A TeZla micromixer for interrogating the early and broad folding landscape of G-quadruplex via multistage velocity descending.

Biomacromolecular folding kinetics involves fast folding events and broad timescales. Current techniques face limitations in either the required time resolution or the observation window. In this study, we developed the TeZla micromixer, integrating Tesla and Zigzag microstructures with a multistage velocity descending strategy. TeZla achieves a significant short mixing dead time (40 µs) and a wide time window covering four orders of magnitude (up to 300 ms). Using this unique micromixer, we explored the folding landscape of c-Myc G4 and its noncanonical-G4 derivatives with different loop lengths or G-vacancy sites. Our findings revealed that c-Myc can bypass folding intermediates and directly adopt a G4 structure in the cation-deficient buffer. Moreover, we found that the loop length and specific G-vacancy site could affect the folding pathway and significantly slow down the folding rates. These results were also cross-validated with real-time NMR and circular dichroism. In conclusion, TeZla represents a versatile tool for studying biomolecular folding kinetics, and our findings may ultimately contribute to the design of drugs targeting G4 structures.

Force-enhanced sensitive and specific detection of DNA-intercalative agents directly from microorganisms at single-molecule level.

Microorganisms can produce a vast array of bioactive secondary metabolites, including DNA-intercalating agents like actinomycin D, doxorubicin, which hold great potential for cancer chemotherapy. However, discovering novel DNA-intercalating compounds remains challenging due to the limited sensitivity and specificity of conventional activity assays, which require large-scale fermentation and purification. Here, we introduced the single-molecule stretching assay (SMSA) directly to microbial cultures or extracts for discovering DNA-intercalating agents, even in trace amounts of microbial cultures (5 µl). We showed that the unique changes of dsDNA in contour length and overstretching transition enable the specific detection of intercalators from complex samples without the need for extensive purification. Applying force to dsDNA also enhanced the sensitivity by increasing both the binding affinity Ka and the quantity of ligands intercalation, thus allowing the detection of weak intercalators, which are often overlooked using traditional methods. We demonstrated the effectiveness of SMSA, identified two DNA intercalator-producing strains: Streptomyces tanashiensis and Talaromyces funiculosus, and isolated three DNA intercalators: medermycin, kalafungin and ligustrone B. Interestingly, both medermycin and kalafungin, classified as weak DNA intercalators (Ka ∼103 M-1), exhibited potent anti-cancer activity against HCT-116 cancer cells, with IC50 values of 52 ± 6 and 70 ± 7 nM, respectively.

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.

Detecting the Formation Kinetics of Doxorubicin-DNA Interstrand Cross-link at the Single-Molecule Level and Clinically Relevant Concentrations of Doxorubicin.

Doxorubicin (DOX) ranks among the most effective anticancer agents. Increasing the formation of covalent DOX-DNA interstrand cross-links can improve the anticancer activity of DOX. However, due to the low stability of the DOX-DNA cross-links to heat and alkali, DOX can be extensively lost during isolation procedures of biochemical methods, thus reducing the apparent clinical relevance of this mechanism. Here, we developed a drug label-free, single-molecule magnetic tweezers assay that can detect a single DOX-DNA cross-link on the basis of the significant increase of the unzipping forces of DNA hairpins upon drug binding. Using this assay, we measured the DOX concentration-dependent cross-linking rates at clinically relevant concentrations of DOX. We report an ∼26-fold higher formaldehyde concentration dependence of cross-linking rates than previously reported and 0.9 ± 0.8 cross-links/103 bp at the clinically relevant concentrations of 70 nM DOX and 50 µM formaldehyde. Our results suggest a much higher cross-link formation ability than previous bulk measurements have reported and suggest that the cross-linking mechanism has promising therapeutic potential. This general method can be used to detect the formation kinetics of other DNA lesions or DNA adducts that affect DNA duplex stability.

ZYH005, a novel DNA intercalator, overcomes all-trans retinoic acid resistance in acute promyelocytic leukemia.

Despite All-trans retinoic acid (ATRA) has transformed acute promyelocytic leukemia (APL) from the most fatal to the most curable hematological cancer, there remains a clinical challenge that many high-risk APL patients who fail to achieve a complete molecular remission or relapse and become resistant to ATRA. Herein, we report that 5-(4-methoxyphenethyl)-[1, 3] dioxolo [4, 5-j] phenanthridin-6(5H)-one (ZYH005) exhibits specific anticancer effects on APL and ATRA-resistant APL in vitro and vivo, while shows negligible cytotoxic effect on non-cancerous cell lines and peripheral blood mononuclear cells from healthy donors. Using single-molecule magnetic tweezers and molecule docking, we demonstrate that ZYH005 is a DNA intercalator. Further mechanistic studies show that ZYH005 triggers DNA damage, and caspase-dependent degradation of the PML-RARa fusion protein. As a result, APL and ATRA-resistant APL cells underwent apoptosis upon ZYH005 treatment and this apoptosis-inducing effect is even stronger than that of arsenic trioxide and anticancer agents including 5-fluorouracil, cisplatin and doxorubicin. Moreover, ZYH005 represses leukemia development in vivo and prolongs the survival of both APL and ATRA-resistant APL mice. To our knowledge, ZYH005 is the first synthetic phenanthridinone derivative, which functions as a DNA intercalator and can serve as a potential candidate drug for APL, particularly for ATRA-resistant APL.

