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Single-stranded DNA as a cleavable linker for bioorthogonal click chemistry-based proteomics.

ABSTRACT: In this communication, we report a new class of cleavable linker based on automatically synthesized, single-stranded DNAs. We incorporated a DNA oligo into an azide-functionalized biotin (biotin-DNA-N3) and used the probe to enrich for alkyne-tagged glycoproteins from mammalian cell lysates. Highly efficient and selective release of the captured proteins from streptavidin agarose resins was achieved using DNase treatment under very mild conditions. A total of 36 sialylated glycoproteins were identified from the lysates of HL60 cells, an acute human promyeloid leukemia cell line. These sialylated glycoproteins were involved in many different biological processes ranging from glycan biosynthesis to cell adhesion events.

SUBMITTER: Zheng T

PROVIDER: S-EPMC4078914 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Single-stranded DNA as a cleavable linker for bioorthogonal click chemistry-based proteomics.

Zheng Tianqing T Jiang Hao H Wu Peng P

Bioconjugate chemistry 20130517 6

In this communication, we report a new class of cleavable linker based on automatically synthesized, single-stranded DNAs. We incorporated a DNA oligo into an azide-functionalized biotin (biotin-DNA-N3) and used the probe to enrich for alkyne-tagged glycoproteins from mammalian cell lysates. Highly efficient and selective release of the captured proteins from streptavidin agarose resins was achieved using DNase treatment under very mild conditions. A total of 36 sialylated glycoproteins were ide ...[more]

PMID: 23627610

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Project description:UnlabelledThe specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacologic half-lives necessitate the use of radioisotopes with long physical half-lives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodology for pretargeted PET imaging based on the bioorthogonal Diels-Alder click reaction between tetrazine and transcyclooctene.MethodsA proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and (64)Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with (64)Cu. Pretargeted in vivo biodistribution and PET imaging experiments were performed with athymic nude mice bearing A33 antigen-expressing, SW1222 colorectal cancer xenografts.ResultsThe huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the (64)Cu-NOTA-labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9-10 MBq/μg. For in vivo experiments, mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with (64)Cu-NOTA-labeled tetrazine for PET imaging and biodistribution experiments. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per gram), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution experiments performed using A33 directly labeled with either (64)Cu or (89)Zr revealed that although absolute tumor uptake was higher with the directly radiolabeled antibodies, the pretargeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calculations revealed that the (64)Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with (89)Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, respectively).ConclusionThe high quality of the images produced by this pretargeting approach, combined with the ability of the methodology to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clinical translation.

Project description:Background:Phospholipids are important structural and signaling molecules in plant membranes. Some fluorescent dyes can stain general lipids of membranes, but labeling and visualization of specific lipid classes have yet to be developed for most components of the membrane. New techniques for visualizing membrane lipids are needed to further delineate their dynamic structural and signaling roles in plant cells. In this study we examined whether propargylcholine, a bioortholog of choline, can be used to label the major membrane lipid, phosphatidylcholine, and other choline phospholipids in plants. We established that propargylcholine is readily taken up by roots, and that its incorporation is not detrimental to plant growth. After plant tissue is harvested and fixed, a click-chemistry reaction covalently links the alkyne group of propargylcholine to a fluorescently-tagged azide, resulting in specific labeling of choline phospholipids. Results:Uptake of propargylcholine, followed by click chemistry with fluorescein or Alexa Fluor 594 azide was used to visualize choline phospholipids in cells of root, leaf, stem, silique and seed tissues from Arabidopsis thaliana. Co-localization with various subcellular markers indicated coinciding fluorescent signals in cell membranes, such as the tonoplast and the ER. Among different cell types in the leaf epidermis, guard cells displayed strong labeling. Mass spectrometry-based lipidomic analysis of the various plant tissues revealed that incorporation of propargylcholine was strongest in roots with approximately 50% of total choline phospholipids being labeled, but it was also incorporated in the other tissues including seeds. Phospholipid profiling confirmed that, in each tissue analyzed, incorporation of the bioortholog had little impact on the pool of choline plus choline-like phospholipids or other lipid species. Conclusion:We developed and validated a click-chemistry based method for fluorescence imaging of choline phospholipids using a bioortholog of choline, propargylcholine, in various cell-types and tissues from Arabidopsis. This click-chemistry method provides a direct way to metabolically tag and visualize specific lipid molecules in plant cells. This work paves the way for future studies addressing in situ localization of specific lipids in plants.

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Single-stranded DNA as a cleavable linker for bioorthogonal click chemistry-based proteomics.

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Single-stranded DNA as a cleavable linker for bioorthogonal click chemistry-based proteomics.

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