Biotechnol Lett DOI 10.1007/s10529-015-1839-8
ORIGINAL RESEARCH PAPER
Selecting DNA aptamers for endotoxin separation GuoQing Ying . FangFang Zhu . Yu Yi . JianShu Chen . JianFeng Mei . YanLu Zhang . ShuQing Chen
Received: 4 February 2015 / Accepted: 11 April 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objectives To select aptamers for endotoxin separation from a 75-nucleotide single-stranded DNA random library using systematic evolution of ligands by exponential enrichment. Results After 15 rounds of selection, the final pool of aptamers was specific to endotoxin. Structural analysis of aptamers that appeared more than once suggested that one aptamer can form a G-quartet structure. Tests for binding affinity and specificity showed that this aptamer exhibited a high affinity for endotoxin. Using this aptamer, aptamer-magnetic beads were designed to separate endotoxin.
G. Ying S. Chen College of Pharmaceutical Science, ZheJiang University, Hangzhou 310013, China e-mail: [email protected]; [email protected] G. Ying F. Zhu Y. Yi (&) J. Chen J. Mei Y. Zhang College of Pharmaceutical Science, ZheJiang University of Technology, Hangzhou 310014, China e-mail: [email protected]; [email protected] F. Zhu e-mail: [email protected] J. Chen e-mail: [email protected] J. Mei e-mail: [email protected] Y. Zhang e-mail: [email protected]
Conclusions Using these aptamer-magnetic beads, a new method to separate endotoxin was developed to enable specific separation of endotoxin that can be applied to drug and food products. Keywords Aptamer Endotoxin Magnetic beads Systematic evolution of ligands by exponential enrichment (SELEX) Separation
Introduction Endotoxins are lipopolysaccharides found in the outer cell walls of Gram-negative bacteria (Wilson et al. 2001). The unit of endotoxin is EU and is measured using a gel reaction test. One such test is that conducted by the American national standard endotoxin and standard limulus reagent. The lowest gel activity value of endotoxin to limulus reagent is 0.2 ng on average. This value is defined as 1 EU endotoxin, and 1 ng = 5 EU. The lowest gel activity value using the Chinese national standard endotoxin is lower than that of the American standard, and is 1 ng = 2.5 EU. In the blood stream, endotoxin shows strong biological effects on animals and humans even at low concentrations. These biological effects include affecting the structures and functions of cells and organs, raising body temperature, triggering the coagulation cascade, altering metabolic functions, and causing hemodynamic shock (Yamamoto et al. 2002). Serious
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Biotechnol Lett
endotoxin intoxication will cause endotoxemia (Ikeda et al. 2014). Because of its toxicity, removing endotoxin from drugs and food is of great importance. Although many methods have been developed to remove endotoxin, such as two-phase extraction, HPLC, and ultrafiltration (Petsch and Anspach 1999; Li et al. 2003), examples of affinity adsorption under mild conditions are relatively few. Affinity techniques, especially aptamer-based affinity techniques, have exhibited favorable results and have been studied extensively in recent years. Aptamers have a high affinity for their targets and are single-strand DNA (ssDNA) or RNA oligonucleotides (Nutiu and Li 2005). Because of their three-dimensional structure, aptamers have the ability to bind to their target with high selectivity and specificity. Systematic evolution of ligands by exponential enrichment (SELEX) is an extensively used technique for the selection of aptamers that can bind to desired targets (Chauveau et al. 2006). The reported targets of aptamers include a large range of substances such as small molecules, metal ions, proteins, peptides, and cells (Missailidis et al. 2005). In recent years, increasing numbers of important functional DNAs have been found and are promising for use in endotoxin removal.
Materials and methods Materials All DNA sequences were synthesized by Sangon Co. Ltd. (Shanghai, China) and purified by HPLC. Endotoxin was purchased from Chinese Horseshoe Crab Reagent Manufactory, Co. Ltd., Xiamen, China. Other reagents were of analytical grade.
