scholarly journals Three-dimensional tracking of tethered particles for probing nanometer-scale single-molecule dynamics using plasmonic microscope

2021 ◽  
Author(s):  
Guangzhong Ma ◽  
Zijian Wan ◽  
Yunze Yang ◽  
Wenwen Jing ◽  
Shaopeng Wang
Keyword(s):  
Single Molecule ◽  
High Spatial Resolution ◽  
Imaging Technique ◽  
Three Dimensional ◽  
Axial Direction ◽  
Nanometer Scale ◽  
3D Tracking ◽  
Single Molecule Level ◽  
Plasmonic Imaging ◽  
Molecule Dynamics

Three-dimensional (3D) tracking of surface-tethered single-particle reveals the dynamics of the molecular tether. However, most 3D tracking techniques lack precision, especially in axial direction, for measuring the dynamics of biomolecules with spatial scale of several nanometers. Here we present a plasmonic imaging technique that can track the motion of ~100 tethered particles in 3D simultaneously with sub-nanometer axial precision at millisecond time resolution. By tracking the 3D coordinates of tethered particle with high spatial resolution, we are able to determine the dynamics of single short DNA and study its interaction with enzyme. We further show that the particle motion pattern can be used to identify specific and non-specific interactions in immunoassays. We anticipate that our 3D tracking technique can contribute to the understanding of molecular dynamics and interactions at the single-molecule level.

scholarly journals Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry

2021 ◽  
Vol 7 (6) ◽  
pp. eabe3902
Author(s):  
Martin Rieu ◽  
Thibault Vieille ◽  
Gaël Radou ◽  
Raphaël Jeanneret ◽  
Nadia Ruiz-Gutierrez ◽  
...  
Keyword(s):  
High Precision ◽  
High Throughput ◽  
Single Molecule ◽  
Particle Tracking ◽  
Focal Plane ◽  
Tracking System ◽  
Three Dimensional ◽  
3D Tracking ◽  
Single Molecule Force Spectroscopy ◽  
A New Technique

While crucial for force spectroscopists and microbiologists, three-dimensional (3D) particle tracking suffers from either poor precision, complex calibration, or the need of expensive hardware, preventing its massive adoption. We introduce a new technique, based on a simple piece of cardboard inserted in the objective focal plane, that enables simple 3D tracking of dilute microparticles while offering subnanometer frame-to-frame precision in all directions. Its linearity alleviates calibration procedures, while the interferometric pattern enhances precision. We illustrate its utility in single-molecule force spectroscopy and single-algae motility analysis. As with any technique based on back focal plane engineering, it may be directly embedded in a commercial objective, providing a means to convert any preexisting optical setup in a 3D tracking system. Thanks to its precision, its simplicity, and its versatility, we envision that the technique has the potential to enhance the spreading of high-precision and high-throughput 3D tracking.

scholarly journals 3D tracking of extracellular vesicles by holographic fluorescence imaging

2020 ◽  
Vol 6 (45) ◽  
pp. eabc2508
Author(s):  
Matz Liebel ◽  
Jaime Ortega Arroyo ◽  
Vanesa Sanz Beltrán ◽  
Johann Osmond ◽  
Ala Jo ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Fluorescent Detection ◽  
Live Cells ◽  
Fluorescent Light ◽  
3D Tracking ◽  
Anisotropic Motion ◽  
Lag Times

Fluorescence microscopy is the method of choice in biology for its molecular specificity and super-resolution capabilities. However, it is limited to a narrow z range around one observation plane. Here, we report an imaging approach that recovers the full electric field of fluorescent light with single-molecule sensitivity. We expand the principle of digital holography to fast fluorescent detection by eliminating the need for phase cycling and enable three-dimensional (3D) tracking of individual nanoparticles with an in-plane resolution of 15 nm and a z-range of 8 mm. As a proof-of-concept biological application, we image the 3D motion of extracellular vesicles (EVs) inside live cells. At short time scales (<4 s), we resolve near-isotropic 3D diffusion and directional transport. For longer lag times, we observe a transition toward anisotropic motion with the EVs being transported over long distances in the axial plane while being confined in the horizontal dimension.

