Binding modes of thrombin binding aptamers investigated by simulations
and experiments
A. Trapaidze1,2, A. Bancaud1,2, M. Brut1,3,a)
1
CNRS, LAAS, 7 avenue du colonel Roche, F-31400 Toulouse, France
Université de Toulouse, LAAS, F-31400 Toulouse, France
3
Université de Toulouse, UPS, LAAS, F-31400 Toulouse, France
a) Author to whom correspondence should be addressed. Electronic mail: marie.brut@laas.fr.
2
SUPPLEMENTARY MATERIAL
Functionalization of AuNP
80 nm diameter colloidal gold nanoparticles (BBinternation, UK) were functionalized either
by HD1 or HD22, bearing thiol modification and 20-thymine spacer for flexibility at 5’ end
(Eurogentec, Belgium), following the protocol described by Liu et al. [1]. Briefly, first, the
disulfide bonds on thiolated ends of 50 µM aptamers were activated by cleaving with 0.1 M
of dithiothreitol (DTT) in 0.2 mM phosphate buffer (PB) over a period of 1 hour. DTT was
then removed using a desalting NAP-5 column (GE Healthcare), and the freshly deprotected
50 µL aptamers were added to 950 µL gold nanoparticles of concentration 1 O.D. Incubating
for 20 minutes aptamer-NP solution was then buffered with phosphate buffer pH 7.4
(NaH2PO4/K2HPO) and Triton20 to reach the concentration of 0.01 M and 0.01% respectively. Then, small volumes of NaCl, PB and Triton20 were added in stepwise manner every 20
minutes, to increase the NaCl concentration till 0.7 M but maintain the same concentration of
PB and Triton20. Each salting step was followed by 1 min sonication. After overnight incubation on a microtube rotator, excess DNA was removed by multiple centrifugations at 14000
rpm (5 min), and the NPs were resuspended in PBS buffer containing 145 mM NaCl, 5 mM
KCl, and 10 mM PB of pH 7.4 to conduct the aggregation experiments. Average size change
of the gold particles measured before and after functionalization with DLS (Zetasizer Nano S,
Malvern technologies) indicated successful functionalization step. DNA-functionalized gold
particles were stored at 4°C.
References
[1] J. Liu and Y. Lu. Nature Protocols 1, 246 (2006)
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