Aptamer loaded superparamagnetic beads for selective capturing and gentle release of activated protein C

Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities which make it an attractive target for diagnostic and therapeutic applications. In this work, we present one-step activation of APC from a commercial source of protein C (PC, Ceprotin) followed by rapid and efficient purification using an APC-specific aptamer, HS02-52G, loaded on MyOne superparamagnetic beads. Due to the Ca2+-dependent binding of APC to HS02-52G, an efficient capturing of APC was applied in the presence of Ca2+ ions, while a gentle release of captured APC was achieved in the elution buffer containing low EDTA concentration (5 mM). The captured and eluted APC showed more than 95% purity according to SDS-PAGE gel analysis and an enzyme-linked fluorescent assay (VIDAS Protein C). The purification yield of 45% was calculated when 4.2 µg APC was used, however this yield reduced to 21% if the starting amount of APC increased to 28.5 µg. Altogether, this method is recommended for rapid and efficient PC activation and APC purification. The purified APC can be used directly for downstream processes where high concentration of pure and active APC is needed.

In order to reduce discrepancies in PC content and to minimize batch to batch variations, Ceprotin which was reconstituted in a NaCl solution provided by the manufacturer at a final concentration of 250 IU/ml was chosen as the starting solution. The concentration of PC in the used batch of Ceprotin was determined by measurement of PC activity (Berichrom Protein C Kit; Siemens Healthcare Diagnostics, Marburg, Germany) and PC antigen (VIDAS Protein C Kit; Biomerieux, Nürtingen, Germany) levels in parallel with characterized PC preparations [HTI], whereat one unit of Ceprotin corresponded to 3.675 µg PC.
Thrombin is the most physiologically relevant activator of PC. In vivo, thrombin binds to its receptor thrombomodulin (TM), while PC binds to endothelial PC receptor (EPCR) which in turn orients and localizes PC to the endothelium. Localization of the thrombin-TM complex to the adjacent proximity of PC-EPCR complex accelerates activation of PC ~ 2000-fold in a Ca 2+ dependent manner compared to incubation of thrombin and PC in free solution [27][28][29] . Ca 2+ ions are required for PC activation in vivo, whereas in the absence of TM, binding of Ca 2+ to the 70-80 loop of PC inhibits PC activation by thrombin 30 . Accordingly, to avoid the inhibitory effect of Ca 2+ in thrombin-mediated PC activation in the purified system, a PC activation buffer containing 1 mM EDTA was used. Following further optimization in the PC activation process, for 1 µM (62.5 µg/ml) PC, the optimal thrombin concentration was 85 nM or 3.12 µg/ml (8.5% molar ratio or 5% w/w ratio to PC), whereas higher thrombin concentrations did not result in higher APC generation (see Supplementary Fig. S1a online). This data correlates well with previous work focusing on PC activation and purification from prothrombin complex concentrate (PCC) in which 5% w/w human thrombin to PC was incubated at 37 °C for 3 h to achieve optimum APC generation 13  www.nature.com/scientificreports/ As the next step, the optimum incubation time of thrombin and PC, in which the balance between PC activation and APC degradation is in favor of PC activation, was determined. According to the Supplementary  Fig. S1b, APC generation was increased within the first hour of incubation followed by a plateau of high and constant activity of APC up to 24 h. However, the stable APC activity might have resulted either from comparable activation and inactivation rates of APC or from complete activation and negligible inactivation of APC in the absence of its inhibitors.
To evaluate APC stability in the activation buffer at different time points, APC was incubated in the activation buffer up to 5 h and the APC activity was measured in the sub-samples. The data shown in Supplementary  Fig. S1c confirmed the stability of APC in activation buffer up to 5 h. Overall, an activation time of 3 h was set for further experiments, however in special occasions which fast APC production is in favor, reduction of the incubation time to 1 h might result in the same APC production.
