Protein–DNA Chimeras for Nano Assembly

Pippig, Diana A.; Baumann, Fabian; Strackharn, Mathias; Aschenbrenner, Daniela; Gaub, Hermann E.

doi:10.1021/nn501644w

Cited by 35 publications

(35 citation statements)

References 34 publications

(51 reference statements)

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“…When pulled apart in zipper orientation, the basepair‐by‐basepair unzipping of DNA is described quantitatively by an equilibrium thermodynamic model, leading to a plateau of constant force. During a pickup event from the depot area, the 40 bp DNA duplex melts at ≈25 pN, which is consistent with previous SMC&P experiments using this same DNA duplex . The non‐equilibrium unbinding of the SII:monoST interaction occurs at forces significantly greater than this value at the given loading rates .…”

supporting

confidence: 88%

“…The resulting duplexes formed aptamers that stabilized the structure of a target dye molecule, enabling it to produce a fluorescent signal upon binding the SMC&P‐arranged constructs . Proteins have also been integrated into SMC&P, including constructs containing modified green fluorescent protein (GFP) with different surface‐immobilization strategies and a protein‐based handle for the cantilever . Directed placement of molecules within the nanoapertures of zero‐mode waveguides with SMC&P is also possible.…”

mentioning

confidence: 99%

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Strep -Tag II and Monovalent Strep -Tactin as Novel Handles in Single-Molecule Cut-and-Paste

et al. 2017

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Directed spatial assembly of single molecules on a surface presents an opportunity to precisely control the positioning, density, and geometry of molecules of interest within an ensemble. In contrast to bulk averaging, this enables detection and analysis of individual behavior within such a designed ensemble. The atomic force microscopy (AFM)‐based technique of single‐molecule cut‐and‐paste (SMC&P) facilitates the arrangement of a variety of biomolecules on a surface through different handling strategies. This technique requires cantilever‐ and surface‐handles that simultaneously adhere to a prerequisite rupture force hierarchy, and also do not cross‐interact with each other or the transported molecules. As the molecules of interest diversify, so too must the handling methods to accommodate their unique characteristics. Here, it is demonstrated that a previously developed monovalent variant of Strep‐Tactin and its corresponding Strep‐Tag II peptide ligand comprise a viable cantilever handling complex for SMC&P. Ultimately, this expansion to the SMC&P toolbox increases the system's versatility for new molecules of interest yet to be studied.

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confidence: 88%

mentioning

confidence: 99%

Strep -Tag II and Monovalent Strep -Tactin as Novel Handles in Single-Molecule Cut-and-Paste

et al. 2017

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“…Remarkably, the difference in binding strength when using SII on either the N-or C-terminus could only be identified as a consequence of the high specificity of our tethering system and the superb understanding and control its pulling geometry provides. As this renders the SII:monoST interaction a tunable rupture force system, other implementations may arise, for example, in 'single-molecule cut & paste' 42 . Finally, the modification of ST to hold a unique immobilization and single functional SII binding site boosts the robustness and applicability of the system.…”

Section: Discussionmentioning

confidence: 99%

Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy

Baumann¹,

Bauer²,

Milles³

et al. 2015

Nature Nanotech

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Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin:streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.

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“…On the other hand, we investigated the performance of the “ybbR tag” coupling system, which is based on the Sfp (surfactin production) phosphopantetheinyl transferase‐catalyzed ligation of proteins bearing the 11‐residue ybbR peptide (Asp‐Ser‐Leu‐Glu‐Phe‐Ile‐Ala‐Ser‐Lys‐Leu‐Ala) with small‐molecule‐Coenzyme A (CoA) conjugates . While this system has previously been used for the covalent attachment of CoA‐modified oligonucleotides to ybbR‐tagged proteins, the direct ligation on the surface of DONs has not yet been explored.…”

Section: Figurementioning

confidence: 99%

Solid‐Phase Synthesis and Purification of Protein–DNA Origami Nanostructures

Burgahn

Garrecht

Rabe

et al. 2019

Chemistry A European J

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We present a facile method for the combined synthesis and purification of protein‐decorated DNA origami nanostructures (DONs). DONs bearing reductively cleavable biotin groups in addition to ligands for ligation of recombinant proteins are bound to magnetic beads. Protein immobilization is conducted with a large protein excess to achieve high ligation yields. Subsequent to cleavage from the solid support, pure sample solutions are obtained which are suitable for direct AFM analysis of occupation patterns. We demonstrate the method's utility using three different orthogonal ligation methods, the “halo‐based oligonucleotide binder” (HOB), a variant of Halo‐tag, the “SpyTag/SpyCatcher” (ST/SC) system, and the enzymatic “ybbR tag” coupling. We find surprisingly low efficiency for ST/SC ligation, presumably due to electrostatic repulsion and steric hindrance, whereas the ybbR method, despite its ternary nature, shows good ligation yields. Our method is particularly useful for the development of novel ligation methods and the synthesis of mechanically fragile DONs that present protein patterns for surface‐based cell assays.

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Protein–DNA Chimeras for Nano Assembly

Cited by 35 publications

References 34 publications

Strep -Tag II and Monovalent Strep -Tactin as Novel Handles in Single-Molecule Cut-and-Paste

Strep -Tag II and Monovalent Strep -Tactin as Novel Handles in Single-Molecule Cut-and-Paste

Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy

Solid‐Phase Synthesis and Purification of Protein–DNA Origami Nanostructures

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