Journal of Chromatography A, 1364 (2014) 289–294
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Low pH capillary electrophoresis application to improve capillary electrophoresis-systematic evolution of ligands by exponential enrichment Qian Li a , Xinying Zhao b , Hongyang Liu a , Feng Qu a,∗ a b
School of Life Science, Beijing Institute of Technology, Beijing 100081, China Beijing Centre for Physical and Chemical Analysis, Beijing 100089, China
a r t i c l e
i n f o
Article history: Received 22 April 2014 Received in revised form 21 August 2014 Accepted 22 August 2014 Available online 27 August 2014 Keywords: Protein Aptamers selection Low pH capillary electrophoresis Low pH CE-SELEX
a b s t r a c t In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) as a CE-SELEX variant is proposed. Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein are incubated with a FAM labeled ssDNA library, respectively. Incubation mixture is separated in low pH CE (pH 2.6), where positively charged protein, protein–ssDNA complex and negatively charged ssDNA library migrate oppositely without EOF driven. Analysis of protein–ssDNA complex under positive voltage and unbound ssDNA library under negative voltage by CE–UV are applied for interactive evaluation. By increasing injection time, larger amount protein–ssDNA complex can be collected conveniently at the cathode end whereas ssDNA migrates to anode. Finally, stability of protein–ssDNA complex in low pH CE separation is discussed.
1. Introduction Aptamers are single-stranded DNA(ssDNA) or RNA, which are selected from a random synthesized ssDNA library through the process of systematic evolution of ligands by exponential enrichment (SELEX) [1,2]. Aptamers bind targets with high affinity and specificity, which is comparable to interactive between antigen and antibody. Moreover, aptamers have a wider range of targets from small molecules to large proteins and composite targets of cell and bacteria, so they have great potential in medical and pharmacy [3,4] as well as in biosensor analysis as recognition molecules [5,6]. Since Ellington [1] and Tuerk [2] reported SELEX in 1990, many SELEX variants have been developed, a recent review has outlined various modified SELEX methods [7]. Usually, aptamers selection from a random ssDNA library requires multiple cycles (4–20 rounds) of partitioning, separation and PCR amplification steps. High efficiency separation of targetssDNA complex from unbound ssDNA library is an important step in SELEX. Many separation methods have been applied in SELEX, such as affinity chromatography, membrane filtration etc. Recent years, some advantageous separation methods such as polyacrylamide gel electrophoresis [8], magnetic beads on microchip [9,10] and
∗ Corresponding author. Tel.: +0086 10 68918015; fax: +0086 10 68915956. E-mail address: [email protected] (F. Qu). http://dx.doi.org/10.1016/j.chroma.2014.08.073 0021-9673/© 2014 Elsevier B.V. All rights reserved.
© 2014 Elsevier B.V. All rights reserved.
capillary electrophoresis (CE) [11–26] have been applied to improve separation in SELEX. Unlike others, CE is a fast and high resolution separation method, allows protein–ssDNA complex separation in free solution without immobilization of target to solid support and other solid media involvement. Some aptamers against important functional proteins have been selected in only 1–4 rounds by CE-SELEX, which greatly reduce selection time and selection cost. Aptamers of HIV-1 reverse transcriptase [12], farnesyltransferase [13], signal transduction protein[14], bovine catalase [15], neuropeptide Y [17], human IgE [18], MutS [19,20] and h-Ras [21], ricin toxin [22], protein kinase C [23], leptin [24], Ara h 1 protein [25], rhVEGF165 [26] have been reported based on CE-SELEX. Above aptamers