-quadruplex and duplex DNA. The different studies emphasize the substantial influence of the position and the number of non-chelating nitrogen atoms on the interaction with both types of DNA secondary structures.

Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion.

Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of RecQ along the DNA and conformational changes of the helicase associated with stepping. Our data reveal the mechanochemical coupling between physical domain motions and chemical reactions that together produce directed motion of the helicase along DNA. Nanopore Tweezers measurements are performed under either assisting or opposing force applied directly on RecQ, shedding light on how RecQ responds to such forces in vivo. Determining the rates of translocation and physical conformational changes under a wide range of assisting and opposing forces reveals the underlying dynamic energy landscape that drives RecQ motion. We show that RecQ has a highly asymmetric energy landscape that enables RecQ to maintain velocity when encountering molecular roadblocks such as bound proteins and DNA secondary structures. This energy landscape also provides a mechanistic basis making RecQ an ‘active helicase,’ capable of unwinding dsDNA as fast as it translocates on ssDNA. Such an energy landscape may be a general strategy for molecular motors to maintain consistent velocity despite opposing loads or roadblocks.

G-quadruplexes Mark Sites of Methylation Instability Associated with Ageing and Cancer.

Regulation of the epigenome is critical for healthy cell function but can become disrupted with age, leading to aberrant epigenetic profiles including altered DNA methylation. Recent studies have indicated that DNA methylation homeostasis can be compromised by the formation of DNA secondary structures known as G-quadruplexes (G4s), which form in guanine-rich regions of the genome. G4s can be recognised and bound by certain methylation-regulating enzymes, and in turn perturb the surrounding methylation architecture. However, the effect G4 formation has on DNA methylation at critical epigenetic sites remains elusive and poorly explored. In this work, we investigate the association between G4 sequences and prominent DNA methylation sites, termed ‘ageing clocks’, that act as bona fide dysregulated regions in aged and cancerous cells. Using a combination of in vitro (G4-seq) and in cellulo (BG4-ChIP) G4 distribution maps, we show that ageing clocks sites are significantly enriched with G4-forming sequences. The observed enrichment also varies across species and cell lines, being least significant in healthy cells and more pronounced in tumorigenic cells. Overall, our results suggest a biological significance of G4s in the realm of DNA methylation, which may be important for further deciphering the driving forces of diseases characterised by epigenetic abnormality, including ageing.

S1-END-seq reveals DNA secondary structures in human cells

DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulate in microsatellite unstable human cancer cell lines and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine mirror repeats common across a variety of cell lines. We show that H-DNA is enriched during replication, that its genomic location is highly conserved, and that H-DNA formed by (GAA)n repeats can be disrupted by treatment with a (GAA)n-binding polyamide. Finally, we show that triplex-forming repeats are hotspots for mutagenesis. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.

Silver Nanoclusters Serve as Fluorescent Rivets Linking Hoogsteen Triplex DNA and Hairpin-Loop DNA Structures.

Greater understanding of the mutual influence between DNA and the associated nanomaterial on the properties of each other can provide alternative strategies for designing and developing DNA nanomachines. DNA secondary structures are essential for encapsulating highly emissive silver nanoclusters (DNA/AgNCs). Likewise, AgNCs stabilize secondary DNA structures, such as hairpin DNA, duplex DNA, and parallel-motif DNA triplex. In this study, we found that the fluorescence of AgNCs encapsulated within a Hoogsteen triplex DNA structure can be turned on and off in response to pH changes. We also show that AgNCs can act as nanoscale rivets, linking two functionally distinctive DNA nanostructures. For instance, we found that a Hoogsteen triplex DNA structure with a seven-cytosine loop encapsulates red fluorescent AgNCs. The red fluorescence faded under alkaline conditions, whereas the fluorescence was restored in a near-neutral environment. Hairpin DNA and random DNA structures did not exhibit this pH-dependent AgNCs fluorescence. A fluorescence lifetime measurement and a small-angle X-ray scattering analysis showed that the triplex DNA-encapsulated AgNCs were photophysically convertible between bright and dark states. An in-gel electrophoresis analysis indicated that bright and dark convertibility depended on the AgNCs-riveted dimerization of the triplex DNAs. Moreover, we found that AgNCs rivet the triplex DNA and hairpin DNA to form a heterodimer, emitting orange fluorescence. Our findings suggest that AgNCs between two cytosine-rich loops can be used as nanorivets in designing noncanonical DNA origami beyond Watson-Crick base pairing.

