Duplex DNA Research Articles

Ribose Sugar Alters Conformational Sampling of G•T Mismatched Duplex DNA.

Polymerases erroneously incorporate Guanine-Thymine (dG•dT) mismatches in genomic DNA that further evades repair by transient sampling of tautomeric/ionic states compromising fidelity of repairing dG•dT mismatches. In conjunction, significant frequency of ribose (mis)incorporation in duplex DNA permits for misincorporated-mismatch in the genome. Ribose incorporated G(rG) mismatched with T(rG•dT) is the most stable across all misincorporated-mismatch calling into question the conformational consequences of the ribose sugar in addition to the mismatch. In this work, the effects of single rG•dT is investigated within a dodecamer DNA duplex employing solution-state NMR spectroscopy, partial anisotropic measurements in conjunction with molecular dynamics simulations to evaluate the impact on base pairs and the overall duplex structure. It is observed that rG•dT pairs exhibit enhanced flexibility in both base-pair and sugar dynamics compared to dG•dT, and the perturbations are enhanced in comparison to a ribose incorporated adenine-thymine (rA•dT) pair. The structural perturbations compared between rG•dT and dG•dT provides clues on plausible recognition modes of ribonucleotide excision repair (RER) pathway that looks for misincorporated ribose and mismatch repair (MMR) enzymes that scout for a mismatch.

Micrometer-sized liposomes self-aggregate by forming DNA duplexes on their surfaces more sensitively than metallic nanoparticles

Abstract We demonstrate that liposomes with diameters of more than 10 μm (giant unilamellar vesicles, GUVs) self-aggregate in a non-crosslinking manner by forming DNA duplexes with blunt ends on their surfaces much more sensitively than gold nanoparticles (AuNPs) by virtue of their unique features.

Atomistic Insights Into Interaction of Doxorubicin WithDNA: From Duplex to Nucleosome.

Doxorubicin (DOX) is a widely used chemotherapeutic agent known for intercalating into DNA. However, the exact modes of DOX interactions with various DNA structures remain unclear. Using molecular dynamics (MD) simulations, we explored DOX interactions with DNA duplexes (dsDNA), G-quadruplex, and nucleosome. DOX predominantly stacks on terminal bases of dsDNA and occasionally binds into its minor groove. In the G-quadruplex, DOX stacks on planar tetrads but does not spontaneously intercalate into these structures. Potential of mean force calculations indicate that while intercalation is the most energetically favorable interaction mode for DOX in dsDNA, the process requires overcoming a significant energy barrier. In contrast, DOX spontaneously intercalates into bent nucleosomal DNA, due to the increased torsional stress. This preferential intercalation of DOX into regions with higher torsional stress provides new insights into its mechanism of action and underscores the importance of DNA tertiary and quaternary structures in therapies utilizing DNA intercalation.

Development and functional evaluation of a psoralen-conjugated nucleoside mimic for triplex-forming oligonucleotides

Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been employed for the photodynamic regulation of gene expression by the photo-cross-linking of psoralen with the target DNA. However, stable triplex formation requires a consecutive purine base sequence in one strand of the target DNA duplexes. The pyrimidine-base interruption in the consecutive purine base sequence drastically decreases the thermodynamic stability of the corresponding triplex, which hampers the TFO application. Here, we propose a design of the Ps-TFO for stable triplex formation with target DNA sequences containing pyrimidine-base interruptions under physiological conditions. This Ps-TFO, named 1’(one)-psoralen-conjugated triplex-forming oligonucleotide (OPTO), incorporates a synthesized nucleoside mimic 1’-psoralen-conjugated deoxyribose to increase the thermodynamic stability of the corresponding triplex by the intercalation of psoralen. The triplex-forming abilities of the OPTO were successfully demonstrated in combination with LNA and 5-methylcytosine, indicating that the use of OPTO will expand the range of the target sequences of TFO for photodynamic gene regulation.

Repurposing the Antihypertensive Agent Hydralazine As an Inhibitor of the Base Excision Repair Enzyme APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a central enzyme in the base excision repair (BER) pathway. APE1 catalyzes incision of the phosphodiester linkage on the 5'-side of apurinic/apyrimidinic (AP) sites during the repair of damaged nucleobases in cellular DNA. Inhibition of this enzyme can potentiate the action of DNA-damaging chemotherapeutic agents. The antihypertensive drug hydralazine generates covalent AP adducts that block the catalytic action of APE1. Hydralazine was found to be superior to the investigational drug methoxyamine in its capacity to covalently capture AP sites in duplex DNA and inhibit the action of APE1. It was further shown that hydralazine sensitized SF295 glioblastoma cells to the cytotoxic action of the anticancer drug Temozolomide, which generates alkylpurine residues requiring APE1 for repair. The results suggest that the FDA-approved drug hydralazine might be repurposed in oncology to potentiate the activity of existing chemotherapeutic agents that induce AP sites in cellular DNA.