In vitro selection of the endotoxin-binding aptamer An oligonucleotide library of 75 nucleotides in length with 35 random nucleotides in the center was designed (Tabarzad et al. 2014). The library was amplified over 25 cycles of PCR using primer 1 and primer 2 shown in Table 1. The ssDNA library was obtained from the double-stranded DNAs (dsDNAs) by an additional 40 cycles of asymmetric PCR, where the concentration of primer 1 was 100 times that of primer 2. The PCR products, ssDNAs, were purified using 5 % agarose gel electrophoresis. The selection of DNA aptamers by SELEX was conducted following the process in Fig. 1. Endotoxin was immobilized on polymyxin B (PMB) coupled sepharose (Warren 1982). Structural analysis of ssDNAs The dsDNAs obtained after 15 rounds of selection were cloned into the PMD-18 T vector (Takara, Dalian, China), which is an efficient and dedicated carrier for cloning PCR products using TA cloning that is constructed from the pUC18 vector, and has the same functionality of pUC18. The obtained PMD-18 T vectors were transformed into Escherichia coli (Takara, Dalian, China), and 26 colonies (no. 1–26) were randomly selected and sequenced. We analyzed the primary structure and secondary structure of these 26 sequences and the repeated sequences using DNAMAN 6.0.3.99. Binding affinity and specificity of aptamers to endotoxin The binding potential of the three repeated sequences were evaluated by PMB ELISA (Hu et al. 2013). The
Table 1 Oligonucleotide sequences used in this study Name
Oligonucleotides
DNA aptamer library
50 -ATGAGAGCGTCGGTGTGGTA-N35-TGTAGGAGGGTGCGGAAGTA-30
Primer 1
50 -ATGAGAGCGTCGGTGTGGTA-30
Primer 2 Biotin-primer 1
50 -TACTTCCGCACCCTCCTACA-30 50 -Biotin-ATGAGAGCGTCGGTGTGGTA-30
Aptamer EAQ1 (random region)
50 -CCACGCGCGGACAACAAGACAGCGTGCCTGCAGTG-30
Aptamer EAQ2 (random region)
50 -GCCATGCGCCGACCCAGGTCTAAGAGCCTCGGCCG-30
Aptamer EAQ3 (random region)
50 -TCGCCGCGCTCGCCGATCGTTCCTCCCCCCCCGTG-30
Primer 1, primer 2 and biotin-primer 1 are primers synthesized by Sangon Co. Ltd. (Shanghai, China). EAQ1, EAQ2, and EAQ3 are the aptamer sequences that appeared frequently in the sequencing results
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Results and discussion Selection of aptamers After 15 rounds of selection and five rounds of reverse selection, 26 clones were sequenced from the pool of remaining ssDNAs, and three high frequency sequences, named EAQ1, EAQ2, and EAQ3 were obtained. After reducing the amount of ssDNAs each round and reverse screening, the ssDNAs were evaluated efficiently. Fig. 1 SELEX process for aptamer selection against endotoxin. The endotoxin was fixed on agarose gel using polymyxin B. The SELEX process was 15 rounds of selection using endotoxin as the target
endotoxin was immobilized on 96-well plates by PMB and incubated with biotin-labeled aptamer for 1 h at 37 °C. After washing, the 96-well plates were incubated with HRP-conjugated immunopure streptavidin, and the HRP activity was detected using a microplate reader at 450 nm after development to evaluate the affinity of the aptamers to endotoxin. The specificity of aptamers to endotoxin was measured using bovine serum albumin and egg albumin as control experiments because they contain lipoid, polysaccharide and polypeptide components, which have structural features similar to those of endotoxin.
