Abstract
Accurate and stable site-specific attachment of DNA molecules to proteins is a requirement for many single-molecule force spectroscopy techniques. The most commonly used method still relies on maleimide chemistry involving cysteine residues in the protein of interest. Studies have consequently often focused on model proteins that either have no cysteines or with a small number of cysteines that can be deleted so that cysteines can then be introduced at specific sites. However, many proteins, especially in eukaryotes, contain too many cysteine residues to be amenable to this strategy, and therefore there is tremendous need for new and broadly applicable approaches to site-specific conjugation. Here we present bioorthogonal approaches for making DNA-protein conjugates required in force spectroscopy experiments. Unnatural amino acids are introduced site-specifically and conjugated to DNA oligos bearing the respective modifications to undergo either strain-promoted azidealkyne cycloaddition (SPAAC) or inverse-electron-demand Diels-Alder (IE-DA) reactions. We furthermore show that SPAAC is compatible with a previously published peptide-based attachment approach. By expanding the available toolkit to tag-free methods based on bioorthogonal reactions, we hope to enable researchers to interrogate the mechanics of a much broader range of proteins than is currently possible.
Single-molecule Study of Site-specific DNA Recombination by γδ Resolvase
