A Theoretical Exploration of the Photoinduced Breaking Mechanism of the Glycosidic Bond in Thymine Nucleotide.

DNA glycosidic bond cleavage may induce cancer under the ultraviolet (UV) effect. Yet, the mechanism of glycosidic bond cleavage remains unclear and requires more detailed clarification. Herein, quantum chemical studies on its photoinduced mechanism are performed using a 5'-thymidine monophosphate (5'-dTMPH) model. In this study, four possible paths were examined to study the glycosidic bond cleavage. The results showed that, upon excitation, the electronic transition from the π bonding to π antibonding orbitals of the thymine ring leads to the damage of the thymine ring. Afterwards, the glycosidic bond is cleaved. At first, the doublet ground state (GS) path of glycosidic bond cleavage widely studied by other groups is caused by free electron generated by photoirradiation, with a kinetically feasible energy barrier of ~23 kcal/mol. Additionally, then, the other three paths were proposed that also might cause the glycosidic bond cleavage. The first one is the doublet excited state (ES) path, triggered by free electron along with UV excitation, which can result in a very-high-energy barrier ~49 kcal/mol that is kinetically unfavorable. The second one is the singlet ES path, induced by direct UV excitation, which assumes DNA is directly excited by UV light, which features a very low-energy barrier ~16 kcal/mol that is favored in kinetics. The third one is the triplet ES path, from the singlet state via intersystem crossing (ISC), which refers to a feasible ~27 kcal/mol energy barrier. This study emphasizes the pivotal role of the DNA glycosidic bond cleavage by our proposed direct UV excitation (especially singlet ES path) in addition to the authorized indirect free-electron-induced path, which should provide essential insights to future mechanistic comprehension and novel anti-cancer drug design.

Interaction between polycyclic aromatic hydrocarbons and thymine (T)-base induces double-strand DNA distortion in different species.

Polycyclic aromatic hydrocarbons (PAHs) are potent inhibitors of DNA that can induce genetic damage, abnormal gene expression, and metabolic disorders upon interfacing with biological macromolecules. However, the mechanism of their interactions with DNA remains elusive. Therefore, this study selected three representative PAHs, including phenanthrene (Phen), pyrene (Pyre), and benzo[a]pyrene (B[a]P), and explored their binding mechanisms with the double-strand DNA (dsDNA) from different species, including 1J1V (Escherichia coli), 6J5B (Arabidopsis thaliana), and 6Q1V (Homo sapiens). The results revealed that binding between PAHs and dsDNA occurred in the groove via van der Waals forces and π-π stacking, with the carboxyl oxygen atom of the thymine (T)-base within dsDNA being the key binding site. This result was further confirmed by the spectroscopic experiments, where significant changes in the peak of the T-base were observed after PAHs-dsDNA binding. More interestingly, the total binding energies of Pyre with the three dsDNA were -138.800 kJ/mol (Pyre-1J1V), -105.523 kJ/mol (Pyre-6J5B), and -127.567 kJ/mol (Pyre-6Q1V), respectively, all of which were higher than those of Phen and B[a]P. This suggests that that Pyre has the strongest dsDNA binding ability. Additionally, analysis of the thermodynamic parameters indicated that the interactions between the three PAHs and dsDNA were exothermic reactions. In contrast, the Pyre-dsDNA interaction predominantly involved van der Waals forces and hydrogen bonding due to the enthalpy change (∆H) < 0 and entropy change (∆S) < 0, while the Phen-dsDNA and B[a]P-dsDNA interactions predominantly involved hydrophobic forces due to ∆H > 0 and ∆S > 0. Furthermore, Pyre caused local distortion of dsDNA, which was more pronounced under atomic force microscopy (AFM). In summary, this study has unveiled a new phenomenon of binding between PAHs and dsDNA. This sheds light on the carcinogenic potential and environmental impacts of PAHs pollution.

Flanking Effect on the Structure and Stability of Human Telomeric G-Quadruplex in Varying Salt Concentrations.

