Distinguishing G-Quadruplexes Stabilizer and Chaperone for c-MYC Promoter G-Quadruplexes through Single-Molecule Manipulation.

G-quadruplex (G4) selective stabilizing ligands can regulate c-MYC gene expression, but the kinetic basis remains unclear. Determining the effects of ligands on c-MYC promoter G4s' folding/unfolding kinetics is challenging due to the polymorphic nature of G4s and the high energy barrier to unfold c-MYC promoter G4s. Here, we used single-molecule magnetic tweezers to manipulate a duplex hairpin containing a c-MYC promoter sequence to mimic the transiently denatured duplex during transcription. We measured the effects of six commonly used G4s binding ligands on the competition between quadruplex and duplex structures, as well as the folding/unfolding kinetics of G4s. Our results revealed two distinct roles for G4s selective stabilization: CX-5461 is mainly acting as c-MYC G4s stabilizer, reducing the unfolding rate (ku) of c-MYC G4s, whereas PDS and 360A also act as G4s chaperone, accelerating the folding rates (kf) of c-MYC G4s. qRT-PCR results obtained from CA46 and Raji cell lines demonstrated that G4s stabilizing ligands can downregulate c-MYC expression, while G4s stabilizer CX-5461 exhibited the strongest c-MYC gene suppression. These results shed light on the potential of manipulating G4s' folding/unfolding kinetics by ligands for precise regulation of promoter G4-associated biological activities.

Mechanical diversity and folding intermediates of parallel-stranded G-quadruplexes with a bulge.

A significant number of sequences in the human genome form noncanonical G-quadruplexes (G4s) with bulges or a guanine vacancy. Here, we systematically characterized the mechanical stability of parallel-stranded G4s with a one to seven nucleotides bulge at various positions. Our results show that G4-forming sequences with a bulge form multiple conformations, including fully-folded G4 with high mechanical stability (unfolding forces > 40 pN), partially-folded intermediates (unfolding forces < 40 pN). The folding probability and folded populations strongly depend on the positions and lengths of the bulge. By combining a single-molecule unfolding assay, dimethyl sulfate (DMS) footprinting, and a guanine-peptide conjugate that selectively stabilizes guanine-vacancy-bearing G-quadruplexes (GVBQs), we identified that GVBQs are the major intermediates of G4s with a bulge near the 5' or 3' ends. The existence of multiple structures may induce different regulatory functions in many biological processes. This study also demonstrates a new strategy for selectively stabilizing the intermediates of bulged G4s to modulate their functions.

Force-Dependent Intercalative Bulky DNA Adduct Formation Detected by Single-Molecule Stretching.

Quantitatively analyzing the binding topology and reactivity is essential for understanding the cytotoxic or tumorigenic activities of bulky DNA adducts formed by chemotherapeutic drugs or carcinogens. Biochemical methods require purification of DNA and discontinuous steps to digest or label the adducts and thus have difficulties in identifying the binding topology and are not suitable for detecting unstable adducts. Herein, we used a single-molecule stretching assay to characterize the number of intercalative adducts, the formation kinetics, and the mechanical properties of intercalative DNA adducts based on measuring adduct-induced DNA elongation. We analyzed various reactive conditions, including formaldehyde-mediated anthracycline-DNA adducts, UV light-catalyzed psoralen-DNA adducts, and liver S9 fraction-catalyzed aflatoxin B1-DNA adducts. We showed that adduct formation abilities are correlated with the noncovalent intercalation binding ability. External forces on double-stranded DNA increased the intercalation of ligands and can result in a 1.8- to 5.3-fold increase in DNA adduct formation.

Folding/unfolding kinetics of G-quadruplexes upstream of the P1 promoter of the human BCL-2 oncogene.

The G-rich Pu39 region of the P1 promoter of the oncogene BCL-2, an apoptosis regulator, can fold into multiple G-quadruplex (G4) structures. Bcl2-2345 and Bcl2-1245 are two major G4 species forming with high thermal stability and distinct topologies in the Pu39 region, but their folding/unfolding kinetics have not yet been investigated. Here, we used magnetic tweezers to measure the mechanical stability and the folding/unfolding kinetics of the Bcl2-2345 and Bcl2-1245 G4 structures. We report that the hybrid-stranded Bcl2-2345 G4 had a lower mechanical stability than the parallel-stranded Bcl2-1245 G4. We observed that the Bcl2-2345 G4 is a kinetically favored structure, whereas the Bcl2-1245 G4, with a slow unfolding rate, may function as a kinetic barrier for transcription. We also determined that in addition to the Bcl2-2345 and Bcl2-1245 G4s, other stable DNA secondary structures, such as a hybrid-stranded Bcl2-1234 G4, can also form in the Pu39 sequence. The characterization of the folding/unfolding kinetics of specific G4s reported here sheds light on the participation of G4s during gene transcription and provides information for designing G4-targeting small molecules that could modulate BCL-2 gene expression.

DPIF-Net: a dual path network for rural road extraction based on the fusion of global and local information.

