Download Tetrazine−trans-cyclooctene Mediated Conjugation of Antibodies to

Communication
pubs.acs.org/bc
Tetrazine−trans-cyclooctene Mediated Conjugation of Antibodies to
Microtubules Facilitates Subpicomolar Protein Detection
Samata Chaudhuri,†,‡ Till Korten,†,‡ and Stefan Diez*,†,‡
†
B CUBE  Center for Molecular Bioengineering, Technische Universität Dresden, 01069 Dresden, Germany
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
‡
S Supporting Information
*
ABSTRACT: Engineering cargo-loading strategies is crucial to
developing nanotechnological applications of microtubule-based
biomolecular transport systems. Here, we report a highly efficient
and robust bioconjugation scheme to load antibodies to microtubules. Our method takes advantage of the inverse-electrondemand Diels−Alder addition reaction between tetrazine and transcyclooctene: the fastest known bioorthogonal reaction, characterized
by its excellent selectivity and biocompatibility. As proof of concept,
we performed kinesin-1 gliding motility assays with antibodyconjugated microtubules and demonstrated the highly sensitive
detection of fluorescent protein analyte down to 0.1 pM in
microliter sample volumes. Importantly, the detection selectivity was
retained in the presence of other fluorescent background proteins.
We envision the applicability of our fast, simple, and robust conjugation method to a wide range of biosensing platforms based on
biomolecular transport systems.
T
(I)-catalyzed alkyne−azide cycloaddition (CuAAC) classes of
“click chemistry” were recently applied to modify MTs.26,27
While the toxicity of copper to gliding motility assays26
precludes the usage of CuAAC reactions for nanotechnological
applications, the SPAAC reactions are limited by their slow
kinetics (k < 1 M−1 s−1 compared to kon ≈ 105−106 M−1 s−1 for
affinity-ligand-based reactions).28,29 For practical applications of
bioorthogonal reactions, kinetic rates of k ≈ 103 M−1 s−1 and
higher are deemed critical.30
Here, we present a novel bioconjugation strategy to
covalently couple antibodies as model cargo to MTs using
the recently developed inverse-electron-demand Diels−Alder
addition (iEDDA)31,32 reaction between tetrazine and transcyclooctene (TCO) (Scheme 2). Besides retaining the
advantages of “click chemistry” (including high reaction
specificity and biocompatibility), iEDDA reactions proceed at
unprecedented speed (k > 800 M−1 s−1, faster than any other
known bioorthogonal reaction) in a wide range of solvents and
do not require catalysts. Moreover, the recent commercial
availability of a wide range of tetrazine and TCO reagents has
made iEDDA reactions a popular choice for bioconjugation
applications. Using gliding motility assays with antibodyconjugated MTs gliding on kinesin-coated surfaces, we report
the specific detection of fluorescent protein analyte down to 0.1
pM, with excellent selectivity.
ransport systems based on biomolecular motors are
promising components for developing nanoscale devices1,2 due to their small size, energy efficiency,3 and detection
sensitivity.4,5 Recently, first proof-of-principle devices employing kinesin-1 and microtubules (MTs) have been fabricated to
transport, sort, detect and manipulate cargo.6−16 In these
examples, it became obvious that effective MT-functionalization
schemes are crucial for reliable cargo loading. Ideally, such
schemes are simple, robust, and applicable to a wide range of
cargo. Traditionally, the most common method of cargo
attachment to MTs has been biotin−streptavidin linkage,6,7,9,10,12−16 a noncovalent, affinity-ligand-based conjugation
scheme. Despite its ubiquitous usage, this method suffers from
several limitations, including aggregation due to the multimeric
nature of streptavidin,17,18 cargo loss due to a decreased lifetime
of the bonds under tension19 as well as at high gliding
velocities,20 and reduced velocity of transport.21 The noncovalent biotin−streptavidin interaction is short-ranged and
reversible, limiting its practical applications: examples are an
almost million fold increase in the dissociation constant (Kd) of
biotinylated peptide when streptavidin was attached to beads22
and very transient bond lifetimes of 0.15 to 20 s in the presence
of shear forces.23 Covalent bonding can circumvent these
drawbacks (Scheme 1). However, early attempts to modify
MTs using nonspecific covalent conjugation schemes were
found to be limited by undesired side reactions and low
reaction efficiencies.11,24,25 To overcome these limitations,
highly specific, bioorthogonal conjugation schemes using strainpromoted alkyne−azide cycloadditions (SPAAC) and copper© 2017 American Chemical Society
Received: March 1, 2017
Published: March 7, 2017
918
DOI: 10.1021/acs.bioconjchem.7b00118
Bioconjugate Chem. 2017, 28, 918−922
Communication
Bioconjugate Chemistry
Scheme 1. Comparison of Schemes to Conjugate Antibodies
to MTs Using Biotin−Streptavidin (A) and Covalent
Bonding (B)a
a
In (A), the antibodies are attached to the MT via two non-covalent
bonds between biotin−streptavidin, each with its characteristic
dissociation constant. In (B), the antibodies are attached to the MT
via a single covalent bond, yielding a more-stable conjugate.