The stability of the human telomere G-quadruplex (G4) is directly linked to cancer disease. The human telomere is mostly associated with the flanking nucleobases, which can affect the stability of G4. Hence, in this study, the effect of the flanking nucleobases in the context of their chemical nature, number, and position on the structure and stability of G4 has been investigated in varying concentrations of KCl mimicking the normal and cancer KCl microenvironments. The addition of flanking nucleobases does not alter the G4 topology. However, the presence of merely a single flanking nucleobase destabilizes the telomeric G4. This destabilizing effect is more prominent for thymine than adenine flanking nucleobase, probably due to the formation of the intermolecular G4 topology by thymine. Interestingly, the change in the stability of the telomeric G4 in the presence of thymine flanking nucleobase is sensitive to the concentration of KCl relevant to the normal and cancerous microenvironments, in contrast to adenine. Flanking nucleobases have a greater impact at the 5' end compared to the 3' end, particularly noticeable in KCl concentrations resembling the normal microenvironment rather than the cancerous one. These findings indicate that the effect of the flanking nucleobases on telomeric G4 is different in the KCl salt relevant to normal and cancerous microenvironments. This study may be helpful in attaining molecular-level insight into the role of G4 in telomeric length regulation under normal and cancerous KCl salt conditions.

Ultrafast and Multichannel Generation of the Triplet State in DNA-Intercalated Gilvocarcin V for an Enhanced Photoaddition Reaction.

Gilvocarcin V (GV) is a natural antibiotic exhibiting excellent antitumor activities and remarkably low toxicity in near-ultraviolet or visible light-dependent treatment. Notwithstanding, the [2 + 2] cycloaddition reaction between GV and thymine has been proven to be the key for its function in photodynamic therapy, and crucial mechanistic details about such a reaction are poorly understood. In this study, the electronic relaxation pathways and photoaddition reaction are characterized by femto- to nanosecond time-resolved spectroscopy combined with quantum chemical calculation. Our results reveal that ultrafast intersystem crossing (<3 ps) leads to the population of a local triplet excited state in DNA-intercalated GV. Such a state can further induce the formation of a biradical state, which is identified as the important reactive precursor for photoaddition between GV and thymine. The overall photoaddition quantum efficiency is determined to be 11.57 ± 1.0%. These results are essential to the elucidation of the DNA photoaddition mechanism of C-aryl glycoside-based artificial photocytotoxic agents and could help further development of those medicines.

Enhancement of the thermal stability of G-quadruplex structures by urea.

The solute urea has been used extensively as a denaturant in protein folding studies; double-stranded nucleic acid structures are also destabilized by urea, but comparatively less than proteins. In previous research, the solute has been shown to strongly destabilize folded G-quadruplex DNA structures. This contribution demonstrates the stabilizing effect of urea on the G-quadruplex formed by the oligodeoxyribonucleotide (ODN), G3T (d[5'-GGGTGGGTGGGTGGG-3']), and related sequences in the presence of sodium or potassium cations. Stabilization is observed up to 7 M urea, which was the highest concentration we investigated. The folded structure of G3T has three G-tetrads and three loops that consist of single thymine residues. ODNs related to G3T, in which the thymine residues in the loop are substituted by adenosine residues, also exhibit enhanced stability in the presence of molar concentrations of urea. The circular dichroism (CD) spectra of these ODNs in the presence of urea are consistent with that of a G-quadruplex. As the urea concentration increases, the spectral intensities of the peaks and troughs change, while their positions change very little. The heat-induced transition from the folded to unfolded state, Tm, was measured by monitoring the change in the UV absorption as a function of temperature. G-quadruplex structures with loops containing single bases exhibited large increases in Tm with increasing urea concentrations. These data imply that the loop region play a significant role in the thermal stability of tetra-helical DNA structures in the presence of the solute urea.

Coupling of conformation and CPD damage in nucleosomal DNA.

