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

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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.
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M ET HODOLOGY AR TICL E Open Access
Copper-free click chemistry for attachment
of biomolecules in magnetic tweezers
Jorine M. Eeftens, Jaco van der Torre, Daniel R. Burnham and Cees Dekker
*
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: Magnetic tweezers, Copper-free click chemistry, SPAAC reactions, Surface chemistry, DNA immobilization
Background
Single-mole cule methods have become increasingly
popular to study biomolecules [1]. With techniques such
as atomic force spectroscopy, or optical or magnetic
tweezers, one is able to study the mechanical properties
of single DNA molecules, single proteins, or individual
DNA-protein complexes. The effect of applied force on
biomolecules is a particularly relevant topic, as mechan-
ical forces play a crucial role in many cellular processes
[24]. The relevant forces range from a few pN, like the
force produced by an RNA polymerase during transcrip-
tion (14pN) [5], to tens of pN, as in, for instance, viral
packaging motors that use forces of 40pN to compact
genomes [6]. Even higher forces are needed in the
process of chromosome segregation in eukaryotic cells,
where microtubules pull on sister chr omatids to segre-
gate them to opposite sides of the spindle pole [710].
Many studies using magnetic tweezers have been
published that probe the behavior of DNA-protein com-
plexes under applied force and torque [1116]. For
studying biomolecules across the full relevant force
range, it is necessary to also measure at higher forces
(>20pN). In this regime, however, many traditional an-
choring methods fail, thus limiting such single-molecule
experiments.
For efficient tethering of biomolecules, it is essential
to use orthogonal anchoring chemistries on both ends of
the molecule, i.e. at the surface and at the bead. To
achieve this, a DNA molecule is constructed that has
different reactive groups incorporated, on both ends. To
complete the anchoring, the bead and surface are func-
tionalized with the corresponding reacting group. A
commonly used techniqu e is the binding of biotin to
streptavidin. The bond between these functional groups
has been shown to resist forces of 150pN [17, 18]. This
is a high rupture force compared to a second commonly
used method; the binding of a digoxygenin (dig) function-
alized nucleotide and a surface coated with antibodies
against digoxygenin (anti-dig) (Fig. 1a). This forms a stable
* Correspondence: c.dekker@tudelft.nl
Department of Bionanoscience, Delft University of Technology, Kavli Institute
of Nanoscience Delft, Delft, The Netherlands
© 2015 Eeftens et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Eeftens et al. BMC Biophysics (2015) 8:9
DOI 10.1186/s13628-015-0023-9
non-covalent bond, but a limitation of this binding tech-
nique is its low stability under an applied force [19]. De-
pending on the force-loading rate applied to such a
molecule, the dig/anti-dig bond breaks at around 20pN.
Other, much stronger, anchoring methods have been
developed [2024] by functionalizing DNA with amine
(Fig. 1b) or thiol groups (Fig. 1c) that are covalently
linked to the surface or bead. Although these bonds in-
deed resist high forces, these techniques have an import-
ant limitation in that significant nonspecific binding
occurs when studying systems that are more complicated
than bare DNA. For example, when studying proteins, na-
tive lysines (amine) or cysteines (thiol) in the protein can
bind nonspecifically (blue arrows in Fig. 1b, c). For con-
trolled single-molecule measurements, it is however im-
portant that the force is being applied at a consistent and
known location [25].
A new and exciting challenge is to study DNA-protein
complexes that are extracted from cell lysate. For con-
trolled single-molecule experiments, it is essential to an-
chor these complexes in a stable, strong, and specific
way. As the anchoring methods developed so far are un-
suitable, studying DNA-protein complexes or complexes
from cell lysate remains challenging [26].
Here, we present a novel method for covalent attach-
ment of a DNA tether to a surface, based on copper-free
click chemistry. Click reactions are defined as those that
are sele ctive, with favorable reaction kinetics, a high
yield, and good physiological stability. Early click chem-
istry reactions required copper as a catalyst [27]. Copper
is cytotoxic and thus limits application of click reactions
in cells. More recently, copper-free methods became
available, for instance the Strain Promoted Azide-Alkyne
Click (SPAAC) reaction, of which the reaction between
dibenzocyclooctyl (DBCO) and azide is an example [28].
These click reactions are bio-orthogonal, i.e. they can
occur within organisms without interfering with native
biochemical processes.
As mentioned above, a spe cific and high-force-
compatible anchoring technique is essential for studying
DNA-protein complexes in magnetic tweezers. The reac-
tions have to be specific, biocompatible, and able to
withstand experimental conditions such as an applied
high force. We develop a novel technique for covalent
attachment that meets these criteria using copper-free
click chemistry, based on the reaction of DBCO with
azide (Scheme 1). By functionalizing DNA with DBCO
on one end (R1), we can covalently link it to an azide-
functionalized surface (R2). As we will show below, this
protocol results in a high-yield of DNA tethers, that are
torsionally constrained and able to withstand very high
forces (>100pN). This method is thus found to be suit-
able for specifically anchoring DNA-protein complexes
and measuring in the relevant force regime.
