Assembly of gold nanoparticles by ribosomal molecular machines

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Pavel, Ioana Simona

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The goal of this work is to develop novel method for organizing gold nanoparticles by linking them to chemically reactive side chains amino acids (e.g., lysine and cysteine) and then incorporating these modified amino acids into a polypeptide through in vitro translation of a synthetic mRNA template. The use of this system not only provides a method for the supramolecular assembly of new bio-inorganic heterostructures, using ribosomes as natural molecular machines and an artificial coding template, but also provides a new tool for probing fundamental biological processes (e.g., translation). Transfer RNA (tRNA) is transcribed in vitro from a plasmid-template containing the tRNA gene under control of the T7 promoter. A novel coupled transcription– aminoacylation (CTA) system which prepares large amounts of pure aminoacyl-tRNA in a single reaction mixture was developed. Radiolabeled [32P]-UTP and [14C] or [32S]- amino acid are used to monitor the coupled transcription- aminoacylation reactions. The ability to further modify the tRNA with a biotinylated probe is also demonstrated. The tRNA synthesized by CTA, or further modified with biotin is shown to retain its functionality in in vitro translation, allowing the synthesis and detection of biotin-labeled protein. Following the charging of the tRNA, the side chain of the amino acid is covalently linked to a gold nanoparticle, i.e. Monomaleido or Mono-sulfo-Nhydroxysuccinimide Nanogolds, either directly or through a heterobifunctional linker, which may vary in length and/or chemical properties, depending on the system used. The result is a novel gold nanoparticle-conjugated tRNA. Each of the steps is verified by mass spectroscopy (ESI and/or MALDI). The attachment of the gold nanoparticle to modified aminoacyl-tRNA is confirmed using High angle annular dark field images (HTEM) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The incorporation of the quantum dot-conjugated tRNA into a polypeptide is measured using an in vitro E. coli S30 cell-free coupled transcription-translation system, and verified by electrophoresis, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), High angle annular dark field (HAADF) images and electron loss spectroscopy .




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