An Improved Surface Passivation Method for Single-Molecule Studies

Hua, Bingzhu; Zhou, Ruobo; Kim, Hajin; Shi, Xinghua; Jain, Ankur; Singh, Digvijay; Aggarwal, Vasudha; Ha, Taekjip

doi:10.1016/j.bpj.2013.11.2218

Cited by 34 publications

(53 citation statements)

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“…Single-molecule data acquisition and analysis were performed with the following method 23,44 . Briefly, fluorescently labeled samples were immobilized on DT20 or PEG surfaces through a biotin-NeutrAvidin linkage 45,46 . Samples were imaged with a prism-based total internal reflection fluorescence (TIRF) imaging microscope 47 .…”

Section: Methodsmentioning

confidence: 99%

Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

Hua¹,

Jones

Mitra

et al. 2020

Nat Commun

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RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5′-to-3′ direction, at ~60 nt s−1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.

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Section: Methodsmentioning

confidence: 99%

Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

Hua¹,

Jones

Mitra

et al. 2020

Nat Commun

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“…GNP imaging experiments were performed inside a flow chamber assembled with a dichlorodimethylsilane (DDS)-coated glass 49 as follows. Coverslips were cleaned by low-pressure plasma for 3 min with a plasma system (Zepto, Diener electronic, Germany).…”

Section: Sample Preparation For Gnp Imagingmentioning

confidence: 99%

Direct visualization of human myosin II force generation using DNA origami-based thick filaments

Fujita

Ohmachi

Ikezaki

et al. 2019

Preprint

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Muscle contraction can be explained by the swinging lever-arm model. However, the dynamic features of how the myosin head swings the lever-arm and its initial interactions with actin are not well understood even though they are essential for the muscle force generation, contraction speed, heat production, and response to mechanical perturbations. This is because myosin heads during force generation have not been directly visualized. Here, we engineered thick filaments composed of DNA origami and recombinant human muscle myosin, and directly visualized the heads during force generation using nanometer-precision single-molecule imaging.We found that when the head diffuses, it weakly interacts with actin filaments and then strongly binds preferentially to the forward region as a Brownian ratchet.Upon strong binding, the head two-step lever-arm swing dominantly halts at the first step and occasionally reverses direction. These results can explain all mechanical characteristics of muscle contraction and suggest that our DNA origami-based assay system can be used to dissect the mechanistic details of motor proteins.

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“…[41][42][43][44] However, all these applications require a proper surface passivation and/or functionalization of the ZMW in order to avoid the unwanted adsorption of the fluorescent molecules onto the ZMW surface that would impede the experiments. While extensive literature exists for passivating glass surfaces for single molecule fluorescence microscopy, [45][46][47][48][49][50][51][52] little is known about the surface passivation of ZMWs and the comparative analysis of different passivation strategies. For aluminum ZMWs, most studies reproduce the protocol based on polyvinylphosphonic acid (PVPA) as initially introduced by Korlach and coworkers in Ref.…”

mentioning

confidence: 99%

Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels

Patra

Baibakov

Claude

et al. 2020

Sci Rep

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Zero mode waveguide (ZMW) nanoapertures efficiently confine the light down to the nanometer scale and overcome the diffraction limit in single molecule fluorescence analysis. However, unwanted adhesion of the fluorescent molecules on the ZMW surface can severely hamper the experiments. Therefore a proper surface passivation is required for ZMWs, but information is currently lacking on both the nature of the adhesion phenomenon and the optimization of the different passivation protocols. Here we monitor the influence of the fluorescent dye (Alexa Fluor 546 and 647, Atto 550 and 647N) on the non-specific adhesion of double stranded DNA molecule. We show that the nonspecific adhesion of DNA double strands onto the ZMW surface is directly mediated by the organic fluorescent dye being used, as Atto 550 and Atto 647N show a pronounced tendency to adhere to the ZMW while the Alexa Fluor 546 and 647 are remarkably free of this effect. Despite the small size of the fluorescent label, the surface charge and hydrophobicity of the dye appear to play a key role in promoting the DNA affinity for the ZMW surface. Next, different surface passivation methods (bovine serum albumin BSA, polyethylene glycol PEG, polyvinylphosphonic acid PVPA) are quantitatively benchmarked by fluorescence correlation spectroscopy to determine the most efficient approaches to prevent the adsorption of Atto 647N labeled DNA. Protocols using PVPA and PEG-silane of 1000 Da molar mass are found to drastically avoid the non-specific adsorption into ZMWs. Optimizing both the choice of the fluorescent dye and the surface passivation protocol are highly significant to expand the use of ZMWs for single molecule fluorescence applications.

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An Improved Surface Passivation Method for Single-Molecule Studies

Cited by 34 publications

References 0 publications

Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

Direct visualization of human myosin II force generation using DNA origami-based thick filaments

Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels

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