Catalytic Relaxation of Kinetically Trapped Intermediates by DNA Chaperones.

Common in biomacromolecules, kinetically trapped misfolded intermediates are often detrimental to the structures, properties, or functions of proteins or nucleic acids. Nature employs chaperone proteins but not nucleic acids to escort intermediates to correct conformations. Herein, we constructed a Jablonski-like diagram of a mechanochemical cycle in which individual DNA hairpins were mechanically unfolded to high-energy states, misfolded into kinetically trapped states, and catalytically relaxed back to ground-state hairpins by a DNA chaperone. The capacity of catalytic relaxation was demonstrated in a 1D DNA hairpin array mimicking nanoassembled materials. At ≥1 µM, the diffusive (or self-walking) DNA chaperone converted the entire array of misfolded intermediates to correct conformation in less than 15 s, which is essential to rapidly prepare homogeneous nanoassemblies. Such an efficient self-walking amplification increases the signal-to-noise ratio, facilitating catalytic relaxation to recognize a 1 fM DNA chaperone in 10 min, a detection limit comparable to the best biosensing strategies.

Dynamic Structures and Fast Transition Kinetics of Oxidized G-Quadruplexes.

8-oxoguanines (8-oxoG) in cells form compromised G-quadruplexes (GQs), which may vary GQ mediated gene regulations. By mimicking molecularly crowded cellular environment using 40% DMSO or sucrose, here it is found that oxidized human telomeric GQs have stabilities close to the wild-type (WT) GQs. Surprisingly, while WT GQs show negative formation cooperativity between a Pt(II) binder and molecularly crowded environment, positive cooperativity is observed for oxidized GQ formation. Single-molecule mechanical unfolding reveals that 8-oxoG sequence formed more diverse and flexible structures with faster folding/unfolding transition kinetics, which facilitates the Pt(II) ligand to bind the best-fit structures with positive cooperativity. These findings offer new understanding on structures and properties of oxidized G-rich species in crowded environments. They also provide insights into the design of better ligands to target oxidized G-rich structures formed under oxidative cell stress.

Chemo-mechanical forces modulate the topology dynamics of mesoscale DNA assemblies.

The intrinsic complexity of many mesoscale (10-100 nm) cellular machineries makes it challenging to elucidate their topological arrangement and transition dynamics. Here, we exploit DNA origami nanospring as a model system to demonstrate that tens of piconewton linear force can modulate higher-order conformation dynamics of mesoscale molecular assemblies. By switching between two chemical structures (i.e., duplex and tetraplex DNA) in the junctions of adjacent origami modules, the corresponding stretching or compressing chemo-mechanical stress reversibly flips the backbone orientations of the DNA nanosprings. Both coarse-grained molecular dynamics simulations and atomic force microscopy measurements reveal that such a backbone conformational switch does not alter the right-handed chirality of the nanospring helix. This result suggests that mesoscale helical handedness may be governed by the torque, rather than the achiral orientation, of nanospring backbones. It offers a topology-based caging/uncaging concept to present chemicals in response to environmental cues in solution.

Single-molecule displacement assay reveals strong binding of polyvalent dendrimer ligands to telomeric G-quadruplex.

Binding between a ligand and a receptor is a fundamental step in many natural or synthetic processes. In biosensing, a tight binding with a small dissociation constant (Kd) between the probe and analyte can lead to superior specificity and sensitivity. Owing to their capability of evaluating competitors, displacement assays have been used to estimate Kd at the ensemble average level. At the more sensitive single-molecule level, displacement assays are yet to be established. Here, we developed a single-molecule displacement assay (smDA) in an optical tweezers instrument and used this innovation to evaluate the binding of the L2H2-6OTD ligands to human telomeric DNA G-quadruplexes. After measuring Kd of linear and dendrimer L2H2-6OTD ligands, we found that dendrimer ligands have enhanced binding affinity to the G-quadruplexes due to their polyvalent geometry. This increased binding affinity enhanced inhibition of telomerase elongation on a telomere template in a Telomerase Repeated Amplification Protocol (TRAP). Our experiments demonstrate that the smDA approach can efficiently evaluate binding processes in chemical and biological processes.

DNA origami nano-mechanics.

Invention of DNA origami has transformed the fabrication and application of biological nanomaterials. In this review, we discuss DNA origami nanoassemblies according to their four fundamental mechanical properties in response to external forces: elasticity, pliability, plasticity and stability. While elasticity and pliability refer to reversible changes in structures and associated properties, plasticity shows irreversible variation in topologies. The irreversible property is also inherent in the disintegration of DNA nanoassemblies, which is manifested by its mechanical stability. Disparate DNA origami devices in the past decade have exploited the mechanical regimes of pliability, elasticity, and plasticity, among which plasticity has shown its dominating potential in biomechanical and physiochemical applications. On the other hand, the mechanical stability of the DNA origami has been used to understand the mechanics of the assembly and disassembly of DNA nano-devices. At the end of this review, we discuss the challenges and future development of DNA origami nanoassemblies, again, from these fundamental mechanical perspectives.