UV-light can cause photodimerization and hence damages in DNA. Most frequent are cyclobutane pyrimidine dimer (CPD) damages, which predominantly form at TpT (thymine-thymine) steps. It is well known that CPD damage probability is different for single-stranded or double stranded DNA and depends on the sequence context. However, DNA deformation due to packing in nucleosomes can also influence CPD formation. Quantum mechanical calculations and Molecular Dynamics simulations indicate little CPD damage probability for DNA's equilibrium structure. We find that DNA needs to be deformed in a specific way to allow the HOMO → LUMO transition required for CPD damage formation. The simulation studies further show that the periodic CPD damage patterns measured in chromosomes and nucleosomes can be directly explained by the periodic deformation pattern of the DNA in the nucleosome complex. It supports previous findings on characteristic deformation patterns found in experimental nucleosome structures that relate to CPD damage formation. The result may have important implications for our understanding of UV-induced DNA mutations in human cancers.

Enriching adenosine by thymine-rich DNA oligomers.

Adenosine levels are important in various physiological and pathological activities, but detecting them is difficult because of interference from a complex matrix. This study designed a series of DNA oligomers rich in thymine to enrich adenosine. Their binding affinity (Kd range: 1.25-5.0 mM) to adenosine varied based on the DNA secondary structures, with a clamped hairpin structure showing the highest binding affinity. Compared to other designs, this clamped DNA hairpin underwent the least conformational change during adenosine binding. These DNAs also suppressed the precipitation of supersaturated adenine. Taken together, these results suggest that thymine-rich DNAs could be used to enrich and separate adenosine.

Resolution of Identity in Gas-Phase Dissociations of Mono- and Diprotonated DNA Trinucleotide Codons by 15N-Labeling and Computational Structure Analysis.

Dissociations of DNA trinucleotide codons as gas-phase singly and doubly protonated ions were studied by tandem mass spectrometry using 15N-labeling to resolve identity in the nucleobase loss and backbone cleavages. The monocations showed different distributions of nucleobase loss from the 5'-, middle, and 3'-positions depending on the nucleobase, favoring cytosine over guanine, adenine, and thymine in an ensemble-averaged 62:27:11:<1 ratio. The distribution for the loss of the 5'-, middle, and 3'-nucleobase was 49:18:33, favoring the 5'-nucleobase, but also depending on its nature. The formation of sequence w2+ ions was unambiguously established for all codon mono- and dications. Structures of low-Gibbs-energy protomers and conformers of dAAA+, dGGG+, dCCC+, dTTT+, dACA+, and dATC+ were established by Born-Oppenheimer molecular dynamics and density functional theory calculations. Monocations containing guanine favored classical structures protonated at guanine N7. Structures containing adenine and cytosine produced classical nucleobase-protonated isomers as well as zwitterions in which two protonated bases were combined with a phosphate anion. Protonation at thymine was disfavored. Low threshold energies for nucleobase loss allowed extensive proton migration to occur prior to dissociation. Loss of the nucleobase from monocations was assisted by neighboring group participation in nucleophilic addition or proton abstraction, as well as allosteric proton migrations remote from the reaction center. The optimized structures of diprotonated isomers for dAAA2+ and dACA2+ revealed combinations of classical and zwitterionic structures. The threshold and transition-state energies for nucleobase-ion loss from dications were low, resulting in facile dissociations involving cytosine, guanine, and adenine.

Tailoring the Structure of Cell Penetrating DNA and RNA Binding Nucleopeptides.

Synthetic nucleic acid interactors represent an exciting research field due to their biotechnological and potential therapeutic applications. The translation of these molecules into drugs is a long and difficult process that justifies the continuous research of new chemotypes endowed with favorable binding, pharmacokinetic and pharmacodynamic properties. In this scenario, we describe the synthesis of two sets of homo-thymine nucleopeptides, in which nucleobases are inserted in a peptide structure, to investigate the role of the underivatized amino acid residue and the distance of the nucleobase from the peptide backbone on the nucleic acid recognition process. It is worth noting that the CD spectroscopy investigation showed that two of the reported nucleopeptides, consisting of alternation of thymine functionalized L-Orn and L-Dab and L-Arg as underivatized amino acids, were able to efficiently bind DNA and RNA targets and cross both cell and nuclear membranes.

