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.

Single-Molecular Dissection of Liquid-Liquid Phase Transitions.

Interaction between peptides and nucleic acids is a ubiquitous process that drives many cellular functions, such as replications, transcriptions, and translations. Recently, this interaction has been found in liquid-liquid phase separation (LLPS), a process responsible for the formation of newly discovered membraneless organelles with a variety of biological functions inside cells. In this work, we studied the molecular interaction between the poly-l-lysine (PLL) peptide and nucleic acids during the early stage of an LLPS process at the single-molecule level using optical tweezers. By monitoring the mechanical tension of individual nucleic acid templates upon PLL addition, we revealed a multistage LLPS process mediated by the long-range interactions between nucleic acids and polyelectrolytes. By varying different types (ssDNA, ssRNA, and dsDNA) and sequences (A-, T-, G-, or U-rich) of nucleic acids, we pieced together transition diagrams of the PLL-nucleic acid condensates from which we concluded that the propensity to form rigid nucleic acid-PLL complexes reduces the condensate formation during the LLPS process. We anticipate that these results are instrumental in understanding the transition mechanism of LLPS condensates, which allows new strategies to interfere with the biological functions of LLPS condensates inside cells.

Amalgamation of DNAzymes and Nanozymes in a Coronazyme.

Artificial enzymes such as nanozymes and DNAzymes are economical and stable alternatives to natural enzymes. By coating Au nanoparticles (AuNPs) with a DNA corona (AuNP@DNA), we amalgamated nanozymes and DNAzymes into a new artificial enzyme with catalytic efficiency 5 times higher than AuNP nanozymes, 10 times higher than other nanozymes, and significantly greater than most of the DNAzymes on the same oxidation reaction. The AuNP@DNA demonstrates excellent specificity as its reactivity on a reduction reaction does not change with respect to pristine AuNP. Single-molecule fluorescence and force spectroscopies and density functional theory (DFT) simulations indicate a long-range oxidation reaction initiated by radical production on the AuNP surface, followed by radical transport to the DNA corona, where the binding and turnover of substrates take place. The AuNP@DNA is named coronazyme because of its natural enzyme mimicking capability through the well-orchestrated structures and synergetic functions. By incorporating different nanocores and corona materials beyond DNAs, we anticipate that the coronazymes represent generic enzyme mimics to carry out versatile reactions in harsh environments.

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.

Mechanical Stability of DNA Corona Phase on Gold Nanospheres.

Noncovalent adsorption of biopolymers on the surface of gold nanoparticles (AuNPs) forms a corona phase that drastically diversify AuNP functions. However, mechanical stabilities of such corona phase are still obscure, hindering the application of biopolymer-coated AuNPs. Here, using optical tweezers, we have observed, for the first time, that DNA corona phase adsorbed on a 5 nm AuNP via two (dA)21 strands in proximity can withstand an average desorption force of 40 pN, which is higher than the stall force of DNA/RNA polymerases. This suggests a new role for AuNPs to modulate replications or transcriptions after binding to prevalent poly(dA) segments in eukaryotic genomes. We have also revealed that with increasing AuNP size (1.8-10 nm), DNA corona becomes harder to remove, likely due to the larger surfaces and flatter facets on bigger AuNPs. These findings provide guidance to design AuNP corona that can withstand harsh environments for biological and materials applications.

Ensemble Force Spectroscopy of a G-Quadruplex Cluster on a Single-Molecule Platform.

Single-molecule methods offer high sensitivities with precisions superior to bulk assays. However, these methods are low in throughput and cannot repetitively interrogate the same cluster of molecular units. In this work, we investigate a tandem array of G-quadruplexes on a single-molecule DNA template with a throughput of at least two orders of magnitude higher than single-molecule force spectroscopy. During mechanical unfolding by optical tweezers, the array of G-quadruplexes experiences identical force, temperature, and ionic conditions, which not only reduce environmental noise but also render unfolding transitions indistinguishable among individual G-quadruplexes. The resultant ensemble behaviors are analyzed by scanning force diagrams, which reveals accurate F1/2 values, where 50% of G-quadruplexes are unfolded. Independent of the number of G-quadruplexes (n > 15) contained in a cluster, F1/2 can effectively evaluate G-quadruplex ligands in a new method called differential scanning forcemetry. When the same G-quadruplex cluster is subject to a series of constant forces in force-jump experiments, unfolding rate constants of G-quadruplexes can be effectively evaluated as a function of force. The high precision demonstrated in all of these measurements reflects the power of repetitive sampling on the same cluster of single-molecule entities under identical conditions. Since biomolecules such as DNA, RNA, and proteins can be conveniently incorporated in a tandem array, we anticipate that this ensemble assay on single-molecule entities (EASE) provides a generic means of ensemble force spectroscopy to amalgamate the accuracy of ensemble measurements with the precision of single-molecule methods.