Dumbbell probe-bridged CRISPR/Cas13a and nicking-mediated DNA cascade reaction for highly sensitive detection of colorectal cancer-related microRNAs.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, necessitating the development of sensitive and minimally invasive diagnostic approaches. In this study, we present a novel diagnostic strategy by integrating dumbbell probe-mediated CRISPR/Cas13a with nicking-induced DNA cascade reaction (DP-bridged Cas13a/NDCR) for highly sensitive microRNA (miRNA) detection. Target miRNA triggers Cas13a-mediated cleavage of the dumbbell probe, releasing an intermediate strand that hybridizes with a methylene blue-labeled hairpin probe on the electrode surface. Nicking enzyme cleaves the formed duplex DNA, triggering a cascade reaction that amplifies the electrochemical signal. Under optimized conditions, the method demonstrates a detection limit of 8.26fM for miRNA-21, with reliable specificity and long-term stability. Furthermore, integration with machine learning models using multiple miRNA markers improved diagnostic accuracy, differentiating CRC from colorectal polyps and healthy controls with 100% accuracy in clinical validation cohorts. This study highlights the potential of DP-bridged Cas13a/NDCR as a sensitive and accurate diagnostic tool for CRC.

Unidirectional MCM translocation away from ORC drives origin licensing

The MCM motor of the eukaryotic replicative helicase is loaded as a double hexamer onto DNA by the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ATP binding supports formation of the ORC-Cdc6-Cdt1-MCM (OCCM) helicase-recruitment complex where ORC-Cdc6 and one MCM hexamer form two juxtaposed rings around duplex DNA. ATP hydrolysis by MCM completes MCM loading but the mechanism is unknown. Here, we used cryo-EM to characterise helicase loading with ATPase-dead Arginine Finger variants of the six MCM subunits. We report the structure of two MCM complexes with different DNA grips, stalled as they mature to loaded MCM. The Mcm2 Arginine Finger-variant stabilises DNA binding by Mcm2 away from ORC/Cdc6. The Arginine Finger-variant of the neighbouring Mcm5 subunit stabilises DNA engagement by Mcm5 downstream of the Mcm2 binding site. Cdc6 and Orc1 progressively disengage from ORC as MCM translocates along DNA. We observe that duplex DNA translocation by MCM involves a set of leading-strand contacts by the pre-sensor 1 ATPase hairpins and lagging-strand contacts by the helix-2-insert hairpins. Mutating any of the MCM residues involved impairs high-salt resistant DNA binding in vitro and double-hexamer formation assessed by electron microscopy. Thus, ATPase-powered duplex DNA translocation away from ORC underlies MCM loading.

A DNA phosphorothioation pathway via adenylated intermediate modulates Tdp machinery.

In prokaryotes, the non-bridging oxygen in the DNA sugar-phosphate backbone can be enzymatically replaced by a sulfur atom, resulting in phosphorothioate (PT) modification. However, the mechanism underlying the oxygen-to-sulfur substitution remains enigmatic. In this study, we discovered a hypercompact DNA phosphorothioation system, TdpABC, in extreme thermophiles. This DNA sulfuration process occurs through two sequential steps: an initial activation step by ATP to form an adenylated intermediate, followed by a substitution step where the adenyl group is replaced with a sulfur atom. Together with the TdpA-TdpB, the TdpABC system provides anti-phage defense by degrading PT-free phage DNA. Cryogenic electron microscopy structural analysis revealed that the TdpA hexamer binds one strand of encircled duplex DNA via hydrogen bonds arranged in a spiral staircase conformation. Nevertheless, the TdpAB-DNA interaction was sensitive to the hydrophobicity of the PT sulfur. PTs inhibit ATP-driven translocation and nuclease activity of TdpAB on self-DNA, thereby preventing autoimmunity.

Binuclear ruthenium complex linker length tunes DNA threading intercalation kinetics.

Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA base pairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semi-rigid linker relative to the model complex. Equilibrium results suggest a DNA affinity that is an order of magnitude higher than the parent binuclear ruthenium complex, likely due to a sterically-relieved DNA threading intercalation mechanism. Notably, kinetics analysis shows that less DNA elongation is required for threading intercalation compared to the parent complex, and the association rate is two orders of magnitude faster. The ruthenium complex elongates the DNA duplex by ∼0.3 nm per bound ligand to reach the equilibrium intercalated state, with a significantly different energy landscape relative to the parent complex. Mechanical properties of the ligand-saturated DNA duplex show a higher persistence length, indicating that the longer semi-rigid linker provides enough molecular spacing to allow a single monomer to fully stack with base pairs, comparable to the monomeric parent ruthenium complex. The DNA base pairs in the equilibrium threading intercalated state are likely intact and the ruthenium complex is shielded from the polar solution, providing measurable single-molecule confocal fluorescence signals. The obtained confocal fluorescence imaging of the bound dye confirms mostly uniform intercalation along the tethered DNA, consistent with other intercalators. The results of this study, along with previously examined ruthenium complex variants, illustrate tunable intercalation mechanisms guided by rational design of therapeutic and diagnostic small molecules to target and modify the DNA duplex.

Refinement of the Sugar Puckering Torsion Potential in the AMBER DNA Force Field.

The transition from B-DNA to A-DNA occurs in many protein-DNA interactions or in DNA/RNA hybrid duplexes, and thus plays a role in many important biomolecular processes that convey the biological function of DNA. However, the stability of A-DNA is severely underestimated in current AMBER force fields such as OL15, OL21 or bsc1, potentially leading to unstable or deformed protein-DNA complexes. In this study, we refine the deoxyribose dihedral potential to increase the stability of the north (N) puckering present in A-DNA. The new parameters, termed OL24, model A/B equilibrium in B-DNA duplexes in water in good agreement with nuclear magnetic resonance (NMR) experiment. They also improve the description of DNA/RNA hybrids and the transition of the DNA duplex to the A-form in concentrated ethanol solutions. These refinements significantly improve the modeling of protein-DNA complexes, increasing their structural stability and A-form population, while maintaining accurate representation of canonical B-DNA duplexes. Overall, the new parameters should allow more reliable modeling of the thermodynamic equilibrium between A- and B-DNA forms and the interactions of DNA with proteins.

In situ detection of piRNA‐651 in exosomes and cells for cancer diagnosis by a new gold nanoparticle nucleic acid probe

AbstractIncreasing studies have demonstrated that PIWI‐interacting RNAs (piRNAs) in circulating exosomes can serve as novel molecular biomarkers for tumor liquid biopsy. However, methods for in situ detection of piRNAs encased in exosomes are limited. In this study, we designed a spherical nucleic acid probe named piR‐651, which can enter exosomes simply by incubating with them for 2 h and in situ detect piR‐651 with a detection limit of 5 × 107 particles/μL. Based on this probe, we established a liquid biopsy method for the in situ detection of piR‐651 in plasma exosomes. The assay could distinguish the expression levels of piR‐651 between 21 breast cancer patients and 22 healthy individuals. The receiver operating characteristic curve shows an area under the curve as 0.9931 and the diagnostic sensitivity and specificity at the best cutoff are 85.7% and 100%, respectively. The probe can also easily perform in situ imaging of piR‐651 in living cells. To avoid low sensitivity and kinetics in detecting large‐sized PIWI‐interacting RNA complexes, we rationally designed the structure and detection scheme of piR‐651 probe, which was synthesized by modifying 13‐nm gold particles with high‐density Anchor‐Report DNA duplexes through the butanol dehydration method. The new design of the gold nanoparticle nucleic acid probe can be applied to the fabrication of nucleic acid probes targeting other large‐volume nucleic acids for developing more molecular biomarker‐based liquid biopsy for cancer diagnosis.

Supramolecular multiplexes from collagen mimetic peptide-PNA(GGG)3 conjugates and C-rich DNA: pH-induced reversible switching from triplex-duplex to triplex-i-motif.