Although the peptide substrate PCa-5791 is rather specific for APC, this substrate is hydrolyzed in the presence of low nanomolar concentration of thrombin which might conceal the detection of APC generation in the activation mixture (see Supplementary Fig. S2 online). To use PCa-5791 in the activity assay for monitoring of the APC generation, it is important to assure that the substrate is converted only by APC in the activation mixture. Therefore, to prohibit thrombin-mediated PCa-5791 substrate conversion, thrombin was inhibited by the addition of hirudin to the activation mixture after 3 h of PC activation, or to the substrate buffer 31 . The optimum hirudin concentration was determined by incubation of 2.5 µg/ml, 12.5 µg/ml, and 20 µg/ml thrombin (corresponding to 68 nM, 341 nM, and 545 nM thrombin, respectively) with increasing concentrations of hirudin. The IC 50 values for the used thrombin concentrations were calculated as 0.17 µM, 1.96 µM, and 2.26 µM hirudin, respectively. In total, the hirudin concentration was set as 5-time higher molar ratio in comparison to thrombin to assure the inhibition of thrombin at the end of the activation process (see Supplementary Fig. S1d online).
In order to separate APC from the other ingredients of the PC activation mixture such as thrombin, hirudin, human albumin, and residual PC, aptamer-based affinity separation was used. To achieve this goal, five previously selected and characterized APC-specific aptamers (HS02-52G, NB1-83, NB1-46, NB2-81, and NB2-57G), were chosen.
All of these aptamers showed binding affinity to APC in the low nanomolar range and high specificity towards APC whereas zymogenic PC and thrombin were not captured using these aptamers 18,26 . To identify the best aptamer for APC capturing and elution, Biolayer Interferometry (BLI) assay was performed by loading the streptavidin-coated (SAX) biosensors with each biotinylated aptamer, followed by APC capturing and dissociation in the binding buffer. The data showed that among all aptamers tested, HS02-52G appeared to be the best capturing aptamer as the highest capturing signal was achieved using this aptamer (see Supplementary Fig. S3 and Table S1 online).  www.nature.com/scientificreports/ The binding forces mediating the aptamer-target interactions are the electrostatic interactions such as hydrogen bonding, electrostatic bonding, the hydrophobic effect, π-π stacking, and van der Waals forces 32 . In addition, it was previously demonstrated that HS02-52G aptamer binds to APC in a Ca 2+ -dependent manner 26 . Therefore, EDTA which was present in the final concentration of 1 mM in activation mixture might interfere with APC capturing by HS02-52G. To avoid this, a buffer-exchange step was added between PC activation and APC capturing steps to remove EDTA.
High salt concentration might either interfere with intermolecular electrostatic interactions between aptamer and target protein or cause structural disruption of aptamer resulting in destabilization of aptamer-target protein bond 33 . Hence, high NaCl concentration was used previously in elution buffers in which detachment of the protein from its specific ligand is required 24,34 . In addition, it was previously demonstrated that HS02-52G aptamer binds to APC in a Ca 2+ -dependent manner 26 therefore, aptamer-mediated captured APC may be eluted using chelating agent, EDTA. To assess the efficiency of different elution strategies, the BLI method was used in which APC was captured by HS02-52G aptamer immobilized on the SAX-biosensors and the dissociation step was performed in the binding buffer containing either 1 M NaCl or 5 mM EDTA.
The obtained data showed that although the calculated association constant was comparable in all three experiments, the dissociation constant (kd) was 9 times higher when the elution buffer 2 containing EDTA was used as dissociation buffer compared to the experiment in which binding buffer was used as elution buffer. This ratio was only 3.4 times higher when elution buffer 1 containing 1 M NaCl was used (Fig. 2). Hence the elution buffer was prepared as 10 mM Tris-HCl, 144 mM NaCl, pH 7.4 containing 5 mM EDTA. This method is applicable only because of the inherent characteristic of HS02-52G and APC binding which is Ca 2+ dependent and is diminished in the presence of high concentration of chelating agents.