The Mechanical Properties of RNA-DNA Hybrid Duplex Stretched by Magnetic Tweezers.

RNA can anneal to its DNA template to generate an RNA-DNA hybrid (RDH) duplex and a displaced DNA strand, termed R-loop. RDH duplex occupies up to 5% of the mammalian genome and plays important roles in many biological processes. The functions of RDH duplex are affected by its mechanical properties, including the elasticity and the conformation transitions. The mechanical properties of RDH duplex, however, are still unclear. In this work, we studied the mechanical properties of RDH duplex using magnetic tweezers in comparison with those of DNA and RNA duplexes with the same sequences. We report that the contour length of RDH duplex is ∼0.30 nm/bp, and the stretching modulus of RDH duplex is ∼660 pN, neither of which is sensitive to NaCl concentration. The persistence length of RDH duplex depends on NaCl concentration, decreasing from ∼63 nm at 1 mM NaCl to ∼49 nm at 500 mM NaCl. Under high tension of ∼60 pN, the end-opened RDH duplex undergoes two distinct overstretching transitions; at high salt in which the basepairs are stable, it undergoes the nonhysteretic transition, leading to a basepaired elongated structure, whereas at low salt, it undergoes a hysteretic peeling transition, leading to the single-stranded DNA strand under force and the single-stranded RNA strand coils. The peeled RDH is difficult to reanneal back to the duplex conformation, which may be due to the secondary structures formed in the coiled single-stranded RNA strand. These results help us understand the full picture of the structures and mechanical properties of nucleic acid duplexes in solution and provide a baseline for studying the interaction of RDH with proteins at the single-molecule level.

RHAU helicase stabilizes G4 in its nucleotide-free state and destabilizes G4 upon ATP hydrolysis.

The DEAH-box ATP-dependent RHAU helicases specifically unfold RNA and DNA G-quadruplexes (G4s). However, it remains unclear how the RHAU's G4 unfolding activity is coupled to different stages of the ATPase cycle. Here, using a single-molecule manipulation approach, we show that binding of Drosophila RHAU stabilizes an intramolecularly folded parallel DNA G4 against mechanical unfolding in its nucleotide-free and in its AMP-PNP or ADP bound states, while it destabilizes the G4 when coupled to ATP hydrolysis. Importantly, our results show that the ADP·AlF[Formula: see text]-bound RHAU does not stabilize the G4. We also found that both a single-stranded 3' DNA tail and the RSM domain of RHAU that binds specifically to the G4 structure, are dispensable for the stabilization of the G4, but both are required for G4 destabilization. Our study provides the first evidence that the unfolding kinetics of a G-quadruplex can be modulated by different nucleotide-bound states of the helicase.

Single-molecule imaging and manipulation of biomolecular machines and systems.

BACKGROUND: Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration. SCOPE OF REVIEW: We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems. MAJOR CONCLUSIONS: Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines. GENERAL SIGNIFICANCE: Quantitative, high-resolution single-molecule analyses of biomolecular systems at the various hierarchies of life will help to answer our fundamental question: "What is life?" This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.

Dynamics and stability of polymorphic human telomeric G-quadruplex under tension.

As critical DNA structures capping the human chromosome ends, the stability and structural polymorphism of human telomeric G-quadruplex (G4) have drawn increasing attention in recent years. This work characterizes the equilibrium transitions of single-molecule telomeric G4 at physiological K(+) concentration. We report three folded states of telomeric G4 with markedly different lifetime and mechanical stability. Our results show that the kinetically favored folding pathway is through a short-lived intermediate state to a longer-lived state. By examining the force dependence of transition rates, the force-dependent transition free energy landscape for this pathway is determined. In addition, an ultra-long-lived form of telomeric G4 structure with a much stronger mechanical stability is identified.

Torque transmission mechanism via DELSEED loop of F1-ATPase.