Figure 1. Functionalization of MTs with trans-cyclooctene (TCO).
(A) Solvent-accessible NH2-groups of tubulin were modified by
TCO−(PEG)4−NHS. (B and C) The presence of TCO groups on the
MTs was confirmed by fluorescence microscopy. Fluorescent
tetrazine−Cy5 was added in gliding assays containing TCO−MTs
(red) and Alexa Fluor 488-labeled control MTs (cyan). Exclusive
colocalization of the tetrazine−Cy5 signal (green) to TCO-MTs
confirms the specific functionalization of the MTs with TCO. Scale bar
5 μm. See also Supporting Movie 1. (D) TCO functionalization of the
MTs, as well as subsequent addition of tetrazine−Cy5 to the TCOMTs, did not affect the gliding velocity of the MTs. Gliding velocity
(mean ± standard deviation) of control MT: 721 ± 97 nm/s (n =
233); TCO-MT: 719 ± 150 nm/s (n = 469); TCO-MT + tetrazine−
Cy5:743 ± 129 nm/s (n = 368).
Scheme 2. Inverse-Electron-Demand Diels−Alder Addition
Reaction between trans-Cyclooctene Functionalized
Biomolecule A and Methyl-tetrazine Functionalized
Biomolecule B
■
control, Alexa Fluor 488-labeled MTs lacking the TCO
functionalization. No co-localization of the tetrazine-Cy5
fluorescent signal with these control MTs was observed (Figure
1C). Notably, the TCO−MTs exhibited a similar gliding
velocity as the control MTs (Figure 1D), in agreement with
previous reports showing that functionalization with small
amine-reactive reagents did not significantly affect the gliding
motility of MTs.11,24,26
To generate antibody-conjugated MTs (Ab−MTs), we
covalently coupled tetrazine-modified mouse IgG antibodies
to TCO−MTs using the bioorthogonal iEDDA reaction
(Figure 2A). To confirm successful conjugation, 100 nM
Alexa Fluor 488 antimouse secondary antibodies were added to
Ab−MTs surface-immobilized by antirhodamine antibodies
(Figure 2B). Rhodamine−Cy5 co-labeled, non-TCO-functionalized MTs that lacked the tetrazine-modified antibodies were
used as control. After 30 min of incubation and subsequent
washing, specific co-localization of the fluorescent Alexa Fluor
488 signal from the secondary antibodies with the Ab−MTs
confirmed the successful assembly of the Ab−MT (Figure 2C).
RESULTS AND DISCUSSION
For developing gliding-assay-based applications with functionalized MTs, two important criteria need to be fulfilled: (i) the
chemically modified tubulin should retain its ability to
polymerize into MTs and (ii) the polymerized MTs, in turn,
should retain their ability to glide over the kinesin surface. We
first tested the capacity of trans-cyclooctene (TCO) functionalized MTs (TCO-MTs) to fulfill these requirements. We
polymerized taxol-stabilized TCO-MTs from a mixture of
TCO-modified, rhodamine-modified and unmodified tubulin
(Figure 1A). The presence and accessibility of the TCO groups
was checked by adding 0.5 μM tetrazine−Cy5 dye to the
TCO−MTs gliding on a surface covered by kinesin-1 motors
(Figure 1B). After an incubation time of 10 min and washing
off of the excess tetrazine−Cy5 dye, the fluorescent Cy5 signal
from the tetrazine was found to co-localize with the fluorescent
rhodamine signal of the TCO−MTs (Figure 1C). Specificity of
tetrazine−Cy5 attachment was verified by similar treatment of
919
DOI: 10.1021/acs.bioconjchem.7b00118
Bioconjugate Chem. 2017, 28, 918−922
Communication
Bioconjugate Chemistry
Figure 2. Covalent conjugation of tetrazine-modified antibodies to
TCO−MTs. (A) Mouse IgG antibodies were modified with tetrazine−
(PEG)5−NHS and covalently conjugated to TCO−MTs via iEDDA
reaction to assemble antibody-conjugated MTs (Ab−MTs). (B and C)
Successful assembly of Ab−MTs was confirmed by fluorescence
microscopy. Fluorescent Alexa Fluor 488 antimouse secondary
antibodies were added to surface-immobilized Ab−MTs (red) and
Cy5-labeled control MTs (cyan). Exclusive colocalization of the Alexa
Fluor 488 signal (green) to Ab−MTs confirms the specific conjugation
of the tetrazine-modified antibodies to TCO−MTs. The high intensity
and distribution of the Alexa Fluor 488 signal indicates high-densitylabeling due to efficient Ab−MT conjugation. Scale bar 10 μm.