A folded aptamer has a relatively compact structure in which the 3' or 5' end might be embedded inside the tertiary structure. In addition, chemical grafting to the 3' or 5' end can alter the tertiary structure of the aptamer which might be necessary for target binding. It is therefore essential to introduce a spacer arm between the aptamer sequence and the grafted functional group to reduce the structural changes and to facilitate the coupling to the sorbent. We previously showed that grafting of small molecules such as biotin with a spacer arm (tetraethylene glycol, TEG) did not interfere with the binding affinity and specificity of the aptamers to APC as the 3'-end biotinylated aptamers, which were immobilized on the streptavidin-coated biosensors for the BLI assay (see Supplementary Fig. S3 online) or coated in the maxisorb plates, easily captured their target molecule, APC 18,26 . In addition, these studies showed that the functional groups such as biotin grafted to the 3' end of aptamer sequence retain their functionality, demonstrating that the 3' end of the aptamer is accessible after tertiary structure formation 26,35,36 . Hence, in the present study, the HS02-52G aptamer was modified on the 3'-end by attachment of first a hexaethylene glycol (HEG) spacer and then a primary amine (-NH 2 ) functional group.
Due to predicaments about how to select the most appropriate grafting to immobilized aptamer on the solidphase carrier, loading of biotinylated oligonucleotides on streptavidin-coated SMBs was investigated previously in our lab 35 . This study showed that although the biotin-streptavidin binding is the strongest non-covalent bond in nature, a femtomole amount of streptavidin was released from the SMB during the first elution step 35 . This not only introduces a source of protein contamination to the downstream processes, but also reduces the efficiency of capturing by successive use of the loaded beads. This problem can be easily removed by grafting aptamer www.nature.com/scientificreports/ molecules to the solid carrier using covalent binding 37 . In this respect, an amide-bond formation strategy was used by grafting of NH 2 -modified aptamer to the COOH-coated magnetic beads.
According to the fact that the oligonucleotide structure does not contain a carboxylic acid group, the onestep protocol for activation and loading of primary amine-modified aptamers on magnetic beads containing carboxylic acid groups on the surface was used. Five hundred microlitres of Dynabeads MyOne Carboxylic Acid beads (MyOne) containing 5 mg beads or 500 µl of Dynabeads M-270 Carboxylic Acid beads (M-270) containing 15 mg beads were mixed with 25 nmol amino-modified HS02-52G aptamer in the presence of 500 mM EDC and incubated overnight. The residual functional groups on the surface of magnetic beads were blocked using 250 mM Tris-HCl blocking buffer. The coupling of aptamer molecules on SMBs was confirmed by flow cytometry experiement in which loaded SMBs were first incubated with SYBR Green (1:10,000 dilution) and then identified by corresponding FCS/SSC-or SYBR Green-positive events (FL1) using a Navios EX flow cytometer (Beckman Coulter, Krefeld, Germany). The data showed in Supplementary Fig. S4 confirmed coupling of HS02-52G or AD02-52 aptamer molecules to the MyOne beads. The coupled beads showed a peak shift after incubation with SYBR Green while non-coupled beads showed overlapping peaks bearing minimum peak displacement.
MyOne beads was provided in a suspension of 10 mg beads/ml, bead density of 7-12e9 beads/ml and bead diameter of 1 µm; therefore, 5 mg of MyOne beads offers ~ 142 cm 2 surface for aptamer binding. On the other hand, M-270 beads was delivered in suspension form having 30 mg beads/ml, bead density of 2e9 beads/ml and bead diameter of 2.8 µM; hence 15 mg of M-270 beads offer 246 cm 2 interacting surface due to their longer diameter and larger spherical shape. Coupling of NH 2 -modified HS02-52G (25 nmol) on MyOne beads resulted in 66.4 µg (4.1 nmol, 16.4% of starting aptamer amount, 820 pmol/mg bead) aptamer immobilization on the beads corresponding to 13.3 µg aptamer/mg of the beads. On the other hand, trying to immobilize the same amount of aptamer on M-270 beads resulted in 54 µg (3.34 nmol, 13.4% of starting aptamer amount, 223 pmol/mg bead) aptamer immobilization on the beads corresponding to 5.38 µg aptamer/mg of the beads (see Supplementary  Table S2 online). This means that, although the M-270 beads offer a larger interactive surface, the yield of aptamer immobilization on their surface is rather lower than MyOne beads. This may show that smaller spherical particles with higher density offer a more accessible interactive surface with less steric hindrance for aptamer coupling.