Heterogeneous migration routes of DNA triplet repeat slip-outs

It is unclear how the length of a repetitive DNA tract determines the onset and progression of repeat expansion diseases, but the dynamics of secondary DNA structures formed by repeat sequences are believed to play an important role. It was recently shown that three-way DNA junctions containing slip-out hairpins of CAG or CTG repeats and contiguous triplet repeats in the adjacent duplex displayed single-molecule FRET (smFRET) dynamics that were ascribed to both local conformational motions and longer-range branch migration. Here we explore these so-called "mobile" slip-out structures through a detailed kinetic analysis of smFRET trajectories and coarse-grained modeling. Despite the apparent structural simplicity, with six FRET states resolvable, most smFRET states displayed biexponential dwell-time distributions, attributed to structural heterogeneity and overlapping FRET states. Coarse-grained modeling for a (GAC)10 repeat slip-out included trajectories that corresponded to a complete round of branch migration; the structured free energy landscape between slippage events supports the dynamical complexity observed by smFRET. A hairpin slip-out with 40 CAG repeats, which is above the repeat length required for disease in several triplet repeat disorders, displayed smFRET dwell times that were on average double those of 3WJs with 10 repeats. The rate of secondary-structure rearrangement via branch migration, relative to particular DNA processing pathways, may be an important factor in the expansion of triplet repeat expansion diseases.

Ensemble Force Spectroscopy by Shear Forces.

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the lack of high throughput capabilities, the application of these techniques is limited in biophysics. Ensemble force spectroscopy (EFS) has demonstrated high throughput in the investigation of a massive set of molecular structures by converting mechanochemical studies of individual molecules into those of molecular ensembles. In this protocol, the DNA secondary structures (i-motifs) were unfolded in the shear flow between the rotor and stator of a homogenizer tip at shear rates up to 77796/s. The effects of flow rates and molecular sizes on the shear forces experienced by the i-motif were demonstrated. The EFS technique also revealed the binding affinity between DNA i-motifs and ligands. Furthermore, we have demonstrated a click chemistry reaction that can be actuated by shear force (i.e., mechano-click chemistry). These results establish the effectiveness of using shear force to control the conformation of molecular structures.

Nucleic Acid Thermodynamics Derived from Mechanical Unzipping Experiments.

Force-spectroscopy techniques have led to significant progress in studying the physicochemical properties of biomolecules that are not accessible in bulk assays. The application of piconewton forces with laser optical tweezers to single nucleic acids has permitted the characterization of molecular thermodynamics and kinetics with unprecedented accuracy. Some examples are the hybridization reaction between complementary strands in DNA and the folding of secondary, tertiary, and other heterogeneous structures, such as intermediate and misfolded states in RNA. Here we review the results obtained in our lab on deriving the nearest-neighbor free energy parameters in DNA and RNA duplexes from mechanical unzipping experiments. Remarkable nonequilibrium effects are also observed, such as the large irreversibility of RNA unzipping and the formation of non-specific secondary structures in single-stranded DNA. These features originate from forming stem-loop structures along the single strands of the nucleic acid. The recently introduced barrier energy landscape model quantifies kinetic trapping effects due to stem-loops being applicable to both RNA and DNA. The barrier energy landscape model contains the essential features to explain the many behaviors observed in heterogeneous nucleic-acid folding.

Short LNA-modified oligonucleotide probes as efficient disruptors of DNA G-quadruplexes.

G-quadruplexes (G4s) are well known non-canonical DNA secondary structures that can form in human cells. Most of the tools available to investigate G4-biology rely on small molecule ligands that stabilise these structures. However, the development of probes that disrupt G4s is equally important to study their biology. In this study, we investigated the disruption of G4s using Locked Nucleic Acids (LNA) as invader probes. We demonstrated that strategic positioning of LNA-modifications within short oligonucleotides (10 nts.) can significantly accelerate the rate of G4-disruption. Single-molecule experiments revealed that short LNA-probes can promote disruption of G4s with mechanical stability sufficient to stall polymerases. We corroborated this using a single-step extension assay, revealing that short LNA-probes can relieve replication dependent polymerase-stalling at G4 sites. We further demonstrated the potential of such LNA-based probes to study G4-biology in cells. By using a dual-luciferase assay, we found that short LNA probes can enhance the expression of c-KIT to levels similar to those observed when the c-KIT promoter is mutated to prevent the formation of the c-KIT1 G4. Collectively, our data suggest a potential use of rationally designed LNA-modified oligonucleotides as an accessible chemical-biology tool for disrupting individual G4s and interrogating their biological functions in cells.

