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

The recent discovery of ultra-high binding affinities in cucurbit[7]uril (CB7)-based host-guest pairs in an aqueous environment has rendered CB7 a rather attractive material in analytical and biomedical applications. Due to the lack of a molecular platform that can follow the same host-guest complex during repetitive mechanical measurements, however, mechanical stabilities of the CB7 system have not been revealed, hindering its potential to rival widely used conjugation pairs, such as streptavidin-biotin. Here, we assembled a DNA template in which a flexible DNA linker was exploited to keep the host (CB7) and guest (adamantane) in proximity. This platform not only increased the efficiency of the single-molecule characterization in optical tweezers but also clearly revealed mechanical features of the same host-guest complex. We found that positively charged adamantane guest demonstrated higher mechanical stability (49 pN) than neutral adamantane (44 pN), a trend consistent with the chemical affinity between guest molecules and the CB7 host. Surprisingly, we found that a hexyl group adjacent to the adamantane served as a chaperone to assist the formation of the adamantane-CB7 pairs. The discovery of an unprecedented chaperone-assisted interaction mechanism provides new approaches to efficiently assemble host-guest-based supramolecules with increased mechanical stabilities, which can be exploited in various biomedical, biosensing, and materials fields.

Chaperone-Assisted Host-Guest Interactions Revealed by Single-Molecule Force Spectroscopy.

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.

Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins.

Chirality transmission in macromolecular domains.

Single-molecule mechanochemical sensing (SMMS) is a novel biosensing technique using mechanical force as a signal transduction mechanism. In the mechanochemical sensing, the chemical binding of an analyte molecule to a sensing template is converted to mechanical signals, such as tensile force, of the template. Since mechanical force can be conveniently monitored by single-molecule tools, such as optical tweezers, magnetic tweezers, or Atomic Force Microscopy, mechanochemical sensing is often carried out at the single molecule level. In traditional format of ensemble sensing, sensitivity can be achieved via chemical or electrical amplifications, which are materials intensive and time-consuming. To address these problems, in 2011, we used the principle of mechanochemical coupling in a single molecular template to detect single nucleotide polymorphism (SNP) in DNA fragments. The single-molecule sensitivity in such SMMS strategy allows to removing complex amplification steps, drastically conserving materials and increasing temporal resolution in the sensing. By placing many probing units throughout a single-molecule sensing template, SMMS can have orders of magnitude better efficiency in the materials usage (i.e., high ) with respect to the ensemble biosensing. The SMMS sensing probes also enable topochemical arrangement of different sensing units. By placing these units in a spatiotemporally addressable fashion,  have been demonstrated in our lab to detect an expandable set of microRNA targets. Because of the stochastic behavior of single-molecule binding, however, it is challenging for the SMMS to accurately report analyte concentrations in a fixed time window. While multivariate analysis has been shown to rectify background noise due to stochastic nature of single-molecule probes, a template containing an array of sensing units has shown ensemble average behaviors to address the same problem. In this so-called , collective mechanical transitions of many sensing units occur in the SMMS sensing probes, which allows accurate quantification of analytes. For the SMMS to function as a viable sensing approach readily adopted by biosensing communities, the future of the SMMS technique relies on the reduction in the complexity and cost of instrumentation to report mechanical signals. In this account, we first explain the mechanism and main features of the SMMS. We then specify basic elements employed in SMMS. Using DNA as an exemplary SMMS template, we further summarize different types of SMMS which present multiplexing capability and increased throughput. Finally, recent efforts to develop simple and affordable high throughput methods for force generation and measurement are discussed in this Account for potential usage in the mechanochemical sensing.

Single-Molecule Mechanochemical Sensing.

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
Chaperone-Assisted Host-Guest Interactions Revealed by Single-Molecule Force Spectroscopy. Journal of the American Chemical Society\| PubMed ID: 31679339	2019
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
Chirality transmission in macromolecular domains. Nature communications\| PubMed ID: 35013247	2022
Single-Molecule Mechanochemical Sensing. Accounts of chemical research\| PubMed ID: 35420417	2022
Single-molecule displacement assay reveals strong binding of polyvalent dendrimer ligands to telomeric G-quadruplex. Analytical biochemistry\| PubMed ID: 35500657	2022

Changpeng Hu

Biography

JoVE Journal Publications

Other Publications