Methods
Magnetic tweezers
We used multiplexed magnetic tweezers [29], as illus-
trated in Fig. 2a. Two 5 mm cube magnets (Supermag-
nete, N50) are mounted in vertical orientation [30], with
a very small (0.3 mm) gap in between them. A red LED
provides illum ination through the magnet holder onto
the flow cell. We use a 50x objective (Nikon) with an
achromatic doublet tube lens (200 mm) to provide 50x
magnification and image the focal plane onto a CCD
camera (Dalsa Falcon 4 M60). Beads are tracked in real
time with custom software (Labview, National Instru-
ments) and images are also saved for later analysis [31].
Reference beads are used to correct for drift. The ap-
plied force is determined from the Brownian motion of
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 gro ups in the protein (bl ue 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 t he sur face
Eeftens et al. BMC Biophysics (2015) 8:9 Page 2 of 7
the magnetic bead [32, 33]. For force-extension curves,
we perform dynamic force microscopy where the force
is increased over time with a constant loading rate of 1
pN/second.
DNA constructs
A 20678 bp pSupercos1 plasmid was made by removal
of the MluI fragment from pSupercos1 (Stratagene) and
insertion of two lambda fragments. This Plasmid DNA
was isolated with midiprep (Qiagen), restricted with
XhoI and NotI.HF (New England Biolabs), and purified
(Wizard® SV Gel and PCR Clean-Up System, Promega),
resulting in a 20 kb fragment.
DB CO and biotin labeled handles were prepared by
PCR on a pbluescriptIISK+ template (Stratagene) with a
taq polymerase (GoTaq, Promega) and the addition of
Biotin-16-dUTP (Roche), or 5-DBCO-dUTP (Jena-
bioscience) to the nucleotide mixture respectively. The
forward primer was: GACCGAGATAGGGTTGAGTG,
and reverse primer: CAGGGTCGGAACAGGAGAGC.
The biotin-handle was digested with XhoI resulting in
554 bp and 684 bp fragme nts. The DBCO-handle was
digested with NotI.HF resulting in 624 bp and 614 bp
fragments. The handles were purified (Wizard® SV Gel
and PCR Clean-Up System, Promega), combined with
the restricted plasmid DNA and ligated with T4 DNA
ligase (Promega) overnight at 16 °C. The tweezer-
construct wa s then purified again (Wizard® SV Gel and
PCR Clean-Up System, Promega).
Surface functionalization and flow cell assembly
For making amine-coated flow cells, coverslips (Menzel
Glaser, 24x60mm, thickness #1) were cleaned in an O
2
plasma cleaner for 30 s, which ensures activation of the
silanol groups. Coverslips were then treated with 2 %
APTES in acetone for 10 min, rinsed with MilliQ and
air-dried. Before flow cell assembly, polystyrene beads
(Polysciences Europe GmbH) were pipetted onto the
coverslip and spread with the side of a pipette tip. These
non-motile surface-bound beads serve as reference
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
Scheme 1 Cycloaddition between dibenzocyclooctyl and azide
Eeftens et al. BMC Biophysics (2015) 8:9 Page 3 of 7
beads for drift correction. The amine-coated coverslips
were then aligned with a pre-cut parafilm gasket and an-
other cover slip (Fig. 2b). The assembled flow cell was
put on a hot plate at 90 °C until the parafilm was suffi-
ciently melted to prevent fluid leakage. The applied heat
also firmly binds the polystyrene reference beads to the
surface.
DNA Anchoring
To anchor the DBCO-functionalized DNA to the amine-
coated flow cell, we used bifunctionalized PEG
4
-linkers
with an N-hydroxysuccimide (NHS) ester on one end and
an azide group on the other (CLK-AZ103, Jenabioscience
GmbH, Germany). We mixed azide-functionalized PEG-
linkers with CH
3
-terminated PEG-linkers of the same
length (MS(PEG)4, Life technologies) in PBS buffer to
passivate the surface and prevent aspecific binding. Both
PEG-linkers were dissolved in DMSO before further
diluting in PBS. To prevent hydrolysis of the NHS ester,
the PEG mixture in PBS was prepared shortly before fill-
ing the flow cell via capillary action through pipetting the
fluid into one flow cell hole of the amine-coated flow cell.
The MS-PEG-linker concentration was held constant at
50 mM, while the Azide-PEG concentration was varied
(0-50 mM). PEG-linkers incubated in the amine-coated
flow cell for 1 h at room temperature, to allow the NHS-
ester group to attach to the amine groups in the flow cells
(Fig. 3). Next, the flow cell was flushed with washing buf-
fer (20 mM Tris, 5 mM EDTA, pH7.4), to stop the reac-
tion and remove excess PEG. Streptavidin-coated beads
(M270 Streptavidin coated, Life Technologies) were incu-
bated with the biotin-functionalized DNA for 20 min.
After incubation, the beads were washed 3 times with
washing buffer with 0.05 % Tween. An overabundance of
DNA-bound beads was then dissolved in 50 μl washing
buffer with 0.05 % Tween and flushed into the flow cell.
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
CH
3
-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
Eeftens et al. BMC Biophysics (2015) 8:9 Page 4 of 7
Beads were incubated for 1 h, to allow the DBCO to click
with the azide (Fig. 3). Finally, the flow cell was washed
with washing buffer until no more unbound beads were
visible.