Structural analysis of ssDNAs using SELEX The similarity of the 26 sequences was 75.2 % after analysis with DNAMAN 6.0.3.99. The bases in the random regions were mainly C-rich and G-rich sequences. Moreover, 25 sequences ended with T or G in their random region. Therefore, the C-rich or G-rich parts, and the T or G loops in a random region may affect target combing. The sequences that appeared frequently showed the same characteristics and are shown in Table 1. The 26 sequences all formed stem-loop structures, and a few had GC-rich sequences. The stem-loop structure and GC-rich sequence have been shown to affect target binding (Satoru and Noburu 2011). The secondary structure of the sequences that appeared frequently are shown in Fig. 2 and suggested that one of the high frequency sequences, EAQ3, can form a G-quartet structure.
Aptamer-magnetic bead preparation The biotin-labeled aptamers with the highest affinity to endotoxin were mixed with streptavidin-immobilized magnetic beads. A magnetic frame was used for the separation of magnetic beads in 1.5-ml tubes. The streptavidin-coated magnetic beads (50 ll) were washed three times with a solution containing 10 mM Tris/HCl pH 7.5, 1 mM EDTA, and 2 M NaCl. A 50 ll 0.1 lM biotin-labeled aptamer solution was mixed with the streptavidin coated magnetic beads for 30 min at room temperature. After the aptamers were immobilized, 50 ll 0.5 EU endotoxin/ ml was mixed with the modified beads. After this step, the endotoxin-aptamer-coated magnetic beads were separated and collected as a supernatant to detect the concentration of endotoxin using the limulus amebocyte lysate test. The endotoxin adsorption was calculated from the A545 value.
Binding affinity and specificity of aptamers to endotoxin The biotin-labeled aptamers EAQ1, EAQ2, and EAQ3 were synthesized, and their binding ability to endotoxin was investigated in 96-well plates. EAQ1, EAQ2, and EAQ3 were three sequences that appeared frequently. These three aptamers showed different degrees of affinity for endotoxin, and EAQ3 was the highest. Moreover, these three aptamers showed no affinity for bovine serum albumin and egg albumin, which demonstrated their specificity to endotoxin (Fig. 3). Designing aptamer-magnetic beads for endotoxin separation Aptamer EAQ3 showed the highest affinity for endotoxin, and after endotoxin was mixed with
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Biotechnol Lett b Fig. 2 Predicted structures contain stem-loops containing parts
of the 75 nucleotides. EAQ1, EAQ2, and EAQ3 are the aptamers that appeared frequently in the sequencing results
Fig. 3 Binding affinity and specificity of aptamers against endotoxin. Each aptamer, 0.1 lM, was incubated with endotoxin. Unbound aptamers were rinsed off, and the concentration of bound aptamers was measured by the OD value. EAQ1, EAQ2 and EAQ3 were aptamers that appeared frequently in the sequencing results. EAQ3 showed specific affinity to endotoxin
EAQ3-modified magnetic beads, we measured the concentration of endotoxin in the supernatant at different incubation times to evaluate adsorption kinetics and capacity. Figure 4 shows that the adsorption quantity reached the highest level in the first 10 min, which was 1199 EU endotoxin/g, indicating that EAQ3 was effective for endotoxin adsorption. The removal rate of endotoxin was 98.3/100 EU at 10 min. However, after 10 min and further increases of adsorption time, the adsorption quantity was reduced, which demonstrated that the adsorption process was relatively fast. The adsorption isotherm of different initial concentrations of endotoxin incubated with EAQ3 are
Fig. 4 Adsorption kinetics curve of aptamer EAQ3 and endotoxin. Full adsorption was reached in the first 10 min, corresponding to 1199 EU endotoxin/g, and indicated that the aptamer EAQ3 was efficient for endotoxin adsorption
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separation, and separated endotoxin from different samples. The process of separation was relatively fast and efficient. The optimum separating time of aptamermagnetic beads used to remove endotoxin is within 10–20 min. The maximum adsorption quantity was 1199 EU endotoxin/g, and the removal rate of endotoxin reached 98.3/100 EU in the first 10 min. All these results demonstrate that our separation system is an alternative way to enable rapid and highly specific separation of endotoxin, and has the potential to separate endotoxin from drug and food products. Fig. 5 Adsorption isotherm of aptamer EAQ3 and endotoxin. With the increase of the initial concentration of endotoxin, the adsorption capacity of EAQ3 increased as well. The adsorption equilibrium was reached at 0.4 EU endotoxin/ml, and the adsorption capacity was 2980 EU endotoxin/g
