A Combined In Vitro and In Silico Approach to Estimate the Molecular Arrangement Within a Fibronectin Fiber

ASME 2011 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2011-53809 ◽

2011 ◽

Author(s):

Mark J. Bradshaw ◽

Michael L. Smith

Keyword(s):

Single Molecule ◽

Strain State ◽

Cell Adhesion Molecule ◽

Tertiary Structure ◽

Cell Behavior ◽

Single Molecule Force Spectroscopy ◽

Binding Partner ◽

Molecular Arrangement ◽

Cellular Mechanotransduction

It has become apparent that the extracellular matrix (ECM) is a powerful modulator of cell behavior. Fibronectin (Fn) is of particular interest because it is a requisite cell adhesion molecule for development and wound healing and it is a promiscuous binding partner for many soluble signaling molecules. It was recently shown that the binding affinity of some molecules is dependent on the strain state of the Fn [2,3], reinvigorating our interest in the molecular mechanism of Fn fiber extension. The tertiary structure of the approximately 30 Fn type III domains of the protein has been shown to be capable of unfolding in single molecule force spectroscopy experiments, although evidence that unfolding occurs in Fn fibers has been indirect and has not been quantified. Nevertheless, unfolding of Fn molecules predicts a possible mechanism of strain dependant binding in Fn matrix and commensurate strain feedback to attached cells, contributing to the cellular mechanotransduction toolbox [3].

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Is mechanical receptor ligand dissociation driven by unfolding or unbinding?

10.1101/593335 ◽

2019 ◽

Cited By ~ 1

Author(s):

Lukas F. Milles ◽

Hermann E. Gaub

Keyword(s):

Single Molecule ◽

Type I ◽

Mechanical Forces ◽

Single Molecule Force Spectroscopy ◽

Receptor Ligand ◽

Binding Partner ◽

Ligand Dissociation ◽

Protein Protein Interaction ◽

Binding Partners ◽

Mechanical Receptor

ABSTRACTMechanical force can play a pivotal role in biological systems. Single Molecule Force Spectroscopy, is a powerful tool to probe the mechanics of proteins and their binding partners. Yet, it remains unclear how complex dissociation of a protein-protein interaction under mechanical forces occurs. Are receptor and ligand unbinding, or are they unfolding? We utilize an approach wherein receptor and ligand are expressed as a single molecule fused by a long flexible linker. Force is applied to the complex via an ultrastable handle. Consequently, the events during and following complex dissociation can be monitored. We investigate two high-affinity systems: The cohesin-dockerin type I interaction in which we find that a binding partner unfolds upon complex dissociation, and a colicin-immunity protein complex in which both proteins unfold completely upon unbinding. Mechanical receptor ligand dissociation thus can encompass unfolding of one or both binding partners.

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Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking

10.1101/2020.09.30.321232 ◽

2020 ◽

Author(s):

Martin Rieu ◽

Jessica Valle-Orero ◽

Bertrand Ducos ◽

Jean-François Allemand ◽

Vincent Croquette

Keyword(s):

Single Molecule ◽

Functional Characterization ◽

Nucleic Acid Binding ◽

Single Molecule Force Spectroscopy ◽

E Coli ◽

Natural Function ◽

Protein Nucleic Acid ◽

Free Quantification

ABSTRACTFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.

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Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding

Communications Biology ◽

10.1038/s42003-021-02606-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Martin Rieu ◽

Jessica Valle-Orero ◽

Bertrand Ducos ◽

Jean-François Allemand ◽

Vincent Croquette

Keyword(s):

Single Molecule ◽

Functional Characterization ◽

Nucleic Acid Binding ◽

Single Molecule Force Spectroscopy ◽

E Coli ◽

Natural Function ◽

Protein Nucleic Acid ◽

Free Quantification

AbstractFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.

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Single-Molecule Force Spectroscopy of the Aplysia Cell Adhesion Molecule Reveals Two Homophilic Bonds

Biophysical Journal ◽

10.1016/j.bpj.2012.07.004 ◽

2012 ◽

Vol 103 (4) ◽

pp. 649-657 ◽

Cited By ~ 30

Author(s):

E. Martines ◽

J. Zhong ◽

J. Muzard ◽

A.C. Lee ◽

B.B. Akhremitchev ◽

...

