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      A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers

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      PLoS ONE
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          Abstract

          Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an efficient, rapid and specific manner, based on the recently developed linkage between the protein StrepTactin (STN) and the peptide StrepTag II (ST). We introduce a two-step approach, in which we first construct a hybrid between DNA and a tandem of two STs peptides (tST). In a second step, this hybrid is linked to polystyrene bead surfaces and Maltose Binding Protein (MBP) using STN. Furthermore, we show the STN-tST linkage is more stable against forces applied by optical tweezers than the commonly used biotin-Streptavidin (STV) linkage. It can be used in conjunction with Neutravidin (NTV)-biotin linkages to form DNA tethers that can sustain applied forces above 65 pN for tens of minutes in a quarter of the cases. The method is general and can be applied to construct other surface-DNA and protein-DNA hybrids. The reversibility, high mechanical stability and specificity provided by this linking procedure make it highly suitable for single molecule mechanical studies, as well as biosensing and lab on chip applications.

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          Most cited references32

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          DNA-templated assembly and electrode attachment of a conducting silver wire.

          Recent research in the field of nanometre-scale electronics has focused on two fundamental issues: the operating principles of small-scale devices, and schemes that lead to their realization and eventual integration into useful circuits. Experimental studies on molecular to submicrometre quantum dots and on the electrical transport in carbon nanotubes have confirmed theoretical predictions of an increasing role for charging effects as the device size diminishes. Nevertheless, the construction of nanometre-scale circuits from such devices remains problematic, largely owing to the difficulties of achieving inter-element wiring and electrical interfacing to macroscopic electrodes. The use of molecular recognition processes and the self-assembly of molecules into supramolecular structures might help overcome these difficulties. In this context, DNA has the appropriate molecular-recognition and mechanical properties, but poor electrical characteristics prevent its direct use in electrical circuits. Here we describe a two-step procedure that may allow the application of DNA to the construction of functional circuits. In our scheme, hybridization of the DNA molecule with surface-bound oligonucleotides is first used to stretch it between two gold electrodes; the DNA molecule is then used as a template for the vectorial growth of a 12 microm long, 100 nm wide conductive silver wire. The experiment confirms that the recognition capabilities of DNA can be exploited for the targeted attachment of functional wires.
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            Escherichia coli K-12: a cooperatively developed annotation snapshot—2005

            The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.
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              Nanotube molecular transporters: internalization of carbon nanotube-protein conjugates into Mammalian cells.

              The interactions between various functionalized carbon nanotubes and several types of human cancer cells are explored. We have prepared modified nanotubes and have shown that these can be derivatized in a way that enables attachment of small molecules and of proteins, the latter through a novel noncovalent association. The functionalized carbon nanotubes enter nonadherent human cancer cells as well as adherent cell lines (CHO and 3T3) and by themselves are not toxic. While the fluoresceinated protein streptavidin (MW approximately 60 kD) by itself does not enter cells, it readily enters cells when complexed to a nanotube-biotin transporter and exhibits dose-dependent cytotoxicity. The uptake pathway is consistent with adsorption-mediated endocytosis. The use of carbon nanotubes as molecular transporters could be exploited for various cargos. The biocompatibility and unique physical, electrical, optical, and mechanical properties of nanotubes provide the basis for new classes of materials for drug, protein, and gene delivery applications.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                15 January 2013
                : 8
                : 1
                : e54440
                Affiliations
                [1 ]AMOLF Institute, Amsterdam, The Netherlands
                [2 ]Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands
                Consejo Superior de Investigaciones Cientificas, Spain
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AM SJT. Performed the experiments: AM FM. Analyzed the data: AM FM. Wrote the paper: AM FM SJT.

                Article
                PONE-D-12-24415
                10.1371/journal.pone.0054440
                3545873
                23336001
                9da69561-7b44-467a-81eb-54d049d1ca5d
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 15 August 2012
                : 13 December 2012
                Page count
                Pages: 7
                Funding
                This work was supported by the research programme of the Foundation for Fundamental Research on Matter (FOM), which is part of the Netherlands Organisation for Scientific Research (NWO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Nucleic Acids
                DNA
                Proteins
                Biophysics
                Nucleic Acids
                Synthetic Nucleic Acids
                Biomechanics
                Biotechnology
                Bioengineering
                Engineering
                Bioengineering
                Materials Science
                Biomaterials
                Nanotechnology
                Bionanotechnology
                Physics
                Biophysics
                Biomacromolecule-Ligand Interactions

                Uncategorized
                Uncategorized

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