Oligonucleotide Hybridization Combined with Competitive Antibody

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Oligonucleotide Hybridization Combined with Competitive Antibody Binding for the Truncation of a High-Affinity Aptamer Cong Quang Vu, Pichayanoot Rotkrua, Yuthana Tantirungrotechai, and Boonchoy Soontornworajit ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.6b00163 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 22, 2017

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Oligonucleotide Hybridization Combined with Competitive Antibody Binding for the Truncation of a High-Affinity Aptamer Cong Quang Vu†, Pichayanoot Rotkrua‡, Yuthana Tantirungrotechai†, and Boonchoy Soontornworajit†,* †

Division of Chemistry, Faculty of Science and Technology, Thammasat University,

Pathumthani 12120, Thailand ‡

Division of Biochemistry, Department of Preclinical Science, Faculty of Medicine, Thammasat

University, Pathumthani 12120, Thailand

ABSTRACT:

Truncation can enhance the affinity of aptamers for their targets by limiting nonessential segments and therefore limiting the molecular degrees of freedom that must be overcome in the binding process. This study demonstrated a truncation protocol relying on competitive antibody binding and the hybridization of complementary oligonucleotides, using platelet derived growth factor BB (PDGF-BB) as the model target. Based on immunoassay results, an initial long aptamer was truncated to a number of sequences with lengths of 36-40 nucleotides (nt). These sequences showed apparent KD values in the picomolar range, with the best case being a 36-nt


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truncated aptamer with a 150-fold increase in affinity over the full-length aptamer. The observed binding energies correlated well with relative energies calculated by molecular dynamics simulations. The effect of the truncated aptamer on PDGF-BB-stimulated fibroblasts was found to be equivalent to that of the full-length aptamer. Keywords: aptamers, PDGF-BB, ELISA, free energy calculation, fibroblast proliferation INTRODUCTION Platelet-derived growth factor BB (PDGF-BB) is a potent mitogen that stimulates cell division in many cell types through its receptor, PDGFR-β.1 PDGF-BB and PDGFR-β are part of a ligandtyrosine kinase receptor system that plays a significant role in angiogenesis, contributing to blood vessel formation.2 Recombinant PDGF-BB has been approved by the FDA for the treatment of chronic wounds.3 The regulation of PDGF-BB bioactivity by adding binding agents could provide new approaches to both treating cancer and speeding wound healing. Recently, nucleic acid aptamers have been developed and used as recognition elements in a number of biomedical products and therapies, such as biosensors,4-5 drug delivery systems, active therapeutics,6-7 and biomaterials.8-9,10-11 Nucleic acid aptamers are single-stranded oligonucleotides selected or evolved from combinatorial libraries for binding to a wide variety of targets by the general process of systematic evolution of ligands by exponential enrichment (SELEX).12 Aptamers have many desirable

properties,

including

chemical

tailorability

to

enhance

stability,13,14

low

immunogenicity or toxicity,15 routine or commercial synthesis,13 and general applicability to limitless numbers and types of target.16 An aptamer against PDGF-BB was identified and characterized by Green et al., and was further truncated by partial fragmentation to yield a sequence with an apparent binding affinity of 100 pM.17


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It is the process of truncation that we focus on here. While numerous approaches to aptamer selection have been developed,18 these procedures usually produce sequences of 60-80 nucleotides containing fixed and randomized regions. The fixed region used for primer binding in the amplification step may reduce the binding capability of the aptamer sequence, due to selfhybridization.19 Moreover, the practicality and cost of oligonucleotide synthesis are highly dependent on sequence length.19 The identification of essential shorter sequences in selected aptamers to achieve effective truncated species in a simple, rapid, and cost-effective manner would significantly enhance aptamer development. Aptamer truncation is usually done by deleting the primer region in the SELEX process,20 followed by analysis of binding of the truncated vs. original aptamers21 and comparison of predicted secondary structures using tools such as M-fold software.22 Binding interactions between aptamers and their targets can be interrupted by hybridization between the aptamers and their complementary oligonucleotides (CO),23 thereby releasing the targets from aptamer complexation.24-25 This molecular interruption, coupled with a cell labeling technique, has been used to successfully identify aptamer sequences.5 We adapted this protocol using aptamer-CO hybridization for the aptamer truncation, starting with the parent PDGF-BB binding aptamer of Green and coworkers.