scholarly journals Nucleic acid-based nanoengineering: novel structures for biomedical applications

2011 ◽  
Vol 1 (5) ◽  
pp. 702-724 ◽  
Author(s):  
Hanying Li ◽  
Thomas H. LaBean ◽  
Kam W. Leong
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Single Molecule ◽  
Biomedical Applications ◽  
Three Dimensional ◽  
Base Pairs ◽  
Single Molecule Level ◽  
Stimulus Responsive ◽  
Bioactive Agents ◽  
Novel Structures

Nanoengineering exploits the interactions of materials at the nanometre scale to create functional nanostructures. It relies on the precise organization of nanomaterials to achieve unique functionality. There are no interactions more elegant than those governing nucleic acids via Watson–Crick base-pairing rules. The infinite combinations of DNA/RNA base pairs and their remarkable molecular recognition capability can give rise to interesting nanostructures that are only limited by our imagination. Over the past years, creative assembly of nucleic acids has fashioned a plethora of two-dimensional and three-dimensional nanostructures with precisely controlled size, shape and spatial functionalization. These nanostructures have been precisely patterned with molecules, proteins and gold nanoparticles for the observation of chemical reactions at the single molecule level, activation of enzymatic cascade and novel modality of photonic detection, respectively. Recently, they have also been engineered to encapsulate and release bioactive agents in a stimulus-responsive manner for therapeutic applications. The future of nucleic acid-based nanoengineering is bright and exciting. In this review, we will discuss the strategies to control the assembly of nucleic acids and highlight the recent efforts to build functional nucleic acid nanodevices for nanomedicine.

scholarly journals Nanopore microscope identifies RNA isoforms with structural colors

2021 ◽  
Author(s):  
Filip N Boskovic ◽  
Ulrich Felix Keyser
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Rna Structures ◽  
Transcript Isoforms ◽  
Single Molecule Level ◽  
Structural Colors ◽  
Rna Targets ◽  
Dna Strands ◽  
One Step ◽  
Simultaneous Identification

Identifying RNA transcript isoforms requires intricate protocols that suffer from various enzymatic biases. Here we design three-dimensional molecular constructs that enable identification of transcript isoforms at the single-molecule level using solid-state nanopore microscopy. We refold target RNA into RNA identifiers (IDs) with designed sets of complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colors. Structural colors consist of DNA structures, protein labels, native RNA structures, or a combination of all three. The sequence of structural colors of RNA IDs enables simultaneous identification and relative quantification of multiple RNA targets without prior amplification. Our Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) reveals structural arrangements in native transcripts in agreement with published variants. ARTEMIS discriminates circular and linear transcript isoforms in a one step, enzyme-free reaction in a complex human transcriptome using single-molecule readout.

scholarly journals De Novo Design of a Nanopore for DNA Detection that Incorporates a β-Hairpin Peptide

2021 ◽  
Author(s):  
Keisuke Shimizu ◽  
Batsaikhan Mijiddorj ◽  
Masataka Usami ◽  
Shuhei Yoshida ◽  
Shiori Akayama ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Design ◽  
Lipid Membrane ◽  
De Novo ◽  
Three Dimensional ◽  
Bilayer Lipid Membrane ◽  
De Novo Design ◽  
Dimensional Structure ◽  
Practical Applications ◽  
Single Molecule Level

Abstract The amino acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo protein design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device with practical applications. This peptide, named SV28, forms nanopore structures ranging from 1.6 to 6.2 nm in diameter assembled from 7 to 18 monomers. The nanopore formed with a diameter of 5 nm is able to detect long double-stranded DNA (dsDNA) with 1 kbp length. Moreover, the larger sized nanopore can discriminate and human telomeric DNA (G-quadruplex, G4). The blocking current signals allowed us to investigate the translocation behavior of dsDNA or G4 structure at the single molecule level. Such de novo design of peptide sequences has the potential to create novel nanopores, which would be applicable in molecular transporter between across lipid membrane.

