Efficient functional neutralization of lethal peptide toxins in vivo by oligonucleotides

Medical means to save the life of human patients affected by drug abuse, envenomation or critical poisoning are currently limited. While the compounds at risks are most often well identified, particularly for bioterrorism, chemical intervention to counteract the toxic effects of the ingested/injected compound(s) is restricted to the use of antibodies. Herein, we illustrate that DNA aptamers, targeted to block the pharmacophore of a poisonous compound, represent a fast-acting and reliable method of neutralization in vivo that possesses efficient and long-lasting life-saving properties. For this proof of concept, we used one putative bioweapon, αC-conotoxin PrXA, a marine snail ultrafast-killing paralytic toxin, to identify peptide-binding DNA aptamers. We illustrate that they can efficiently neutralize the toxin-induced (i) displacement of [125I]-α-bungarotoxin binding onto nicotinic receptors, (ii) inhibition of diaphragm muscle contraction, and (iii) lethality in mice. Our results demonstrate the preclinical value of DNA aptamers as fast-acting, safe and cheap antidotes to lethal toxins at risk of misuse in bioterrorism and offer hope for an alternative method than donor sera to treat cases of envenomation.


Aptamer selection against αC-conotoxin PrXA using the CE-SELEX approach
Aptamer selection -CE-SELEX was performed using a 77-nucleotide single-stranded DNA (ssDNA) library containing a randomized region of 30-nt central region flanked by two conserved primer hybridization regions (23-nt at 5' end position and 24-nt at 3' end position). Selection was performed on a Beckman Coulter P/ACE MDQ system (Fullerton, California, USA) with exchangeable UV absorbance and laser-induced fluorescence (LIF) detectors (λ ex 488 nm and λ em 520 nm). The capillary was 60 cm in length (48.5 cm from inlet to detection window) with an inner diameter of 50 µm and an outer diameter of 360 µm (Polymicro Technologies Inc., Phoenix, Arizona, USA). The fused-silica capillaries were conditioned by performing the following washes at 20 psi: 1 M NaOH for 5 min, water for 5 min and TGK buffer (25 mM Tris, 192 mM Glycine, 5 mM KH 2 PO 4 (pH 7.34)) for 30 min. The washing process between runs was performed at 20 psi with 1 M NaOH (2 min), water (2 min) and TGK buffer (5 min). DNA library was heated at 80°C for 5 min and left at room temperature for 15 min. αC-conotoxin PrXA (750 nM) was then added to the DNA library for the first round of selection. The mixture was incubated at room temperature for 20 min. For each selection round, DNA sequences, target peptide and TGK buffer were combined in 30 µL total volume. Toxin concentrations were 20 nM, 6 nM and 0.5 nM for rounds 2, 3 and 4, respectively. The equilibrated sample was injected at 0.7 psi for 20 sec and separated under 20 kV voltage. UV detection was used to monitor the separation. During a selection round, the eluate was collected into 300 µL TGK buffer until the unbound DNA peak began to elute. The injection, separation and collection process was repeated five more times for round 1, 10 times for round 2 and 9 times for rounds 3 and 4. Input DNA concentration was determined by absorbance at 260 nm on a Shimadzu UV mini-1240 spectrophotometer with a Tray Cell from Hellma Analytics (Müllheim, Germany). For the first round of selection, the unselected library was used with 40 µM concentration in the incubated sample. Subsequent rounds of selection used collected DNA, amplified and purified from the previous round as the input DNA. 3 The concentration of input DNA in later selection rounds was 40, 1, 3 and 2.5 µM, respectively.
Approximately 10 12 sequences were introduced into the capillary in the first round of selection.
PCR amplification and single-stranded DNA Production -Single-stranded DNA candidates were generated using a reverse primer and a 5'-FAM-labeled forward primer during each round of the selection procedure.
The reverse primer is made heavier with a succession of six C3 links extended with a 5' DNA stretch of 20 nucleotides (5'-ACTGACTGACTGACTGACTA-6C3-GGGAGACAAGAATAAACGCTCAA). During PCR, this six C3 region and the 20-nucleotide stretch cannot be amplified by the Taq DNA polymerase. A PCR product with two strands of unequal length is consequently synthesized. Each strand is then easily purified on a denaturing polyacrylamide gel. All PCR were performed using a Biometra cycler from After mixing, 10 µL (5 U/µL) of AmpliTaq Gold DNA polymerase was added. To finish, 200 µL of DNA collected during selection were added. This mixed solution was divided equally over thin-walled tubes that were subjected to PCR. The thermal cycling regime was: initial denaturation for 10 min at 95°C, and then cycling for 60 sec at 95°C, 60 sec at 60°C and 90 sec at 72°C for 20 cycles. After a Nanosep 3K purification (Pall, Washington, New York, USA), the samples, which contained different amounts of amplified products, were resolved on a 12% acrylamide gel at an applied voltage of 300 V. The band corresponding to the library molecular weight and corresponding to selected aptamers was visualized by UV-shadow method at 254 nm, cut and eluted for 1 h at 65°C and 1 h at 4°C, in 1 mL of the extraction buffer (100 mM Tris-HCl (pH 7.4), 500 mM NaCl, 1 mM EDTA). To remove acrylamide, the extracted product was transferred into a Nanosep device, the retention membrane was replaced by glass wool and the system was centrifuged during 10 min at 14,000 rpm. Filtrate was removed, transferred in another native Nanosep 3K device and centrifuged for 90 min at 5,000 rpm at 15°C. The retentate was washed with 100 µL of water and centrifuged again. The amount of single strand was quantified by UV absorbance at 260 nm.
Binding Affinity, cloning and sequencing -Four rounds of selection were performed and the progress of selection was monitored using non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) to estimate the bulk affinity. Capillary conditions (length capillary, preconditioning, migration buffer, voltage, temperature) were identical to those used for the capillary electrophoresis aptamer selection, except for sample preparation. Each sample contained 100 nM of enriched DNA pools and αC-conotoxin

