Journal: Science Advances

Article Title: Recapitulating complex biological signaling environments using a multiplexed, DNA-patterning approach

doi: 10.1126/sciadv.aay5696

Figure Lengend Snippet: ( A ) The heterobifunctional linker DBCO-PEG 4 -maleimide enables covalent labeling of ligands of interest with an oligonucleotide label. A free sulfhydryl group on the protein is reacted first with the maleimide moiety on the cross-linker, introducing a DBCO functional group to the ligand that then reacts via click chemistry to an azide-terminated oligonucleotide. SH, thiol group. ( B ) The incorporation of an oligonucleotide label having a fluorescent tag enables imaging and monitoring of DNA-directed ligand patterns. For proof of concept, eGFP with a Cy5 tag was assembled using DNA surface patterns (top). Trends in fluorescence intensity profiles for the patterned protein and the fluorescent tag closely matched one other when tuning surface DNA concentrations (2, 4, and 10 μM, from left to right), suggesting that the fluorescent oligonucleotide label can also be used as a relative readout of patterned protein concentration (bottom). ( C ) Multicomponent DNA surface patterns enable tunable control over each ligand concentration as evident in the DNA assembly of eGFP and mCherry. mCherry concentration was held constant as eGFP concentration was tuned as quantified by the change in eGFP fluorescence intensity (bottom) and visualized in the fluorescent composite images (top). Surface DNA concentration for mCherry was 20 μM. Surface DNA concentration for eGFP was 1, 2, and 20 μM (left to right). Scale bars, 500 μm.

Article Snippet: Immediately before use, a 10 mM solution of DBCO-PEG 4 -maleimide (Jena Bioscience) was prepared in anhydrous dimethyl sulfoxide and reacted, at a fourfold molar excess, with the protein of interest (i.e., eGFP, mCherry, or FGF-2) diluted to 0.1 mM in conjugation buffer [1× PBS with 1 mM EDTA (pH 7)].

Techniques: Labeling, Functional Assay, Imaging, Fluorescence, Protein Concentration, Concentration Assay