De Novo Design of a Nanopore for DNA Detection Incorporating a β-hairpin Peptide

2020 ◽  
Author(s):  
Keisuke Shimizu ◽  
Batsaikhan Mijiddorj ◽  
Shuhei Yoshida ◽  
Shiori Akayama ◽  
Yoshio Hamada ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Design ◽  
Lipid Membrane ◽  
De Novo ◽  
Three Dimensional ◽  
Bilayer Lipid Membrane ◽  
De Novo Design ◽  
Dimensional Structure ◽  
Practical Applications ◽  
Single Molecule Level

The amino acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo protein design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device with practical applications. This peptide, named SV28, forms nanopore structures ranging from 1.6 to 6.2 nm in diameter assembled from 7 to 18 monomers. The nanopore formed with a diameter of 5 nm is able to detect long double-stranded DNA (dsDNA) with 1 kbp length, and measurement of current signals allowed us to investigate the translocation behavior of dsDNA at the single molecule level. Such de novo design of peptide sequences has the potential to create assembled structure in lipid membrane such as novel nanopores, which would also be applicable in molecular transporter between inside and outside of lipid membrane.

scholarly journals De Novo Design of a Nanopore for DNA Detection Incorporating a β-hairpin Peptide

2020 ◽  
Author(s):  
Keisuke Shimizu ◽  
Batsaikhan Mijiddorj ◽  
Shuhei Yoshida ◽  
Shiori Akayama ◽  
Yoshio Hamada ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Design ◽  
Lipid Membrane ◽  
De Novo ◽  
Three Dimensional ◽  
Bilayer Lipid Membrane ◽  
De Novo Design ◽  
Dimensional Structure ◽  
Practical Applications ◽  
Single Molecule Level

The amino acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo protein design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device with practical applications. This peptide, named SV28, forms nanopore structures ranging from 1.6 to 6.2 nm in diameter assembled from 7 to 18 monomers. The nanopore formed with a diameter of 5 nm is able to detect long double-stranded DNA (dsDNA) with 1 kbp length, and measurement of current signals allowed us to investigate the translocation behavior of dsDNA at the single molecule level. Such de novo design of peptide sequences has the potential to create assembled structure in lipid membrane such as novel nanopores, which would also be applicable in molecular transporter between inside and outside of lipid membrane.

De Novo Design of a Nanopore for DNA Detection Incorporating a β-hairpin Peptide

2020 ◽  
Author(s):  
Keisuke Shimizu ◽  
Batsaikhan Mijiddorj ◽  
Shuhei Yoshida ◽  
Shiori Akayama ◽  
Yoshio Hamada ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Design ◽  
Lipid Membrane ◽  
De Novo ◽  
Three Dimensional ◽  
Bilayer Lipid Membrane ◽  
De Novo Design ◽  
Dimensional Structure ◽  
Practical Applications ◽  
Single Molecule Level

The amino acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo protein design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device with practical applications. This peptide, named SV28, forms nanopore structures ranging from 1.6 to 6.2 nm in diameter assembled from 7 to 18 monomers. The nanopore formed with a diameter of 5 nm is able to detect long double-stranded DNA (dsDNA) with 1 kbp length, and measurement of current signals allowed us to investigate the translocation behavior of dsDNA at the single molecule level. Such de novo design of peptide sequences has the potential to create assembled structure in lipid membrane such as novel nanopores, which would also be applicable in molecular transporter between inside and outside of lipid membrane.

Simultaneous single-molecule discrimination of cysteine and homocysteine with a protein nanopore

2019 ◽  
Vol 55 (63) ◽  
pp. 9311-9314 ◽  
Author(s):  
Yao Lu ◽  
Xue-Yuan Wu ◽  
Yi-Lun Ying ◽  
Yi-Tao Long
Keyword(s):  
Amino Acid ◽  
Spatial Resolution ◽  
Single Molecule ◽  
High Spatial Resolution ◽  
Amino Acid Difference ◽  
Confined Space ◽  
Single Molecule Level

Discrimination between cysteine and homocysteine at the single-molecule level is achieved within a K238Q mutant aerolysin nanopore, which provides a confined space for high spatial resolution to identify the amino acid difference.

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