Correction: DNA origami nano-mechanics.

Correction for 'DNA origami nano-mechanics' by Jiahao Ji et al., Chem. Soc. Rev., 2021, DOI: 10.1039/d1cs00250c.

Mechanical unfolding of ensemble biomolecular structures by shear force.

Mechanical unfolding of biomolecular structures has been exclusively performed at the single-molecule level by single-molecule force spectroscopy (SMFS) techniques. Here we transformed sophisticated mechanical investigations on individual molecules into a simple platform suitable for molecular ensembles. By using shear flow inside a homogenizer tip, DNA secondary structures such as i-motifs are unfolded by shear force up to 50 pN at a 77 796 s-1 shear rate. We found that the larger the molecules, the higher the exerted shear forces. This shear force approach revealed affinity between ligands and i-motif structures. It also demonstrated a mechano-click reaction in which a Cu(i) catalyzed azide-alkyne cycloaddition was modulated by shear force. We anticipate that this ensemble force spectroscopy method can investigate intra- and inter-molecular interactions with the throughput, accuracy, and robustness unparalleled to those of SMFS methods.

Mechanochemical properties of DNA origami nanosprings revealed by force jumps in optical tweezers.

By incorporating pH responsive i-motif elements, we have constructed DNA origami nanosprings that respond to pH changes in the environment. Using an innovative force jump approach in optical tweezers, we have directly measured the spring constants and dynamic recoiling responses of the DNA nanosprings under different forces. These DNA nanosprings exhibited 3 times slower recoiling rates compared to duplex DNA backbones. In addition, we observed two distinct force regions which show different spring constants. In the entropic region below 2 pN, a spring constant of ∼0.03 pN nm-1 was obtained, whereas in the enthalpic region above 2 pN, the nanospring was 17 times stronger (0.5 pN nm-1). The force jump gave a more accurate measurement on nanospring constants compared to regular force ramping approaches, which only yielded an average spring constant in a specific force range. Compared to the reported DNA origami nanosprings with a completely different design, our nanospring is up to 50 times stiffer. The drastic increase in the spring constant and the pH responsive feature allow more robust applications of these nanosprings in many mechanobiological processes.

Chemo-Mechanical Modulation of Cell Motions Using DNA Nanosprings.

Cell motions such as migration and change in cellular morphology are essential activities for multicellular organism in response to environmental stimuli. These activities are a result of coordinated clustering/declustering of integrin molecules at the cell membrane. Here, we prepared DNA origami nanosprings to modulate cell motions by targeting the clustering of integrin molecules. Each nanospring was modified with arginyl-glycyl-aspartic acid (RGD) domains with a spacing such that when the nanospring is coiled, the RGD ligands trigger the clustering of integrin molecules, which changes cell motions. The coiling or uncoiling of the nanospring is controlled, respectively, by the formation or dissolution of an i-motif structure between neighboring piers in the DNA origami nanodevice. At slightly acidic pH (<6.5), the folding of the i-motif leads to the coiling of the nanospring, which inhibits the motion of HeLa cells. At neutrality (pH 7.4), the unfolding of the i-motif allows cells to resume mechanical movement as the nanospring becomes uncoiled. We anticipate that this pH-responsive DNA nanoassembly is valuable to inhibit the migration of metastatic cancer cells in acidic extracellular environment. Such a chemo-mechanical modulation provides a new mechanism for cells to mechanically respond to endogenous chemical cues.

Ensemble Sensing Using Single-Molecule DNA Copolymers.

While single-molecule sensing has offered ultimate mass sensitivity at the precision of individual molecules, it requires a longer time to detect analytes at lower concentrations when analyte binding to single-molecule probes becomes diffusion-limited. Here, we solved this accuracy problem in the concentration sensitivity determination by using single-molecule DNA homopolymers, in which up to 473 identical sensing elements (DNA hairpins) were introduced by rolling circle amplification. Surprisingly, the DNA homopolymers containing as few as 10 tandem hairpins displayed ensemble unfolding/refolding transitions, which were exploited to recognize microRNAs (miRNAs) that populated unfolded hairpins. Within 20 min, the femtomolar detection limit for miRNAs was observed, 6 orders of magnitude better than standalone hairpins. By incorporating different hairpin probes in an alternating DNA copolymer, multiplex recognition of different miRNAs was demonstrated. These DNA co-polymers represent new materials for innovative sensing strategies that combine the single-molecule precision with the accuracy of ensemble assays to determine concentration sensitivities.