Background: Automatic extraction of roads from remote sensing images can facilitate many practical applications. However, thus far, thousands of kilometers or more of roads worldwide have not been recorded, especially low-grade roads in rural areas. Moreover, rural roads have different shapes and are influenced by complex environments and other interference factors, which has led to a scarcity of dedicated low level category road datasets. Methods: To address these issues, based on convolutional neural networks (CNNs) and tranformers, this article proposes the Dual Path Information Fusion Network (DPIF-Net). In addition, given the severe lack of low-grade road datasets, we constructed the GaoFen-2 (GF-2) rural road dataset to address this challenge, which spans three regions in China and covers an area of over 2,300 km, almost entirely composed of low-grade roads. To comprehensively test the low-grade road extraction performance and generalization ability of the model, comparative experiments are carried out on the DeepGlobe, and Massachusetts regular road datasets. Results: The results show that DPIF-Net achieves the highest IoU and F1 score on three datasets compared with methods such as U-Net, SegNet, DeepLabv3+, and D-LinkNet, with notable performance on the GF-2 dataset, reaching 0.6104 and 0.7608, respectively. Furthermore, multiple validation experiments demonstrate that DPIF-Net effectively preserves improved connectivity in low-grade road extraction with a modest parameter count of 63.9 MB. The constructed low-grade road dataset and proposed methods will facilitate further research on rural roads, which holds promise for assisting governmental authorities in making informed decisions and strategies to enhance rural road infrastructure.

Nonselective Intercalation of G-Quadruplex-Targeting Ligands into Double-Stranded DNA Quantified by Single-Molecule Stretching.

Most G-quadruplex (G4)-targeting ligands reported so far contain planar heteroaromatic groups and can intercalate into adjacent base pairs of double-stranded DNA (dsDNA). However, quantitative data on the binding number γ (ligands/bp) of G4 ligands that intercalate into long dsDNA remain lacking, which are essential for understanding the selectivity of G4 ligands. Here, using a single-molecule stretching assay based on the lengthening of dsDNA, we analyzed the dissociation constants and the binding number of eight most commonly used G4 ligands that intercalate into dsDNA. We showed that five ligands (CX-5461, BRACO-19, RHPS4, TrisQ, and Phen-DC3) intercalate into dsDNA avidly (Kd = 0.5-2.1 µM, saturated γ > 0.2 ligands/bp), which was similar to the typical dsDNA intercalator EB. Two bisquinolines, PDS and 360A, showed moderate intercalation ability (Kd = 22.5 and 48.7 µM) and γ < 0.01 ligands/bp in the presence of 1 µM ligands. Porphyrin NMM showed no intercalative binding even at 200 µM. Molecular docking and molecular dynamics simulations were carried out to further evaluate the intercalative binding of these G4 ligands with dsDNA by calculating the binding energies and π-π stacking probability.

Naringenin prevents non-alcoholic steatohepatitis by modulating the host metabolome and intestinal microbiome in MCD diet-fed mice.

Non-alcoholic steatohepatitis (NASH) is a severe inflammatory phase of the non-alcoholic fatty liver disease (NAFLD) spectrum and can progress to advanced stages of NAFLD if left untreated. This study uses multi-omics data to elucidate the underlying mechanism of naringenin's reported benefit in alleviating (NASH). Male mice were fed a NASH-inducing (methionine-choline-deficient) MCD diet with or without naringenin supplementation for 6 weeks. Naringenin prevented NASH-induced histopathological liver damage and reversed the abnormal levels of hepatic triglyceride (TG)/total cholesterol (TC), serum TG/TC, serum alanine aminotransferase/aspartate transaminase, and hepatic malondialdehyde and glutathione. Importantly, naringenin intervention significantly modulated the relative abundance of gut microbiota and the host metabolomic profile. We detected more than 700 metabolites in the serum and found that the gut genus levels of Anaeroplasma and the [Eubacterium] nodatum group were closely associated with xanthine, 2-picoline, and securinine, respectively. Tuzzerella alterations showed the highest number of associations with host endogenous metabolites such as FAHFA (8:0/10:0), FFA (20:2), carnitine C8:1, tridecanedioic acid, securinine, acetylvaline, DL-O-tyrosine, and Phe-Asn. This study indicates that the interplay between host serum metabolites and gut microbiota may contribute to the therapeutic effect of naringenin against NASH.

Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy.

G-quadruplexes (G4s) are stable secondary nucleic acid structures that play crucial roles in many fundamental biological processes. The folding/unfolding dynamics of G4 structures are associated with the replication and transcription regulation functions of G4s. However, many DNA G4 sequences can adopt a variety of topologies and have complex folding/unfolding dynamics. Determining the dynamics of G4s and their regulation by proteins remains challenging due to the coexistence of multiple structures in a heterogeneous sample. Here, in this mini-review, we introduce the application of single-molecule force-spectroscopy methods, such as magnetic tweezers, optical tweezers, and atomic force microscopy, to characterize the polymorphism and folding/unfolding dynamics of G4s. We also briefly introduce recent studies using single-molecule force spectroscopy to study the molecular mechanisms of G4-interacting proteins.

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.