Control experiment
For control experiments, we used a dig-functionalized
DNA construct. The dig handle was constructed in the
same matter as the DBCO handle described above, but in-
stead dig-11-dUTP was used (Digoxygenin-11-dUTP,
Roche). Coverslips were cleaned in acetone for 30 min in
a sonicator for creating the flow cells. After air-drying,
they were coated with 1 % nitrocellulose (Invitrogen) in
amylacetate (Sigma Aldrich). Application of reference
beads and assembly of flow cells proceeded as described
above. Next, nitrocellulose-coated flow cells were incu-
bated with 100 mM anti-dig antibodies (Fab-fragment,
Roche) for 30 min. After washing as described above, the
surface was passivated with 10 mg/ml BSA (Bioke) for 1 h.
Preparation of beads proceeded as described above. Beads
with digoxygenin-functionalized DNA then incubated in
the flow cell for 10 min. Finally, the flow cell was washed
with washing buffer until no more unbound beads were
visible.
Results and Discussion
We developed a protocol to covalently attach biomole-
cules in a magnetic tweezers flow cell using copper-free
click chemistry. As described in Methods, we coat the
glass surface with azide-functionalized PEG-linkers, and
attach DBCO-tagged DNA through the azide-group,
thereby covalently linking the DNA molecule at one end
to the surfa ce.
The DBCO-functionalized DNA thus covalently at-
taches to the azide-coated flow cell while the biotin
groups at the other end of the DNA attach to the bead.
The amount of these DNA tethers is expected to scale
with the amount of clickable groups on the surface. To
verify the protoco l, we varied the density of the azide
groups on the surface by using different concentrations
of the PEG-linking groups. We determined the tether
density by manually counting the number of successful
DNA tethers in our field of view (0.02 mm
2
), for differ-
ent azide-PEG concentrations. As expected, we found
that the number of tethers increased linearly with in-
creasing azide-PEG concentrations, see Fig. 4. Import-
antly, when no azide-functionalized PEG-linkers were
added, no tethers of the expected length were observed.
This shows that the steps in the protocol are specific
and that, conveniently, the tether density is tunable.
Our DNA tethers anchored with copper-free click
chemistry are able to withstand high forc e. We an-
chored 20 kb DNA molecules using copper-free click
chemistry and tracked the position of the magnetic
beads (corresponding to the end-to-end length of the
DNA) while applying a force ramp of 1pN/sec. A s
showninFig.5,thetethered double-stranded DNA
molecules show the expected behavior, viz., with in-
creasing end-to-end distance we observe a strongly ris-
ing force, a plateau as the DNA overstretches , and a
further rise. As expected, for torsionally unconstrained
molecules , overstretching of the double-stranded DNA
is observed at about 65pN [34]. Torsionally constrained
DNAmolecules(depictedingreyinFig.5a)areex-
pected to show overstretching at a force of about
110pN, a force that, unfortu na tely, is just beyond the
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
Eeftens et al. BMC Biophysics (2015) 8:9 Page 5 of 7
reach of our set-up [35]. We find an avera ge contour
length of 6.75 ± 0.04 μm (as measured from the exten-
sion just before the overstretching plateau), indicating
correct attachment of the DNA molec ules at the func-
tional end groups. Most importantly , the tethers can
withstand a force of >100pN (Fig. 5a). The tethers re-
main stable at this high force for over 12 h, allowing
ample time for measurements. By contrast, DNA mole-
cules attache d with the conventional anti-dig tag break
off well before the overstretching force (cf. the black
lineinFig.5a).Inaddition,asshowninFig.5b,the
click-chemistry-assembled DNA tethers can be torsion -
ally constrained, which allows for DNA supercoiling
studies with magnetic tweezers. For the described con-
ditions , we found half of the tethers to be coilable. Loss
of torsional constrain is likely induced by nicking of the
DNA. The new attachment strategy is thus found to be
suitable for both high force and torque measurements.
In contrast to the binding of DBCO to azide, the bond
between biotin and streptavidin on the other end of the
DNA is not covalent. Yet, as can be observed from Fig. 5,
this bond also withsta nds forces of >100pN, which is
consistent with earlier reports [17, 18]. For a wide range
of applications , the current method, with tethers that
contain a mutually orthogonal DBCO/azide bond on
one end and biotin/streptavidin on the other, will suffice.
Double copper-free click chemistry (with orthogonal
click reactions at both bead and surface) can be consid-
ered in future applications if even much higher forces
are desired.
The copper-free click chemistry attachment strategy
presents many advantages. The reaction between DBCO
and azide is relatively fast, specific, it does not require a
catalyst, and, importantly for some applications, it can
be performed in physiological conditions. Furthermore,
azide and DBCO groups are relatively small and inert to
biological moieties [27] and thus easy to incorporate.
There are already numerous examples of the application
of SPAAC reactions in biological systems and even living
cells [28, 36]. Examples include use of copper-free click
chemistry in non-canonical amino acids [37], imaging in
live cells [38], joining of DNA strands [39], and DNA-
functionalized nanoparticles [40].