Figure 3. Detection of fluorescent protein analyte using Ab−MTs. (A)
Fluorescent protein analyte (Alexa Fluor 488 antimouse secondary
antibodies) was detected by Ab−MTs in gliding motility assays using
TIRF microscopy. (B) TIRF images of Ab−MTs (red) transporting
fluorescent protein analyte (green) at 0.1, 1, and 10 pM
concentrations. Scale bar: 5 μm. (C) Plotting the increase in the
fluorescence intensity of the analyte per unit length of Ab−MTs as a
function of analyte concentration demonstrates the sensitivity of the
detection method down to 0.1 pM (red data). The sensitivity of the
assay was not affected by the presence of equal concentrations of
background fluorescent protein analyte (Cy3 anti-rabbit antibody; “BG
protein”, blue line). Control MTs without conjugated antibodies
showed no increase in the fluorescence intensity upon addition of
analyte (black line). For each data point, between 127 to 529 MTs
were analyzed (mean ± standard deviation). The detected signal on
Ab−MTs at 0.1 pM analyte concentration was significantly different
from that on the control MTs (Mann−Whitney U = 0, p < 0.0001).
The high intensity and uniform distribution of the colocalized
signal indicate a high labeling density of the antibodies to the
MTs. This can be ascribed to the high reactivity of the iEDDA
reaction, enabling an efficient conjugation of the antibodies to
the MTs. Additionally, we believe that the presence of the short
PEG linkers to both TCO and tetrazine (see the Materials and
Methods section) accentuated the conjugation efficiency by
increasing the accessibility to the binding sites.
Detection of fluorescent protein analyte was performed by
using Ab−MTs in gliding motility assays (Figure 3A).
Fluorescent Alexa Fluor 488 anti-mouse secondary antibodies
were used as model protein analytes to specifically bind to the
Ab−MTs. Using total internal reflection fluorescence (TIRF)
microscopy, the protein analyte was found to specifically bind
to (and to be transported by) the Ab−MTs. After a short
incubation incubation time of 10 min, we were able to detect
protein analyte at concentrations as low as 0.1 pM using
microliter sample volumes (Figure 3B). In our assay, 0.1 pM of
analyte corresponds to about 30 000 molecules in the volume
of our flow chamber or an average of three molecules per field
of view (about 80 μm × 80 μm). Analyte detection at this low
concentration demonstrates the high sensitivity of our
detection method. Analyte binding and transport was not
observed with control MTs that lacked the covalently
conjugated antibodies, confirming the selectivity of our method.
Additionally, the sensitivity of the detection scheme was
unaffected by the presence of equivalent concentrations of
fluorescent Cy3 antirabbit secondary antibodies as background
proteins (Figure 3C).
Our method of analyte detection by gliding Ab−MTs offers
several advantages over methods based on surface-immobilized
antibodies: first, active transport allows the distinguishing of the
specific transport of analyte bound to the gliding MTs from
background molecules nonspecifically bound to the surfaces of
the detection chamber. Second, as active transport obviates
elaborate washing steps, the detection time can be significantly
decreased, and robust detection devices working with microliter
sample volumes can be fabricated.
■
CONCLUSIONS
We employ the fastest known bioorthogonal reaction between
tetrazine and trans-cyclooctene to covalently conjugate antibodies to MTs. As proof-of-concept, we use Ab−MTs in gliding
motility assays to demonstrate the detection of fluorescent
920
DOI: 10.1021/acs.bioconjchem.7b00118
Bioconjugate Chem. 2017, 28, 918−922
Bioconjugate Chemistry
■
proteins down to 0.1 pM concentration with excellent
selectivity. Because antibodies are gold-standard affinity probes,
the commercial availability of antibodies allows our method to
be extended to the setup of various other biosensing platforms.