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EXPERIMENTAL PROCEDURES

Scheme 1. Schematic presentation of the concept. (A) A full-length aptamer hybridizes with CO. (B) Interaction between hybridized aptamer and PDGF-BB causes reduction of immunoassay signal. Identification of PDGF-BB aptamer binding region To determine the binding region, a competition ELISA was conducted to compare PDGF-BB, PDGF-BB antibody, and the original PDGF-BB aptamer (designated FullApt) (Scheme 1). Each well of a microtiter plate was coated with PDGF-BB antibodies, washed, and further blocked by BSA. FullApt was incubated with several complementary oligonucleotide sequences at designated concentrations, allowing hybridization at either the 5ʹ or 3ʹ end of the aptamer


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sequence. The hybridized aptamers were then mixed with PDGF-BB solution, and the mixtures transferred to the antibody-coated wells. After incubation, the wells were washed and the ELISA protocol completed by the sequential addition of human PDGF-BB biotinylated antibody, streptavidin-HRP, and development reagents (see Supporting Information (SI) for details). Examination of binding functionality After identifying the binding region of the PDGF-BB aptamer, the aptamer was truncated. The binding functionality of the truncated sequences was examined by ELISA, using the competitive binding assay. Experiments were performed as described in the previous section, using the truncated aptamers in place of the hybridized aptamers. To evaluate the binding affinity of the truncated aptamers, the apparent dissociation constants (KD,app) of each sequence were estimated from a one binding site model (equation 1).

(1 − ) = (1 − ) ×

[] [] + ,

(1)

where [apt] is the aptamer concentration, (1-OD)cplx represents a signal response from the aptamer-PDGF-BB complex, and (1-OD)max represents a signal response from the total amount of PDGF-BB added to the assay.26 Examination of binding specificity The binding specificity of the truncated aptamer was tested using a competitive assay with PDGF-AA and VEGF as the target molecules. The experimental procedures were the same as those used for investigating binding functionality, but the PDGF-BB was replaced by PDGF-AA and VEGF.


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Surface plasmon resonance PDGF-BB was immobilized onto a carboxylic overhung surface of a sensor chip using EDC/NHS as the coupling agent. Solutions containing FullApt, 36aApt, and 36bApt were then flowed over the sensor chip to study the binding interaction. The dissociation constant (KD) was determined by kinetic analysis.27 (Experimental details are elaborated in the SI). Molecular dynamics simulation Preparation of initial structures Aptamer models were generated and optimized using software available on webservers.28-31 The crystal structure of PDGF-BB was taken from the PDB database (PDB Code: 3MJG).32 To reduce the cost, the calculation was performed on only one chain of the homodimeric PDGF-BB. A molecular dynamics simulation was then carried out separately for the PDGF-BB and aptamer models in water, using the GROMACS 5.0 package.33 The simulation conditions and constraints are summarized in the SI. After equilibration, the structures of the PDGF-BB and aptamers were extracted for use as the initial structures in the molecular dynamics simulation of the aptamerPDGF-BB complexes. The binding sites of the PDGF-BB and aptamer models were pre-defined using AUTODOCK4.34 Simulation procedure The aptamer-PDGF-BB complexes were placed in the center of a dodecahedral box containing water molecules, and sodium and chloride ions, at 300 K and 1 bar. The VMD package was used for trajectory and structural visualization.35 Next, the atomic coordinates and energy-related parameters were saved for use in the free energy calculations, using the molecular mechanics


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Poisson-Boltzmann/Generalized-Bonn surface area (MMPB(GB)/SA) model. (Details of the MD calculation are given in the SI.) Free energy calculations The experimental free energy (∆Gexp) was evaluated using equation (2), where R is the gas constant, T was set to 298 K, and KD,app was set to correspond to the immunoassay results. ∆ = −,

(2)