F1-ATPase (F1) is an ATP-driven rotary motor in which the three catalytic ß subunits in the stator ring sequentially induce the unidirectional rotation of the rotary γ subunit. Many lines of evidence have revealed open-to-closed conformational transitions in the ß subunit that swing the C-terminal domain inward. This conformational transition causes a C-terminal protruding loop with conserved sequence DELSEED to push the γ subunit. Previous work, where all residues of DELSEED were substituted with glycine to disrupt the specific interaction with γ and introduce conformational flexibility, showed that F1 still rotated, but that the torque was halved, indicating a remarkable impact on torque transmission. In this study, we conducted a stall-and-release experiment on F1 with a glycine-substituted DELSEED loop to investigate the impact of the glycine substitution on torque transmission upon ATP binding and ATP hydrolysis. The mutant F1 showed a significantly reduced angle-dependent change in ATP affinity, whereas there was no change in the equilibrium for ATP hydrolysis. These findings indicate that the DELSEED loop is predominantly responsible for torque transmission upon ATP binding but not for that upon ATP hydrolysis.

Stability and kinetics of c-MYC promoter G-quadruplexes studied by single-molecule manipulation.

A DNA G-quadruplex (G4) formed at the oncogene c-MYC promoter region functions as a gene silencer. Due to its high stability at physiological K(+) concentrations, its thermodynamics and kinetic properties have not been characterized in physiological solution conditions. In this work, we investigated the unfolding and folding transitions of single c-MYC G4 and several of its truncated or point mutants at 100 mM KCl concentration under mechanical force. We found that the wild type could fold into multiple species, and the major specie has a slow unfolding rate of (1.4 ± 1.0) × 10(-6) s(-1). The force-dependent thermodynamics and kinetic properties of the major specie were obtained by studying a truncated mutant, Myc2345, that contains the G-tracts 2, 3, 4, and 5. As the c-MYC G4 is a prototype of many other intermolecular parallel-stranded G4's, our results provide important insights into the stability of a broad class of promoter G4's which also play a role in transcription regulation and are potential anticancer targets.

Winding single-molecule double-stranded DNA on a nanometer-sized reel.

A molecular system of a nanometer-sized reel was developed from F(1)-ATPase, a rotary motor protein. By combination with magnetic tweezers and optical tweezers, single-molecule double-stranded DNA (dsDNA) was wound around the molecular reel. The bending stiffness of dsDNA was determined from the winding tension (0.9-6.0 pN) and the diameter of the wound loop (21.4-8.5 nm). Our results were in good agreement with the conventional worm-like chain model and a persistence length of 54 ± 9 nm was estimated. This molecular reel system offers a new platform for single-molecule study of micromechanics of sharply bent DNA molecules and is expected to be applicable to the elucidation of the molecular mechanism of DNA-associating proteins on sharply bent DNA strands.

Unfolding rates of 1:1 and 2:1 complex of CX-5461 and c-MYC promoter G-quadruplexes revealed by single-molecule force spectroscopy.

CX-5461, also known as pidnarulex, is a strong G4 stabilizer and has received FDA fast-track designation for BRCA1- and BRCA2- mutated cancers. However, quantitative measurements of the unfolding rates of CX-5461-G4 complexes which are important for the regulation function of G4s, remain lacking. Here, we employ single-molecule magnetic tweezers to measure the unfolding force distributions of c-MYC G4s in the presence of different concentrations of CX-5461. The unfolding force distributions exhibit three discrete levels of unfolding force peaks, corresponding to three binding modes. In combination with a fluorescent quenching assay and molecular docking to previously reported ligand-c-MYC G4 structure, we assigned the ~69 pN peak corresponding to the 1:1 (ligand:G4) complex where CX-5461 binds at the G4's 5'-end. The ~84 pN peak is attributed to the 2:1 complex where CX-5461 occupies both the 5' and 3'. Furthermore, using the Bell-Arrhenius model to fit the unfolding force distributions, we determined the zero-force unfolding rates of 1:1, and 2:1 complexes to be (2.4 ± 0.9) × 10-8 s-1 and (1.4 ± 1.0) × 10-9 s-1 respectively. These findings provide valuable insights for the development of G4-targeted ligands to combat c-MYC-driven cancers.

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.

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.

Using Magnetic Tweezers to Unravel the Mechanism of the G-quadruplex Binding and Unwinding Activities of DHX36 Helicase.

Single-molecule manipulation methods are useful techniques to probe the interactions of proteins and nucleic acid structures. Here, we describe the magnetic tweezers-based single-molecule investigation of the binding of helicases to G-quadruplex structures and their ATP-dependent unwinding activity, using DHX36 (also known as RHAU and G4R1) helicase and a DNA G-quadruplex structure for an example. We specifically emphasize on the principle and method to probe the interactions between DHX36 and the DNA G-quadruplex in different intermediate states during an ATPase cycle of DHX36, based on detecting the DHX36-induced changes in the lifetime of the DNA G-quadruplex under tension. The principle of the measurement can be broadly extended to the studies of other DNA or RNA G-quadruplex helicases.