A low yield of coupling (≤ 20%) was also described previously by Liu et al., where the coupling ratio of aptamer immobilization on CNBr-activated Sepharose beads was only 17% 21 . In addition, Lim et al. described the efficiency of immobilization of His-tag-specific aptamer on aminomethylated polystyrene resin as 510 fmol/mg bead 38 . One possible explanation is that the acidic character of aptamers, due to the ionizable phosphate groups and their negative net charge, likely produce charges repulsion between the aptamer and COOH-modified solid sorbent. This electrostatic repulsion might slow down the aptamer-bead grafting reaction and affect the grafting yield negatively 39 . However, by loading of 6H7 aptamer on BioMag Carboxyl-terminated magnetic beads, Zhu et al. reported an increased grafting efficiency of 157 pmol/mg to 1037 pmol/mg when aptamer concentrations increased from 10 µM to 40 µM, respectively. The loading efficiency was superior to the yield we achieved, but it did not necessarily reflect the yield of capturing of His-tag protein because a 1:1 protein:aptamer molar ratio of capturing was achieved only by using the beads with the lowest surface aptamer density (157 pmol/mg beads) and the protein:aptamer ratio was reduced to 1:5 when using beads with higher aptamer density of 1037 pmol/mg 34 .
The capturing capacity of aptamer-loaded magnetic beads is not only dominated by the aptamer density coupled on the surface of magnetic beads but also by the steric hindrance among neighbored aptamers; thus, the capturing capacity varied from lower amount to maximum 1:1 molar ratio of ligand:target. Therefore, the efficiency of capturing and elution of 10 µg APC was determined using both MyOne and M-270 beads. MyOne beads showed higher efficiency of capturing as in the first elution step a higher amount of APC was released indicating more efficient capturing capacity (70 µg/ml APC in E1 using MyOne compared to 9.5 µg/ml APC concentration in E1 by using M-270 beads) (Fig. 3). Hence, MyOne beads were chosen for further characterization. www.nature.com/scientificreports/ In order to exclude the unspecific binding of APC to the beads, non-loaded SMB or SMB loaded with an antisense sequence of HS02-52G, AD02-52 were introduced to the same capturing and elution system. As shown in Fig. 4, only the application of HS02-52G-loaded beads enabled the isolation of APC from binding buffer, demonstrating the specificity of loaded aptamer for APC capturing. In addition, the release of high APC amount from non-loaded SMB in the first washing step might demonstrate higher unspecific adsorption of APC to nonloaded beads in comparison to loaded beads either with the specific or unspecific aptamer.
To evaluate the capturing capacity of the selected MyOne beads, increasing amounts of APC were spiked to 500 µl of binding buffer and the amount of APC released during consecutive washing steps or eluted in elution steps was monitored. Data showed that the loss of APC observed during washing steps increased with the amount of APC added to the starting solution. This might be explained by the overload of loosely-bound APC molecules on the beads (Table 1, Fig. 5) or by trapping the binding buffer which contained a high concentration of non-bound APC, within the free spaces among the beads. The amount of captured APC increased with the initial amount of APC introduced to the beads. However, the yield of capturing in relation to the initial APC amount decreased drastically, showing ineffectiveness of subjecting high initial APC concentrations to capturing procedure.