In vitro study of cell proliferation Cell culture Fibroblasts were cultured and maintained in DMEM complete media at 37 ℃ and 5% CO2 in an incubator. The complete medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were washed with phosphate buffer saline (PBS) and their culture media were changed every 2-3 days. Fibroblast proliferation Fibroblasts with an approximate number of 2000 cells were seeded into each well of a 96-well plate and incubated for 24 h, then treated with either PDGF-BB alone or aptamer PDGF-BB complex. After 48 h, a solution containing tetrazolium compound (MTS) was added and the mixture was incubated for one hour. The optical density (OD) at 490 nm was measured using a microplate reader (BioTek) to determine the proliferation of the fibroblasts. To evaluate the effect of the aptamers on these PDGF-BB-stimulated fibroblasts, the OD of each sample was normalized to the OD of the sample without growth factor treatment, and the percentage stimulation was calculated using the following equation:


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'

!"#$"%" (%) = ' × 100%. (

(3)

Here, OD is the optical density of each treated fibroblast sample, and ODc is the optical density of the corresponding fibroblast sample without PDGF-BB treatment RESULTS AND DISCUSSION Identification of PDGF-BB aptamer binding region As reported in the literature, the full-length PDGF-BB contains 86 oligonucleotides.17 Certain nucleotides in the aptamer sequence fold into a specific structure in response to the binding functionality of the aptamer. We developed a protocol based on hybridization and competitive binding assay to identify the most likely locations of these nucleotides. The complementary oligonucleotides used here (15, 17, and 31 nt in length, denoted as in “5CO15” for the sequence complementary to the first 15 nucleotides at the 5’-end of FullApt) were shown in preliminary experiments not to bind to the growth factor protein (Figure 1A), nor to disrupt protein-antibody binding (Figure S1). The sandwich ELISA protocol for competitive binding analysis of FullApt vs. antibody for PDGF-BB was validated as shown in Figure 1B, in which it can be seen that the ELISA signal was diminished by aptamer in a dose-dependent fashion. No further statistically significant increase was found when the aptamer concentration was increased beyond 60 pM, as the PDGF-BB became fully blocked by the aptamer. In consequence, the signals were comparable to those from the blank sample. These experiments established the standard assay condition of 1:3:100 for the molar ratio of PDGF-BB:FullApt:CO, at a PDGF-BB concentration of 20 pM.


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Figure 1. Optimization of hybridization condition. (A) 20 pM of PDGF-BB was incubated with 2000 pM (1:100 ratio) of indicated CO prior to sandwich ELISA analysis. (B) Effect of FullApt concentration on competitive binding (sandwich ELISA) assay. Control and blank represent the presence or absence of PDGF-BB at 20 pM, respectively. *P < 0.05 against FullApt (100 pM) (two-tailed Student’s t-test). Error bar indicates SD (n=6). To identify the binding region of the PDGF-BB aptamer, the hybridized aptamer was prepared by incubating FullApt with the designated COs for 30 minutes. The hybridized aptamers were then treated with PDGF-BB, and antibody binding was tested using ELISA. For 5′-end hybridization, aptamer binding remained unaffected until the CO length exceeded 21 nt (Figure 2A). This result suggested that approximately 23 nucleotides at the 5’-end of FullApt were unnecessary for target binding. Similarly, approximately 27 nucleotides at the 3’-end of the full aptamer could be complexed without sacrificing binding affinity (Figure 2B). Initially, 5CO31 did not completely abrogate aptamer binding, but this was found to be due to insufficient formation of hybridized duplexes. Upon increasing the CO:FullApt molar ratio to 200:1, full inhibition of binding to PDGF-BB was observed (Figure S2). The alteration of aptamer binding functionality by CO hybridization suggested that the increase of the hybridization length over the essential nucleotide region caused changes in aptamer


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folding, leading to the loss of aptamer binding functionality. Three FullApt regions were distinguished, based on the effectiveness of binding interruption by CO: no loss, moderate loss, and complete loss. The corresponding hybridization lengths were 21, 23, and 25 nt for the 5’end and 25, 27, and 29 nt for the 3ʹ-end. The aptamer was therefore truncated, corresponding to these regions. The truncated sequences were named 36aApt, 36bApt, 36cApt, 38aApt, 38bApt, and 40Apt (Table S2). Their binding affinities were then further examined and compared with that of the FullApt sequence.

