Determination of the size of lipid rafts studied through single-molecule FRET simulations

ABSTRACTSingle-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object. The limited number of available FRET pair fluorophores and complicated data analysis makes it challenging to apply single-molecule FRET for structural analysis of biomolecules. Currently, only a couple of FRET pairs can be reliably measured on a single object. Here we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the FRET efficiency and pair distance with sub-nanometer resolution. We determine the distance between other pairs by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. We envision that our FRET X technology will be a tool for the high-resolution structural analysis of biomolecules and other nano-structures.

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The Study of Protein Folding and Dynamics by Determination of Intramolecular Distance Distributions and Their Fluctuations Using Ensemble and Single-Molecule FRET Measurements

ChemPhysChem ◽

10.1002/cphc.200400617 ◽

2005 ◽

Vol 6 (5) ◽

pp. 858-870 ◽

Cited By ~ 84

Author(s):

Elisha Haas

Keyword(s):

Protein Folding ◽

Single Molecule ◽

Single Molecule Fret ◽

Distance Distributions ◽

Intramolecular Distance

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Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4–P6 RNA by smFRET

Biochemical Society Transactions ◽

10.1042/bst20140268 ◽

2015 ◽

Vol 43 (2) ◽

pp. 172-178 ◽

Cited By ~ 11

Author(s):

Namita Bisaria ◽

Daniel Herschlag

Keyword(s):

Single Molecule ◽

Specific Binding ◽

Specific Interaction ◽

Monovalent Cations ◽

Folding Kinetics ◽

Rna Molecules ◽

Single Molecule Fret ◽

Wide Range ◽

Monovalent Ion

Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4–P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na+ and K+, to the RNA tetraloop–tetraloop receptor (TL–TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state.

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Encoding multiple virtual signals in DNA barcodes with single-molecule FRET

10.1101/2020.11.12.379479 ◽

2020 ◽

Author(s):

Sung Hyun Kim ◽

Hyunwoo Kim ◽

Hawoong Jeong ◽

Tae-Young Yoon

Keyword(s):

Single Molecule ◽

Dna Barcode ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Emission Spectra ◽

Biological Molecules ◽

Dna Barcodes ◽

Signal Space ◽

Single Molecule Fret

ABSTRACTDNA barcoding provides a way to label a huge number of different biological molecules using the extreme programmability in DNA sequence synthesis. Fluorescence imaging is an easy-to-access method to detect individual DNA barcodes, which can be scaled up to a massively high-throughput format. Large overlaps between emission spectra of fluorescence dyes, however, severely limit the numbers of DNA barcodes–and thus its signal space–that can be detected in a simultaneous manner. We here demonstrate the use of single-molecule fluorescence resonance energy transfer (FRET) to encode virtual signals in DNA barcodes using conventional two-color fluorescence microscopy. By optimizing imaging and biochemistry conditions for weak hybridization events for DNA barcodes, we markedly enhanced accuracy in our determination of the efficiency by which single-molecule FRET occurred. This allowed us to unambiguously differentiate six DNA barcodes exhibiting different FRET values without involving probe sequence exchanges. Our method can be directly incorporated with previous DNA barcode techniques, and may thus be widely adopted to expand the signal space of the DNA barcode techniques.

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