Small Molecules Modulate Liquid-to-Solid Transitions in Phase-Separated Tau Condensates.

In Tau protein condensates formed by the Liquid-Liquid Phase Separation (LLPS) process, liquid-to-solid transitions lead to the formation of fibrils implicated in Alzheimer's disease. Here, by tracking two contacting Tau-rich droplets using a simple and nonintrusive video microscopy, we found that the halftime of the liquid-to-solid transition in the Tau condensate is affected by the Hofmeister series according to the solvation energy of anions. After dissecting functional groups of physiologically relevant small molecules using a multivariate approach, we found that charged groups facilitate the liquid-to-solid transition in a manner similar to the Hofmeister effect, whereas hydrophobic alkyl chains and aromatic rings inhibit the transition. Our results not only elucidate the driving force of the liquid-to-solid transition in Tau condensates, but also provide guidelines to design small molecules to modulate this important transition for many biological functions for the first time.

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.

Methylene blue accelerates liquid-to-gel transition of tau condensates impacting tau function and pathology.

Preventing tau aggregation is a potential therapeutic strategy in Alzheimer's disease and other tauopathies. Recently, liquid-liquid phase separation has been found to facilitate the formation of pathogenic tau conformations and fibrillar aggregates, although many aspects of the conformational transitions of tau during the phase transition process remain unknown. Here, we demonstrate that the tau aggregation inhibitor methylene blue promotes tau liquid-liquid phase separation and accelerates the liquid-to-gel transition of tau droplets independent of the redox activity of methylene blue. We further show that methylene blue inhibits the conversion of tau droplets into fibrils and reduces the cytotoxicity of tau aggregates. Although gelation slows down the mobility of tau and tubulin, it does not impair microtubule assembly within tau droplets. These findings suggest that methylene blue inhibits tau amyloid fibrillization and accelerates tau droplet gelation via distinct mechanisms, thus providing insights into the activity of tau aggregation inhibitors in the context of phase transition.

Ensemble Force Spectroscopy by Shear Forces.

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the lack of high throughput capabilities, the application of these techniques is limited in biophysics. Ensemble force spectroscopy (EFS) has demonstrated high throughput in the investigation of a massive set of molecular structures by converting mechanochemical studies of individual molecules into those of molecular ensembles. In this protocol, the DNA secondary structures (i-motifs) were unfolded in the shear flow between the rotor and stator of a homogenizer tip at shear rates up to 77796/s. The effects of flow rates and molecular sizes on the shear forces experienced by the i-motif were demonstrated. The EFS technique also revealed the binding affinity between DNA i-motifs and ligands. Furthermore, we have demonstrated a click chemistry reaction that can be actuated by shear force (i.e., mechano-click chemistry). These results establish the effectiveness of using shear force to control the conformation of molecular structures.

Machine-Learning-Assisted Analysis of Colorimetric Assays on Paper Analytical Devices.

Paper-based analytical devices (PADs) employing colorimetric detection and smartphone images have gained wider acceptance in a variety of measurement applications. PADs are primarily meant to be used in field settings where assay and imaging conditions greatly vary, resulting in less accurate results. Recently, machine-learning (ML)-assisted models have been used in image analysis. We evaluated a combination of four ML models-logistic regression, support vector machine (SVM), random forest, and artificial neural network (ANN)-as well as three image color spaces, RGB, HSV, and LAB, for their ability to accurately predict analyte concentrations. We used images of PADs taken at varying lighting conditions, with different cameras and users for food color and enzyme inhibition assays to create training and test datasets. The prediction accuracy was higher for food color than enzyme inhibition assays in most of the ML models and color space combinations. All models better predicted coarse-level classifications than fine-grained concentration classes. ML models using the sample color along with a reference color increased the models' ability to predict the result in which the reference color may have partially factored out the variation in ambient assay and imaging conditions. The best concentration class prediction accuracy obtained for food color was 0.966 when using the ANN model and LAB color space. The accuracy for enzyme inhibition assay was 0.908 when using the SVM model and LAB color space. Appropriate models and color space combinations can be useful to analyze large numbers of samples on PADs as a powerful low-cost quick field-testing tool.

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.

Detecting the Coronavirus (COVID-19).