Fig. 6 Langmuir-fitted curve of Fig. 5, the Langmuir formula m bC , (Qm and b are constants, C is for endotoxin Q ¼ Q1þbC concentrations) was used to model the adsorption data and the result was shown. The adsorption model was in agreement with the Langmuir model
shown in Fig. 5. Figures 5 and 6 show that with the increase of the initial concentration of endotoxin, the adsorption capacity of EAQ3 also increased. When the initial concentration of endotoxin exceeded certain values, each EAQ3 aptamer was bound to endotoxin, and the adsorption capacity plateaued. The maximum adsorption capacity was 2980 EU endotoxin/g.
Conclusions Aptamers that recognize endotoxin that were selected using a SELEX process with PMB-sepharose are described. Three DNA aptamers were obtained and, among them, EAQ3 exhibited the highest affinity for endotoxin. The binding specificity of aptamers for endotoxin was measured, and the result demonstrated that the EAQ3 aptamer has specific affinity to endotoxin. We also made aptamer-magnetic beads for endotoxin
References Chauveau F, Pestourie C, Tavitian B (2006) Aptamers: selection and scope of applications. Pathol Biol 4:251–258 Hu P, Liu ZS, Tian RY, Ren HL, Wang XX, Lin C, Gong S, Meng XM, Wang GM, Zhou Y, Lu SY (2013) Selection and identification of a DNA aptamer that mimics saxitoxin in antibody binding. J Agric Food Chem 14:3533–3541 Ikeda T, Ikeda K, Suda S, Ueno T (2014) Usefulness of the endotoxin activity assay as a biomarker to assess the severity of endotoxemia in critically ill patient. Innate Immun 8:881–887 Li Y, Lander R, Manger W, Lee A (2004) Determination of lipid profile in meningococcal polysaccharide using reversedphase liquid chromatography. J Chromatogr B 804:353–358 Missailidis S, Thomaidou D, Borbas KE, Price MR (2005) Selection of aptamers with high affinity and high specificity against C595, an anti-MUC1 IgG3 monoclonal antibody, for antibody targeting. J Immunol Methods 1:45–62 Nutiu R, Li YF (2005) Aptamers with fluorescence-signaling properties. Methods 1:16–25 Petsch D, Anspach FB (2000) Endotoxin removal from protein solutions. J Biotechnol 76:97–119 Satoru N, Noburu I (2011) Loop residues of thrombin-binding DNA aptamer impact G-quadruplex stability and thrombin binding. Biochimie 93:1231–1238 Tabarzad M, Kazemi B, Vahidi H, Aboofazeli R, Shahhosseini S, Nafissi-Varcheh N (2014) Challenges to design and develop of DNA aptamers for protein targets. I. Optimization of asymmetric PCR for generation of a single stranded DNA library. Iran J Pharm Res 13:133–141 Warren JR (1982) Polymyxin B suppresses the endotoxin inhibition of concanavalin a-mediated erythrocyte agglutination. Infect Immun 2:594–599 Wilson MJ, Haggart CL, Gallagher SP, Walsh D (2001) Removal of tightly bound endotoxin from biological products. J Biotechnol 88:67–75 Yamamoto A, Ochiai M, Kataoka M, Toyoizumi H, Horiuchi Y (2002) Development of a highly sensitive in vitro assay method for biological activity of endotoxin contamination in biological products. Biologicals 2:85–92
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ORIGINAL RESEARCH PAPER
Selecting DNA aptamers for endotoxin separation GuoQing Ying . FangFang Zhu . Yu Yi . JianShu Chen . JianFeng Mei . YanLu Zhang . ShuQing Chen
Received: 4 February 2015 / Accepted: 11 April 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objectives To select aptamers for endotoxin separation from a 75-nucleotide single-stranded DNA random library using systematic evolution of ligands by exponential enrichment. Results After 15 rounds of selection, the final pool of aptamers was specific to endotoxin. Structural analysis of aptamers that appeared more than once suggested that one aptamer can form a G-quartet structure. Tests for binding affinity and specificity showed that this aptamer exhibited a high affinity for endotoxin. Using this aptamer, aptamer-magnetic beads were designed to separate endotoxin.
