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. 2013;8(1):e54440.
doi: 10.1371/journal.pone.0054440. Epub 2013 Jan 15.

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

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Free PMC article

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

Fatemeh Moayed et al. PLoS One. .
Free PMC article

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.

Conflict of interest statement

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

Figures

Figure 1
Figure 1. Hierarchical synthesis of protein-DNA hybrids.
(a) Schematic drawing of the building blocks (b) 1% agarose gel demonstrating construction of tST-DNA-biotin hybrid at 2553 bps (c) SDS-PAGE analysis illustrating production of tST-MBP in Ecoli BL21.1 (d) SDS-PAGE characterization of STN-tST-MBP hybrid after amylose column purification. STN decomposes into monomers upon boiling. The schematic represents the expected dominant stoichiometry of the complex but does not exclude the possibility of minor amounts of complexes with other stoichiometries. (e) 1% agarose gel confirming the formation of multi protein-DNA hybrid (f) 1% agarose gel showing the presence and absence of DNA strand in the supernatant of incubated NTV beads by tST-DNA and biotin-DNA respectively. Biotinylated DNA easily binds to NTV, tST labelled DNA does not and remains in the supernatant.
Figure 2
Figure 2. Mechanical stability analysis.
(a) Optical tweezers setup (b) Force-extension curve of dsDNA showing overstretching at 65 pN, as well as the characteristic step-wise relaxation. The measured DNA stretching curves did not display additional steps that might have arisen from STN unfolding or its detachment from the surface. (c) Fraction of tethers that resisted 60 pN in first and second pull, compared between several commonly used linkage strategies and our proposed linkage strategies based on STN. For the (STN)biotin-DNA-Dig(AntiDig) system, almost all tethers broke at the first pull, and hence the subsequent pulls are not indicated.
Figure 3
Figure 3. Mechanical stability analysis at constant force.
(a) An example of stretched tether kept under a constant force of 60 pN in the second pulling cycle (2) by means of a force feedback for more than one hour. Stretching and relaxation cycles in the beginning (1) and at the end of the experiment (3) display a typical behaviour of dsDNA. (b) Fraction of the tethers resisting more than 10 min at 60 pN.

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Grant support

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.
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