The COVID-19 pandemic has created huge damage to society and brought panic around the world. Such panic can be ascribed to the seemingly deceptive features of COVID-19: Compared to other deadly viral outbreaks, it has medium transmission and mortality rates. As a result, the severity of the causative coronavirus, SARS-CoV-2, was deeply underestimated by society at the beginning of the COVID-19 outbreak. Based on this, in this review, we define the viruses with features similar to those of SARS-CoV-2 as the Panic Zone viruses. To contain those viruses, accurate and fast diagnosis followed by effective isolation and treatment of patients are pivotal at the early stage of virus breakouts. This is especially true when there is no cure or vaccine available for a transmissible disease, which is the case for the current COVID-19 pandemic. As of July 2020, more than 100 kits for COVID-19 diagnosis on the market have been surveyed in this review, while emerging sensing techniques for SARS-CoV-2 are also discussed. It is of critical importance to rationally use these kits for efficient management and control of the Panic Zone viruses. Therefore, we discuss guidelines to select diagnostic kits at different outbreak stages of the Panic Zone viruses, SARS-CoV-2 in particular. While it is of utmost importance to use nucleic acid based detection kits with low false negativity (high sensitivity) at the early stage of an outbreak, the low false positivity (high specificity) gains importance at later stages of the outbreak. When society is set to reopen from the lockdown stage of the COVID-19 pandemic, it becomes critical to have immunoassay based kits with high specificity to identify people who can safely return to society after their recovery from SARS-CoV-2 infections. Finally, since a massive attack from a viral pandemic requires a massive defense from the whole society, we urge both government and the private sector to research and develop affordable and reliable point-of-care testing (POCT) kits, which can be used massively by the general public (and therefore called massive POCT) to contain Panic Zone viruses in the future.

Selection of appropriate protein assay method for a paper microfluidics platform.

BACKGROUND: Paper-analytical devices (PADs) have gained popularity as a simple and low-cost alternative for determining a wide range of analytes including proteins. Even though several colorimetric PADs methods for protein estimation are reported in literature, they lack justification for the chosen method and parameters therein. AIM: Major aim of this work was to thoroughly evaluate the most commonly used colorimetric protein assays and recommend the most appropriate method for PADs platform. METHOD: We performed following six colorimetric protein assays on PADs: biuret, lowry, bicinchoninic acid, bradford, bromocresol green, and tetrabromophenol blue. We obtained assay signal by analyzing images of the PADs and then assessed analytical figures of merit. RESULT: Precision, accuracy, LOD, and LOQ of PADs protein assay methods ranged from 1.2 to 6.4%, 73.3-102.4%, 0.3-3.8 â€‹mg/mL, and 1.2-12.8 â€‹mg/mL, respectively. Out of six methods, we determined bromocresol green and tetrabromophenol blue as the best methods for serum and urine samples, respectively based on their optimized parameters and analytical figures of merit. The total average serum and urine protein in human samples were found to be 94.6 â€‹± â€‹16.2 â€‹mg/mL and 2.1 â€‹± â€‹1.5 â€‹mg/mL, respectively using PADs methods. The PADs result on human samples moderately correlated with the results from spectrophotometric determination (r2 â€‹> â€‹0.6). CONCLUSION: Paper-based protein assays were easy to perform and were completed with thousand-fold less volume of samples/reagents without a spectrophotometer compared to conventional assay methods. After testing human samples, we found one protein assay method may not be appropriate for all types of samples.

Evaluation of secondary metabolites, antioxidant activity, and color parameters of Nepali wines.

We evaluated the quality of wines produced in Nepal in terms of phenolic, flavonoid, anthocyanin and tannin content, antioxidant capacity, and color parameters using spectrophotometric methods. The total phenolic content, total flavonoid content, and total antioxidant activities in Nepali wines ranged from 85.5 to 960.0 (mean = 360.5 ± 268.7) mg/L GAE, 40.9-551.3 (mean = 188.9 ± 161.5) mg/L QE, and 66.6-905.0 (mean = 332.8 ± 296.5) mg/L AAE, respectively. These parameters were significantly higher in red wines compared to white wines. The phenolic and flavonoid content showed strong correlation with each other as well as with antioxidant activities. Additional parameters measured included various color parameters and carbohydrates. The wine color showed strong correlation with phenol, flavonoid, and antioxidant activity, whereas this correlation was not significant with anthocyanin content. Multivariate analysis was carried out to better describe and discriminate the wine samples. Finally, we compared Nepali wines with wines from other countries.