G. Ying S. Chen College of Pharmaceutical Science, ZheJiang University, Hangzhou 310013, China e-mail: [email protected]; [email protected] G. Ying F. Zhu Y. Yi (&) J. Chen J. Mei Y. Zhang College of Pharmaceutical Science, ZheJiang University of Technology, Hangzhou 310014, China e-mail: [email protected]; [email protected] F. Zhu e-mail: [email protected] J. Chen e-mail: [email protected] J. Mei e-mail: [email protected] Y. Zhang e-mail: [email protected]
Conclusions Using these aptamer-magnetic beads, a new method to separate endotoxin was developed to enable specific separation of endotoxin that can be applied to drug and food products. Keywords Aptamer Endotoxin Magnetic beads Systematic evolution of ligands by exponential enrichment (SELEX) Separation
Introduction Endotoxins are lipopolysaccharides found in the outer cell walls of Gram-negative bacteria (Wilson et al. 2001). The unit of endotoxin is EU and is measured using a gel reaction test. One such test is that conducted by the American national standard endotoxin and standard limulus reagent. The lowest gel activity value of endotoxin to limulus reagent is 0.2 ng on average. This value is defined as 1 EU endotoxin, and 1 ng = 5 EU. The lowest gel activity value using the Chinese national standard endotoxin is lower than that of the American standard, and is 1 ng = 2.5 EU. In the blood stream, endotoxin shows strong biological effects on animals and humans even at low concentrations. These biological effects include affecting the structures and functions of cells and organs, raising body temperature, triggering the coagulation cascade, altering metabolic functions, and causing hemodynamic shock (Yamamoto et al. 2002). Serious
123
Biotechnol Lett
endotoxin intoxication will cause endotoxemia (Ikeda et al. 2014). Because of its toxicity, removing endotoxin from drugs and food is of great importance. Although many methods have been developed to remove endotoxin, such as two-phase extraction, HPLC, and ultrafiltration (Petsch and Anspach 1999; Li et al. 2003), examples of affinity adsorption under mild conditions are relatively few. Affinity techniques, especially aptamer-based affinity techniques, have exhibited favorable results and have been studied extensively in recent years. Aptamers have a high affinity for their targets and are single-strand DNA (ssDNA) or RNA oligonucleotides (Nutiu and Li 2005). Because of their three-dimensional structure, aptamers have the ability to bind to their target with high selectivity and specificity. Systematic evolution of ligands by exponential enrichment (SELEX) is an extensively used technique for the selection of aptamers that can bind to desired targets (Chauveau et al. 2006). The reported targets of aptamers include a large range of substances such as small molecules, metal ions, proteins, peptides, and cells (Missailidis et al. 2005). In recent years, increasing numbers of important functional DNAs have been found and are promising for use in endotoxin removal.
Materials and methods Materials All DNA sequences were synthesized by Sangon Co. Ltd. (Shanghai, China) and purified by HPLC. Endotoxin was purchased from Chinese Horseshoe Crab Reagent Manufactory, Co. Ltd., Xiamen, China. Other reagents were of analytical grade.