Keyword(s):

Cell Adhesion ◽

Single Molecule ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Force Spectroscopy ◽

Single Molecule Force Spectroscopy

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Insights into chromatin fibre structure by in vitro and in silico single-molecule stretching experiments

Biochemical Society Transactions ◽

10.1042/bst20120349 ◽

2013 ◽

Vol 41 (2) ◽

pp. 494-500 ◽

Cited By ~ 8

Author(s):

Rosana Collepardo-Guevara ◽

Tamar Schlick

Keyword(s):

Gene Regulation ◽

Single Molecule ◽

Fibre Structure ◽

Detailed Structure ◽

Single Molecule Force Spectroscopy ◽

Linker Histones ◽

Chromatin Fibre ◽

Structure And Dynamics ◽

Experimental Findings

The detailed structure and dynamics of the chromatin fibre and their relation to gene regulation represent important open biological questions. Recent advances in single-molecule force spectroscopy experiments have addressed these questions by directly measuring the forces that stabilize and alter the folded states of chromatin, and by investigating the mechanisms of fibre unfolding. We present examples that demonstrate how complementary modelling approaches have helped not only to interpret the experimental findings, but also to advance our knowledge of force-induced events such as unfolding of chromatin with dynamically bound linker histones and nucleosome unwrapping.

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Torus Circumference and Thickness Parameters From a Linear DNA Size Series Suggest How Toruses Self-Assemble

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119430 ◽

1985 ◽

Vol 43 ◽

pp. 522-523

Author(s):

George C. Ruben ◽

Kenneth A. Marx

Keyword(s):

Tertiary Structure ◽

Dna Complexes ◽

Tertiary Structures ◽

Self Assembling ◽

Double Stranded Dna ◽

Calf Thymus ◽

Dna Organization ◽

Condensed Dna ◽

Dna Size

Certain double stranded DNA bacteriophage and viruses are thought to have their DNA organized into large torus shaped structures. Morphologically, these poorly understood biological DNA tertiary structures resemble spermidine-condensed DNA complexes formed in vitro in the total absence of other macromolecules normally synthesized by the pathogens for the purpose of their own DNA packaging. Therefore, we have studied the tertiary structure of these self-assembling torus shaped spermidine- DNA complexes in a series of reports. Using freeze-etch, low Pt-C metal (10-15Å) replicas, we have visualized the microscopic DNA organization of both calf Thymus( CT) and linear 0X-174 RFII DNA toruses. In these structures DNA is circumferentially wound, continuously, around the torus into a semi-crystalline, hexagonal packed array of parallel DNA helix sections.

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In Silico Study of Potential Cross-Kingdom Plant MicroRNA Based Regulation in Chronic Myeloid Leukemia

Current Pharmacogenomics and Personalized Medicine ◽

10.2174/1875692118666200106113610 ◽

2020 ◽

Vol 17 (2) ◽

pp. 125-132

Author(s):

Marjanu Hikmah Elias ◽

Noraziah Nordin ◽

Nazefah Abdul Hamid

Keyword(s):

Targeted Therapy ◽

In Silico ◽

Structure Prediction ◽

Tertiary Structure ◽

Target Prediction ◽

In Silico Analysis ◽

Microrna Target ◽

Good Target

Background: Chronic Myeloid Leukaemia (CML) is associated with the BCRABL1 gene, which plays a central role in the pathogenesis of CML. Thus, it is crucial to suppress the expression of BCR-ABL1 in the treatment of CML. MicroRNA is known to be a gene expression regulator and is thus a good candidate for molecularly targeted therapy for CML. Objective: This study aims to identify the microRNAs from edible plants targeting the 3’ Untranslated Region (3’UTR) of BCR-ABL1. Methods: In this in silico analysis, the sequence of 3’UTR of BCR-ABL1 was obtained from Ensembl Genome Browser. PsRNATarget Analysis Server and MicroRNA Target Prediction (miRTar) Server were used to identify miRNAs that have binding conformity with 3’UTR of BCR-ABL1. The MiRBase database was used to validate the species of plants expressing the miRNAs. The RNAfold web server and RNA COMPOSER were used for secondary and tertiary structure prediction, respectively. Results: In silico analyses revealed that cpa-miR8154, csi-miR3952, gma-miR4414-5p, mdm-miR482c, osa-miR1858a and osa-miR1858b show binding conformity with strong molecular interaction towards 3’UTR region of BCR-ABL1. However, only cpa-miR- 8154, osa-miR-1858a and osa-miR-1858b showed good target site accessibility. Conclusion: It is predicted that these microRNAs post-transcriptionally inhibit the BCRABL1 gene and thus could be a potential molecular targeted therapy for CML. However, further studies involving in vitro, in vivo and functional analyses need to be carried out to determine the ability of these miRNAs to form the basis for targeted therapy for CML.

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The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Biomolecules ◽

10.3390/biom11030360 ◽

2021 ◽

Vol 11 (3) ◽

pp. 360

Author(s):

Pieterjan Debie ◽

Noemi B. Declerck ◽

Danny van Willigen ◽

Celine M. Huygen ◽

Bieke De Sloovere ◽

...

Keyword(s):

Single Molecule ◽

Gamma Ray ◽

Nuclear Imaging ◽

Primary Amines ◽

Resection Margins ◽

Fluorescent Light ◽

Single Structure ◽

Fluorescence Images

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

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