Moreover, the described conjugation scheme can be easily
adapted for loading other types of cargos, as iEDDA reactions
have been shown to be compatible with a wide range of
biomolecules. Because the iEDDA chemistry is orthogonal to
other “click chemistry” schemes, it will open new opportunities
for the simultaneous and sequential assembly of multiple
modular units to MTs in gliding motility assays.
■
Communication
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00118.
A movie showing gliding motility assay of TCO−MTs
(red) and control MTs (blue) after the addition of
tetrazine−Cy5 dye (green). (AVI)
A movie showing detection of fluorescent protein analyte
(green) at various concentrations by Ab−MTs (red).
(AVI)
■
MATERIALS AND METHODS
AUTHOR INFORMATION
Corresponding Author
Conjugation of Antibodies to MTs. Lyophilized mouse
anti-CD45 IgG antibodies (R&D Biosystems, Germany) were
reconstituted in phosphate buffer saline at 2.5 mg/mL and
conjugated to NHS−(PEG)5−tetrazine (Jena Biosciences,
Germany) at a 5-fold molar excess to give tetrazine-modified
antibodies. After overnight incubation at 4 °C, excess NHS−
(PEG)5−tetrazine was removed using Zeba spin desalting
columns (ThermoFisher). Purified tubulin33 was modified with
NHS−(PEG)4−trans-cyclooctene (Jena Biosciences, Germany)
based on established protocols.34 Kinesin-1 expressed in insect
cells was purified as recently described.35 TCO-MTs were
polymerized in BRB80 (80 mM PIPES, 1 mM EGTA, 1 mM
MgCl2; pH 6.9), supplemented with 5 mM MgCl2, 1 mM Mg−
GTP, and 5% DMSO, at 37 °C from a 4 mg/mL tubulin
mixture containing TCO-modified, rhodamine-modified, and
unmodified tubulin. Polymerized TCO−MTs were diluted 40
fold and stabilized in BRB80T (BRB80 containing 10 μM
taxol). TCO−MTs were concentrated 20-fold by centrifugation
through a 60% glycerol cushion, resuspended in BRB80T, and
added to an equal volume of tetrazine-modified antibodies.
After 15 min of incubation at room temperature, the conjugate
mixture was diluted 50 fold to obtain antibody-conjugated
MTs.
Gliding Motility Assays. MT gliding motility assays were
performed as previously described36 in flow cells separated by
Parafilm M stripes. A 0.5 mg/mL solution of casein in BRB80
was flowed in and incubated for 2 min. Kinesin-1 solution (12.5
nM) was then added and incubated for 2 min. Finally, a
motility solution (1 mM ATP, 20 mM D-glucose, 20 mM
glucose oxidase, 10 mM catalase, 10 mM DTT, and 10 μM
taxol in BRB80) with the MTs was added. Excess unbound
MTs were removed by flushing motility solution without MTs.
Image Acquisition and Data Analysis. Imaging was
performed using a fluorescence microscopy setup as previously
described37 with 100 ms exposure time. Rhodamine, Cy5, and
Alexa Fluor 488-labeled MTs were observed using epifluorescence. Fluorescent protein analyte was imaged with
488 nm laser line using total internal reflection fluorescence
(TIRF) microscopy. Data analysis was performed using ImageJ,
MATLAB, FIESTA,38 and GraphPad Prism. The integrated
intensity in the fluorescent protein analyte channel was
measured using the MTs as the mask. The same mask was
used to read out the background from an adjacent empty area,
which was then subtracted from the original signal to obtain
background-subtracted fluorescence intensities.
*E-mail: [email protected].
ORCID
Samata Chaudhuri: 0000-0003-4245-4302
Stefan Diez: 0000-0002-0750-8515
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors thank Corina Bräuer for technical support, Sumeet
Pal Singh for comments on the manuscript, and the entire Diez
lab for fruitful discussions. Financial support from the German
Research Foundation (Cluster of Excellence Center for
Advancing Electronics Dresden and the Dresden International
Graduate School for Biomedicine and Bioengineering, DIGSBB) and the European Union Seventh Framework and Horizon
2020 Programs (under grant agreements 613044 (ABACUS)
and 732482 (Bio4Comp)) is acknowledged.
■
REFERENCES
(1) Hess, H., and Vogel, V. (2001) Molecular shuttles based on
motor proteins: active transport in synthetic environments. Rev. Mol.
