• Journal of the Optical Society of Korea
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• 제12권2호
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• pp.107-111
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• 2008

We demonstrated single-molecule fluorescence resonance energy transfer (FRET) from single donor-acceptor dye pair attached to a DNA with a setup based on a confocal microscope. Singlestrand DNAs were immobilized on a glass surface with suitable inter-dye distance. Energy transfer efficiency between the donor and the acceptor dyes attached to the DNA was measured with different lengths of DNA. Photobleaching of single dye molecule was observed and used as a sign of single-molecule detection. We could achieve high enough signal-to-noise ratio to detect the fluorescence from a single-molecule, which allows real-time observation of the distance change between single dye pairs in nanometer scale.

• Bulletin of the Korean Chemical Society
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• 제33권3호
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• pp.958-962
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• 2012

In single-molecule fluorescence experiment, the oxygen scavenging system is indispensable for avoiding photo-bleaching of fluorescent dyes. Here we report that the gloxy-based oxygen scavenging system commonly used in single molecule fluorescence experiments can disturb the solution pH considerably. To track in situ pH change, we utilized the pH-sensitive conformational transition of i-motif and examined the transition with ensemble and single-molecule FRET measurements. Based on our results, we also suggested several practical remedies for the stability of the solution pH.

• Bulletin of the Korean Chemical Society
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• 제34권9호
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• pp.2725-2730
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• 2013

Single C-reactive protein (CRP) molecules, which are non-specific acute phase markers and products of the innate immune system, were quantitatively detected on a gold-nanopatterned biochip using evanescent field-enhanced fluorescence imaging. The $4{\times}5$ gold-nanopatterned biochip (spot diameter of 500 nm) was fabricated by electron beam nanolithography. Unlabeled CRP molecules in human serum were identified with single-molecule sandwich immunoassay by detecting secondary fluorescence generated by total internal reflection fluorescence (TIRF) microscopy. With decreased standard CRP concentrations, relative fluorescence intensities reduced in the range of 33.3 zM-800 pM. To enhance fluorescence intensities in TIRF images, the distance between biochip surface and CRP molecules was optimally adjusted by considering the quenching effect of gold and the evanescent field intensity. As a result, TIRF only detected one single-CRP molecule on the biochip the first time.

• 한국정밀공학회:학술대회논문집
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• 한국정밀공학회 2008년도 춘계학술대회 논문집
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• pp.697-698
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• 2008

• Molecules and Cells
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• 제45권1호
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• pp.33-40
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• 2022

The various DNA-protein interactions associated with the expression of genetic information involve double-stranded DNA (dsDNA) bending. Due to the importance of the formation of the dsDNA bending structure, dsDNA bending properties have long been investigated in the biophysics field. Conventionally, DNA bendability is characterized by innate averaging data from bulk experiments. The advent of single-molecule methods, such as atomic force microscopy, optical and magnetic tweezers, tethered particle motion, and single-molecule fluorescence resonance energy transfer measurement, has provided valuable tools to investigate not only the static structures but also the dynamic properties of bent dsDNA. Here, we reviewed the single-molecule methods that have been used for investigating dsDNA bendability and new findings related to dsDNA bending. Single-molecule approaches are promising tools for revealing the unknown properties of dsDNA related to its bending, particularly in cells.

• Journal of Photoscience
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• 제11권2호
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• pp.47-53
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• 2004

A laser scanning fluorescence microscope system has been fabricated for single molecule detection (SMD). Problems associated with the system set-up have been discussed along with proper suggestions. Based on the SMD results obtained by using the apparatus, a statistical method has been suggested to determine the minimum number of required molecules to form a group of uniform average in a selected error range.

• BMB Reports
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• 제46권2호
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• pp.65-72
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• 2013

In situ detection of RNAs is becoming increasingly important for analysis of gene expression within and between intact cells in tissues. International genomics efforts are now cataloging patterns of RNA transcription that play roles in cell function, differentiation, and disease formation, and they are demon-strating the importance of coding and noncoding RNA transcripts in these processes. However, these techniques typically provide ensemble averages of transcription across many cells. In situ hybridization-based analysis methods complement these studies by providing information about how expression levels change between cells within normal and diseased tissues, and they provide information about the localization of transcripts within cells, which is important in understanding mechanisms of gene regulation. Multi-color, single-molecule fluorescence in situ hybridization (smFISH) is particularly useful since it enables analysis of several different transcripts simultaneously. Combining smFISH with immunofluorescent protein detection provides additional information about the association between transcription level, cellular localization, and protein expression in individual cells.

• BMB Reports
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• 제50권2호
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• pp.55-57
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• 2017

Toll-like receptor 4 (TLR4) together with MD2, one of the key pattern recognition receptors for a pathogen-associated molecular pattern, activates innate immunity by recognizing lipopolysaccharide (LPS) of Gram-negative bacteria. Although LBP and CD14 catalyze LPS transfer to the TLR4/MD2 complex, the detail mechanisms underlying this dynamic LPS transfer remain elusive. Using negative-stain electron microscopy, we visualized the dynamic intermediate complexes during LPS transfer-LBP/LPS micelles and ternary CD14/LBP/LPS micelle complexes. We also reconstituted the entire cascade of LPS transfer to TLR4/MD2 in a total internal reflection fluorescence (TIRF) microscope for a single molecule fluorescence analysis. These analyses reveal longitudinal LBP binding to the surface of LPS micelles and multi-round binding/unbinding of CD14 to single LBP/LPS micelles via key charged residues on LBP and CD14. Finally, we reveal that a single LPS molecule bound to CD14 is transferred to TLR4/MD2 in a TLR4-dependent manner. These discoveries, which clarify the molecular mechanism of dynamic LPS transfer to TLR4/MD2 via LBP and CD14, provide novel insights into the initiation of innate immune responses.

• BMB Reports
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• 제48권12호
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• pp.643-644
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• 2015

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection.

• Bulletin of the Korean Chemical Society
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• 제31권4호
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• pp.1021-1024
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• 2010