scholarly journals Single-Molecule FRET Detection of Sub-Nanometer Distance Changes in the Range below a 3-Nanometer Scale

Biosensors ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 168
Author(s):  
Heyjin Son ◽  
Woori Mo ◽  
Jaeil Park ◽  
Joong-Wook Lee ◽  
Sanghwa Lee
Keyword(s):  
Single Molecule ◽  
Short Distance ◽  
High Sensitivity ◽  
Nanometer Scale ◽  
Biological Processes ◽  
Dna Duplexes ◽  
Detection Range ◽  
Distance Range ◽  
Single Molecule Fret ◽  
Fluorescence Energy

Single-molecule fluorescence energy transfer (FRET) detection has become a key technique to monitor intra- and intermolecular distance changes in biological processes. As the sensitive detection range of conventional FRET pairs is limited to 3–8 nm, complement probes are necessary for extending this typical working range. Here, we realized a single-molecule FRET assay for a short distance range of below 3 nm by using a Cy2–Cy7 pair having extremely small spectral overlap. Using two DNA duplexes with a small difference in the labeling position, we demonstrated that our assay can observe subtle changes at a short distance range. High sensitivity in the range of 1–3 nm and compatibility with the conventional FRET assay make this approach useful for understanding dynamics at a short distance.

Quantitative structural information from single-molecule FRET

2015 ◽  
Vol 184 ◽  
pp. 117-129 ◽  
Author(s):  
M. Beckers ◽  
F. Drechsler ◽  
T. Eilert ◽  
J. Nagy ◽  
J. Michaelis
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Monte Carlo Sampling ◽  
Dye Molecules ◽  
Distance Information ◽  
Single Molecule Fret ◽  
Single Molecule Studies ◽  
Archaeal Transcription ◽  
Short Time ◽  
Fluorescence Energy

Single-molecule studies can be used to study biological processes directly and in real-time. In particular, the fluorescence energy transfer between reporter dye molecules attached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be used to determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertainties are highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short time and yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dye molecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcription pre-initiation complex, whose architecture has recently been unravelled by smFRET measurements.

scholarly journals ABEL-FRET: tether-free single-molecule FRET with hydrodynamic profiling

2019 ◽  
Author(s):  
Hugh Wilson ◽  
Quan Wang
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Biological Processes ◽  
Resolution Limit ◽  
Single Molecule Fret ◽  
Experimental Protocols ◽  
Fret Efficiency ◽  
Nanoscale Metrology ◽  
Observation Times

ABSTRACTSingle-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental protocols often resort to molecule immobilization for long observation times and rarely approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with near shot-noise limited, Angstrom-level resolution in FRET efficiency. Furthermore, ABEL-FRET naturally integrates hydrodynamic profiling, which harnesses single-molecule diffusion coefficient to enhance FRET sensing of biological processes.

scholarly journals Conformational pathway provides unique sensitivity to a synaptic mGluR

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Chris H. Habrian ◽  
Joshua Levitz ◽  
Vojtech Vyklicky ◽  
Zhu Fu ◽  
Adam Hoagland ◽  
...  
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Dynamic Range ◽  
High Sensitivity ◽  
G Protein Coupled Receptors ◽  
Metabotropic Glutamate ◽  
Single Molecule Fret ◽  
Broad Dynamic Range ◽  
Maximal Activation ◽  
G Protein Coupled

AbstractMetabotropic glutamate receptors (mGluRs) are dimeric G-protein–coupled receptors that operate at synapses. Macroscopic and single molecule FRET to monitor structural rearrangements in the ligand binding domain (LBD) of the mGluR7/7 homodimer revealed it to have an apparent affinity ~4000-fold lower than other mGluRs and a maximal activation of only ~10%, seemingly too low for activation at synapses. However, mGluR7 heterodimerizes, and we find it to associate with mGluR2 in the hippocampus. Strikingly, the mGluR2/7 heterodimer has high affinity and efficacy. mGluR2/7 shows cooperativity in which an unliganded subunit greatly enhances activation by agonist bound to its heteromeric partner, and a unique conformational pathway to activation, in which mGluR2/7 partially activates in the Apo state, even when its LBDs are held open by antagonist. High sensitivity and an unusually broad dynamic range should enable mGluR2/7 to respond to both glutamate transients from nearby release and spillover from distant synapses.

scholarly journals A versatile platform for single-molecule enzymology of restriction endonuclease

2019 ◽  
Vol 12 (01) ◽  
pp. 1841002 ◽  
Author(s):  
Xin Wang ◽  
Jingyuan Nie ◽  
Yi Li ◽  
Hai Pan ◽  
Peng Zheng ◽  
...  
Keyword(s):  
Experimental Design ◽  
Enzymatic Activity ◽  
Single Molecule ◽  
Holliday Junction ◽  
Biological Processes ◽  
Surface Anchoring ◽  
Single Molecule Level ◽  
Enzyme Binding ◽  
Single Molecule Fret ◽  
Single Molecule Enzymology

