Bioorthogonal protein-DNA conjugation methods for force spectroscopy

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.

Consensus tetratricopeptide repeat proteins are complex superhelical nanosprings

“…Angles between repeat planes were calculated essentially as published previously [13]. Constructs were prepared for force spectroscopy using sitespecific modification of either terminal ybbR-tags or cysteine residues [72,73]. All single-molecule force spectroscopy data was collected on a custom-built instrument [74], processed using custom scripts developed in Igor Pro (WaveMetrics) and further analysed using Igor Pro or Python [75][76][77][78][79][80][81].…”

Bioconjugation Strategies for Connecting Proteins to DNA-Linkers for Single-Molecule Force-Based Experiments

“…Classic examples of click chemistry are copper(I)-catalyzed cycloaddition between an azide and alkyne, and strain-promoted azide-alkyne cycloaddition, using strained alkynes such as DBCO. In general, a higher reaction rate can be achieved with copper(I)-catalyzed click chemistry [57], though conjugation of protein and DNA is possibly inhibited by chelation of Cu 2+ by the oligonucleotide itself [58]. Strain-promoted cycloaddition avoids the requirement for metal ions due to the ring-strain-driven reaction and is an efficient way to introduce covalent linkages.…”

Optical tweezers in single-molecule experiments

“…The force jump relative to the unfolding of the 169 aminoacids is evident, and at the standard sample rate of 1kHz the presence of intermediates states are not observable; on the contrary, by using a higher temporal sampling (up to 100kHz) it was possible to highlight the occurrence of an intermediate state in the unfolding pathway of the protein. Very recently new biological methods have been presented [93] to create DNA-protein bounds without the need for cystein residues in the polypeptide chain, thus broadening the range of proteins which can be investigated by OT.…”