A localized molecular automaton for <i>in situ</i> visualization of proteins with specific chemical modifications

Liu, Lu; Li, Siqiao; Mao, Anwen; Wang, Guyu; Liu, Yiran; Ju, Huangxian; Ding, Lin

doi:10.1039/c9sc04161c

Cited by 10 publications

(7 citation statements)

References 32 publications

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“…As shown in Figure b, no significant change in the imaging signal intensities on the Cy5 channel was observed, indicating a similar amount of SiaNAz. Compared with the single labeling results, , F 0.5 PNP, F 1 PNP, and F 2 PNP enhanced imaging intensity up to approximately 2.5, 6.8, and 13.6 times on the Cy3 channel, and approximately 5.3, 6.7, and 8.8 times via FRET, respectively. The greater the number of dyes loaded on the probe, the higher the fluorescence intensity observed in the imaging results on the Cy3 and FRET channels.…”

Section: Resultsmentioning

confidence: 59%

High Dye-Loaded and Thin-Shell Fluorescent Polymeric Nanoparticles for Enhanced FRET Imaging of Protein-Specific Sialylation on the Cell Surface

et al. 2020

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Nanoparticle-based probes have great potential for imaging specific biomolecules in signal distinguishing and amplification via Forster resonance energy transfer (FRET). Protein-specific sialylation plays key roles in the regulation of protein structure and function, as well as in various pathophysiological processes. Here, we developed a fluorescent polymeric nanoparticle with a biocompatible hydrophilic thin shell loaded with plentiful dye and used it as the donor to enhance the FRET imaging of cell surface proteinspecific sialylation. The hydrophobic core decreased the self-quenching of loaded fluorescent molecules, while the hydrophilic thin shell ensured that the nanoparticles remained on the extracellular surface and guaranteed the FRET effect. Thus, the thin-shell polymeric nanoparticles enhanced the FRET imaging of protein tyrosine kinase-7-specific sialylation on the CCRF-CEM cell surface and showed high sensitivity under drug treatment. This nanoparticle has great potential for elucidating the relationship between dynamic specific glycosylation states and disease processes, as well as for the study of new cell surface imaging methodologies.

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Section: Resultsmentioning

confidence: 59%

High Dye-Loaded and Thin-Shell Fluorescent Polymeric Nanoparticles for Enhanced FRET Imaging of Protein-Specific Sialylation on the Cell Surface

et al. 2020

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“…After NaIO 4 treatment, the incubation of MCF-7 cells with Hz−FAM−H resulted in an obvious fluorescence on the cell membrane (Figure 2, parts B and D, case d). As expected, the elimination Considering that the following Nt.BbvCI cleavage step should be performed at 37 °C for 2 h, 27,28 the cells, after probe installation, were fixed with 4% PFA 28 to (1) preclude the fast entry of CTxB conjugates into cells since CTxB can mostly enter cells within 30 min at 37 °C and 37 (2) eliminate the influence from the Nt.BbvCI cleavage buffer on cellular morphology. Maintaining the fluorescence-quenched state of the Sia probe after probe assembly is a prerequisite for achieving raft specificity, which was verified by the background-leveled fluorescence in Figure 3, parts A and C.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…After probe assembly on the cell surface, the fluorophores on the raft probes indicate the distribution of the rafts. A proximity cascade DNA reaction , was performed to ensure that only the Sia probe linked to LR-Sia, rather than those on nonraft-Sia, can be lighted up, which represents the sialylation level of the rafts. The obtained LR-Sia signals showed excellent raft and sugar specificities.…”

Section: Introductionmentioning

confidence: 99%

Hierarchical Fluorescence Imaging Strategy for Assessment of the Sialylation Level of Lipid Rafts on the Cell Membrane

Shi

Chen

et al. 2021

Anal. Chem.

