Article

Photocrosslinking of glycoconjugates using metabolically incorporated diazirine-containing sugars.

Department of Chemistry, Stanford University, Stanford, California, USA.
Nature Protocol (Impact Factor: 8.36). 02/2009; 4(7):1044-63. DOI: 10.1038/nprot.2009.85
Source: PubMed

ABSTRACT Transient interactions among glycoconjugates underlie developmental, immunological and metastatic recognition. Glycan-mediated interactions have low binding affinities and rapid dissociation rates. As a result, these complexes dissociate when removed from their cellular context, complicating characterization. Photocrosslinkers introduce a covalent bond between glycoconjugates and their binding partners, allowing physiologically relevant complexes to be isolated. This protocol describes metabolic incorporation of a diazirine photocrosslinker into sialic acids in cellular glycoconjugates. Subsequent irradiation results in photocrosslinking of sialic acid to neighboring macromolecules, providing a photochemical 'snapshot' of binding events. As photocrosslinking sugars are light activated, these reagents have the potential to be used for temporally and/or spatially restricted crosslinking. We provide instructions for the synthesis of photocrosslinking sugar precursors, cell culture for metabolic incorporation, flow cytometry to evaluate metabolic incorporation, photoirradiation and analysis of the crosslinked complexes. Synthesis of photocrosslinking sugars requires 4-6 d, and photocrosslinking experiments can be completed in an additional 6 d.

1 Bookmark
 · 
263 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The roles played by many ncRNAs remain largely unknown. Similarly, relatively little is known about the RNA binding proteins involved in processing ncRNA. Identification of new RNA/RNA binding protein (RBP) interactions may pave the way to gain a better understanding of the complex events occurring within cells during gene expression and ncRNA biogenesis. The development of chemical tools for the isolation of RBPs is of paramount importance. In this context, we report on the synthesis of the uridine phosphoramidite UDz that bears a diazirine moiety on the nucleobase. RNA probes containing UDz units were irradiated in the presence of single-stranded DNA binding protein (SSB), which is also known to bind ssRNAs, and shown to efficiently (15% yield) and selectively cross-link to the protein. The corresponding diazirine-modified uridine triphosphate UDzTP was synthesized and its capacity to act as a substrate for the T7 RNA polymerase was tested in transcription assays. UDzTP was accepted with a maximum yield of 38% for a 26mer RNA containing a single incorporation and 28% yield for triple consecutive incorporations. Thus, this uridine analogue represents a convenient biochemical tool for the identification of RNA binding proteins and unraveling the role and function played by ncRNAs.
    RSC Advances 09/2014; 4(89). DOI:10.1039/C4RA08682A · 3.71 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We previously reported the identification and development of novel inhibitors of streptokinase (SK) expression by Group A Streptococcus (GAS), originating from a high throughput cell-based phenotypic screen. Although phenotypic screening is well-suited to identifying compounds that exert desired biological effects in potentially novel ways, it requires follow-up experiments to determine the macromolecular target(s) of active compounds. We therefore designed and synthesized several classes of chemical probes for target identification studies, guided by previously established structure-activity relationships. The probes were designed to first irreversibly photolabel target proteins in the intact bacteria, followed by cell lysis and click ligation with fluorescent tags to allow for visualization on SDS-PAGE gels. This stepwise, 'tag-free' approach allows for a significant reduction in molecular weight and polar surface area compared to full-length fluorescent or biotinylated probes, potentially enhancing membrane permeability and the maintenance of activity. Of the seven probes produced, the three most biologically active were employed in preliminary target identification trials. Despite the potent activity of these probes, specific labeling events were not conclusively observed due to a considerable degree of nonspecific protein binding. Nevertheless, the successful synthesis of potent biologically active probe molecules will serve as a starting point for initiating more sensitive methods of probe-based target identification.
    Bioorganic & medicinal chemistry letters 02/2014; DOI:10.1016/j.bmcl.2014.01.079 · 2.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Around 300 people from 18 countries took part in the fourth biennial Chemical Biology conference at The European Molecular Biology Laboratory (EMBL) in Heidelberg, from August 20 to 23, 2014. Many advances in the field of chemical biology were presented in talks and poster sessions. Picture: Petra Riedinger (EMBL).
    ChemBioChem 12/2014; 15(18). DOI:10.1002/cbic.201402527 · 3.06 Impact Factor

Full-text (2 Sources)

Download
34 Downloads
Available from
Jun 1, 2014