Figure 2. Investigation of binding capability of hybridized aptamers. Effect of hybridization length on binding functionality of PDGF-BB aptamer at 5ʹ-end (A) and 3’-end regions (B). The aptamer allowed formation of hybridization at both regions. Lines to the left of each panel illustrate hybridization at 5ʹ-end and 3ʹ-end. *P < 0.05 against FullApt (two-tailed Student’s ttest). Control and blank denote the presence or absence of PDGF-BB at 20 pM, respectively. Error bar indicates SD (n=6).


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Binding affinity of truncated aptamers Secondary structural prediction of the truncated aptamers (Figure 3A) showed the folding regions of 40Apt, 38aApt, 38bApt, and 36aApt to be comparable with those of FullApt, whereas 36bApt and 36cApt showed a change in structural conformation. Competitive binding assays were performed by treating PDGF-BB with the truncated sequences before allowing the growth factor to interact with its antibody (Figure 3B), and showed dose-response inhibition of antibody binding for the predicted sequences (FullApt, 40Apt, 38aApt, 38bApt, and 36aApt) but not for those predicted to have an altered secondary structure (36bApt and 36cApt).

Figure 3. Binding affinity of FullApt and truncated aptamers. (A) Secondary structure of FullApt and all truncated sequences. (B) Competitive binding assay. (C) Dissociation constants (KD,app). ND: KD,app could not be determined. Error bars indicate SD (n=6).


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Interestingly, truncation appeared by this test to give enhanced affinity relative to FullApt in two cases: 38aApt and 36aApt. This was most clearly seen at 1 pM. The competitive binding curves were used to derive the apparent dissociation constants (KD,app) (Figure 3C). These showed approximately 60- and 150-fold increases in sub-picomolar affinity over FullApt. Enhancement of aptamer binding affinity by truncation has been previously reported. For example, a full length and truncated thrombin aptamer exhibited KD values of 278 and 99 nM, respectively.18

The

binding affinity of a truncated aptamer targeting acetylcholinesterase was reported to show a 12fold increase over the original sequence.36 Examination of binding specificity Key characteristics of aptamers are the affinity and specificity of binding. The immunoassay results demonstrated an enhancement of binding affinity of PDGF-BB aptamers after truncation. To confirm the binding specificity of the truncated aptamers, FullApt, 36aApt, and 36bApt (representing the original, the highest binding, and non-binding sequences, respectively) were tested for competitive binding with PDGF-AA and VEGF, using ELISA. No blocking of antibody binding to these proteins was found (Figure 4), confirming the retention of binding specificity to PDGF-BB. In addition, both FullApt and 36aApt were able to bind to PDGF-BB even in the presence of BSA (Figure S3).


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Figure 4. Examination of the aptamer specificity by competitive binding assay. The truncated aptamer was treated with (A) PDGF-AA and (B) VEGF. 20 pM of the protein molecules was incubated with the aptamers at the designated concentration. Error bars indicate SD (n=6). Surface plasmon resonance SPR analysis of the binding of the three representative aptamers (Figure 5) showed no interaction of immobilized PDGF-BB with 36bApt, but strong interactions with FullApt and 36aApt. The apparent KD value of 36aApt represented an approximately 2.5-fold increase over FullApt, suggesting that FullApt and 36aApt bound to PDGF-BB within the same order of magnitude. The apparent dissociation constants (KD) determined by immunoassay and SPR indicated very strong binding interaction due to avidity effects. The avidity or chelating effects were more pronounced in the immunoassay, because PDGF-BB is a homodimeric molecule containing two identical aptamer binding sites. It has been reported that PDGF-BB is able to bind to two molecules of the MOR8457 antibody, which could enhance the binding interaction.37 In addition, the binding sites of PDGF-BB in the binding media had more opportunity to interact with the