Lignin Nanoparticles Deliver Novel Thymine Biomimetic Photo-Adducts with Antimelanoma Activity.

We report here the synthesis of novel thymine biomimetic photo-adducts bearing an alkane spacer between nucleobases and characterized by antimelanoma activity against two mutated cancer cell lines overexpressing human Topoisomerase 1 (TOP1), namely SKMEL28 and RPMI7951. Among them, Dewar Valence photo-adducts showed a selectivity index higher than the corresponding pyrimidine-(6-4)-pyrimidone and cyclobutane counterpart and were characterized by the highest affinity towards TOP1/DNA complex as evaluated by molecular docking analysis. The antimelanoma activity of novel photo-adducts was retained after loading into UV photo-protective lignin nanoparticles as stabilizing agent and efficient drug delivery system. Overall, these results support a combined antimelanoma and UV sunscreen strategy involving the use of photo-protective lignin nanoparticles for the controlled release of thymine dimers on the skin followed by their sacrificial transformation into photo-adducts and successive inhibition of melanoma and alert of cellular UV machinery repair pathways.

Exploiting the optical sensing of fluorophore-tagged DNA nucleobases on hexagonal BN and Al-doped BN sheets: a computational study.

Hexagonal boron nitride (h-BN) sheets possess high fluorescence quenching ability and high affinity towards DNA/RNA, and they can be used as a sensing platform for rapid detection. We report the absorption and emission properties of DNA nucleobases such as adenine (A), cytosine (C), guanine (G), and thymine (T) tagged with benzoxazole on h-BN and aluminium-doped h-BN (Al_hBN) sheets. The binding affinity of studied nucleobases on h-BN sheets at the M062X/6-31G* level of theory showed the following adsorption trend: G ≥ T ≥ A > C, which is in good agreement with the previous results. The calculated stability trend of nucleobases on the Al_hBN sheet follows as C > G > A > T at the same level of theory. The physically adsorbed behavior of nucleobases to h-BN sheets was confirmed by the non-covalent interactions (NCIs) and the total density of states (TDOS) plots. The NCI results indicated that van der Waals interactions contribute significantly to the adsorption of nucleobases on h-BN sheets. Atoms in molecules (AIM) calculations revealed the electrostatic interactions between nucleobases and the Al_hBN sheet. The quenching phenomenon of nucleobase-tagged fluorophores on h-BN and Al_hBN sheets was investigated by TD-DFT calculations using the same level of theory. The thymine-tagged fluorophore upon adsorption to the pristine h-BN sheet was found to be blue-shifted (∼43 nm); however, the guanine-tagged fluorophore with Al_hBN showed a remarkable difference from other nucleobase-tagged fluorophores in the absorption and emission spectrum. Guanine-tagged fluorophores showed a smaller blue shift (∼7 nm) in the absorption spectrum; however, it showed a larger red shift (∼55 nm) than the other nucleobase-tagged fluorophores on Al_hBN sheets and can be useful in recognizing a sequence-specific phenomenon as a fluorescent biosensor of DNA and RNA to ascertain the presence of such nucleobases.

Molecular basis for DarT ADP-ribosylation of a DNA base.

ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.

Structural basis for glucocorticoid receptor recognition of both unmodified and methylated binding sites, precursors of a modern recognition element.

The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. Genomes from more primitive organisms are more abundant in CpG sites that, through the process of methylation, deamination and subsequent mutation to thymine-phosphate-guanine (TpG) sites, can produce new transcription factor binding sites. Here, we examined the evolutionary history of the over 36 000 glucocorticoid receptor (GR) consensus binding motifs in the human genome and identified a subset of them in regulatory regions that arose via a deamination and subsequent mutation event. GR can bind to both unmodified and methylated pre-GR binding sequences (GBSs) that contain a CpG site. Our structural analyses show that CpG methylation in a pre-GBS generates a favorable interaction with Arg447 mimicking that made with a TpG in a GBS. This methyl-specific recognition arose 420 million years ago and was conserved during the evolution of GR and likely helps fix the methylation on the relevant cytosines. Our study provides the first genetic, biochemical and structural evidence of high-affinity binding for the likely evolutionary precursor of extant TpG-containing GBS.