Enzymes are the major players for many biological processes. Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level. Yet, these single-molecule experiments are technically difficult and generally require complicated experimental design. Here, we develop a Holliday junction (HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET (sm-FRET). We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events. Thanks to the multiple-arms structure of HJ, the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm. Therefore, it is possible to independently change the substrate arm to study different enzymes with similar functions. Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations. Moreover, this method can be easily extended to study other types of DNA-binding enzymes without too much modification of the design. We anticipate it can find broad applications in single-molecule enzymology.

scholarly journals Single-Molecule Sandwich Immunoassay for Quantification of Alpha-Fetoprotein Based on Evanescent Field-Enhanced Fluorescence Imaging

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Seungah Lee ◽  
Seong Ho Kang
Keyword(s):  
Single Molecule ◽  
Accurate Determination ◽  
Protein A ◽  
High Sensitivity ◽  
Evanescent Field ◽  
Alpha Fetoprotein ◽  
Fluorescence Signal ◽  
Detection Range ◽  
Enhanced Sensitivity ◽  
Fluorescence Dye

A highly sensitive immunosensor based on a gold nanopatterned chip was developed for accurate determination of alpha-fetoprotein (AFP)viatotal internal refection fluorescence microscopy (TIRFM). The surface of the gold nanopatterned chips was modified with dithiobis(succinimidyl propionate) and protein A/G for immobilization of the AFP antibody. The immunoassay created a sandwich of antigen between the AFP antibody on the chip that was modified with protein A/G, and the secondary antibody, a monoclonal anti-human-AFP labeled with biotin (biotin-labeled anti-AFP). AFP concentration was determined based on evanescent field fluorescence signal, which was generated by interaction between biotin-labeled anti-AFP and a streptavidin-labeled fluorescence dye. AFP concentration could be measured in a wide dynamic linear range of 720 zM–10 nM with a detection limit of 720 zM. A significant enhanced sensitivity (~40,000-fold) was achieved with the AFP-nanoarray chip compared to conventional chemiluminescence immunosensors. The immunoassay exhibited a wide detection range and high sensitivity and accuracy, qualities valuable for clinical assay of AFP.

scholarly journals Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yong Wang ◽  
Yanxin Liu ◽  
Hannah A DeBerg ◽  
Takeshi Nomura ◽  
Melinda Tonks Hoffman ◽  
...  
Keyword(s):  
Single Molecule ◽  
Model System ◽  
Channel Structure ◽  
Fluorescence Energy Transfer ◽  
Mechanosensitive Channels ◽  
Dynamics Model ◽  
Single Molecule Fret ◽  
Direct Observations ◽  
Exact Size ◽  
Fluorescence Energy

The mechanosensitive channel of large conductance, which serves as a model system for mechanosensitive channels, has previously been crystallized in the closed form, but not in the open form. Ensemble measurements and electrophysiological sieving experiments show that the open-diameter of the channel pore is >25 Å, but the exact size and whether the conformational change follows a helix-tilt or barrel-stave model are unclear. Here we report measurements of the distance changes on liposome-reconstituted MscL transmembrane α-helices, using a ‘virtual sorting’ single-molecule fluorescence energy transfer. We observed directly that the channel opens via the helix-tilt model and the open pore reaches 2.8 nm in diameter. In addition, based on the measurements, we developed a molecular dynamics model of the channel structure in the open state which confirms our direct observations.

Direct Read and Quantify Damage Nucleotide from an Oligonucleotide Using a Single Molecule Interface

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Meng-Yin Li ◽  
Jie Yang ◽  
Ya-Qian Wang ◽  
Xue-Yuan Wu ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Genetic Diseases ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Lesion Detection ◽  
The Novel ◽  
Structural Variations ◽  
Dna Lesion ◽  
Base Lesion

DNA lesion such as metholcytosine(<sup>m</sup>C), 8-OXO-guanine(<sup>O</sup>G), inosine(I) <i>etc</i> could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (<sup>m</sup>C, <sup>O</sup>G, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

Spectroscopy-Based Mapping with Scanning Microwave Impedance Microscopy

2018 ◽  
Author(s):  
Peter De Wolf ◽  
Zhuangqun Huang ◽  
Bede Pittenger
Keyword(s):  
Single Point ◽  
Electrical Characterization ◽  
Principal Component ◽  
High Sensitivity ◽  
Data Cube ◽  
Nanometer Scale ◽  
Learning Approaches ◽  
Data Set ◽  
3D Data ◽  
Higher Dimensional

Abstract Methods are available to measure conductivity, charge, surface potential, carrier density, piezo-electric and other electrical properties with nanometer scale resolution. One of these methods, scanning microwave impedance microscopy (sMIM), has gained interest due to its capability to measure the full impedance (capacitance and resistive part) with high sensitivity and high spatial resolution. This paper introduces a novel data-cube approach that combines sMIM imaging and sMIM point spectroscopy, producing an integrated and complete 3D data set. This approach replaces the subjective approach of guessing locations of interest (for single point spectroscopy) with a big data approach resulting in higher dimensional data that can be sliced along any axis or plane and is conducive to principal component analysis or other machine learning approaches to data reduction. The data-cube approach is also applicable to other AFM-based electrical characterization modes.

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