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aptamer than in the SPR experiment, due to the molecular immobilization. The avidity effects played a role in increasing the binding capability via multimerization of the binding ligands, including antibodies and aptamers.38-39

Figure 5 SPR sensorgrams of the aptamer-PDGF-BB interaction. The vertical dashed line indicates the time point at which the aptamer solution was replaced by the running buffer. Molecular dynamics simulation To verify the immunoassay results, a molecular dynamics simulation, a useful tool for studying the interaction of biomolecules, was carried out for the aptamer-PDGF-BB complexes. FullApt, 40Apt, 38aApt, 38bApt, 36aApt, and 36bApt were chosen as the aptamer models. The initial binding configurations of these complexes were predicted using AUTODOCK4 (Figure 6A). Using these initial configurations, the molecular dynamics simulation was continued until equilibrium was reached. Then, the trajectory of the complex was sampled for another 100 ns. Throughout the process, the energy of the system (Figure S4) and the root mean square deviation (RMSD) (Figure 6B) were monitored, to confirm the stability of the simulation. The


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RMSD values of the free PDGF-BB and aptamers in complexes were in the range 3-7 Å. This suggested some flexibility in the complex configuration. However, 36bApt exhibited the greatest fluctuation in RMSD values, because of weak interaction.

Figure 6. Root mean square deviation (RMSD) of aptamer-PDGF-BB complexes. (A) Initial binding conformations of the complexes predicted by AUTODOCK4. Pink represents the fixed region. (B) RMSD of the complexes as a function of time. To understand the change in binding affinity of the studied complexes, the binding affinity of the truncated aptamers measured by immunoassay and MD were compared and correlated. For the MD results, the binding free energies were extracted from the last 50 ns of the MD trajectory with 1000 snapshots, and were calculated using an MMPB(GB)/SA model. The calculated binding energies (∆GPB/∆GGB) are listed in Table 1.


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Table 1. Calculated binding energies of FullApt and truncated aptamers. Entries

KD,app (pM)

∆Gexp (kcal/mol) ∆GPB (kcal/mol) ∆GGB (kcal/mol)

FullApt

5.33 ± 2.36

-15.37

-44.28 ± 14.99

-72.09 ± 10.36

40Apt

5.92 ± 1.13

-15.31

-42.14 ± 19.15

-68.19 ± 13.29

38aApt

0.094 ± 0.008

-17.76

-54.05 ± 12.38

-90.13 ± 10.83

38bApt

7.03 ± 1.28

-15.21

-42.82 ± 12.67

-68.18 ± 12.15

36aApt

0.036 ± 0.012

-18.33

-63.18 ± 17.56

-92.91 ± 11.08

36bApt

-

-

-26.70 ± 18.16

-42.35 ± 18.88

We do not expect this implicit model to yield quantitatively accurate free energies, but we believe that it can accurately describe the change in free energies of the target complexes. 36aApt exhibited a binding free energy of -63.18 ± 17.56 (PB) and -92.91 ± 11.08 (GB) kcal/mol. 38aApt had a binding free energy of -54.05 ± 12.38 (PB) and -90.13 ± 10.83 (GB) kcal/mol, lower (greater in magnitude) than that of FullApt. The ∆GPB/GB of 40Apt and 38bApt were higher than that of FullApt, with 36bApt having the highest value (lowest in magnitude), indicating the weakest binding interaction. The MD results suggested that 36aApt and 38aApt were more inclined to bind with PDGF-BB than the other sequences. The correlation between the binding energies calculated from immunoassay results (∆Gexp) and those from MD simulation is presented in Figure 7. Interestingly, all ∆Gexp showed good correlation with the calculated ∆G, with R2 values of 0.9435 for MMPB/SA, and 0.9868 for MMGB/SA. We are therefore confident that the simulation described the aptamer-protein binding reasonably accurately. In addition, the MD results supported the notion that both essential and non-essential nucleotides play a role in aptamer binding.