Above, we demonstrated the use of a new DNA-
attachment method in magnetic tweezers. We note that
it can easily be applied to other single-molecule methods
as well. For example, in the same manner, polystyrene
beads could be coated with click chemistry functional
groups for use in optical tweezers. By immobilizing the
PEG linkers on the surface, the same copper-free click
chemistry can also be used in atomic force microscopy
[41], flow stretching and DNA combing.
Single-mole cule force spectroscopy opens up the pos-
sibility to apply and measure forces on biomolecules,
and study DNA-protein interactions. These in vitro ex-
periments with bare DNA and purified protein give great
insights into the cell machinery, but purified complexes
are taken out of their cellular context. As our new
method does not cross-react, it is possible to anchor and
measure complexes that are directly extracted from cell
lysate. Measuring on this native state of biomolecules
can be expected to yield new insight into interactions
between biomolecules.
Conclusions
Traditional methods for anchoring biomolecules have
encountered limitations in studying DNA-protein com-
plexes in magnetic tweezers related to low force stability
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
Eeftens et al. BMC Biophysics (2015) 8:9 Page 6 of 7
and cross reactivity. Here, we developed a method for
covalently anchoring biomolecules with copper-free click
chemistry, using the reaction between DBCO and azide.
This reaction is bio-orthogonal and no catalyst is
needed. Furthermore, it is highly specific and it resists
high force (>100pN). The protocol is reproducible, fast
and uses commercially available reagents. Perhaps most
excitingly, covalently linking molecules with copper-free
click chemistry opens up the possibility to measure on a
wide variety of DNA-protein complexes and complexes
isolated from cell lysate.
Competing insterests
The authors declare that they have no competing interests.
Authors contributions
JE, JvdT: conceived and designed the experiments. JE: performed the
experiments, analyzed the data. JE, JvdT, DB: contributed materials/analy sis
tools. JE, JvdT, DB, CD: wrote the paper. All authors read and approved the
final manuscript.
Authors information
Not applicable.
Acknowledgements
We thank Jacob Kerssemakers for technical support and Richard Janissen for
discussions. This work was supported by the ERC Advanced Grant
NanoforBio (No. 247072) and by The Netherlands Organization for Scientific
Research (NWO/OCW), as part of the Frontiers of Nanoscience program.
Received: 24 April 2015 Accepted: 16 September 2015
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Eeftens et al. BMC Biophysics (2015) 8:9 Page 7 of 7
... Going beyond non-covalent attachment, introduction of one or more covalent linkages for biomolecular attachment provides force stabilities up to nN [22]. Several approaches for covalent attachment have been developed [22][23][24][25] and provide stability for long measurements and experiments over a broad force spectrum [8,[26][27][28][29][30]. For DNA attachment via dibenzocyclooctyne (DBCO), covalent binding to an azide-functionalized surface has been developed. ...
... For DNA attachment via dibenzocyclooctyne (DBCO), covalent binding to an azide-functionalized surface has been developed. This copper-free click chemistry method is specific, highly efficient and yields tethers that are able to withstand very high forces (>100 pN) [25]. ...
... To obtain a high density of labels on the DNA, we use 50% Biotin-dUTP and 50% DBCO-dUTP megaprimers for amplification and test the resulting labeled DNA construct in our single-molecule magnetic tweezers set up (Figure 2a and Methods). In the magnetic tweezers flow cell, the 2823 bp DNA tethers are anchored with copper-free click chemistry [25] to the surface and via biotin-streptavidin coupling to magnetic beads. We track the position of the magnetic beads while applying calibrated forces [43,54]. ...
Preprint
Full-text available
Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCR reactions: first ~380 bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR reaction to generate ~kbp long double-stranded DNA constructs with multiple labels at the respective ends. We use DBCO-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under force, with an average lifetime of 60 +/- 3 hours at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.
... For the surface-binding end, there are two choices of NA tethering: a fast and relatively weak digoxygenin/anti-digoxygenin (DIG/AntiDIG) binding ( Fig. 2A and B) (fluorescein/anti-fluorescein have also been used, (Bryant et al., 2003)) or a slow ($1 h) but strong covalent binding, the most biocompatible and specific being dibenzylcyclooctyne (DBCO)-azide Click chemistry (Eeftens, van der Torre, Burnham, & Dekker, 2015) (Fig. 2C and D). DIG/AntiDIG binding can be reinforced by functionalizing the NA with several DIG groups but this is not compatible with high-resolution experiments such as helicase stepping. ...
... Here, we describe the two main ways to tether hairpins (or adaptors) to the surface: through Click chemistry or DIG/AntiDIG interaction. Click chemistry corresponds to the attachment of DNA oligonucleotides coupled to a DCBO group on an azide-functionalized surface by producing a stable triazole link (Eeftens et al., 2015). The hairpins (or adaptors) can be then indirectly tethered to the DNA-coated surface by hybridization through complementary sequences and washed away with NaOH, rendering reusable and long-lasting surfaces. ...