Peptides are well known for forming nanoparticles, while DNA duplexes, triplexes and tetraplexes create rigid nanostructures. Accordingly, the covalent conjugation of peptides to DNA/RNA produces hybrid self-assembling features and may lead to interesting nano-assemblies distinct from those of their individual components. Herein, we report the preparation of a collagen mimetic peptide incorporating lysine in its backbone, with alkylamino side chains radially conjugated with G-rich PNA [collagen-(PNA-GGG)3]. In the presence of complementary C-rich DNA (dCCCTTTCCC) at neutral pH, the collagen mimetic triplexes were interconnected by PNA-GGG : DNA-CCC duplexes, leading to the formation of larger assemblies of nanostructures. Upon decreasing the pH to 4.5, the dissociation of the triplex-duplex assembly released the protonated C-rich DNA, which immediately folded into an i-motif. With an increase in the pH to 7.2 (neutral), the i-motif unfolded into linear DNA, which reformed the PNA-GGG : DNA-CCC duplex interconnecting the collagen triplexes. The pH-induced switching of the assembly and disassembly was reversible over a few cycles. The hybrid collagen-(PNAGGG)3 : DNA-C3T3C3 triplex-duplex and the individual components of the assembly including the i-motif were characterized by UV and CD melting, fluorescence, TEM and gel electrophoresis. The pH-induced reversible switching was established by the changes in the CD and fluorescence properties. Peptide-DNA conjugates have wide applications in both biology and materials science, ranging from therapeutics and drug delivery to diagnostics and molecular switches. Thus, the prototype ensemble of the triplex peptide-PNA conjugate and its duplex with DNA described herein has potential for elaboration into rationally designed systems by varying the PNA/DNA sequences to trap functional ligands/drugs for release in pH-controlled environments.

Resolution of complex mixtures of duplex and antiparallel triplex DNA structures by capillary electrophoresis and multivariate analysis

Resolution of complex mixtures of duplex and antiparallel triplex DNA structures by capillary electrophoresis and multivariate analysis

DNA crossover flexibilities upon discrete spacers revealed by single-molecule FRET.

In this study, we utilized the origami technique to integrate various types of spacers into the double-stranded crossover and examined their flexibilities using single-molecule fluorescence resonance energy transfer (smFRET). We discovered that for the traditional Holliday Junction connection with zero-base spacers, the inter-structural angle measures 58.7 degrees, which aligns well with previous crystallographic research. When introducing non-complementary double-stranded spacers as a free leash, we observed that longer spacers resulted in a more relaxed connection. In contrast, when using complementary segments, the two origami structures rotated as the number of base pairs increased, reflecting the structural characteristics of the B-duplex. Our findings indicate that a stable intramolecular duplex requires a minimum of 5 base pairs. Overall, our results highlight the potential for re-engineering crossovers and designing materials that can change volume with shrink-swell capabilities, as well as applications in torque sensing using short DNA duplexes.

Comparative Studies on Bulky DNA Damage Binding by Nucleotide Excision Repair Proteins Using Surface Plasmon Resonance, Differential Scanning Fluorometry, and DNase I Footprinting.

Nucleotide excision repair is a crucial cellular mechanism that ensures genomic stability, thereby preventing mutations that can lead to cancer. The human XPC and its yeast ortholog Rad4 protein complexes are central to this process and were the focus of the study. We used surface plasmon resonance and differential scanning fluorimetry to study the binding characteristics of XPC and Rad4 when bound to the bulky cluster di-FAAF-containing 55-mer duplex DNA. Our findings revealed that XPC binds 10 times more significant affinity to control and di-FAAF-modified DNA than Rad4 with greater protein-DNA interactions. Differential scanning fluorimetry indicates that Rad4 causes comparatively more significant conformational changes upon complexation with the damaged DNA. We conducted DNase I footprinting of the Rad4/DNA complex for the first time by determining the regions protected from DNase I digestion. The DNA at the lesion is entirely resistant to digestion by DNase I in the absence of Rad4 several nucleotides to the 3'-side of the first FAAF lesion. The lack of DNase I cleavage at the lesions did not change upon adding Rad4. However, in the presence of Rad4, a footprint is observed on the 7-nucleotide region (5'-TGGTGAT-3') of the complementary strand to the 3' side of the lesion.

Indocarbocyanine-Indodicarbocyanine (sCy3-sCy5) Absorptive Interactions in Conjugates and DNA Duplexes.

Sulfonated indocyanines 3 and 5 (sCy3, sCy5) are widely used to label biomolecules. Their high molar absorption coefficients and lack of spectral overlap with biopolymers make them ideal as linker components for rapid assessment of bioconjugate stoichiometry. We recently found that the determination of the sCy3:sCy5 molar ratio in a conjugate from its optical absorption spectrum is not straightforward, as the sCy3:sCy5 absorbance ratio at the maxima tends to be larger than expected. In this work, we have investigated this phenomenon in detail by studying the spectral properties of a series of sCy3-sCy5 conjugates in which the dyes are separated by linkers of various lengths, including DNA duplexes. It was found that when sCy3 and sCy5 are located in close proximity, they consistently exhibit an "abnormal" absorbance ratio. However, when the two dyes are separated by long rigid DNA-based spacers, the absorbance ratio becomes consistent with their individual molar absorption coefficients. This phenomenon should be taken into account when assessing the molar ratio of the dyes by UV-Vis spectroscopy.