selection by CE applies conventional electrolyte solution (pH 7–8) in electrophoresis, both protein–ssDNA complex and unbound ssDNA library migrate to the cathode end under electroosmotic flow (EOF) driven. In order to collect a complete protein–ssDNA complex fraction at cathode capillary outlet end without unbound ssDNA library in it, complex collection time must be strictly controlled. In addition, due to very small injection volume in CE, only a small portion of protein–ssDNA complex is injected, which certainly reduces the possibility of finding sequences with high binding affinity, so injection and collection have to be repeated several times to get more protein–ssDNA complex. Even though enrichment of aptamers can be achieved due to exponentially amplified by PCR [27], the question “ssDNA sequences diversity is lost in CE system” mentioned by Li et al. [8]
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Table 1 Capillary electrophoresis analysis conditions. Items
Protein–ssDNA complex separation and collection under CE–UV
Protein–ssDNA complex identification under CE–LIF
Instruments Capillary length
Agilent 7100 CE 33.5 cm/25 cm (effective)
Running electrolyte
Low pH solution 50 mM NaH2 PO4 /H3 PO4 (pH 2.6) 50 mbar, 10 s 15 ◦ C 10 kV UV 195 nm/254 nm
Beckman P/ACE MDQ 50.2 cm/40 cm (effective) Common solution 50 mM Na2 B4 O7 /H3 BO3 (pH 8.7) 0.5 psi, 5 s 25 ◦ C 20 Kv LIF ex /em 488/520 nm
Injection Temperature Voltage Detection
has to be faced. Therefore, convenient protein–ssDNA complex collection and expansion of injection amount of CE are beneficial to CE-SELEX. In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) is proposed. In order to improve CE-SELEX, low pH CE is applied to interactive evaluation of protein and ssDNA and convenient larger amount protein–ssDNA complex collection. With transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein, under low pH solution (pH 2.6), protein–ssDNA complex and ssDNA library migrate in reverse direction without EOF driven. Binding of protein and ssDNA library is illustrated by protein–ssDNA peak analysis under positive voltage and unbound ssDNA library analysis under negative voltage, respectively. Moreover, protein–ssDNA complex is conveniently collected at the cathode end without unbound ssDNA library involving. Furthermore, larger amount protein–ssDNA complex collection is completed just by increasing injection time. 2. Experimental 2.1. Instruments Agilent 7100 Capillary Electrophoresis equipped with a DAD detector (Agilent Technology, USA) was applied for protein and ssDNA library mixture separation and protein–ssDNA complex collection. Beckman P/ACE MDQ (Beckman-coulter, Fullerton, CA, USA) equipped with a LIF detector was applied for collected protein–ssDNA complex identification, by which FAM labeled ssDNA was detectable. 2.2. Reagents and materials Random synthesized ssDNA library was 6-carboxyfluorescein (FAM) labeled sequences 5 -FAM-AGC AGC ACA GAG GTC AGA TG(N40 )-CCT ATG CGT GCT ACC GTG AA-3 (ssDNA library), which was supplied by Sangon Biological Engineering Technology and Services (Shanghai, China). Transferring (Trf) was purchased from Sigma, bovine serum albumin (BSA) from Amresco, cytochrome c (Cyt c) from Roche. Na2 B4 O7 ·10H2 O, H3 BO3 , NaH2 PO4 , H3 PO4 were of analytical grade, purchased from Beijing Reagent Plant. 75 m fused silica capillary was provided by Sino Sumtech (Handan, Hebei, China). 2.3. Capillary electrophoresis conditions Table 1 showed capillary electrophoresis analysis conditions using Agilent 7100 Capillary Electrophoresis for complex analysis and collection while Beckman P/ACE MDQ for collected protein–ssDNA complex identification.ssDNA library was heated to 94 ◦ C for 5 min, then cooled down to 0 ◦ C quickly. Protein was incubated with ssDNA library on ice for 30 min, the incubation mixture