Biotechnol. 82, 67−85.
(2) van den Heuvel, M. G. L., and Dekker, C. (2007) Motor proteins
at work for nanotechnology. Science 317, 333−6.
(3) Howard, J. (1996) The movement of kinesin along microtubules.
Annu. Rev. Physiol. 58, 703−729.
(4) Katira, P., and Hess, H. (2010) Two-Stage Capture Employing
Active Transport Enables Sensitive and Fast Biosensors. Nano Lett. 10,
567−572.
(5) Korten, T., Månsson, A., and Diez, S. (2010) Towards the
application of cytoskeletal motor proteins in molecular detection and
diagnostic devices. Curr. Opin. Biotechnol. 21, 477−88.
(6) Hess, H., Clemmens, J., Qin, D., Howard, J., and Vogel, V. (2001)
Nano Lett. 1, 235.
(7) Diez, S., Reuther, C., Dinu, C., Seidel, R., Mertig, M., Pompe, W.,
and Howard, J. (2003) Stretching and Transporting DNA Molecules
Using Motor Proteins. Nano Lett. 3, 1251−1254.
(8) Jia, L., Moorjani, S. G., Jackson, T. N., and Hancock, W. O.
(2004) Microscale transport and sorting by kinesin molecular motors.
Biomed. Microdevices 6, 67−74.
(9) Platt, M., Muthukrishnan, G., Hancock, W. O., and Williams, M.
E. (2005) J. Am. Chem. Soc. 127, 15686.
(10) Ramachandran, S., Ernst, K.-H., Bachand, G. D., Vogel, V., and
Hess, H. (2006) Selective Loading of Kinesin-Powered Molecular
Shuttles with Protein Cargo and its Application to Biosensing. Small 2,
330−334.
(11) Bachand, G. D., Rivera, S. B., Carroll-Portillo, A., Hess, H., and
Bachand, M. (2006) Active Capture and Transport of Virus Particles
Using a Biomolecular Motor-Driven, Nanoscale Antibody Sandwich
Assay. Small 2, 381−385.
921
DOI: 10.1021/acs.bioconjchem.7b00118
Bioconjugate Chem. 2017, 28, 918−922
Communication
Bioconjugate Chemistry
(12) Brunner, C., Wahnes, C., and Vogel, V. (2007) Cargo pick-up
from engineered loading stations by kinesin driven molecular shuttles.
Lab Chip 7, 1263.
(13) Hutchins, B. M., Platt, M., Hancock, W. O., and Williams, M. E.
(2007) Directing transport of CoFe2O4-functionalized microtubules
with magnetic fields. Small 3, 126−31.
(14) Lin, C.-T., Kao, M.-T., Kurabayashi, K., and Meyhofer, E.
(2008) Self-contained, biomolecular motor-driven protein sorting and
concentrating in an ultrasensitive microfluidic chip. Nano Lett. 8,
1041−6.
(15) Fischer, T., Agarwal, A., and Hess, H. (2009) A smart dust
biosensor powered by kinesin motors. Nat. Nanotechnol. 4, 162−166.
(16) Schmidt, C., and Vogel, V. (2010) Molecular shuttles powered
by motor proteins: loading and unloading stations for nanocargo
integrated into one device. Lab Chip 10, 2195−8.
(17) Hess, H., Clemmens, J., Brunner, C., Doot, R., Luna, S., Ernst,
K.-H., and Vogel, V. (2005) Molecular self-assembly of “nanowires”and “nanospools” using active transport. Nano Lett. 5, 629−33.
(18) Lam, A. T., VanDelinder, V., Kabir, A. M. R., Hess, H., Bachand,
G. D., and Kakugo, A. (2016) Cytoskeletal motor-driven active selfassembly in in vitro systems. Soft Matter 12, 988−97.
(19) Merkel, R., Nassoy, P., Leung, A., Ritchie, K., and Evans, E.
(1999) Energy landscapes of receptor−ligand bonds explored with
dynamic force spectroscopy. Nature 397, 50−53.
(20) Agarwal, A., Katira, P., and Hess, H. (2009) Millisecond Curing
Time of a Molecular Adhesive Causes Velocity-Dependent CargoLoading of Molecular Shuttles. Nano Lett. 9, 1170−1175.
(21) Korten, T., and Diez (2008) Setting up roadblocks for kinesin-1:
mechanism for the selective speed control of cargo carrying
microtubules. Lab Chip 8, 1441.
