Covalent‐Bond‐Based Immobilization Approaches for Single‐Molecule Fluorescence

Alemán, Elvin A.; Pedini, Heidi S.; Rueda, David

doi:10.1002/cbic.200900640

Cited by 18 publications

(22 citation statements)

References 18 publications

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“…This may be partially overcome by using different types of self-assembled monolayers to passivate the surface and surface-immobilize the molecules (22). However, the only way to completely eliminate any interactions with the slide surface is to isolate the system (i.e., a nucleic acid-protein complex) inside a lipid vesicle, for example.…”

Section: Description Of Methodsmentioning

confidence: 99%

“…Frequently, molecules are surface-immobilized to extend observation times into the minute time scale (22). Molecules of interest can be immobilized through a biotin-BSA, poly(ethylene glycol) (PEG) conjugated to biotin, PEG with Ni 2+ or Cu 2+ ions, or using click chemistry.…”

Section: Introductionmentioning

confidence: 99%

“…Alternatively, Ni-NTA coated surfaces and PEG with Ni 2+ or Cu 2+ ions can be used to immobilize His-tagged proteins; as this method is not generally applicable to all systems, we have not discussed it in detail (27–29). The click chemistry method of immoblization was recently introduced and may be useful in preventing interactions with the slide, but it has not yet been applied to systems that include protein (22). In addition, non-specific surface interactions can be suppressed by trapping the molecules inside of lipid vesicles immobilized on a PEGylated surface (30, 31).…”

Section: Introductionmentioning

confidence: 99%

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Single-molecule FRET of protein–nucleic acid and protein–protein complexes: Surface passivation and immobilization

Lamichhane

Solem

Black

et al. 2010

Methods

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105

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Single-molecule fluorescence spectroscopy reveals the real time dynamics that occur during biomolecular interactions that would otherwise be hidden by the ensemble average. It also removes the requirement to synchronize reactions, thus providing a very intuitive approach to study kinetics of biological systems. Surface immobilization is commonly used to increase observation times to the minute time scale, but it can be detrimental if the sample interacts nonspecifically with the surface. Here, we review detailed protocols to prevent such interactions by passivating the surface or by trapping the molecules inside surface immobilized lipid vesicles. Finally, we discuss recent examples where these methods were applied to study the dynamics of important cellular processes at the single molecule level.

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Section: Description Of Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single-molecule FRET of protein–nucleic acid and protein–protein complexes: Surface passivation and immobilization

Lamichhane

Solem

Black

et al. 2010

Methods

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105

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“…Thus, these methods, especially immobilization-based methods, have potential applications in the amplification-free detection of individual DNA and RNA molecules. The most common immobilization strategies for single-molecule fluorescence experiments utilize biotin-streptavidin linkages (6), thiol crosslinking (7), and click chemistry (7, 8). These strategies give detectable immobilization of molecules from solutions containing 250 amol to 1 pmol of analytes (9, 10).…”

Section: Introductionmentioning

confidence: 99%

Zeptomole detection of DNA nanoparticles by single-molecule fluorescence with magnetic field-directed localization

Cannon

Campos

Lewitz

et al. 2012

Analytical Biochemistry

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Single-molecule fluorescence methods offer the promise of ultrasensitive detection of biomolecules, but the passive immobilization methods commonly employed require analyte concentrations in the picomolar range. Here, we demonstrate that superparamagnetic Fe3O4 nanoparticles (NPs) can be used with an external magnetic field as a simple strategy to enhance the immobilization efficiency and thereby decrease the detection limit. Inorganic nanoparticles functionalized with streptavidin were bound to biotinylated single-stranded DNA oligonucleotides, which were in turn annealed to complementary oligonucleotides labeled with a Cy3 fluorescence dye. Using an external magnetic field, the superparamagnetic nanoparticles were localized to a specific region within the flow chamber surface. From the single-molecule fluorescence time traces, single-step photobleaching indicated that the surface-immobilized NPs were primarily bound with a single Cy3-labeled oligonucleotide. This strategy gave a concentration detection limit for the Cy3-labeled oligonucleotide of 100 aM, 3,000-fold lower than that from an analogous strategy with passive immobilization. With a sample volume of 25 μL, this method achieved a mole detection limit of approximately 2.5 zeptomoles (~1500 molecules). Together, the results support that idea that single-molecule fluorescence methods could be used for biological applications, such as detection and measurements of nucleic acids from biological or clinical samples without PCR amplification.

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“…23 The widely used biotin–streptavidin immobilization approach cannot be used here due to the fluorescence background caused by the numerous tryptophan residues in streptavidin. 24 We linked 2AP at the 5′ terminus of a dsDNA through a triethylene glycol linker to mimic the behavior of free 2AP in solution (FREE-2AP).…”

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confidence: 99%

Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle

Alemán

Silva

Patrick

et al. 2014

J. Phys. Chem. Lett.

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Fluorescent nucleotide analogues, such as 2-aminopurine (2AP) and pyrrolo-C (PyC), have been extensively used to study nucleic acid local conformational dynamics in bulk experiments. Here we present a proof-of-principle approach using 2AP and PyC fluorescence at the single-molecule level. Our data show that ssDNA, dsDNA, or RNA containing both 2AP and PyC can be monitored using single-molecule fluorescence and a click chemistry immobilization method. We demonstrate that this approach can be used to monitor DNA and RNA in real time. This is the first reported assay using fluorescent nucleotide analogs at the single-molecule level. We anticipate that single 2AP or PyC fluorescence will have numerous applications in studies of DNA and RNA, including protein-induced base-flipping dynamics in protein–nucleic acid complexes.

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Covalent‐Bond‐Based Immobilization Approaches for Single‐Molecule Fluorescence

Cited by 18 publications

References 18 publications

Single-molecule FRET of protein–nucleic acid and protein–protein complexes: Surface passivation and immobilization

Single-molecule FRET of protein–nucleic acid and protein–protein complexes: Surface passivation and immobilization

Zeptomole detection of DNA nanoparticles by single-molecule fluorescence with magnetic field-directed localization

Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle

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