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.

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.

Elucidating Molecule–Plasmon Interactions in Nanocavities with 2 nm Spatial Resolution and at the Single‐Molecule Level

2019 ◽  
Vol 131 (35) ◽  
pp. 12261-12265 ◽  
Author(s):  
Fan‐Li Zhang ◽  
Jun Yi ◽  
Wei Peng ◽  
Petar M. Radjenovic ◽  
Hua Zhang ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Single Molecule Level

Prospects for Molecular Spectroscopy at High Spatial Resolution

2001 ◽  
Vol 215 (8) ◽  
Author(s):  
F. Hamann
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
High Spatial Resolution ◽  
Molecular Spectroscopy ◽  
Near Field ◽  
Dark Field ◽  
Optical Methods ◽  
Weak Scattering ◽  
Scanning Optical Microscopy ◽  
Near Field Scanning

This work discusses the prospects and feasibility of optical spectroscopy and microscopy of single molecules at nanometer resolution via apertureless, antenna-based near-field scanning optical microscopy. First, different near-field optical methods are compared, which detect the weak scattering or fluorescence from a probe–single molecule interaction at high spatial resolution. Specifically, ultimate sensitivities of coherent (bright-field) and non-coherent (dark-field) apertureless near-field microscopes for resonant (e.g., scattering, absorption) and non-resonant (e.g.,

scholarly journals On-Site Processing of Single Chromosomal DNA Molecules Using Optically Driven Microtools on A Microfluidic Workbench

2020 ◽  
Author(s):  
Akihito Masuda ◽  
Hidekuni Takao ◽  
Fusao Shimokawa ◽  
KYOHEI TERAO
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Single Cells ◽  
Optical Microscope ◽  
Optical Manipulation ◽  
Confined Space ◽  
Dna Molecules ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Immobilization Of Enzymes

Abstract We developed optically driven microtools for processing single biomolecules using a microfluidic workbench composed of a microfluidic platform that functions under an optical microscope. The optically driven microtools have enzymes immobilized on their surfaces, which catalyze chemical reactions for molecular processing in a confined space. Optical manipulation of the microtools enables them to be integrated with a microfluidic device for controlling the position, orientation, shape of the target sample. Here, we describe the immobilization of enzymes on the surface of microtools, the microfluidics workbench, including its microtool storage and sample positioning functions, and the use of this system for on-site cutting of single chromosomal DNA molecules. We fabricated microtools by UV lithography with SU-8 and selected ozone treatments for immobilizing enzymes. The microfluidic workbench has tool-stock chambers for tool storage and micropillars to trap and extend single chromosomal DNA molecules. The DNA cutting enzymes DNaseI and DNaseII were immobilized on microtools that were manipulated using optical tweezers. The DNaseI tool shows reliable cutting for on-site processing. This pinpoint processing provides an approach for analyzing chromosomal DNA at the single-molecule level. The flexibility of the microtool design allows for processing of various samples, including biomolecules and single cells.

scholarly journals Mechanism of duplex unwinding by coronavirus nsp13 helicases

2020 ◽  
Author(s):  
Xiao Hu ◽  
Wei Hao ◽  
Bo Qin ◽  
Zhiqi Tian ◽  
Ziheng Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Hinge Region ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
List Type ◽  
Single Molecule Fret ◽  
The Difference ◽  
Dna Complex ◽  
Loaded Mechanism

AbstractThe current COVID-19 pandemic urges in-depth investigation into proteins encoded with coronavirus (CoV), especially conserved CoV replicases. The nsp13 of highly pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV exhibit the most conserved CoV replicases. Using single-molecule FRET, we observed that MERS-CoV nsp13 unwound DNA in discrete steps of approximately 9 bp when ATP was used. If another NTP was used, then the steps were only 4 to 5 bp. In dwell time analysis, we detected 3 or 4 hidden steps in each unwinding process, which indicated the hydrolysis of 3 or 4 dTTP. Based on crystallographic and biochemical studies of CoV nsp13 helicases, we modeled an unwinding mechanism similar to the spring-loaded mechanism of HCV NS3 helicase, although our model proposes that flexible 1B and stalk domains, by allowing a lag greater than 4 bp during unwinding, cause the accumulated tension on the nsp13-DNA complex. The hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible than in MERS-CoV nsp13 due to the difference of a single amino acid, which causes the former to induce significantly greater NTP hydrolysis. Our findings thus establish a blueprint for determining the unwinding mechanism of a unique helicase family.When dTTP was used as the energy source, 4 hidden steps in each individual unwinding step after 3 - 4 NTP hydrolysis were observed.An unwinding model of MERS-CoV-nsp13 which is similar to the spring-loaded mechanism of HCV NS3 helicase, except the accumulation of tension on nsp13/DNA complex is caused by the flexible 1B and stalk domains that allow a lag of 4-bp in unwinding.Comparing to MERS-CoV nsp13, the hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible due to a single amino acid difference, which contributes to the significantly higher NTP hydrolysis by SARS-CoV-2 nsp13.

scholarly journals Semisynthetic protein nanoreactor for single-molecule chemistry

2015 ◽  
Vol 112 (45) ◽  
pp. 13768-13773 ◽  
Author(s):  
Joongoo Lee ◽  
Hagan Bayley
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Ionic Current ◽  
Unnatural Amino Acid ◽  
Chemical Ligation ◽  
Single Molecule Level ◽  
Flow Through ◽  
Recombinant Polypeptides

The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach.

scholarly journals On-site processing of single chromosomal DNA molecules using optically driven microtools on a microfluidic workbench

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Akihito Masuda ◽  
Hidekuni Takao ◽  
Fusao Shimokawa ◽  
Kyohei Terao
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Single Cells ◽  
Optical Microscope ◽  
Optical Manipulation ◽  
Confined Space ◽  
Dna Molecules ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Immobilization Of Enzymes

AbstractWe developed optically driven microtools for processing single biomolecules using a microfluidic workbench composed of a microfluidic platform that functions under an optical microscope. The optically driven microtools have enzymes immobilized on their surfaces, which catalyze chemical reactions for molecular processing in a confined space. Optical manipulation of the microtools enables them to be integrated with a microfluidic device for controlling the position, orientation, shape of the target sample. Here, we describe the immobilization of enzymes on the surface of microtools, the microfluidics workbench, including its microtool storage and sample positioning functions, and the use of this system for on-site cutting of single chromosomal DNA molecules. We fabricated microtools by UV lithography with SU-8 and selected ozone treatments for immobilizing enzymes. The microfluidic workbench has tool-stock chambers for tool storage and micropillars to trap and extend single chromosomal DNA molecules. The DNA cutting enzymes DNaseI and DNaseII were immobilized on microtools that were manipulated using optical tweezers. The DNaseI tool shows reliable cutting for on-site processing. This pinpoint processing provides an approach for analyzing chromosomal DNA at the single-molecule level. The flexibility of the microtool design allows for processing of various samples, including biomolecules and single cells.

scholarly journals High spatial resolution nanoslit SERS for single-molecule nucleobase sensing

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Chang Chen ◽  
Yi Li ◽  
Sarp Kerman ◽  
Pieter Neutens ◽  
Kherim Willems ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
High Spatial Resolution

Elucidating Molecule–Plasmon Interactions in Nanocavities with 2 nm Spatial Resolution and at the Single‐Molecule Level

2019 ◽  
Vol 58 (35) ◽  
pp. 12133-12137 ◽  
Author(s):  
Fan‐Li Zhang ◽  
Jun Yi ◽  
Wei Peng ◽  
Petar M. Radjenovic ◽  
Hua Zhang ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Single Molecule Level
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