Bioorthogonal protein-DNA conjugation methods for force spectroscopy

Marie Synakewicz,Matthias Rief,Daniela Bauer,Laura S Itzhaki

doi:10.1038/s41598-019-49843-1

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.

Highlights

Mechanical forces are involved in varied biological processes such as force-bearing proteins in the muscle and tension upon chromosomes separation during cell division, and disruption of the cell’s ability to sense the mechanical properties of its surroundings represents a hallmark of many diseases
A variety of bioorthogonal chemistries have been developed for post-translational modification of bio-molecules which rely on the introduction of unnatural amino acids (UAAs) [reviewed extensively in[16,17]
We employed the amber codon suppression system developed by Chin and co-workers, which uses the amber codon tRNA and aminoacyl tRNA synthetase from Methanosarcina barkeri combined with an orthogonal ribosome to incorporate pyrrolysine derivatives

Summary

Introduction

Mechanical forces are involved in varied biological processes such as force-bearing proteins in the muscle and tension upon chromosomes separation during cell division, and disruption of the cell’s ability to sense the mechanical properties of its surroundings represents a hallmark of many diseases. Current DNA-protein cross-linking protocol for optical tweezers experiments mainly use site-specific introduction of cysteine residues at either termini or internally of the protein of interest. To improve reaction speeds and minimize the excess use of expensive components, Mukhortava and Schlierf[12] developed a two-step protocol, in which cysteines are first functionalised with DBCO-maleimide followed by subsequent conjugation to azide-modified DNA oligos These improvements are limited to proteins where cysteines are not present or where unwanted cysteines can be removed without severely affecting protein stability. We turned to a cysteine-independent method using different bio-orthogonal chemistries These methods require the site-specific introduction of unnatural amino acids (UAAs) bearing desired chemical functionalities to react selectively with the corresponding modified DNA oligo. We designed protein constructs containing ybbR-tags and show that both attachment methods provide equivalent results in this case, and can be combined

Methods

Results

Conclusion