Telomere Length Regulation.

The number of (TTAGGG)n repeats at the ends of chromosomes is highly variable between individual chromosomes, between different cells and between species. Progressive loss of telomere repeats limits the proliferation of pre-malignant human cells but also contributes to aging by inducing apoptosis and senescence in normal cells. Despite enormous progress in understanding distinct pathways that result in loss and gain of telomeric DNA in different cell types, many questions remain. Further studies are needed to delineate the role of damage to telomeric DNA, replication errors, chromatin structure, liquid-liquid phase transition, telomeric transcripts (TERRA) and secondary DNA structures such as guanine quadruplex structures, R-loops and T-loops in inducing gains and losses of telomere repeats in different cell types. Limitations of current telomere length measurements techniques and differences in telomere biology between species and different cell types complicate generalizations about the role of telomeres in aging and cancer. Here some of the factors regulating the telomere length in embryonic and adult cells in mammals are discussed from a mechanistic and evolutionary perspective.

Slow Rotational Dynamics of Cytosine NH2 Groups in Double-Stranded DNA.

Aromatic NH2 groups are essential as hydrogen-bond donors in secondary structures of DNA and RNA. Although rapid rotations of NH2 groups of adenine and guanine bases were previously characterized, there has been a lack of quantitative information about slow rotations of cytosine NH2 groups in Watson-Crick base pairs. In this study, using an NMR method we had recently developed, we determined the kinetic rate constants and energy barriers for cytosine NH2 rotations in a 15-base-pair DNA duplex. Our data show that the rotational dynamics of cytosine NH2 groups depend on local environments. Qualitative correlation between the ranges of 15N chemical shifts and rotational time scales for various NH2 groups of nucleic acids and proteins illuminates a relationship between the partial double-bond character of the C-N bond and the time scale for NH2 rotations.

WRN rescues replication forks compromised by a BRCA2 deficiency: Predictions for how inhibition of a helicase that suppresses premature aging tilts the balance to fork demise and chromosomal instability in cancer.

Hereditary breast and ovarian cancers are frequently attributed to germline mutations in the tumor suppressor genes BRCA1 and BRCA2. BRCA1/2 act to repair double-strand breaks (DSBs) and suppress the demise of unstable replication forks. Our work elucidated a dynamic interplay between BRCA2 and the WRN DNA helicase/exonuclease defective in the premature aging disorder Werner syndrome. WRN and BRCA2 participate in complementary pathways to stabilize replication forks in cancer cells, allowing them to proliferate. Whether the functional overlap of WRN and BRCA2 is relevant to replication at gaps between newly synthesized DNA fragments, protection of telomeres, and/or metabolism of secondary DNA structures remain to be determined. Advances in understanding the mechanisms elicited during replication stress have prompted the community to reconsider avenues for cancer therapy. Insights from studies of PARP or topoisomerase inhibitors provide working models for the investigation of WRN's mechanism of action. We discuss these topics, focusing on the implications of the WRN-BRCA2 genetic interaction under conditions of replication stress.

Electrochemically classifying DNA structure based on the small molecule-DNA recognition

Herein, we reported the differential binding ability of aminoglycosides to DNA structures using electrochemical method through principal component analysis (PCA) to classify different DNA secondary structures and understand the link between secondary structure and DNA conformation. In these analyses, the DNA with different secondary structure motifs: bulge, internal loop, hairpin loop and stem loop were designed. The aminoglycosides as receptors were modified on the surface of electrode. In the presence of DNA, the DNA will be absorbed on the surface of electrode via the recognition of DNA and aminoglycosides, resulting in the electrochemical signal observed in [Fe(CN)6]3-/4-. Furthermore, the DNA structures labeled with 2-aminopurine (2-AP) at the structural motif of interest were also employed to study the binding affinity between aminoglycosides and different DNA motifs. The PCA suggested that this method may achieve nucleotide-specific classification of two independent secondary structure motifs, and the structure and sequence of DNA and the size and structure of small molecule could affect the binding ability of the aminoglycosides and DNA. This approach presents a new approach to classify DNA structure and offers ideas for designing targeted drugs small molecule compounds for wound dressing and drug delivery.