(22) Buranda, T., Lopez, G. P., Keij, J., Harris, R., and Sklar, L. A.
(1999) Peptides, antibodies, and FRET on beads in flow cytometry: A
model system using fluoresceinated and biotinylated ?-endorphin.
Cytometry 37, 21−31.
(23) Pierres, A., Touchard, D., Benoliel, A.-M., and Bongrand, P.
(2002) Dissecting streptavidin-biotin interaction with a laminar flow
chamber. Biophys. J. 82, 3214−23.
(24) Soto, C. M., Martin, B. D., Sapsford, K. E., Blum, A. S., and
Ratna, B. R. (2008) Toward single molecule detection of staphylococcal enterotoxin B: mobile sandwich immunoassay on gliding
microtubules. Anal. Chem. 80, 5433−40.
(25) Carroll-Portillo, A., Bachand, M., and Bachand, G. D. (2009)
Directed attachment of antibodies to kinesin-powered molecular
shuttles. Biotechnol. Bioeng. 104, 1182−8.
(26) Früh, S. M., Steuerwald, D., Simon, U., and Vogel, V. (2012)
Covalent Cargo Loading to Molecular Shuttles via Copper-free “Click
Chemistry. Biomacromolecules 13, 3908−3911.
(27) Kleiner, R. E., Ti, S.-C., and Kapoor, T. M. (2013) Site-specific
chemistry on the microtubule polymer. J. Am. Chem. Soc. 135, 12520−
3.
(28) Agard, N. J., Prescher, J. A., and Bertozzi, C. R. (2004) A StrainPromoted [3 + 2] Azide−Alkyne Cycloaddition for Covalent
Modification of Biomolecules in Living Systems. J. Am. Chem. Soc.
126, 15046−15047.
(29) Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard, N. J., Chang,
P. V., Miller, I. A., Lo, A., Codelli, J. A., and Bertozzi, C. R. (2007)
Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl.
Acad. Sci. U. S. A. 104, 16793−7.
(30) Devaraj, N. K., and Weissleder, R. (2011) Biomedical
applications of tetrazine cycloadditions. Acc. Chem. Res. 44, 816−27.
(31) Blackman, M. L., Royzen, M., and Fox, J. M. (2008) Tetrazine
ligation: fast bioconjugation based on inverse-electron-demand DielsAlder reactivity. J. Am. Chem. Soc. 130, 13518−9.
(32) Devaraj, N. K., Upadhyay, R., Haun, J. B., Hilderbrand, S. A.,
and Weissleder, R. (2009) Fast and sensitive pretargeted labeling of
cancer cells through a tetrazine/trans-cyclooctene cycloaddition.
Angew. Chem., Int. Ed. 48, 7013−6.
(33) Castoldi, M., and Popov, A. V. (2003) Purification of brain
tubulin through two cycles of polymerization-depolymerization in a
high-molarity buffer. Protein Expression Purif. 32, 83−8.
(34) Hyman, A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K.,
Steffen, P., Wordeman, L., and Mitchison, T. (1991) Preparation of
modified tubulins. Methods Enzymol. 196, 478−85.
(35) Korten, T., Chaudhuri, S., Tavkin, E., Braun, M., and Diez, S.
(2016) Kinesin-1 Expressed in Insect Cells Improves Microtubule in
Vitro Gliding Performance, Long-Term Stability and Guiding
Efficiency in Nanostructures. IEEE Trans. Nanobioscience 15, 62−69.
(36) Nitzsche, B., Bormuth, V., Bräuer, C., Howard, J., Ionov, L.,
Kerssemakers, J., Korten, T., Leduc, C., Ruhnow, F., and Diez, S.
(2010) Studying kinesin motors by optical 3D-nanometry in gliding
motility assays. Methods Cell Biol. 95, 247−71.
(37) Grover, R., Fischer, J., Schwarz, F. W., Walter, W. J., Schwille, P.,
and Diez, S. (2016) Transport efficiency of membrane-anchored
kinesin-1 motors depends on motor density and diffusivity. Proc. Natl.
Acad. Sci. U. S. A. 113, E7185−E7193.
(38) Ruhnow, F., Zwicker, D., and Diez, S. (2011) Tracking single
particles and elongated filaments with nanometer precision. Biophys. J.
100, 2820−8.
922
DOI: 10.1021/acs.bioconjchem.7b00118
Bioconjugate Chem. 2017, 28, 918−922