Chapter
Helicases form a universal family of molecular motors that bind and translocate onto nucleic acids. They are involved in essentially every aspect of nucleic acid metabolism: from DNA replication to RNA decay, and thus ensure a large spectrum of functions in the cell, making their study essential. The development of micromanipulation techniques such as magnetic tweezers for the mechanistic study of these enzymes has provided new insights into their behavior and their regulation that were previously unrevealed by bulk assays. These experiments allowed very precise measures of their translocation speed, processivity and polarity. Here, we detail our newest technological advances in magnetic tweezers protocols for high-quality measurements and we describe the new procedures we developed to get a more profound understanding of helicase dynamics, such as their translocation in a force independent manner, their nucleic acid binding kinetics and their interaction with roadblocks.
... Strainpromoted alkyne-azide cycloaddition with the azide-bearing copolymer enables facile GUV membrane functionalization at mild conditions. [35] Using these polymers, we produced monodisperse GUVs with a diameter of 31.8 ± 0.5 µm with typical production rates on the order of 1 kHz (Fig. 2c). ...
Article
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Bottom-up synthetic cells offer the potential to study cellular processes with reduced complexity. Giant unilamellar vesicles (GUVs) can mimic cells in their morphological characteristics because their architecture is precisely controllable. We propose a block copolymer-based GUV system that can be used for high-throughput screening. Through droplet microfluidic methods, we produce double emulsions that then serve as templates for GUVs with adjustable inner, polymer membrane, and outer composition. Using flow cytometry, we are able to analyze tens of thousands of GUVs in a short amount of time, enabling their use for screening assays.
... For instance, a recent study of DNA tether lifetime using multiple digoxigenin-anti-digoxigenin interactions on a nitrocellulose coated surface measured a median value of ∼7 min at 45 pN (41). Covalent chemistries for DNA tethering have been developed but can suffer from slow reaction kinetics or elaborate and time-consuming surface modification requirements (41,42). Therefore, there remains a need for techniques which can enhance tether lifetime without the requirement for complicated experimental procedures. ...
Article
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Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics.
... A possible solution is to covalently capture the genotype on bead via the use of DBCO-modified oligonucleotides that will covalently react with azide-modified magnetic beads. DNA was thus amplified with a DBCOoligonucleotide, and captured on azide-modified magnetic beads (Gift of Michael Herger) via the copper-free click reaction [82]. These DBCO-linked DNA-coated beads were then tested for genotype linkage stability in IVTT, as described for biotin-linked DNA coated beads previously. ...
Thesis
This work establishes a new genotype:phenotype linked display platform: Polyacrylamide Hydrogel bead Display (PHD). Four core elements have been designed into it; compatibility with ultra-high throughput microfluidic workflows, stability of genotype linkage, stability and control of protein display, and compatibility with a diverse range of assays (affinity, catalytic, cell-based) and displayed proteins (scFvs, DARPins, antimicrobial peptides, enzymes). The first chapter presents the development of polyacrylamide hydrogel bead display. To enable protein display on polyacrylamide beads a new molecule is made, methacrylate-PEG-benzyl guanine, which is co-polymerised into the polyacrylamide hydrogel via the methacrylate group. The benzyl guanine group can form a covalent bond with the SNAP-tag. SNAP-tag can be expressed as a fusion to proteins of interest, thus enabling covalent phenotype linkage to beads. Proteins can be displayed at up to 2.8 x 108 molecules per 20 micron bead, and still be detected at only 5000 molecules per bead. Acrydite- modified oligonucleotides can also be co-polymerised into the hydrogel beads, enabling PCR to be used for genotype linkage to the beads. Emulsion in vitro transcription and translation (IVTT) can then be used to monoclonally express the encoded protein, which is then captured on the encoding bead in the droplet, thus establishing on bead genotype:phenotype linkage. The second chapter focusses on the development of library-scale polyacrylamide hydrogel bead display in the context of improving a potential cancer therapeutic agent; a pro-apoptotic, anti-TRAIL-R1 scFv. Emulsion PCR was found to be an unsuitable library amplification technology, and instead amplification of single DNA molecules in microfluidic droplets is achieved by emulsion RCA. Hydrogel bead formation is achieved by “pico-injecting” a polyacrylamide hydrogel mix into each individual droplet, and allowing polymerisation to occur. The polymerised hydrogel beads, functionalised with both genotype and benzyl-guanine, are recovered and used in an emulsion IVTT reaction. The hydrogel beads can be packed on chip, enabling single bead per droplet encapsulation efficiencies far in excess of the Poisson distribution. The in vitro expression of the pro-apoptopic scFv molecules was found to be low yield, limiting its use for cell-based functional screens, thus an alternative protein scaffold, DARPin, was tested and shown to express at high yield in IVTT. Concomitantly it was found that multimerization of the agonistic molecule improves the induction of apoptosis significantly. To enable screening of libraries at desired multimeric states a display construct was made, SNAP- PhoCl-SpyCatcher 3x, that would trimerise library members when expressed as a fusion to SpyTag. Trimerised library members can then be released from bead in droplet through photocleavage of the PhoCl protein. The third chapter focuses on an alternative library construction technique termed splinted ligation. Splinted ligation is a combinatorial technique that achieves diversity through iteration rather than compartmentalisation. Notably combinatorial library construction achieves 100% of beads with a monoclonal genotype. A small test library is created of an antimicrobial peptide LL37, and successful in vitro expression and display confirmed by antibody staining and FACS analysis. Ultra-high throughput compatible bacteriolytic assays based upon either fluorescence or absorbance are shown.