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Glycosylation is one of the most ubiquitous and complicated modifications of proteins and lipids. The revelation of glycosylation-mediated regulation mechanisms of biological processes relies critically on the tools that can reflect the spatial heterogeneity of cell surface glycans, for example, distinguishing glycans exhibited in lipid raft or nonraft domains. To achieve simultaneous visualization of raft and raft-harbored glycans on the cell surface, we combine specific raft recognition, glycan chemoselective labeling, and DNA dynamic hybridization techniques to develop a hierarchical fluorescence imaging strategy using N-acetylneuraminic acid (Sia) as the model sugar. We fabricate a raft probe and Sia probe for rafts and Sia, respectively. After specifically anchoring the two probes on the cell surface, the raft probe can be cyclically utilized to turn on the fluorescence of the Sia probe, only residing in rafts, via a proximity cascade DNA reaction. The duplex imaging capability for spatially relevant levels of biological structures enables the revelation of the reason for raft-confined Sia variation in different biological processes. Thus, this work provides an elegant and powerful tool for interrogation of the glycan regulation mechanisms on raft composition, organization, and functions and also contributes to the development of raft-carried glycoconjugate-based theranostic techniques.

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“…Some studies used lectin, metabolism and other methods to study the overall state of glycosylation of tumor cells (118). In order to achieve more precise analysis, more comprehensive surface accessibility, higher sensitivity, and wider applicability, cell-specific (119-123) and protein-specific (124)(125)(126)(127)(128)(132)(133)(134) glycan in situ analytical methods have been continuously developed in the past five years.…”

Section: Fluorescence Imaging-based In Situ Cellular Glycan Analysismentioning

confidence: 99%

“…Fluorescence resonance energy transfer (FRET) is the main method to analyze protein-specific glycans ( 117 , 124 ). Methods based on site-specific duplexed luminescence resonance energy transfer (D-LRET) ( 125 ), hierarchical coding (HieCo) ( 126 ), localized chemical remodeling (LCM) ( 127 ), and DNA enzymatic reactions ( 128 , 132 ) have also been developed in the past five years. A FRET strategy based on hybridization chain reaction (HCR) amplification was reported ( 124 ).…”

Section: Recent Advances In Glycomics-based Biomarker Discoverymentioning

confidence: 99%

Cancer glycomics offers potential biomarkers and therapeutic targets in the framework of 3P medicine

et al. 2022

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Glycosylation is one of the most important post-translational modifications (PTMs) in a protein, and is the most abundant and diverse biopolymer in nature. Glycans are involved in multiple biological processes of cancer initiation and progression, including cell-cell interactions, cell-extracellular matrix interactions, tumor invasion and metastasis, tumor angiogenesis, and immune regulation. As an important biomarker, tumor-associated glycosylation changes have been extensively studied. This article reviews recent advances in glycosylation-based biomarker research, which is useful for cancer diagnosis and prognostic assessment. Truncated O-glycans, sialylation, fucosylation, and complex branched structures have been found to be the most common structural patterns in malignant tumors. In recent years, immunochemical methods, lectin recognition-based methods, mass spectrometry (MS)-related methods, and fluorescence imaging-based in situ methods have greatly promoted the discovery and application potentials of glycomic and glycoprotein biomarkers in various cancers. In particular, MS-based proteomics has significantly facilitated the comprehensive research of extracellular glycoproteins, increasing our understanding of their critical roles in regulating cellular activities. Predictive, preventive and personalized medicine (PPPM; 3P medicine) is an effective approach of early prediction, prevention and personalized treatment for different patients, and it is known as the new direction of medical development in the 21st century and represents the ultimate goal and highest stage of medical development. Glycosylation has been revealed to have new diagnostic, prognostic, and even therapeutic potentials. The purpose of glycosylation analysis and utilization of biology is to make a fundamental change in health care and medical practice, so as to lead medical research and practice into a new era of 3P medicine.

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A localized molecular automaton for in situ visualization of proteins with specific chemical modifications

Abstract: A localized DNA automaton is reported for in situ visualization of a specific protein subtype with dual chemical modifications on the cell surface, which executes protein-confined computation according to an anticoding–coding propagation algorithm.

Cited by 10 publications

References 32 publications

High Dye-Loaded and Thin-Shell Fluorescent Polymeric Nanoparticles for Enhanced FRET Imaging of Protein-Specific Sialylation on the Cell Surface

High Dye-Loaded and Thin-Shell Fluorescent Polymeric Nanoparticles for Enhanced FRET Imaging of Protein-Specific Sialylation on the Cell Surface

Hierarchical Fluorescence Imaging Strategy for Assessment of the Sialylation Level of Lipid Rafts on the Cell Membrane

Cancer glycomics offers potential biomarkers and therapeutic targets in the framework of 3P medicine

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