N-Terminal Labeling of Filamentous Phage To Create Cancer Marker Imaging Agents
ABSTRACT We report a convenient new technique for the labeling of filamentous phage capsid proteins. Previous reports have shown that phage coat protein residues can be modified, but the lack of chemically distinct amino acids in the coat protein sequences makes it difficult to attach high levels of synthetic molecules without altering the binding capabilities of the phage. To modify the phage with polymer chains, imaging groups, and other molecules, we have developed chemistry to convert the N-terminal amines of the ~4200 coat proteins into ketone groups. These sites can then serve as chemospecific handles for the attachment of alkoxyamine groups through oxime formation. Specifically, we demonstrate the attachment of fluorophores and up to 3000 molecules of 2 kDa poly(ethylene glycol) (PEG2k) to each of the phage capsids without significantly affecting the binding of phage-displayed antibody fragments to EGFR and HER2 (two important epidermal growth factor receptors). We also demonstrate the utility of the modified phage for the characterization of breast cancer cells using multicolor fluorescence microscopy. Due to the widespread use of filamentous phage as display platforms for peptide and protein evolution, we envision that the ability to attach large numbers of synthetic functional groups to their coat proteins will be of significant value to the biological and materials communities.
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ABSTRACT: We report the synthesis, characterization, and protein sensing capabilities of M13 bacteriophage-DNA bioconjugates. DNA oligonucleotides were conjugated to M13 through acyl hydrazone linkages. In one case, DNAzymes retained their catalytic ability when anchored to the virus coat, and in a separate study, the dynamic nature of the hydrazone allowed for liberation of DNA from the phage under mild conditions.Chemical Communications 01/2013; 49(17). DOI:10.1039/c3cc38871a · 6.72 Impact Factor
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ABSTRACT: Increasing demands from nanotechnology require increasingly more rigorous methods to control nanoparticle traits such as assembly, size, morphology, monodispersity, stability, and reactivity. Viruses are a compelling starting point for engineering nanoparticles, as eons of natural biological evolution have instilled diverse and desirable traits. The next step is to reengineer these viruses into something functional and useful. These reengineered particles, or virus-based nanoparticles (VNPs), are the foundation for many promising new technologies in drug delivery, targeted delivery, vaccines, imaging, and biocatalysis. To achieve these end goals, VNPs must often be manipulated genetically and post-translationally. We review prevailing strategies of genetic and noncovalent functionalization and focus on the covalent modifications using natural and unnatural amino acid residues.Current Opinion in Biotechnology 03/2013; DOI:10.1016/j.copbio.2013.01.011 · 8.04 Impact Factor
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ABSTRACT: NMR imaging: Filamentous fd bacteriophage expressing antibodies recognizing the epidermal growth factor receptor (EGFR) were modified to incorporate cage-like xenon-binding molecules (CryA). The resulting contrast agent was shown to bind to an EGFR-positive cell line and detected by hyperpolarized (129) Xe NMR spectroscopy using chemical exchange saturation transfer (hyperCEST, see picture).Angewandte Chemie International Edition 04/2013; 52(18). DOI:10.1002/anie.201300170 · 11.34 Impact Factor