... The H6CM18 peptide (described below) was custom-synthesized by ThermoFisher with a N 3 -PEG 3 -vc-PABC linker attached to the N-terminus. Addition of a terminal azide group allows conjugation to antibody by Cu 2+ -free Click-Chemistry (27). T84.66 was functionalized with dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS, Sigma-Aldrich, St. Louis, MO, 761524) by reacting at either 1:20 (high modification) or 1:5 (low modification) molar equivalents for 15 min at room temperature with constant mixing in PBS. ...
Article
This work describes use of anti-carcinoembryonic antigen antibodies (10H6, T84.66) for targeted delivery of an endosomal escape peptide (H6CM18) and gelonin, a type I ribosome inactivating protein. The viability of colorectal cancer cells (LS174T, LoVo) was assessed following treatment with gelonin or gelonin immunotoxins, with or without co-treatment with T84.66-H6CM18. Fluorescent microscopy was used to visualize the escape of immunoconjugates from endosomes of treated cells, and efficacy and toxicity were assessed in vivo in xenograft tumor-bearing mice following single- and multiple-dose regimens. Application of 25 pM T84.66-H6CM18 combined with T84.66-gelonin increased gelonin potency by ~ 1,000-fold and by ~ 6,000-fold in LS174T and LoVo cells. Intravenous 10H6-gelonin at 1.0 mg/kg was well tolerated by LS174T tumor-bearing mice, while 10 and 25 mg/kg doses led to signs of toxicity. Single-dose administration of PBS, gelonin conjugated to T84.66 or 10H6, T84.66-H6CM18, or gelonin immunotoxins co-administered with T84.66-H6CM18 were evaluated. The combinations of T84.66-gelonin + 1.0 mg/kg T84.66-H6CM18 and 10H6-gelonin + 0.1 mg/kg T84.66-H6CM18 led to significant delays in LS174T growth. Use of a multiple-dose regimen allowed further anti-tumor effects, significantly extending median survival time by 33% and by 69%, for mice receiving 1 mg/kg 10H6-gelonin + 0.1 mg/kg T84.66-H6CM18 (p = 0.0072) and 1 mg/kg 10H6-gelonin + 1 mg/kg T84.66-H6CM18 (p = 0.0017). Combined administration of gelonin immunoconjugates with antibody-targeted endosomal escape peptides increased the delivery of gelonin to the cytoplasm of targeted cells, increased gelonin cell killing in vitro by 1,000-6,000 fold, and significantly increased in vivo efficacy.
... The introduced azide functionality was used to conjugate the alkyne (Diazo-Biotin-DBCO comprising a cleavable linker) via a strain-promoted azide-alkyne cycloaddition (SPAAC), which can be performed in the absence of catalysts, such as Cu(I). 29,30 In the next step, the functionalized antibodies were incubated with the CHO-HCP ELISA standard (antigen), since immunocomplexation in solution proved superior compared to their formation after bead conjugation. 17 After immobilization of the biotinlabeled antibodies onto Streptavidin beads, several washing steps were performed using similar conditions as commonly applied for ELISAs and the Diazo-spacer arm was cleaved reductively facilitating the release of the immunocomplexes from the matrix. ...
Article
In the control strategy for process related impurities in biopharmaceuticals the enzyme linked immunosorbent assay (ELISA) is the method of choice for the quantification of host cell proteins (HCP). Besides two dimensional - western blots (2D-WB), the coverage of ELISA antibodies is increasingly evaluated by affinity purification based liquid chromatography–tandem mass spectrometry (AP-MS) methods. However, all these methods face the problem of unspecific binding issues between antibodies and the matrix, involving the application of arbitrarily defined thresholds during data evaluation. To solve this, a new approach (optimized AP-MS) was developed in this study, for which a cleavable linker was conjugated to the ELISA antibodies enabling the subsequent isolation of specifically interacting HCPs. By comparing both approaches in terms of method variability and the number of false positive or negative hits, we could demonstrate that the optimized AP-MS method is very reproducible and superior in the identification of antibody detection gaps, while previously described strategies suffered from over- or underestimating the coverage. As only antibody associated HCPs were identified, we demonstrated that the method is beneficial for hitchhiker analysis. Overall, the method described herein has proven as a powerful tool for reliable coverage determination of ELISA antibodies, without the need to arbitrarily exclude HCPs during the coverage evaluation.
... Before reacting with DNA samples, cisplatin was fully dissolved for >1 day in reaction buffer at 37 • C. Recombinant histone H2A/H2B dimers and H3.1/H4 tetramers were purchased from NEB (New England Biolabs, USA). The DNA tethers used in our single-molecule studies were prepared as previously (16), except for attachment chemistry, that is, copper-free click chemistry to covalently attach a single DNA molecule to the flow chamber surface (17). A 15-kb DNA plasmid was digested by BamHI and SacI (NEB, USA) and its longer fragment (∼12 kb) was used as a main body of DNA tether. ...