Copper(I)-Catalyzed Intramolecular Tandem Acylation/O-Arylation under Mild Conditions: Synthesis of Benzofuro[3,2-c]quinolin-6(5H)-ones and G-Quadruplex-Targeting Analogues.

This paper presents a copper(I)-catalyzed intramolecular tandem acylation/O-arylation of methyl 2-[2-(2-bromophenyl)acetamido]benzoates for the synthesis of benzofuro[3,2-c]quinolin-6(5H)-ones under mild conditions. The combination of CuI, 1,10-phenanthroline, and K2CO3 in DMSO was found to be the optimal reaction condition, producing the target products in high yields (84-99%) at 70 °C for 16 h. The tandem reaction was applicable to substrates bearing halo, electron-withdrawing, and electron-donating groups at their phenyl moieties with a broad substrate scope. Further derivation produced compounds serving as G-quadruplex DNA (G4 DNA) stabilizers. The most potent analogue, 2,9-bis{[3-(diethylamino)propyl]amino}-5-methylbenzofuro[3,2-c]quinolin-6(5H)-one, significantly increased the melting temperature of G4 DNA by 9.8 °C at 1.0 μM, approximately 4.6 times more selective than duplex DNA. The G4 stabilizer also showed anticancer activity, actively inhibiting MDA-MB-231 cancer cells with a GI50 value of 0.41 μM.

A DNA Origami Pivot Hinge Driven by DNA Intercalators.

The DNA origami nanotechnology has been used to construct nanoscale structures whose shapes can be dynamically reconfigured. Here, we propose a DNA origami hinge with a continuous pivot motion controlled by the concentration of DNA intercalators. It consists of two six-helix bundles connected by two gold nanoparticles. We can control the sensitivity and range of pivot motion by using different types of intercalators and their mixture and by varying the distance between cross-linking gold nanoparticles. The pivot motion is reversible and repeatable. A concept of bending nanoactuators is demonstrated by connecting three pivot hinges. The proposed pivot hinge mechanism may be useful in developing nanosensors or actuators that require amplification of the small deformation of DNA duplexes induced by molecular agents.

Photo-Cross-linked DNA Structures Greatly Improves Their Serum Nuclease Resistances and Gene Knock-In Efficiencies.

The stabilization and structural integrity of DNA architectures remain significant challenges in their biomedical applications, particularly when inserting functional units into the genome using long single-stranded DNA (lssDNA). To address these challenges, a site-specific photo-cross-linking method is employed. Single-stranded oligonucleotides, containing one or two photosensitive cyanovinylcarbazole nucleoside (CNVK) molecules, are precisely incorporated and cross-linked at the specific sites of ssDNA through base-pairing, followed by rapid UV irradiation at 365nm. This interstrand photo-cross-linking improves the thermal stability of DNA duplexes and allows this study to afford a tetrahedral DNA nanostructure in a yield of >94%. Most importantly, the photo-cross-linked DNA architectures exhibit high resistances against serum degradation, especially prevent digestion of exonuclease III (exo III), which is common in conventional lambda-processing method. Meanwhile, this photo-cross-linking treatment can significantly improve the knock-in (KI) efficiencies of lssDNA in different cells including 293T, K562, and HepG2, approximately three to eightfold those of the uncross-linked lssDNA, and remain a low cytotoxicity. Given the significantly enhanced nuclease resistance in serum and improved KI efficiencies, this study anticipates that this photo-cross-linking method will become a valuable tool in technologically advanced biomedical applications, such as nanotechnology and nucleic acid therapy.

DNA Mimic Foldamer Recognition of a Chromosomal Protein

Helical aromatic oligoamide foldamers bearing anionic side chains that mimic the overall shape and charge surface distribution of DNA were synthesized. Their interactions with chromosomal protein Sac7d, a non‐sequence‐selective DNA‐binder that kinks DNA, were investigated by Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), Circular Dichroism spectroscopy (CD), melting curve analysis, Atomic Force Microscopy (AFM), and Nuclear Magnetic Resonance (NMR), as well as by single crystal X‐ray crystallography. The foldamers were shown to bind to Sac7d better than a DNA duplex of comparable length. The interaction is diastereoselective and takes place at the DNA binding site. Crystallography revealed that the DNA mimic foldamers have a binding mode of their own and that they can bind to Sac7d without being kinked.