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-100

Calculated ∆G (kcal/mol)

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-90 -80 -70

GBSA PBSA

-60 -50 -40 -30 -15.00

-16.00

-17.00

-18.00

-19.00

Experimental ∆G (kcal/mol) Figure 7. Correlation between binding energy calculated from immunoassay and from MMPB/SA (R2 = 0.9435) and MMGB/SA (R2 = 0.9868) models. Effect of the truncated aptamers on PDGF-BB stimulated fibroblasts To confirm the binding functionality of the truncated aptamers in an in vitro cell culture model, the proliferation of PDGF-BB-stimulated fibroblasts was investigated in the absence or presence of FullApt, 36aApt, and the nonbinding 36bApt. When fibroblasts were treated with PDGF-BB, the stimulated fibroblast proliferation reached approximately 140% (Figure 8) which was similar to the levels reported in previous works.40-41 Meanwhile, aptamer-PDGF-BB complexes were prepared using a molar ratio of 10 between the aptamer and the growth factor, and their effect on stimulation of fibroblast proliferation was tested. When the fibroblasts were treated with the complexes of PDGF-BB and FullApt, 36aApt, and 36bApt, stimulated proliferations of 104, 104, and 120% were observed (Figure 8). These results demonstrated both stimulation of fibroblast proliferation by the PDGF-BB and inhibition of PDGF-BB functionality by the aptamers. The inhibition indicated a binding interaction between the PDGF-BB and aptamers. Additionally, to verify the correlation between the inhibition of PDGF-BB functionality and the binding affinity


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of the aptamers, the aptamer-PDGF-BB complexes were prepared at different molar ratios and tested using fibroblasts. 36aApt inhibited the stimulated proliferation to 104%, 104%, and 106% for complexes with molar ratios of 1:10, 1:5, and 1:1, respectively. However, FullApt and 36bApt gradually reduced their inhibition of PDGF-BB functionality as their amount in the aptamer-complex formation was reduced (Figure 8). The in vitro cell culture study clearly showed binding affinity to run in the following order, from highest to lowest: 36aApt, FullApt, 36bApt. Moreover, 36aApt exhibited dose-independent suppression of PDGF-BB, implying that an off-target effect arose from interactions with unintended targets. The combination of on-target and off-target activity has been shown to result in highly effective inhibition of PDGF-BB by 36aApt in cell experiments.42 The off-target effect also contributed to the dose-dependent supersession exhibited by 36bApt. These non-specific binding events may be due to the high concentration of the tested aptamers in the cell experiments, even though their molar ratios to PDGF-BB were consistent in the immunoassay and in vitro cell test. Overall, the cell results agreed with both the immunoassay and MD simulation, as reported above.


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Percentage (%)


160 150 140 130 120 110 100 90 80 70 60 50

** **

**

** **

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** FullApt 36aApt 36bApt

Control

PDGF-BB 1:10 PDGF-BB:Apt

1:5

1:1

Figure 8. Inhibitory effect of truncated aptamers on PDGF-BB-stimulated fibroblasts. Measurements were taken after 48 h of treatment. Control and PDGF-BB denote the absence and presence of PDGF-BB at 40 ng/mL. **P < 0.01 against PDGF-BB (two-tailed Student’s t-test). Error bar indicates SD (n=6). We believe that truncation by hybridization and immunoassay offers the following advantages. The hybridization and immunoassay reagent does not require the use of special treatments, or special permission to use. The CO used for hybridization is commercially available, and the plate reader used for the immunoassay is standard in most major laboratories. The truncation protocol presented in this work is therefore more practical than truncation based on the conventional method. Because the separation between binding and non-binding sequences relies on the specific interactions in the immunoassay, it is more effective in identifying the binding sequences. In addition, COs are conventionally more robust than the enzymes used in the conventional method, and their structural characteristics are therefore reversible in a number of conditions, extending their storage life. CO hybridization is a by far preferable strategy, although truncation could be accomplished by directly synthesizing either primer-deleted sequences or