Article
Full-text available
Cisplatin is one of the most potent anti-cancer drugs developed so far. Recent studies highlighted several intriguing roles of histones in cisplatin's anti-cancer effect. Thus, the effect of nucleosome formation should be considered to give a better account of the anti-cancer effect of cisplatin. Here we investigated this important issue via single-molecule measurements. Surprisingly, the reduced activity of cisplatin under [NaCl] = 180 mM, corresponding to the total concentration of cellular ionic species, is still sufficient to impair the integrity of a nucleosome by retaining its condensed structure firmly, even against severe mechanical and chemical disturbances. Our finding suggests that such cisplatin-induced fastening of chromatin can inhibit nucleosome remodelling required for normal biological functions. The in vitro chromatin transcription assay indeed revealed that the transcription activity was effectively suppressed in the presence of cisplatin. Our direct physical measurements on cisplatin-nucleosome adducts suggest that the formation of such adducts be the key to the anti-cancer effect by cisplatin.
Chapter
Single-molecule force spectroscopy can precisely probe the biomechanical interactions of proteins that unwind duplex DNA and bind to and wrap around single-stranded (ss)DNA. Yet assembly of the required substrates, which often contain a ssDNA segment embedded within a larger double-stranded (ds)DNA construct, can be time-consuming and inefficient, particularly when using a standard three-way hybridization protocol. In this chapter, we detail how to construct a variety of force-activated DNA substrates more efficiently. To do so, we engineered a dsDNA molecule with a designed sequence of specified GC content positioned between two enzymatically induced, site-specific nicks. Partially pulling this substrate into the overstretching transition of DNA (~65 pN) using an optical trap led to controlled dissociation of the ssDNA segment delineated by the two nicks. Here, we describe protocols for generating ssDNA of up to 1000 nucleotides as well as more complex structures, such as a 120-base-pair DNA hairpin positioned next to a 33-nucleotide ssDNA segment. The utility of the hairpin substrate was demonstrated by measuring the motion of E. coli. RecQ, a 3′-to-5′ DNA helicase.Key wordsOptical tweezersOptical trapHelicaseDNA overstretchingSingle-molecule force spectroscopy
Article
Cell penetrating peptides conjugated to delivery vehicles, such as nanoparticles or antibodies, can enhance the cytosolic delivery of macromolecules. The present study examines the effects of conjugation to cell penetrating and endosomal escape peptides (i.e., TAT, GALA, and H6CM18) on the pharmacokinetics and distribution of an anti-carcinoembryonic antigen "catch-and-release" monoclonal antibody, 10H6, in a murine model of colorectal cancer. GALA and TAT were conjugated to 10H6 using SoluLINK technology that allowed the evaluation of peptide-to-antibody ratio by ultraviolet spectroscopy. H6CM18 was conjugated to either NHS or maleimide-modified 10H6 using an azide-modified valine-citrulline linker and copper-free click chemistry. Unmodified and peptide-conjugated 10H6 preparations were administered intravenously at 6.67 nmol/kg to mice-bearing MC38CEA+ tumors. Unconjugated 10H6 demonstrated a clearance of 19.9 ± 1.36 mL/day/kg, with an apparent volume of distribution of 62.4 ± 7.78 mL/kg. All antibody-peptide conjugates exhibited significantly decreased plasma and tissue exposure, increased plasma clearance, and increased distribution volume. Examination of tissue-to-plasma exposure ratios showed an enhanced selectivity of 10H6-TAT for the GI tract (+25%), kidney (+24%), liver (+38%), muscle (+3%), and spleen (+33%). 10H6-GALA and 10H6-H6CM18 conjugates demonstrated decreased exposure in all tissues, relative to unmodified 10H6. All conjugates demonstrated decreased tumor exposure and selectivity; however, differences in tumor selectivity between 10H6 and 10H6-H6CM18 (maleimide) were not statistically significant. Relationships between the predicted peptide conjugate isoelectric point (pI) and pharmacokinetic parameters were bell-shaped, where pI values around 6.8-7 exhibit the slowest plasma clearance and smallest distribution volume. The data and analyses presented in this work may guide future efforts to develop immunoconjugates with cell penetrating and endosomal escape peptides.
