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. Author manuscript; available in PMC: 2019 Jan 5.
Published in final edited form as: Chemphyschem. 2017 Dec 4;19(1):19–23. doi: 10.1002/cphc.201701147

Figure 1.

Figure 1

Single-molecule force spectroscopy (SMFS) studies using DIG10.3•Dig. A) Schematic of DIG10.3•Dig being used in an optical-trapping-based SMFS assay. B) Force-extension curve shows DNA being stretched with an optical trap undergoing the overstretching transition at 65 pN. Data smoothed to 500 Hz. C) A force-vs-time trace shows the lifetime of DIG10.3•Dig stretched under constant force by an optical trap. Data smoothed to 40 Hz (light pink) and 10 Hz (dark pink). D) Cartoon of the coupling scheme for AFM-based assay showing force applied to the ligand and a N-terminal cysteine variant of DIG10.3. DIG10.3 was covalently coupled to a maleimide-functionalized, PEG-coated AFM tip via a maleimide-thiol bond. Dig-labeled DNA was covalently coupled to an azide-functionalized, PEG-coated coverslip using dibenzocyclooctyl (DBCO), a copper-free click chemistry reagent. Abbreviations: SiN3, silicon nitride. E) Force-extension curve taken using an AFM also shows the DNA’s overstretching transition. Data smoothed to 50 kHz (light purple) and 50 Hz (dark purple). F) Force-vs-time traces using an AFM show the lifetime of DIG10.3•Dig stretched at constant force. Data smoothed to 1 kHz (light colors) and 50 Hz (dark colors).