scholarly journals Single-molecule, real-time measurement of enzyme kinetics by alternating-laser excitation fluorescence resonance energy transfer

2010 ◽  
Vol 46 (26) ◽  
pp. 4683 ◽  
Author(s):  
Nam Ki Lee ◽  
Hye Ran Koh ◽  
Kyu Young Han ◽  
Jihyun Lee ◽  
Seong Keun Kim
Keyword(s):  
Energy Transfer ◽  
Enzyme Kinetics ◽  
Real Time ◽  
Single Molecule ◽  
Laser Excitation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Measurement ◽  
Real Time Measurement ◽  
Alternating Laser Excitation

Red light, green light: probing single molecules using alternating-laser excitation

2008 ◽  
Vol 36 (4) ◽  
pp. 738-744 ◽  
Author(s):  
Yusdi Santoso ◽  
Ling Chin Hwang ◽  
Ludovic Le Reste ◽  
Achillefs N. Kapanidis
Keyword(s):  
Single Molecule ◽  
Laser Excitation ◽  
Dynamic Range ◽  
Red Light ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Fret ◽  
Alternating Laser Excitation

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein–nucleic acid interactions.

scholarly journals Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

10.3791/60045 ◽  
2019 ◽  
Author(s):  
Mohamed A. Sobhy ◽  
Amer Bralić ◽  
Vlad-Stefan Raducanu ◽  
Muhammad Tehseen ◽  
Yujing Ouyang ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Holliday Junction

scholarly journals Mechanism of DNA surface exploration and operator bypassing during target search

2020 ◽  
Author(s):  
E. Marklund ◽  
B. van Oosten ◽  
G. Mao ◽  
E. Amselem ◽  
K. Kipper ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Single Molecule ◽  
Time Scale ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Signal ◽  
Target Search ◽  
Fluorescence Correlation ◽  
Speed Accuracy

SummaryMany proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA1–5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 μs. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

Towards Single-Molecule Diagnostics Using Microfluidic Manipulation and Quantum Dot Nanosensors

2007 ◽  
Author(s):  
Hsin-Chih Yeh ◽  
Christopher M. Puleo ◽  
Yi-Ping Ho ◽  
Tza-Huei Wang
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mutational Analysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Detection ◽  
Specific Analysis ◽  
Low Volume

In this report, we review several single-molecule detection (SMD) methods and newly developed nanocrystal-mediated single-fluorophore strategies for ultrasensitive and specific analysis of genomic sequences. These include techniques, such as quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) technology and dual-color fluorescence coincidence and colocalization analysis, which allow separation-free detection of low-abundance DNA sequences and mutational analysis of oncogenes. Microfluidic approaches developed for use with single-molecule detection to achieve rapid, low-volume, and quantitative analysis of nucleic acids, such as electrokinetic manipulation of single molecules and confinement of sub-nanoliter samples using microfluidic networks integrated with valves, are also discussed.

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