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DNA wrapping around histone octamers generates nucleosomes, the basic compaction unit of eukaryotic chromatin. Nucleosome stability is carefully tuned to maintain DNA accessibility in transcription, replication, and repair. Using freely orbiting magnetic tweezers, which measure the twist and length of single DNA molecules, we monitor the real-time loading of tetramers or complete histone octamers onto DNA by Nucleosome Assembly Protein-1 (NAP1). Remarkably, we find that tetrasomes exhibit spontaneous flipping between a preferentially occupied left-handed state (ΔLk = -0.73) and a right-handed state (ΔLk = +1.0), separated by a free energy difference of 2.3 kBT (1.5 kcal/mol). This flipping occurs without concomitant changes in DNA end-to-end length. The application of weak positive torque converts left-handed tetrasomes into right-handed tetrasomes, whereas nucleosomes display more gradual conformational changes. Our findings reveal unexpected dynamical rearrangements of the nucleosomal structure, suggesting that chromatin can serve as a "twist reservoir," offering a mechanistic explanation for the regulation of DNA supercoiling in chromatin. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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Magnetic tweezers are a powerful single-molecule technique that allows real-time quantitative investigation of biomolecular processes under applied force. High pulling forces exceeding tens of picoNewtons may be required, e.g. to probe the force range of proteins that actively transcribe or package the genome. Frequently, however, the application of such forces decreases the sample lifetime, hindering data acquisition. To provide experimentally viable sample lifetimes in the face of high pulling forces, we have designed a novel anchoring strategy for DNA in magnetic tweezers. Our approach, which exploits covalent functionalization based on heterobifunctional poly(ethylene glycol) crosslinkers, allows us to strongly tether DNA while simultaneously suppressing undesirable non-specific adhesion. A complete force and lifetime characterization of these covalently anchored DNA-tethers demonstrates that, compared to more commonly employed anchoring strategies, they withstand 3-fold higher pulling forces (up to 150 pN) and exhibit up to 200-fold higher lifetimes (exceeding 24 h at a constant force of 150 pN). This advance makes it possible to apply the full range of biologically relevant force scales to biomolecular processes, and its straightforward implementation should extend its reach to a multitude of applications in the field of single-molecule force spectroscopy.
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We demonstrate a new method to program the ligation of single stranded DNA-modified gold nanoparticles using copper-free click chemistry. Gold nanoparticles functionalized with a discrete number of 30-azide or 50-alkyne modified oligonucleotides, can be brought together via a splint strand and covalently ‘clicked’, in a simple onepot reaction. This new approach to the assembly of gold nanoparticles is inherently advantageous in comparison to the traditional enzymatic ligation. The chemical ligation is specific and takes place at room temperature by simply mixing the particles without the need for special enzymatic conditions. The yield of ‘clicked’ nanoparticles can be as high as 92%. The ease of the copper-free, ‘click-ligation’ method allows for its universal applicability and opens up new avenues in programmed nanoparticle organization.
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While many proteins are involved in the assembly and (re)positioning of nucleosomes, the dynamics of protein-assisted nucleosome formation are not well understood. We study NAP1 (nucleosome assembly protein 1) assisted nucleosome formation at the single-molecule level using magnetic tweezers. This method allows to apply a well-defined stretching force and supercoiling density to a single DNA molecule, and to study in real time the change in linking number, stiffness and length of the DNA during nucleosome formation. We observe a decrease in end-to-end length when NAP1 and core histones (CH) are added to the dsDNA. We characterize the formation of complete nucleosomes by measuring the change in linking number of DNA, which is induced by the NAP1-assisted nucleosome assembly, and which does not occur for non-nucleosomal bound histones H3 and H4. By rotating the magnets, the supercoils formed upon nucleosome assembly are removed and the number of assembled nucleosomes can be counted. We find that the compaction of DNA at low force is about 56 nm per assembled nucleosome. The number of compaction steps and associated change in linking number indicate that NAP1-assisted nucleosome assembly is a two-step process.
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Magnetic tweezers (MT) are a powerful tool for the study of DNA-enzyme interactions. Both the magnet-based manipulation and the camera-based detection used in MT are well suited for multiplexed measurements. Here, we systematically address challenges related to scaling of multiplexed magnetic tweezers (MMT) towards high levels of parallelization where large numbers of molecules (say 10(3)) are addressed in the same amount of time required by a single-molecule measurement. We apply offline analysis of recorded images and show that this approach provides a scalable solution for parallel tracking of the xyz-positions of many beads simultaneously. We employ a large field-of-view imaging system to address many DNA-bead tethers in parallel. We model the 3D magnetic field generated by the magnets and derive the magnetic force experienced by DNA-bead tethers across the large field of view from first principles. We furthermore experimentally demonstrate that a DNA-bead tether subject to a rotating magnetic field describes a bicircular, Limaçon rotation pattern and that an analysis of this pattern simultaneously yields information about the force angle and the position of attachment of the DNA on the bead. Finally, we apply MMT in the high-throughput investigation of the distribution of the induced magnetic moment, the position of attachment of DNA on the beads, and DNA flexibility. The methods described herein pave the way to kilo-molecule level magnetic tweezers experiments.
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The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 -> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.
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Selective chemical reactions that are orthogonal to the diverse functionality of biological systems have become important tools in the field of chemical biology. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catalyzed [3 + 2] cycloaddition of azides and alkynes ("click chemistry"). The Staudinger ligation has sufficient biocompatibility for performance in living animals but suffers from phosphine oxidation and synthetic challenges. Click chemistry obviates the requirement of phosphines, but the Cu(I) catalyst is toxic to cells, thereby precluding in vivo applications. Here we present a strain-promoted [3 + 2] cycloaddition between cyclooctynes and azides that proceeds under physiological conditions without the need for a catalyst. The utility of the reaction was demonstrated by selective modification of biomolecules in vitro and on living cells, with no apparent toxicity.
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Oligonucleotides have been ligated efficiently on solid-phase using CuAAC and SPAAC chemistry to produce up to 186-mer triazole linked DNA products. Multiple sequential ligation reactions can be carried out by using a masked azide approach. This work suggests a novel modular approach to the synthesis of large complex oligonucleotide analogues.