ensemble-force-spectroscopy-by-shear-forces

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 ...

Kent State University

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.

Duplex DNA Is Weakened in Nanoconfinement.

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.

Detecting the Coronavirus (COVID-19).

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 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.

Mechanical unfolding of ensemble biomolecular structures by shear force.

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.

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

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 (K) 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 K 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 K 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.

Article	Year
Duplex DNA Is Weakened in Nanoconfinement. Journal of the American Chemical Society\| PubMed ID: 32383870	2020
Detecting the Coronavirus (COVID-19). ACS sensors\| PubMed ID: 32627534	2020
Mechanical unfolding of ensemble biomolecular structures by shear force. Chemical science\| PubMed ID: 34377405	2021
Small Molecules Modulate Liquid-to-Solid Transitions in Phase-Separated Tau Condensates. Angewandte Chemie (International ed. in English)\| PubMed ID: 35320624	2022
Single-molecule displacement assay reveals strong binding of polyvalent dendrimer ligands to telomeric G-quadruplex. Analytical biochemistry\| PubMed ID: 35500657	2022

Pravin Pokhrel

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