The effect of flanking bases on direct and triplet sensitized cyclobutane pyrimidine dimer formation in DNA depends on the dipyrimidine, wavelength and the photosensitizer.

Cyclobutane pyrimidine dimers (CPDs) are the major products of DNA produced by direct absorption of UV light, and result in C to T mutations linked to human skin cancers. Most recently a new pathway to CPDs in melanocytes has been discovered that has been proposed to arise from a chemisensitized pathway involving a triplet sensitizer that increases mutagenesis by increasing the percentage of C-containing CPDs. To investigate how triplet sensitization may differ from direct UV irradiation, CPD formation was quantified in a 129-mer DNA designed to contain all 64 possible NYYN sequences. CPD formation with UVB light varied about 2-fold between dipyrimidines and 12-fold with flanking sequence and was most frequent at YYYR and least frequent for GYYN sites in accord with a charge transfer quenching mechanism. In contrast, photosensitized CPD formation greatly favored TT over C-containing sites, more so for norfloxacin (NFX) than acetone, in accord with their differing triplet energies. While the sequence dependence for photosensitized TT CPD formation was similar to UVB light, there were significant differences, especially between NFX and acetone that could be largely explained by the ability of NFX to intercalate into DNA.

Lactobionic acid-modified thymine-chitosan nanoparticles as potential carriers for methotrexate delivery.

In order to achieve efficient delivery of methotrexate (MTX), thymine-chitosan nanoparticles (Thy-Cs NPs) were prepared, and further decorated with lactobionic acid (LA) to obtain tumor-targeting nanoparticles (LA-Thy-Cs NPs). These nanoparticles possessed a regular spherical structure with the average size about 190-250 nm and narrow size distribution, which were kinetically stable in the physiological environment. Due to electrostatic interactions and multiple hydrogen-bonding interactions between MTX and carriers, MTX was loaded into Thy-Cs NPs with high drug loading content (~20%). MTX release from Thy-Cs NPs was significantly accelerated in the mildly acidic environment due to the destruction of two types of non-covalent interactions. In vitro cell experiments demonstrated that LA-Thy-Cs NPs could be efficiently internalized into hepatoma carcinoma cells, leading to higher cytotoxicity. Moreover, MTX-loaded LA-Thy-Cs NPs performed an enhanced growth inhibition in three-dimensional multicellular tumor spheroids. Thus, the LA decorated thymine-chitosan nanocarriers can be a promising candidate for efficient delivery of MTX.

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2.

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

Peptide nucleic acid Hoogsteen strand linker design for major groove recognition of DNA thymine bases.

Sequence-specific targeting of double-stranded DNA and non-coding RNA via triple-helix-forming peptide nucleic acids (PNAs) has attracted considerable attention in therapeutic, diagnostic and nanotechnological fields. An E-base (3-oxo-2,3-dihydropyridazine), attached to the polyamide backbone of a PNA Hoogsteen strand by a side-chain linker molecule, is typically used in the hydrogen bond recognition of the 4-oxo group of thymine and uracil nucleic acid bases in the major groove. We report on the application of quantum chemical computational methods, in conjunction with spatial constraints derived from the experimental structure of a homopyrimidine PNA·DNA-PNA hetero-triplex, to investigate the influence of linker flexibility on binding interactions of the E-base with thymine and uracil bases in geometry-optimised model systems. Hydrogen bond formation between the N2 E-base atom and target pyrimidine base 4-oxo groups in model systems containing a ß-alanine linker (J Am Chem Soc 119:11116, 1997) was found to incur significant internal strain energy and the potential disruption of intra-stand aromatic base stacking interactions in an oligomeric context. In geometry-optimised model systems containing a 3-trans olefin linker (Bioorg Med Chem Lett 14:1551, 2004) the E-base swung out away from the target pyrimidine bases into the solvent. These findings are in qualitative agreement with calorimetric measurements in hybridisation experiments at T-A and U-A inversion sites. In contrast, calculations on a novel 2-cis olefin linker design indicate that it could permit simultaneous E-base hydrogen bonding with the thymine 4-oxo group, circumvention and solvent screening of the thymine 5-methyl group, and maintenance of triplex intra-stand base stacking interactions.