was injected for analysis and protein–ssDNA complex collection. protein–ssDNA complex was separated and collected under low pH (50 mM NaH2 PO4 /H3 PO4 pH 2.6) solution by CE–UV. The collected protein–ssDNA complex was identified using a common solution (50 mM Na2 B4 O7 /H3 BO3 pH 8.7) by CE–LIF. Three steps to capillary washing was done before each run. In protein–ssDNA complex separation and collection experiment, capillary was rinsed with methanol, water and low pH electrolyte solution (pH 2.6) each for 3 min. In protein–ssDNA complex identification experiments, capillary was rinsed with 0.1 mol/L NaOH, water and common solution (pH 8.7) each for 3 min. 3. Results and discussions 3.1. Principle of protein–ssDNA complex separation and collection in low pH CE For aptamers selection, one of the critical steps is to isolate protein–ssDNA complex from unbound ssDNA library. In conventional CE-SELEX, protein and ssDNA library incubation mixture is injected at the anode capillary inlet end, three components migrate in the order of protein, protein–ssDNA complex and ssDNA library from anode to cathode driven by EOF [11–26]. When EOF is inhibited in low pH solution (pH < 3), three compounds migrate in a different direction depending on their charge/mass ratio. Positively charged protein and protein–ssDNA complex move to the cathode meanwhile negatively charged ssDNA library moves to the anode. Fig. 1 demonstrates the migration and detection of protein, protein–ssDNA complex and ssDNA library under positive or negative separation voltage, where protein–ssDNA complex collection is always completed at cathode end. As we known, coated capillary is usually applied to inhibit EOF and protein adsorption. However, some proteins adsorption on coated capillary is inevitable. In our experiment, protein and protein–ssDNA complex cannot be identified using coated capillary, so coating media binding protein and protein–ssDNA has to be considered. However, in low pH solution (pH < 3), EOF and protein adsorption are inhibited simply without additives involvement. 3.2. Analysis of protein and ssDNA library incubation mixture Three protein targets Trf (5.6–5.8), BSA (pI 4.7–4.8) and Cyt c (pI 10.2) were incubated with a FAM labeled ssDNA library, respectively, incubation reaction was always in 10 L final volume, then incubation mixtures were analyzed under positive and negative voltage. 3.2.1. Positive voltage analysis Fig. 2 showed incubation mixture analysis under positive voltage. Trf and Trf–ssDNA complex reached baseline separation (Fig. 2A). With ssDNA library concentration increasing, Trf peak decreased accompanied with Trf–ssDNA complex peak increased. Both 195 nm and 254 nm detection showed the change of Trf and Trf–ssDNA complex clearly, which confirmed the interactive of Trf and ssDNA library as well as the formation of Trf–ssDNA complex. Fig. 2B showed the similar results of BSA and ssDNA library. In contrast, with ssDNA library concentration increasing, Cyt c peak decreased significantly, but only a very small peak after Cyt c was detected at 195 nm and 254 nm (Fig. 2C). Results in Fig. 2 indicated Trf, BSA, Cyt c and Trf–ssDNA, BSA–ssDNA complex held positive net charges, and migrated toward cathode and were detectable by UV. Peak area of Trf–ssDNA complex was obviously larger than BSA–ssDNA complex, which illustrated more Trf–ssDNA complex was produced and Trf bound more ssDNA than BSA. So Trf had stronger interactive with this ssDNA library than BSA. For strong basic protein Cyt c, there was no
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Fig. 1. Diagram of protein, protein–ssDNA complex and ssDNA library separation and collection in low pH CE. (A) positive voltage; (B) negative voltage.