Finally, to confirm that the MyOne beads loaded with HS02-52G are able not only to capture APC from a purified system containing only APC, but also from the activation mixture containing generated APC, residual amounts of PC as well as human thrombin and hirudin, different amount of PC (5 µg to 40 µg Ceprotin) were subjected first to activation process (see section PC activation) and then to the optimized capture and elution process. The obtained data showed that the generated APC was captured easily from the activation mixture. Increasing the initial amount of PC from 5 to 40 µg was associated with a slight increase of the captured amount from 4.6 to 7.3 µg, however higher loss of APC during washing steps was observed as well. Increasing the initial amount of PC to more than 20 µg did not increase more the captured yield indicating that the maximum loading capacity of the beads was reached (Fig. 6a). The high purity of captured APC from the activation mixture which contained 10 µg PC was confirmed by loading the activation mixture, the same as products of washing and elution steps, on SDS-PAGE gel followed by silver staining (Fig. 6b, Figure S5 and S6). According to the fact that APC and PC have close molecular weights and discrimination of their corresponding bands on gel might be difficult, samples of the supernatant before and after capturing, the same as first elution fraction resulted from 5 µg, 10 µg, 20 µg, and 40 µg PC activation were subjected to an enzyme-linked fluorescence assay (VIDAS Protein C) which is only reactive to PC and is not interfered by APC. Figure 4. Specificity of aptamer-loaded beads. Capturing and elution results of APC using MyOne beads coupled to APC-specific HS02-52G aptamer, negative control sequence AD02-52, or non-coupled MyOne beads. The amount of APC which was captured from binding buffer containing 10 µg APC in 500 µl of binding buffer (5 µg/ml) and released during consecutive steps of washing (W1 to W3) or elution (E1 to E3) was quantified by an activity assay using an APC standard curve. Data are shown as mean ± SD of three measurements. www.nature.com/scientificreports/ The data showed in Table 2 confirmed that although PC activation yield reduced when higher PC amount was subjected to the activation process, but this increasing residual amount of PC didn't lead to higher co-purification of PC with APC which confirms the specificity of HS02-52G aptamer in capturing APC.

Figure 5.
Capturing and elution of APC using HS02-52G coupled MyOne beads. Different amounts of APC were added to 500 µl binding buffer followed by APC capturing with MyOne beads coupled to HS02-52G aptamer and elution using elution buffer containing 5 mM EDTA. The eluted APC in consecutive elution steps (E1 to E3) was quantified by an activity assay using an APC standard curve. Data are shown as mean ± SD of three measurements.

Conclusions
Oligonucleotide-based affinity purification strategies for selective extraction of a target protein from complex matrices are an emerging field. In this study, we have successfully optimized PC activation and developed and validated an aptamer-facilitated protein purification process using superparamagnetic beads. The gentle elution of aptamer-bound active enzyme using EDTA circumvents protein degradation which might occur using other invasive elution conditions. This method can be utilized for rapid PC activation and isolation of highly purified APC. The purified APC might be used directly for downstream processes in which high concentrations of pure and active APC is needed. Protein C activation. Ceprotin (Baxter AG) lyophilized powder containing 500 IU of PC was dissolved in 2.5 mL water for injection to reach 250 IU/ml concentration. Berichrom Protein C Kit (Siemens Healthcare Diagnostics) was used to determine the PC concentration in comparison to PC standard solutions prepared from commercially available plasma-derived PC (pPC, HTI). To achieve the optimum generation of APC, as the first step, thrombin concentration was optimized. Briefly, Ceprotin (62.5 μg/ml equal to 1 µM) was incubated with different concentrations of human α-thrombin (0.78 µg/ml to 12.5 µg/ml) in activation buffer. After 1 h of incubation, samples were subjected to the APC activity assay (see the APC activity assay section). To avoid the unspecific cleavage of APC-specific substrate by thrombin, residual activity of thrombin was inhibited by addition of recombinant hirudin (Refludan, Bayer Health Care). In order to identify the optimum hirudin concentration, 2.5, 12.5, and 20 µg/ml thrombin was incubated with increasing concentrations of hirudin (14 nM to 14 µM) in activation buffer. After incubation for 30 min, 50 µl of the mixture were transferred to the wells of a black F16 Fluoronunc module (ThermoFisher Scientific, Nunc) containing 50 µl of 10 mM fluorogenic thrombin substrate (Boc-Asp(OBzl)-Pro-Arg-AMC) and thrombin catalyzed substrate hydrolysis was monitored at λ ex of 360 nm and λ em of 460 nm using a Synergy 2 microplate reader (BioTek, Bad Friedrichshall, Germany).