Duplex DNA Is Weakened in Nanoconfinement.

For proteins and DNA secondary structures such as G-quadruplexes and i-motifs, nanoconfinement can facilitate their folding and increase structural stabilities. However, the properties of the physiologically prevalent B-DNA duplex have not been elucidated inside the nanocavity. Using a 17-bp DNA duplex in the form of a hairpin stem, here, we probed folding and unfolding transitions of the hairpin DNA duplex inside a DNA origami nanocavity. Compared to the free solution, the DNA hairpin inside the nanocage with a 15 × 15 nm cross section showed a drastic decrease in mechanical (20 → 9 pN) and thermodynamic (25 → 6 kcal/mol) stabilities. Free energy profiles revealed that the activation energy of unzipping the hairpin DNA duplex decreased dramatically (28 → 8 kcal/mol), whereas the transition state moved closer to the unfolded state inside the nanocage. All of these indicate that nanoconfinement weakens the stability of the hairpin DNA duplex to an unexpected extent. In a DNA hairpin made of a stem that contains complementary telomeric G-quadruplex (GQ) and i-motif (iM) forming sequences, formation of the Hoogsteen base pairs underlining the GQ or iM is preferred over the Watson-Crick base pairs in the DNA hairpin. These results shed light on the behavior of DNA in nanochannels, nanopores, or nanopockets of various natural or synthetic machineries. It also elucidates an alternative pathway to populate noncanonical DNA over B-DNA in the cellular environment where the nanocavity is abundant.

Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3).

Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe3O4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe3O4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe3O4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe3O4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles. Schematic representation of the conjugation, characterization and cytotoxicity analysis of Fe3O4-ZnO magnetic composite particles (MCPs).

Enhanced preferential cytotoxicity through surface modification: synthesis, characterization and comparative in vitro evaluation of TritonX-100 modified and unmodified zinc oxide nanoparticles in human breast cancer cell (MDA-MB-231).

BACKGROUND: Nanoparticles (NPs) are receiving increasing interest in biomedical research owing to their comparable size with biomolecules, novel properties and easy surface engineering for targeted therapy, drug delivery and selective treatment making them a better substituent against traditional therapeutic agents. ZnO NPs, despite other applications, also show selective anticancer property which makes it good option over other metal oxide NPs. ZnO NPs were synthesized by chemical precipitation technique, and then surface modified using Triton X-100. Comparative study of cytotoxicity of these modified and unmodified NPs on breast cancer cell line (MDA-MB-231) and normal cell line (NIH 3T3) were carried out. RESULTS: ZnO NPsof average size 18.67 ± 2.2 nm and Triton-X modified ZnO NPs of size 13.45 ± 1.42 nm were synthesized and successful characterization of synthesized NPs was done by Fourier transform infrared spectroscopy (FT-IR), X-Ray diffraction (XRD), transmission electron microscopy (TEM) analysis. Surface modification of NPs was proved by FT-IR analysis whereas structure and size by XRD analysis. Morphological analysis was done by TEM. Cell viability assay showed concentration dependent cytotoxicity of ZnO NPs in breast cancer cell line (MDA-MB-231) whereas no positive correlation was found between cytotoxicity and increasing concentration of stress in normal cell line (NIH 3T3) within given concentration range. Half maximum effective concentration (EC50) value for ZnO NPs was found to be 38.44 µg/ml and that of modified ZnO NPs to be 55.24 µg/ml for MDA-MB-231. Crystal violet (CV) staining image showed reduction in number of viable cells in NPs treated cell lines further supporting this result. DNA fragmentation assay showed fragmented bands indicating that the mechanism of cytotoxicity is through apoptosis. CONCLUSIONS: Although use of surfactant decreases particle size, toxicity of modified ZnO NPs were still less than unmodified NPs on MDA-MB-231 contributed by biocompatible surface coating. Both samples show significantly less toxicity towards NIH 3T3 in concentration independent manner. But use of Triton-X, a biocompatible polymer, enhances this preferentiality effect. Since therapeutic significance should be analyzed through its comparative effect on both normal and cancer cells, possible application of biocompatible polymer modified nanoparticles as therapeutic agent holds better promise.Graphical abstractSurface coating, characterization and comparative in vitro cytotoxicity study on MDA-MB 231 and NIH 3T3 of ZnO NPs showing enhanced preferentiality by biocompatible surface modification.