Copper-free click chemistry for attachment of biomolecules in magnetic tweezers

Abstract

Background: Single-molecule techniques have proven to be an excellent approach for quantitatively studying DNA-protein interactions at the single-molecule level. In magnetic tweezers, a force is applied to a biopolymer that is anchored between a glass surface and a magnetic bead. Whereas the relevant force regime for many biological processes is above 20pN, problems arise at these higher forces, since the molecule of interest can detach from the attachment points at the surface or the bead. Whereas many recipes for attachment of biopolymers have been developed, most methods do not suffice, as the molecules break at high force, or the attachment chemistry leads to nonspecific cross reactions with proteins.

Results: Here, we demonstrate a novel attachment method using copper-free click chemistry, where a DBCO-tagged DNA molecule is bound to an azide-functionalized surface. We use this new technique to covalently attach DNA to a flow cell surface. We show that this technique results in covalently linked tethers that are torsionally constrained and withstand very high forces (>100pN) in magnetic tweezers.

Conclusions: This novel anchoring strategy using copper-free click chemistry allows to specifically and covalently link biomolecules, and conduct high-force single-molecule experiments. Excitingly, this advance opens up the possibility for single-molecule experiments on DNA-protein complexes and molecules that are taken directly from cell lysate.

Keywords: Copper-free click chemistry; DNA immobilization; Magnetic tweezers; SPAAC reactions; Surface chemistry.

Fig. 1

Common DNA tethering techniques. a Binding of a digoxygenin-functionalized DNA-protein complex to an anti-digoxygenin-coated surface. This reaction is specific, but unstable when high forces are applied. b Binding of an amine-functionalized DNA-protein complex to a carboxyl-coated surface. Both the functionalized DNA (black arrow) and native lysine groups in the protein (blue arrow) bind the surface. c. Binding of a thiol-functionalized DNA-protein complex to a maleimide-coated surface. Both the functionalized DNA (black arrow) and native cysteine groups in the protein (blue arrow) bind the surface

Scheme 1

Cycloaddition between dibenzocyclooctyl and azide

Fig. 2

Magnetic tweezers set-up for measuring on a tethered DNA molecule. a Schematic of the set-up. A LED illuminates the flow cell through a lens and the magnet holder. Imaging is done with a 50x Nikon objective onto a CCD camera. Magnets manipulate a magnetic bead attached to the DNA. b A flow cell is constructed with 24x60mm coverslips. The bottom coverslip is amine-coated and has reference beads bound to it. The top coverslip has sandblasted holes to allow fluid flow. Parafilm is used to seal the coverslips and to create a ˜50 μl flow cell volume. c Schematic of a tethered DNA molecule. A DNA molecule is linked to a streptavidin-coated magnetic bead with biotin, and to azide groups on the surface with DBCO at the other end

Fig. 3

Stepwise linkage of DNA to the surface with copper-free click chemistry. Bifunctionalized PEG-linkers are attached to an amine-coated surface via their NHS group. The NHS ester on the PEG conjugates to the amine on the surface. Non-reactive PEG linkers (terminated with a CH₃-group) are used to passivate the surface. Finally, a DBCO group on DNA clicks with the azide and thus forms a covalent bond between the DNA and the surface

Fig. 4

Tether density as a function of PEG concentration. DNA tether density for different Azide-PEG concentrations. The number of tethers increases linearly with increasing PEG concentration. Inset shows an example of a reference bead (left) and three beads that signal 20 kb DNA molecules tethered with click chemistry

Fig. 5

Anchored DNA molecules can be torsionally constrained and withstand forces of >100pN. a The DNA molecules anchored with click chemistry show the expected behavior (a strongly rising force, and for unconstrained molecules, a plateau near 65pN as DNA overstretches and a further rise) in a slow force ramp of 1pN/sec. Different colors represent different tethers. All tethers that were bonded by click chemistry withstand forces of over 100pN. By contrast, the DNA anchored with digoxygenin/anti-dig (black) breaks off near 40pN, well before the overstretching point. b Rotation curves at constant forces of (light to dark) 0.5, 1, 3 and 5pN, indicating that this 20 kb DNA molecule anchored with click chemistry is torsionally constrained