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

Abstract

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.

Force-Induced Unravelling of DNA Origami

“…Reduced sulfhydryl groups of DNA origami were coupled to the free maleimide groups on the cover slides by forming permanent thiol-ether bonds. 74,94 Maleimide groups on the cantilevers were bound to coenzyme A, and subsequently to a monovalent variant of StrepTactin (monoST) 95,96 or a monomeric variant of streptavidin 97 via Sfp phosphopantetheinyl transferase (SFP) with ybbR tags (amino acid sequence DSLEFIASKLA) for use as specific AFM pulling handles. 98,99 SMFS Measurement Protocol.…”

“…Clean glass cover slides and UV-ozone cleaned silicon nitride AFM cantilevers were covalently functionalized first with aminosilane and subsequently with 5 kDa NHS-PEG-maleimide polymer linkers. Reduced sulfhydryl groups of DNA origami were coupled to the free maleimide groups on the cover slides by forming permanent thiol-ether bonds. , Maleimide groups on the cantilevers were bound to coenzyme A, and subsequently to a monovalent variant of StrepTactin (monoST) , or a monomeric variant of streptavidin via Sfp phosphopantetheinyl transferase (SFP) with ybbR tags (amino acid sequence DSLEFIASKLA) for use as specific AFM pulling handles. , …”

Next Generation Methods for Single-Molecule Force Spectroscopy on Polyproteins and Receptor-Ligand Complexes

“…The biotin-(strept)avidin interaction has a long history in the AFM-SMFS community (Florin et al, 1994(Florin et al, , 1995Lee et al, 1994;Moy et al, 1994;Grubmüller et al, 1996;Wong et al, 1999;Yuan et al, 2000;Pincet and Husson, 2005;de Odrowaz Piramowicz et al, 2006;Rico and Moy, 2007;Erdmann et al, 2008;Guo et al, 2008;Zhang et al, 2009;Chivers et al, 2010;Taninaka et al, 2010a,b;Teulon et al, 2011;Han et al, 2012;Rico et al, 2015Rico et al, , 2019Baumann et al, 2016;Erlich et al, 2017;Sedlak et al, 2017Sedlak et al, , 2019Sedlak et al, , 2020Bauer et al, 2018), and a thorough review of this controversy is beyond the scope of the review here. Much of the irreproducibility of biotinstreptavidin rupture force measurements by AFM-SMFS can be attributed to random lysine-based immobilization of the protein, as well as the fact that the streptavidin tetramer can disassemble during stretching.…”

“…Site-specific biotinylation tags are furthermore available using the biotin-ligase BirA (Beckett et al, 1999;de Boer et al, 2003;Chen et al, 2005), and site-specific biotinylation of recombinant proteins is a valuable method recently reported for magnetic tweezers-based SMFS measurements (Renn et al, 2019). In addition to the biotin ligase acceptor sequence, the Streptag peptide sequence is being commonly used for AFM-SMFS with success (Baumann et al, 2016;Erlich et al, 2017). One limitation of the aforementioned non-covalent interactions as molecular handles for AFM-SMFS is the relatively low forces required to rupture these complexes.…”