In vitro selection of the endotoxin-binding aptamer An oligonucleotide library of 75 nucleotides in length with 35 random nucleotides in the center was designed (Tabarzad et al. 2014). The library was amplified over 25 cycles of PCR using primer 1 and primer 2 shown in Table 1. The ssDNA library was obtained from the double-stranded DNAs (dsDNAs) by an additional 40 cycles of asymmetric PCR, where the concentration of primer 1 was 100 times that of primer 2. The PCR products, ssDNAs, were purified using 5 % agarose gel electrophoresis. The selection of DNA aptamers by SELEX was conducted following the process in Fig. 1. Endotoxin was immobilized on polymyxin B (PMB) coupled sepharose (Warren 1982). Structural analysis of ssDNAs The dsDNAs obtained after 15 rounds of selection were cloned into the PMD-18 T vector (Takara, Dalian, China), which is an efficient and dedicated carrier for cloning PCR products using TA cloning that is constructed from the pUC18 vector, and has the same functionality of pUC18. The obtained PMD-18 T vectors were transformed into Escherichia coli (Takara, Dalian, China), and 26 colonies (no. 1–26) were randomly selected and sequenced. We analyzed the primary structure and secondary structure of these 26 sequences and the repeated sequences using DNAMAN 6.0.3.99. Binding affinity and specificity of aptamers to endotoxin The binding potential of the three repeated sequences were evaluated by PMB ELISA (Hu et al. 2013). The
Table 1 Oligonucleotide sequences used in this study Name
Oligonucleotides
DNA aptamer library
50 -ATGAGAGCGTCGGTGTGGTA-N35-TGTAGGAGGGTGCGGAAGTA-30
Primer 1
50 -ATGAGAGCGTCGGTGTGGTA-30
Primer 2 Biotin-primer 1
50 -TACTTCCGCACCCTCCTACA-30 50 -Biotin-ATGAGAGCGTCGGTGTGGTA-30
Aptamer EAQ1 (random region)
50 -CCACGCGCGGACAACAAGACAGCGTGCCTGCAGTG-30
Aptamer EAQ2 (random region)
50 -GCCATGCGCCGACCCAGGTCTAAGAGCCTCGGCCG-30
Aptamer EAQ3 (random region)
50 -TCGCCGCGCTCGCCGATCGTTCCTCCCCCCCCGTG-30
Primer 1, primer 2 and biotin-primer 1 are primers synthesized by Sangon Co. Ltd. (Shanghai, China). EAQ1, EAQ2, and EAQ3 are the aptamer sequences that appeared frequently in the sequencing results
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Results and discussion Selection of aptamers After 15 rounds of selection and five rounds of reverse selection, 26 clones were sequenced from the pool of remaining ssDNAs, and three high frequency sequences, named EAQ1, EAQ2, and EAQ3 were obtained. After reducing the amount of ssDNAs each round and reverse screening, the ssDNAs were evaluated efficiently. Fig. 1 SELEX process for aptamer selection against endotoxin. The endotoxin was fixed on agarose gel using polymyxin B. The SELEX process was 15 rounds of selection using endotoxin as the target
endotoxin was immobilized on 96-well plates by PMB and incubated with biotin-labeled aptamer for 1 h at 37 °C. After washing, the 96-well plates were incubated with HRP-conjugated immunopure streptavidin, and the HRP activity was detected using a microplate reader at 450 nm after development to evaluate the affinity of the aptamers to endotoxin. The specificity of aptamers to endotoxin was measured using bovine serum albumin and egg albumin as control experiments because they contain lipoid, polysaccharide and polypeptide components, which have structural features similar to those of endotoxin.