Methods
The optimum incubation time of PC and thrombin was determined by incubation of 62.5 μg/ml PC with 3.12 µg/ml thrombin for up to 24 h and the APC activity was monitored using the APC activity assay described below.
In order to subject the generated APC to further downstream experiments, a buffer exchange step was included. Briefly the activation mixture passed through the centrifugal filter units (Amicon Ultra-2 Ultracell-NMWL 10 kDa, Merck, Darmstadt, Germany) and the activation buffer was exchanged with binding buffer.
APC activity assay and PC quantification assay. The activity of APC was assessed using an APCspecific fluorogenic peptide substrate (PCa-5791). Briefly, 50 µl of a 1:50 dilution of the sample in assay buffer were transferred to the wells of white F8 FluoroNunc modules [Thermo Fisher Scientific (Nunc)]. Subsequently, 50 µl of 300 µM PCa-5791 were added and substrate hydrolysis rates were recorded at λ ex of 360 nm and λ em of 460 nm using the Synergy 2 microplate reader. The same activity assay was used to determine the stability of APC in binding buffer. A stock solution containing 160 nM APC was kept on ice and aliquots were removed www.nature.com/scientificreports/ and incubated at 37 °C at selected time points. At the end of the incubation time 50 µl of a 1:10 dilution of each sample were added to the wells of white F8 FluoroNunc modules followed by addition of 50 µl of the 300 µM PCa-5791. The APC activity was compared to the activity of the APC stock solution which was kept for the maximum incubation time on ice and presented as the activity in percentage. PC quantification was performed in an enzyme-linked fluorescent assay (ELFA) using VIDAS Protein C assay. Briefly, samples were recalcified with CaCl 2 and diluted 1:3 to 1:10 in D-PBS buffer containing 1 mM MgCl 2 and 1 mM CaCl 2 . Then diluted samples were incubated in the SPR cuvettes of the system which were coated with PC-specific antibody. Further, captured PC was quantified after subsequent addition of a secondary antibody linked to alkaline phosphatase and 4-methyl umbelliferyl phosphate (4-MUP) substrate.
Biolayer interferometry analysis. Biolayer Interferometry (BLI) technology was applied 40 first to determine the appropriate aptamer to be loaded on beads and then to evaluate the dissociation kinetics of APC from aptamer molecules loaded on a High-Precision Streptavidin (SAX) Biosensor (Pall Life Sciences) when the biosensor immersed in different dissociation buffers (dissociation buffer 1-3). A BLItz system (Pall Life Sciences, Dreieich, Germany) and the Blitz 1.2.1.5 software package were used for corresponding analysis.
To identify the best aptamer for capturing, the SAX biosensor was hydrated for 10 min followed by loading of 3´-biotinylated HS02-52G, NB1-83, NB1-46, NB2-81, and NB2-57G aptamers (using a 500 nM solution of the aptamer in binding buffer) for 2 min. The loaded biosensor was equilibrated for 30 s in binding buffer followed by immersing in the same buffer containing increasing concentrations of APC to calculate the association rate constant (ka [kon]). Thereafter, for determination of dissociation rate constants (kd [koff]), the biosensor was lowered into a tube containing 500 µL of the same binding buffer. The K D was calculated out of ka and kd of using different concentrations of APC.
To determine the optimum dissociation buffer, the SAX biosensor was loaded with the aptamer and was used for APC capturing out of binding buffer containing 500 nM APC. Then, the biosensor was lowered into a tube containing 500 µL of either binding buffer or each dissociation buffer 1 or 2 and the dissociation kinetic was studied. All measurements were performed at a shaking speed of 2,200 rpm.
