Compact and stable SNAP ligand-conjugated quantum dots as a fluorescent probe for single-molecule imaging of dynein motor protein

Ohyanagi, Tatsuya; Shima, Tomohiro; Okada, Yasushi; Tsukasaki, Yoshikazu; Komatsuzaki, Akihito; Tsuboi, Setsuko

doi:10.1039/c5cc05526a

Cited by 7 publications

(6 citation statements)

References 28 publications

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“…The size and monodispersity of aqueous CdSe/CdS QDs were unchanged during >4 months of storage at room temperature (Figure S3). In contrast, our attempts to exchange hydrophobic QDs into water by ligand exchange, in which hydrophobic synthetic ligands are replaced by polar ones, 23,24,30 resulted in QDs with poor colloidal stability that aggregated irreversibly over a matter of days (not shown). Whereas these aqueous QDs are smaller than their polymer-wrapped counterparts, the rapid onset of aggregation makes them unsuitable for many imaging experiments.…”

Section: Resultsmentioning

confidence: 89%

“…Single-particle imaging demands that QD probes have exceptional stability, particularly for long-term tracking or use within live cells. Exchange of native ligands for hydrophilic thiols or other coordinating ligands produces functional QDs with small passivation shells, which is an advantage for many imaging applications. , Our attempts to shrink the QD complex by ligand exchange have been unsuccessful in producing single-molecule probes, and we have found that retention of native ligands within a liposome-like structure is essential for the brightness, chemical stability, and photostability required for single-molecule experiments. Monovalent ligands are rapidly displaced by other ligands, such as the thiols found in the cytosol, and are prone to photo-oxidation under the high photon flux required for single-molecule imaging .…”

Section: Resultsmentioning

confidence: 99%

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Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots

Wichner¹,

Mann

Powers

et al. 2017

ACS Nano

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Semiconductor quantum dots (QDs) have proven to be superior probes for single molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ~10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on state, ~4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor in vitro and the improved labeling efficiency enables tracking of single kinesins in live cells.

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots

Wichner¹,

Mann

Powers

et al. 2017

ACS Nano

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“…The structural data further suggested that the efficiency of alkyl transfer could be reduced by impairing the substrate binding to SNAP‐tag. Based on this, two BG derivatives, that is, BI lacking the 2‐amino group and 7‐deaza‐benzylguanine (deBG) replacing N7 with C7 (Figure 2b), were chosen as the candidates [42,49,50] . Subsequently these substrates were synthesized as described in the Experimental Section and Supporting Information.…”

Section: Resultsmentioning

confidence: 99%

“…Based on this, two BG derivatives, that is, BI lacking the 2-amino group and 7-deaza-benzylguanine (deBG) replacing N7 with C7 (Figure 2b), were chosen as the candidates. [42,49,50] Subsequently these substrates were synthesized as described in the Experimental Section and Supporting Information.…”

Section: Structure-based Design and Synthesis Of Snap-tag Substratesmentioning

confidence: 99%

Tuning the Reactivity of a Substrate for SNAP‐Tag Expands Its Application for Recognition‐Driven DNA‐Protein Conjugation

Zhang

Nakata

Dinh

et al. 2021

Chemistry A European J

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Recognition‐driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site‐specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition‐driven modification of DNA as they consist of both a sequence‐specific DNA‐binding domain (DBD) and a self‐ligating protein‐tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence‐specific reaction. However, combining an MA consisting of a reactive protein‐tag and its substrate, for example, SNAP‐tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP‐tag have been designed to realize sequence‐selective rapid crosslinking reactions of MAs with SNAP‐tag. The reactions of substrates with SNAP‐tag were verified by kinetic analyses to enable the sequence‐selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP‐tag against CLIP‐tag to achieve orthogonal DNA‐protein crosslinking by six unique MAs.

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“…Although existing approaches have added significant understanding of the structure and function of receptors, most involve ensemble measurements that lack the specificity to monitor the dynamics of individual receptor–ligand activity. − Single-molecule studies of membrane proteins are limited by intracellular autofluorescence, weak fluorophore emission, membrane protein mobility on the cell surface, and high protein concentrations that are incompatible with single-molecule measurements. One approach to address the challenge of fluorophore brightness is the use of relatively large but photostable labels such as quantum dots, which can be utilized to monitor membrane receptor mobility and, in some circumstances, ligand interactions. , However, these studies are complicated by the relatively large size of functionalized quantum dots, and their potential toxicity limits their practical use in many applications …”

Section: Introductionmentioning

confidence: 99%

Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices

Martin

Ge²,

Srijanto

et al. 2017

ACS Omega

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The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, allowing us to quantify the ligand turnover in real time. These results demonstrate that this nanoaperture-coupled microfluidic device allows for the spatial isolation of individual membrane proteins while maintaining them in their cellular environment, providing the capability to monitor single-ligand binding events while maintaining receptors in their physiological environment. These methods should be applicable to a wide range of membrane proteins.

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Compact and stable SNAP ligand-conjugated quantum dots as a fluorescent probe for single-molecule imaging of dynein motor protein

Abstract: Compact SNAP ligand-conjugated quantum dots (<10 nm) with high colloidal stability over a wide range of pH (5-9) have been synthesized as fluorescent probe for the single-molecule imaging of dynein motor protein.

Cited by 7 publications

References 28 publications

Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots

Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots

Tuning the Reactivity of a Substrate for SNAP‐Tag Expands Its Application for Recognition‐Driven DNA‐Protein Conjugation

Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices

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