Structural analysis of ssDNAs using SELEX The similarity of the 26 sequences was 75.2 % after analysis with DNAMAN 6.0.3.99. The bases in the random regions were mainly C-rich and G-rich sequences. Moreover, 25 sequences ended with T or G in their random region. Therefore, the C-rich or G-rich parts, and the T or G loops in a random region may affect target combing. The sequences that appeared frequently showed the same characteristics and are shown in Table 1. The 26 sequences all formed stem-loop structures, and a few had GC-rich sequences. The stem-loop structure and GC-rich sequence have been shown to affect target binding (Satoru and Noburu 2011). The secondary structure of the sequences that appeared frequently are shown in Fig. 2 and suggested that one of the high frequency sequences, EAQ3, can form a G-quartet structure.
Aptamer-magnetic bead preparation The biotin-labeled aptamers with the highest affinity to endotoxin were mixed with streptavidin-immobilized magnetic beads. A magnetic frame was used for the separation of magnetic beads in 1.5-ml tubes. The streptavidin-coated magnetic beads (50 ll) were washed three times with a solution containing 10 mM Tris/HCl pH 7.5, 1 mM EDTA, and 2 M NaCl. A 50 ll 0.1 lM biotin-labeled aptamer solution was mixed with the streptavidin coated magnetic beads for 30 min at room temperature. After the aptamers were immobilized, 50 ll 0.5 EU endotoxin/ ml was mixed with the modified beads. After this step, the endotoxin-aptamer-coated magnetic beads were separated and collected as a supernatant to detect the concentration of endotoxin using the limulus amebocyte lysate test. The endotoxin adsorption was calculated from the A545 value.
Binding affinity and specificity of aptamers to endotoxin The biotin-labeled aptamers EAQ1, EAQ2, and EAQ3 were synthesized, and their binding ability to endotoxin was investigated in 96-well plates. EAQ1, EAQ2, and EAQ3 were three sequences that appeared frequently. These three aptamers showed different degrees of affinity for endotoxin, and EAQ3 was the highest. Moreover, these three aptamers showed no affinity for bovine serum albumin and egg albumin, which demonstrated their specificity to endotoxin (Fig. 3). Designing aptamer-magnetic beads for endotoxin separation Aptamer EAQ3 showed the highest affinity for endotoxin, and after endotoxin was mixed with
123
Biotechnol Lett b Fig. 2 Predicted structures contain stem-loops containing parts
of the 75 nucleotides. EAQ1, EAQ2, and EAQ3 are the aptamers that appeared frequently in the sequencing results
Fig. 3 Binding affinity and specificity of aptamers against endotoxin. Each aptamer, 0.1 lM, was incubated with endotoxin. Unbound aptamers were rinsed off, and the concentration of bound aptamers was measured by the OD value. EAQ1, EAQ2 and EAQ3 were aptamers that appeared frequently in the sequencing results. EAQ3 showed specific affinity to endotoxin
EAQ3-modified magnetic beads, we measured the concentration of endotoxin in the supernatant at different incubation times to evaluate adsorption kinetics and capacity. Figure 4 shows that the adsorption quantity reached the highest level in the first 10 min, which was 1199 EU endotoxin/g, indicating that EAQ3 was effective for endotoxin adsorption. The removal rate of endotoxin was 98.3/100 EU at 10 min. However, after 10 min and further increases of adsorption time, the adsorption quantity was reduced, which demonstrated that the adsorption process was relatively fast. The adsorption isotherm of different initial concentrations of endotoxin incubated with EAQ3 are
Fig. 4 Adsorption kinetics curve of aptamer EAQ3 and endotoxin. Full adsorption was reached in the first 10 min, corresponding to 1199 EU endotoxin/g, and indicated that the aptamer EAQ3 was efficient for endotoxin adsorption
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separation, and separated endotoxin from different samples. The process of separation was relatively fast and efficient. The optimum separating time of aptamermagnetic beads used to remove endotoxin is within 10–20 min. The maximum adsorption quantity was 1199 EU endotoxin/g, and the removal rate of endotoxin reached 98.3/100 EU in the first 10 min. All these results demonstrate that our separation system is an alternative way to enable rapid and highly specific separation of endotoxin, and has the potential to separate endotoxin from drug and food products. Fig. 5 Adsorption isotherm of aptamer EAQ3 and endotoxin. With the increase of the initial concentration of endotoxin, the adsorption capacity of EAQ3 increased as well. The adsorption equilibrium was reached at 0.4 EU endotoxin/ml, and the adsorption capacity was 2980 EU endotoxin/g
Fig. 6 Langmuir-fitted curve of Fig. 5, the Langmuir formula m bC , (Qm and b are constants, C is for endotoxin Q ¼ Q1þbC concentrations) was used to model the adsorption data and the result was shown. The adsorption model was in agreement with the Langmuir model
shown in Fig. 5. Figures 5 and 6 show that with the increase of the initial concentration of endotoxin, the adsorption capacity of EAQ3 also increased. When the initial concentration of endotoxin exceeded certain values, each EAQ3 aptamer was bound to endotoxin, and the adsorption capacity plateaued. The maximum adsorption capacity was 2980 EU endotoxin/g.