Experimental and theoretical studies on thymine photodimerization mediated by oxidatively generated DNA lesions and epigenetic intermediates.

Interaction of nucleic acids with light is a scientific question of paramount relevance not only in the understanding of life functioning and evolution, but also in the insurgence of diseases such as malignant skin cancer and in the development of biomarkers and novel light-assisted therapeutic tools. This work shows that the UVA portion of sunlight, not absorbed by canonical DNA nucleobases, can be absorbed by 5-formyluracil (ForU) and 5-formylcytosine (ForC), two ubiquitous oxidatively generated lesions and epigenetic intermediates present in living beings in natural conditions. We measure the strong propensity of these molecules to populate triplet excited states able to transfer the excitation energy to thymine-thymine dyads, inducing the formation of cyclobutane pyrimidine dimers (CPDs). By using steady-state and transient absorption spectroscopy, NMR, HPLC, and theoretical calculations, we quantify the differences in the triplet-triplet energy transfer mediated by ForU and ForC, revealing that the former is much more efficient in delivering the excitation energy and producing the CPD photoproduct. Although significantly slower than ForU, ForC is also able to harm DNA nucleobases and therefore this process has to be taken into account as a viable photosensitization mechanism. The present findings evidence a rich photochemistry crucial to understand DNA damage photobehavior.

Toward G-Quadruplex-Based Anticancer Agents: Biophysical and Biological Studies of Novel AS1411 Derivatives.

Certain G-quadruplex forming guanine-rich oligonucleotides (GROs), including AS1411, are endowed with cancer-selective antiproliferative activity. They are known to bind to nucleolin protein, resulting in the inhibition of nucleolin-mediated phenomena. However, multiple nucleolin-independent biological effects of GROs have also been reported, allowing them to be considered promising candidates for multi-targeted cancer therapy. Herein, with the aim of optimizing AS1411 structural features to find GROs with improved anticancer properties, we have studied a small library of AS1411 derivatives differing in the sequence length and base composition. The AS1411 derivatives were characterized by using circular dichroism and nuclear magnetic resonance spectroscopies and then investigated for their enzymatic resistance in serum and nuclear extract, as well as for their ability to bind nucleolin, inhibit topoisomerase I, and affect the viability of MCF-7 human breast adenocarcinoma cells. All derivatives showed higher thermal stability and inhibitory effect against topoisomerase I than AS1411. In addition, most of them showed an improved antiproliferative activity on MCF-7 cells compared to AS1411 despite a weaker binding to nucleolin. Our results support the hypothesis that the antiproliferative properties of GROs are due to multi-targeted effects.

High Mechanical Stability and Slow Unfolding Rates Are Prevalent in Parallel-Stranded DNA G-Quadruplexes.

Guanine-rich repeat sequences are known to adopt diverse G-quadruplex (G4) topologies. Determining the unfolding rates of individual G4 species is challenging due to the coexistence of multiple G4 conformations in a solution. Here, using single-molecule magnetic tweezers, we systematically measured the unfolding force distributions of 4 oncogene promoter G4s, 12 model sequences with two 1-nucleotide (nt) thymine loops that predominantly adopt parallel-stranded G4 structures, and 6 sequences forming multiple G4 structures. All parallel-stranded G4s reveal an unfolding force peak at 40-60 pN, which is associated with extremely slow unfolding rates on the order of 10-5-10-7 s-1. In contrast, nonparallel G4s and partially folded intermediate states reveal an unfolding force peak <40 pN. These results suggest a strong correlation between the parallel-stranded G4s folding topology and the slow unfolding rates and provide important insights into the mechanism that govern the stability and the transition kinetics of G4s.