Aptamer binding affinity measurement using surface plasmon resonance (SPR)
The binding affinity of the selected aptamer B4 sequence against αC-conotoxin PrXA were analyzed by SPR at 25°C using a Biacore T200 instrument (Biacore, GE Healthcare). Sensor chips were cleaned by UV/ozone treatment (10 min) followed by rinsing with MilliQ water and ethanol. The cleaned gold surfaces were then functionalized according to the following procedure. Firstly, mixed self-assembled monolayers (SAMs) were formed at room temperature by dipping overnight gold sensors in a (9:1) mixture of HS-(CH2)11-EG4-OH and HS-(CH2)11-EG6-Biotin (1 mM total thiol concentration in EtOH). After overnight adsorption, gold sensors were rinsed with EtOH and dried under nitrogen. Conforming to the fluorescence anisotropy experiment, all measurements were conducted in the same TGK buffer containing 0.1% Tween 20. This buffer was filtered in each case and also used as running buffer. Streptavidin (100 ng/mL) was injected (10 µL/min) until saturation of the surface (around 2500 R.U.), following by around 300 RU of 5'biotinylated B4 aptamer at the same flow rate. Binding experiments were conducted at 50 µL/min by injection of αC-conotoxin PrXA (dissolved in the TGK buffer containing 0.1% Tween 20, injection time: 120 sec, dissociation time: 300 sec, stabilization time: 300 sec) at different concentrations (1 nM to 20 µM) by using a multicycle method. A streptavidin surface, prepared as described above, was used as reference.
Curves obtained on the reference surface were deduced from the curves recorded on the recognition one, thereby allowing elimination of refractive index changes owing to buffer effects. The kinetic data were treated independently by using the Bio-Kine software (Biologic, Claix, France) to give the apparent rate constants k obs . The k obs related to the specific association were plotted against the complex concentrations to extract the k on values, while the k obs for the dissociation were averaged to give k off . Supplementary Fig. 1. (a)  D/T = dead/treated mice; None = No toxic symptoms during the observation period; mortality latency = time to death (in minute) after the intraperitoneal injection. αC-conotoxin PrXA or waglerin 1 were administered to group of male and female mice. Mice in each group were carefully examined for any signs of toxicity (behavioural changes or mortality) for 1 h. Control group received distilled water (10 mL/kg, intraperitoneal).

Supplementary Table 2.
Aptamer sequences of the clones isolated after four rounds of selection using CE-SELEX. Primers are in bold script. Aptamer sequences chosen for affinity measurements and functional evaluation are in red. The sequence of scramble aptamers used in various controls are also shown. S1: scramble with same primer sequences, S2: D7 scramble sequence, and S3: random scramble.