Conclusions Aptamers that recognize endotoxin that were selected using a SELEX process with PMB-sepharose are described. Three DNA aptamers were obtained and, among them, EAQ3 exhibited the highest affinity for endotoxin. The binding specificity of aptamers for endotoxin was measured, and the result demonstrated that the EAQ3 aptamer has specific affinity to endotoxin. We also made aptamer-magnetic beads for endotoxin
References Chauveau F, Pestourie C, Tavitian B (2006) Aptamers: selection and scope of applications. Pathol Biol 4:251–258 Hu P, Liu ZS, Tian RY, Ren HL, Wang XX, Lin C, Gong S, Meng XM, Wang GM, Zhou Y, Lu SY (2013) Selection and identification of a DNA aptamer that mimics saxitoxin in antibody binding. J Agric Food Chem 14:3533–3541 Ikeda T, Ikeda K, Suda S, Ueno T (2014) Usefulness of the endotoxin activity assay as a biomarker to assess the severity of endotoxemia in critically ill patient. Innate Immun 8:881–887 Li Y, Lander R, Manger W, Lee A (2004) Determination of lipid profile in meningococcal polysaccharide using reversedphase liquid chromatography. J Chromatogr B 804:353–358 Missailidis S, Thomaidou D, Borbas KE, Price MR (2005) Selection of aptamers with high affinity and high specificity against C595, an anti-MUC1 IgG3 monoclonal antibody, for antibody targeting. J Immunol Methods 1:45–62 Nutiu R, Li YF (2005) Aptamers with fluorescence-signaling properties. Methods 1:16–25 Petsch D, Anspach FB (2000) Endotoxin removal from protein solutions. J Biotechnol 76:97–119 Satoru N, Noburu I (2011) Loop residues of thrombin-binding DNA aptamer impact G-quadruplex stability and thrombin binding. Biochimie 93:1231–1238 Tabarzad M, Kazemi B, Vahidi H, Aboofazeli R, Shahhosseini S, Nafissi-Varcheh N (2014) Challenges to design and develop of DNA aptamers for protein targets. I. Optimization of asymmetric PCR for generation of a single stranded DNA library. Iran J Pharm Res 13:133–141 Warren JR (1982) Polymyxin B suppresses the endotoxin inhibition of concanavalin a-mediated erythrocyte agglutination. Infect Immun 2:594–599 Wilson MJ, Haggart CL, Gallagher SP, Walsh D (2001) Removal of tightly bound endotoxin from biological products. J Biotechnol 88:67–75 Yamamoto A, Ochiai M, Kataoka M, Toyoizumi H, Horiuchi Y (2002) Development of a highly sensitive in vitro assay method for biological activity of endotoxin contamination in biological products. Biologicals 2:85–92
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