apparent Cyt c -ssDNA complex peak, the interactive of Cyt c and ssDNA library could not be confirmed in this way. 3.2.2. Negative voltage analysis Fig. 3 showed incubation mixture analysis under negative voltage. Negatively charged ssDNA library migrated toward anode and was detectable. With Trf, BSA and Cyt c concentration increasing, unbound ssDNA library peak reduced clearly (Fig. 3A–C). 195 nm and 254 nm detection gave the same results. When Trf and BSA concentration increased in the range of 1–5 M, ssDNA library peak decreased obviously. However, when Cyt c concentration increased to 5 M, the decrease of ssDNA library was negligible, which corresponded to the result of Fig. 2C, where a very small complex peak was visible. Protein concentration increase caused unbound ssDNA reduction, which also illustrated the interactive of protein and ssDNA library. 3.2.3. Interactive comparison Fig. 4A showed Trf–ssDNA and BSA–ssDNA complex peak area increased with ssDNA library concentration increasing. Trf–ssDNA peak area larger than BSA–ssDNA illustrated the stronger interactive of Trf with ssDNA library than BSA. Fig. 4B showed unbound ssDNA peak area decreased with Trf, BSA and Cyt c concentration increasing, the decrease trend also indicated their interactive strength was in the order of Trf > BSA> Cyt c. In contrast, the
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In order to confirm protein–ssDNA complex was collected at the outlet end (under positive voltage) and inlet end (under negative voltage), both collection fractions were analyzed by sensitive LIF detection (conditions see Table 1). Fig. 5 showed the results of collected complex identification. Both Trf–ssDNA 100
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evidence based on protein peak area decrease was not as strong as that of complex peak increase or unbound ssDNA peak decrease, even though protein decrease trend (based on slopes value) followed the same order of Trf > BSA> Cyt c (Fig. 4C). Fig. 4 illustrated that under positive or negative voltage analysis, both complex increase and unbound ssDNA decrease could be used for interactive evaluation and comparison. Since the interaction between protein and ssDNA library is an equilibrium reaction, their concentration ratio will determine specific and nonspecific interaction strength. With positive voltage analysis or negative voltage analysis, the total binding strength between protein target and designed ssDNA library can be illustrated, but specific or nonspecific interaction cannot be identified. To improve specific binding, other follow-up work is always needed, for example, (1) to optimized protein and ssDNA library ratio; (2) with other protein target as negative control; (3) increase selection round, etc.
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Fig. 2. Analysis of three proteins and protein–ssDNA complexes in incubation mixture with ssDNA library concentration increasing under positive voltage with 195 nm and 214 nm UV detection.
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Q. Li et al. / J. Chromatogr. A 1364 (2014) 289–294
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Fig. 3. Analysis of unbound ssDNA library in incubation mixture with three proteins concentration increasing under negative voltage with 195 nm and 214 nm UV detection.
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and BSA–ssDNA complex fractions gave clear FAM–ssDNA signal, whereas Cyt c–ssDNA complex fraction only gave a negligible peak. CE–LIF detection results confirmed the formation of Trf–ssDNA and BSA–ssDNA complex and their successful collection without unbound ssDNA. 3.4. Protein–ssDNA complex stability in low pH solution In conventional CE, protein and ssDNA library mixture was incubated and separated in the higher pH solution (pH 7–8). Here, in low pH CE, protein and ssDNA library mixture was incubated in pure H2 O but separated in low pH solution. In order to validate the stability of protein–ssDNA complex in 15 min low pH separation, three
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proteins and ssDNA library mixtures incubated in low pH solution for 30 min, respectively, were compared with those incubated in H2 O. Since affinity binding of protein and ssDNA depended mainly on ssDNA secondary structure, low pH solution could result in part protonation of protein and ssDNA base, but it did not impede Trf–ssDNA and BSA–ssDNA complex formation. Trf–ssDNA complex and BSA–ssDNA complex formed in low pH incubation were showed in Fig. 6, which indicated the stability of Trf–ssDNA and BSA–ssDNA in low pH solution, even though smaller peaks of Trf–ssDNA and BSA–ssDNA complex and a little worse resolution of separation were shown comparing with those in H2 O incubation. Therefore, low pH CE separation process less than 15 min would
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Q. Li et al. / J. Chromatogr. A 1364 (2014) 289–294
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Fig. 6. Comparison of three protein–ssDNA complexes formed in pH 2.6 solution and H2 O.
not damage Trf–ssDNA and BSA–ssDNA complex formed in H2 O. Strong basic Cyt c and its complex peaks, however, could not be identified after low pH incubation.
