Surface Passivation for Single-molecule Protein Studies

Chandradoss, Stanley D.; Haagsma, Anna C.; Lee, Young Kwang; Hwang, Jae‐Ho; Nam, Jwa‐Min; Joo, Chirlmin

doi:10.3791/50549

Cited by 190 publications

(185 citation statements)

References 16 publications

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“…The details of the procedure are described in our video protocol (Chandradoss et al, 2014). A microfluidic chamber was incubated with 20 µL Streptavidin (0.1 mg/mL, Sigma) for 30 sec.…”

Section: Methodsmentioning

confidence: 99%

A Dynamic Search Process Underlies MicroRNA Targeting

Chandradoss

Schirle

Szczepaniak

et al. 2015

Cell

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228

258

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Summary Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target messenger RNAs for repression. Here we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2–4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2–8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.

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“…The details of the procedure are described in our video protocol (Chandradoss et al, 2014). A microfluidic chamber was incubated with 20 µL Streptavidin (0.1 mg/mL, Sigma) for 30 sec.…”

Section: Methodsmentioning

confidence: 99%

A Dynamic Search Process Underlies MicroRNA Targeting

Chandradoss

Schirle

Szczepaniak

et al. 2015

Cell

Self Cite

228

258

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“…Quartz slides and microfluidic chambers were prepared as described (Chandradoss et al, 2014). Cy3 and Cy5 molecules were excited with a solid-state laser at 532 nm and a helium-neon (HeNe) laser at 635 nm, respectively.…”

Section: Methodsmentioning

confidence: 99%

Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade

et al. 2017

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Summary CRISPR-Cas systems defend bacteria and archaea against infection by bacteriophage and other threats. The central component of these systems are surveillance complexes that use guide RNAs to bind specific regions of foreign nucleic acids, marking them for destruction. Surveillance complexes must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear. Here, we used single-molecule FRET to visualize how the Type I–E surveillance complex Cascade searches DNA in real time. Cascade rapidly and randomly samples DNA through nonspecific electrostatic contacts, pausing at short PAM recognition sites that may be adjacent to the target. We identify Cascade motifs that are essential for either nonspecific sampling or positioning and readout of the PAM. Our findings provide a comprehensive structural and kinetic model for the Cascade target-search mechanism, revealing how CRISPR surveillance complexes can rapidly search large amounts of genetic material en route to target recognition.

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“…The most commonly adopted remedy is to tether the molecule to a surface 164 , but the act of immobilization could perturb the system of interest 165 . It is also possible to encapsulate single molecules in nano-containers to spatially limit Brownian motion for extended measurements 166–169 .…”

Section: Single-molecule Photodynamics and Transport Properties Inmentioning

confidence: 99%

Single-molecule spectroscopy and imaging over the decades

2015

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As of 2015, it has been 26 years since the first optical detection and spectroscopy of single molecules in condensed matter. This area of science has expanded far beyond the early low temperature studies in crystals to include single molecules in cells, polymers, and in solution. The early steps relied upon high-resolution spectroscopy of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral fine structure arising directly from the position-dependent fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 1990's, a variety of fascinating physical effects were observed for individual molecules, including imaging of the light from single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency. In the room temperature regime, researchers showed that bursts of light from single molecules could be detected in solution, leading to imaging and microscopy by a variety of methods. Studies of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. All of these early steps provided important fundamentals underpinning the development of super-resolution microscopy based on single-molecule localization and active control of emitting concentration. Current thrust areas include extensions to three-dimensional imaging with high precision, orientational analysis of single molecules, and direct measurements of photodynamics and transport properties for single molecules trapped in solution by suppression of Brownian motion. Without question, a huge variety of studies of single molecules performed by many talented scientists all over the world have extended our knowledge of the nanoscale and microscopic mechanisms previously hidden by ensemble averaging.

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Surface Passivation for Single-molecule Protein Studies

Cited by 190 publications

References 16 publications

A Dynamic Search Process Underlies MicroRNA Targeting

A Dynamic Search Process Underlies MicroRNA Targeting

Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade

Single-molecule spectroscopy and imaging over the decades

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