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Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding.

ABSTRACT: An important goal of nanotechnology is to assemble multiple molecules while controlling the spacing between them. Of particular interest is the phenomenon of multivalency, which is characterized by simultaneous binding of multiple ligands on one biological entity to multiple receptors on another. Various approaches have been developed to engineer multivalency by linking multiple ligands together. However, the effects of well-controlled inter-ligand distances on multivalency are less well understood. Recent progress in self-assembling DNA nanostructures with spatial and sequence addressability has made deterministic positioning of different molecular species possible. Here we show that distance-dependent multivalent binding effects can be systematically investigated by incorporating multiple-affinity ligands into DNA nanostructures with precise nanometre spatial control. Using atomic force microscopy, we demonstrate direct visualization of high-affinity bivalent ligands being used as pincers to capture and display protein molecules on a nanoarray. These results illustrate the potential of using designer DNA nanoscaffolds to engineer more complex and interactive biomolecular networks.

SUBMITTER: Rinker S

PROVIDER: S-EPMC2556356 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding.

Rinker Sherri S Ke Yonggang Y Liu Yan Y Chhabra Rahul R Yan Hao H

Nature nanotechnology 20080622 7

An important goal of nanotechnology is to assemble multiple molecules while controlling the spacing between them. Of particular interest is the phenomenon of multivalency, which is characterized by simultaneous binding of multiple ligands on one biological entity to multiple receptors on another. Various approaches have been developed to engineer multivalency by linking multiple ligands together. However, the effects of well-controlled inter-ligand distances on multivalency are less well underst ...[more]

PMID: 18654566

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Project description:Chemically self-assembled nanorings (CSANs) are made of dihydrofolate reductase (DHFR) fusion proteins and have been successfully used in vitro for cellular cargo delivery and cell surface engineering applications. However, CSANs have yet to be evaluated for their in vivo stability, circulation, and tissue distribution. In an effort to evaluate CSANs in vivo, we engineered a site-specifically PEGylated epidermal growth factor receptor (EGFR) targeting DHFR molecules, characterized their self-assembly into CSANs with bivalent methotrexates (bis-MTX), visualized their in vivo tissue localization by microPET/CT imaging, and determined their ex vivo organ biodistribution by tissue-based gamma counting. A dimeric DHFR (DHFR(2)) molecule fused with a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate a site-specific ketone functionality using unnatural amino acid mutagenesis. Aminooxy-PEG, of differing chain lengths, was successfully conjugated to the protein using oxime chemistry. These proteins were self-assembled into CSANs with bis-MTX DHFR dimerizers and characterized by size exclusion chromatography and dynamic light scattering. In vitro binding studies were performed with fluorescent CSANs assembled using bis-MTX-FITC, while in vivo microPET/CT imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTA[(64)Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by mouse macrophages (RAW 264.7) up to 40% without altering the CSAN's binding affinity toward U-87 MG glioblastoma cells in vitro. A significant time dependent tumor accumulation of (64)Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and biodistribution studies in mice bearing U-87 MG xenografts. PEGylated CSANs demonstrated a reduced uptake by the liver, kidneys, and spleen resulting in high contrast tumor imaging within an hour of intravenous injection (9.6% ID/g), and continued to increase up to 24 h (11.7% ID/g) while the background signal diminished. CSANs displayed an in vivo profile between those of rapidly clearing small molecules and slow clearing antibodies. Thus, CSANs offer a modular, programmable, and stable protein based platform that can be used for in vivo drug delivery and imaging applications.

Dataset Information

Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding.

Publications

Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding.

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