Julian Whelan, “Using Click Chemistry for Binding DNA to SpyTag”
Mentor: Ionel Popa, Physics, Letters & Science (College of)
Poster #95
Single molecules of proteins can be studied using the magnetic tweezer (MT), a force spectroscopy instrument which applies a magnetic force to single protein molecules tethered between a glass surface and a magnetic bead. DNA linkers are becoming increasingly popular in MT experiments because of their characteristic overstretching (B-to-S) transition at 65 pN, where the DNA stretches from its typical helix shape to a ladder shape. Since this transition always occurs at the same force, it can be used to help calibrate the results of an MT experiment. DNA linkers typically attach in series with the protein molecules, connecting them with the magnetic beads, and to withstand larger forces ≥ 65 pN, a covalent attachment between a protein molecule and DNA is necessary. Here, we use a highly specific, copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, also known as click chemistry, to bioconjugate a ~6 kbp DNA linker and single-molecules of two different proteins. This reaction was used to replace the commonly used sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) based bioconjugation technique, which targets sulfhydrl groups. These groups are common in proteins, which sometimes caused DNA linkers to attach at an unwanted section of the protein constructs. We finally validated our reaction by measuring our protein-DNA constructs with the MT.