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any possible truncated sequence, then testing their binding functionality. As CO hybridization results in a shorter truncated sequence, it can be used to identify the essential nucleotides in a randomized region of the studied aptamer. It is also more efficient and more cost effective to synthesize the short COs for hybridization than to synthesize aptamer by randomly removing nucleotides. The results produced by this protocol can be verified using computer simulations, biosensor techniques, or cell assays. However, its success depends on the aptamers and antibodies binding to their corresponding targets at or close to the same binding sites. A further requirement is that the structural conformation of the target changes significantly after binding to an aptamer, and an antibody is not able to identify such a change. CONCLUSIONS PDGF-BB aptamer was successfully truncated using hybridization and immunoassay. The truncation strategy demonstrated that both the nucleotides in the primer sites and those in randomized regions played key roles in aptamer binding, and the shorter aptamer sequences were shown to exhibit a higher affinity than the full-length aptamer. The binding interaction of the truncated sequences was confirmed using SPR and MD simulation. The binding free energy from MD correlated reasonably well with that from the immunoassay. An in vitro cell study further confirmed the binding affinity of the truncated aptamers. Overall, the reliability of the truncation protocol was demonstrated. This protocol shows promise as a standard approach to truncation of a number of aptamer sequences, and could provide a better understanding of the interaction between aptamers and their targets. ASSOCIATED CONTENT


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Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI. Experimental procedures: Figures S1, S2, S3, and S4. Tables S1, S2 give oligonucleotide sequences. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Present Addresses †Division of Chemistry, Faculty of Science and Technology, Thammasat University, Pathumthani 12120, Thailand Author Contributions The manuscript was written through contributions of all authors. Funding Sources This work was granted by Research Grant for New Researcher from Thailand Research Fund and Thammasat University (TRG5880201). An Additional grant was supported by Thammasat University in a fiscal year 2555, BE. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS The authors gratefully acknowledge the partial support provided by Central Scientific Instrument Center (CSIC), Faculty of Science and Technology, Thammasat University. Cong Quang Vu was financially supported by Asean Economics Community (AEC) scholarship from Thammasat University. This work was funded by Research Grant for New Researcher from Thailand Research Fund and Thammasat University (TRG5880201) and the general grant from Thammasat University in a fiscal year 2555, BE. The authors also thank Ms. Suvaraporn Sae-lim for technical support in cell culture, and Mr. John Winward for comments on the manuscript. In addition, the authors would like to express their gratitude to Dr. Toemsak Srikhirin and Ms. Wanida Tangkawsakul for the SPR measurements. REFERENCES 1. Andrae, J.; Gallini, R.; Betsholtz, C., Role of platelet-derived growth factors in physiology and medicine. Genes & Dev. 2008, 22 (10), 1276-1312. 2. Saik, J. E.; Gould, D. J.; Keswani, A. H.; Dickinson, M. E.; West, J. L., Biomimetic hydrogels with immobilized ephrinA1 for therapeutic angiogenesis. Biomacromolecules 2011, 12 (7), 2715-2722. 3. Barrientos, S.; Stojadinovic, O.; Golinko, M. S.; Brem, H.; Tomic-Canic, M., Growth factors and cytokines in wound healing. Wound Repair and Regeneration 2008, 16 (5), 585-601. 4. Numnuam, A.; Chumbimuni-Torres, K. Y.; Xiang, Y.; Bash, R.; Thavarungkul, P.; Kanatharana, P.; Pretsch, E.; Wang, J.; Bakker, E., Aptamer-based potentiometric measurements of proteins using ion-selective microelectrodes. Anal. Chem. 2008, 80 (3), 707-712. 5. Zhou, J.; Soontornworajit, B.; Snipes, M. P.; Wang, Y., Development of a novel pretargeting system with bifunctional nucleic acid molecules. Biochem. Biophys. Res. Commun. 2009, 386 (3), 521-525. 6. Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vermaas, E. H.; Toole, J. J., Selection of single-stranded-dna molecules that bind and inhibit human thrombin. Nature 1992, 355 (6360), 564-566. 7. White, R. R.; Sullenger, B. A.; Rusconi, C. P., Developing aptamers into therapeutics. J. Clin. Invest. 2000, 106 (8), 929-934.


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Oligonucleotide Hybridization Combined with Competitive Antibody

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