Aptamer loading on carboxylic acid magnetic beads. Five milligrams of Dynabeads MyOne carboxylic acid beads (MyOne) or 15 mg of Dynabeads M-270 carboxylic acid beads (M-270) were washed using MES buffer followed by resuspension of the beads in 300 µl of the same buffer. HS02-52G aptamer (25 nmol) or AD02-52 having HEG-Amino at the 3´end was diluted in 150 µl MES buffer and mixed with 150 µl 1 M EDC in MES buffer. The aptamer-EDC mixture was subsequently added to the magnetic beads and mixed by vortexing for 10 s. The first sub-sampling was performed immediately after mixing, and then the mixture was mixed thoroughly overnight followed by the second sub-sampling. The loaded beads were washed 3 × using Tris-Tween20 (TT) buffer and resuspended in 500 µl Tris-EDTA (TE) buffer. The loading yield was calculated out of sub-samples at the beginning and end point of the loading process.
Assessment of loading of aptamers on superparamagnetic beads. The SMBs which were coupled to either HS02-52G aptamer or non-binder AD02-52 aptamer, the same as non-loaded MyOne beads were diluted 1:100 in binding buffer and incubated for 30 min with SYBR Green (1:10,000 dilution) under rigorous shaking. Then the beads were washed and diluted 1:50 in binding buffer. Subsequently, the samples were transferred to 12 × 75 mm polypropylene tubes (Beckman Coulter) and subjected to a Navios EX flow cytometer (Beckman Coulter, Krefeld, Germany) for assay readout. For each reaction, 10,000 bead events were recorded (FSC/SSC or FL1) and associated binding of SYBR Green to the beads was identified by corresponding FCS/ SSC-and positive events at FL1.

Assessment of binding and release of captured molecules to/from loaded magnetic beads.
To evaluate the capturing capacity of loaded beads, different amounts of APC (2.5 µg, 5 µg, 10 µg and 20 µg) were dissolved in 500 µl binding buffer and incubated with loaded beads under a shaking speed of 1,100 rpm at RT. After 30 min of incubation, beads were washed 3 × using washing buffer. Captured APC was eluted from the beads by incubation of beads with 50 µl elution buffer for three times, each 5 min. The starting solution, supernatant of the beads after capturing and, three washing and elution fractions were subjected to the APC activity assay. In order to evaluate the unspecific binding of APC to non-loaded beads as well as beads loaded with non-specific oligonucleotides, the capturing and releasing of 10 µg APC in binding buffer to/from beads in both conditions were tested. The optimum adjustment was applied for capturing of generated APC out of the activation mixture using Ceprotin.
Purity evaluation on SDS-PAGE gel. Yield and quality of captured APC either from binding buffer or from activation mixture were tested on gradient SDS-PAGE gel followed by silver staining.
Briefly, sample containing approximately 2 µg protein in Tris-HCl buffer was mixed with the same volume of 2 × Laemmli loading buffer containing 5% α-mercaptoethanol and heated for 8 min followed by cooling down on ice. Subsequently, samples were loaded on a gradient SDS-PAGE gel (Bio-rad) and subjected to electrophoresis. The bands were visualized by silver staining following the manufacturer's instructions.

Data availability
The data that supports the generation of oligonucleotides used in this study are openly available in Zenodo.org at https:// zenodo. org/ record/ 58788 73#. Yege9 lVKiJA. The molecular evolutionary relations, ligand tracing and tracking of defined sequences through successive selection rounds were performed using COMPAS software www.nature.com/scientificreports/ (AptalT GbmH, Planneg, Germany) 41,42 . The motif similarity and finding of consensus sequence was done using multiple sequence alignment program Clustal Omega from EMBL-EBI (https:// www. ebi. ac. uk/ Tools/ msa/ clust alo/; December 2016) 43 .