3.5. Collection amount expansion of protein–ssDNA complex In conventional CE, to get high separation efficiency, injection amount of the incubation mixture was very limited. For example, common 5 s injection volume at 50 mbar in Agilent 7100 corresponded to 66 nL, accounted for 6% of total capillary volume. In this work, to collect more protein–ssDNA complex, injection amount was enlarged by increasing injection time. For a 33.5 cm capillary length (effective 25 cm) in Agilent 7100, the maximum injection time 30 s was tried, which corresponded to injection volume 396 nL, accounted for 36% of total capillary volume. Fig. 7 showed peak area (PA) increase of Trf and Trf–ssDNA when injection time increased from 5 s to 25 s, the increase of PA of Trf and Trf–ssDNA was linear with injection time (inj-t), PATrf = 261Xinj-t − 817.4 (R2 = 0.9990) and PATrf-ssDNA = 844.32Xinj-t − 1524.8 (R2 = 0.9992). Comparing with common 5 s injection, 25 s injection obtained 7.26 times more Trf–ssDNA complex. With longer injection time, the electric current became unstable. Above results showed that larger amount of protein–ssDNA complex could be collected just by simply increasing injection time.
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4. Conclusion Low pH CE for SELEX provides a CE-SELEX variant (LpH-CESELEX), which can be used for fast interactive evaluation of protein and ssDNA library and convenient larger amount protein–ssDNA complex collection, improve protein–ssDNA complex separation and collection for CE-SELEX. Comparing with conventional CESELEX, LpH-CE-SELEX has the following advantages: (1) both positive and negative voltage separation can be applied for interactive evaluation before aptamers selection to reduce risk and blindness of aptamers selection; (2) protein–ssDNA complex collection amount can be amplified easily by increasing injection time; (3) reverse migration of protein–ssDNA complex and unbound ssDNA makes complete protein–ssDNA complex collection at cathode without unbound ssDNA involvement, there is no need to exactly control collection time; (4) low pH collection does not affect the recovery of ssDNA in subsequent PCR due to larger amount pH 7.4 HCl–Tris buffer is used in PCR. Acknowledgements This work is supported by the National Natural Science Foundation of China (no. 21175011, 21375008), National Basic Research Program of China (973 program, no. 2012CB910603). References [1] A.D. Ellington, J.W. Szostak, In vitro selection of RNA molecules that binds specific ligands, Nature 346 (1990) 818–822. [2] C. Tuerk, L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 249 (1990) 505–510. [3] P. Sundaram, H. Kurniawan, M.E. Byrne, J. Wower, Therapeutic RNA aptamer in clinical trials, Eur. J. Pharm. Sci. 48 (2013) 259–271. ´ Aptamers molecules [4] F. Radom, P.M. Jurek, M.P. Mazurek, J. Otlewski, F. Jelen, of great potential, Biotechnol. Adv. 31 (2013) 1260–1274. [5] M. Citartan, S.C.B. Gopinath, J. Tominaga, S-C. Tan, T-H. Tang, Assays for aptamer-based platforms, Biosens. Bioelectron. 34 (2012) 1–11. [6] J. Huang, X. Yang, X. He, K. Wang, J. Liu, H. Shi, Q. Wang, Q. Guo, D. He, Design and bioanalytical applications of DNA hairpin-based fluorescent probes, Trends Anal. Chem. 53 (2014) 11–20. [7] G. Aquino-Jarquin, J.D. Toscano-Garibay, RNA aptamer evolution: two decades of SELEction, Int. J. Mol. Sci. 12 (2011) 9155–9171. [8] Y. Liu, C. Wang, F. Li, S. Shen, D.L.J. Tyrrell, X.C. Le, X-F. Li, DNase-mediated single-cycle selection of aptamers for proteins blotted on a membrane, Anal. Chem. 84 (2012) 7603–7606. [9] J. Qian, X. Lou, Y. Zhang, Y. Xiao, H.T. Soh, Generation of highly specific aptamers via micromagnetic selection, Anal. Chem. 81 (2009) 5490–5495. [10] X. Lou, J. Qian, Y. Xiao, L. Viel, A.E. Gerdon, E.T. Lagally, P. Atzberger, T.M. Tarasow, A.J. Heeger, H.T. Soh, Micromagnetic selection of aptamers in microfluidic channels, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 2989–2994. [11] S.D. Mendonsa, M.T. Bowser, In vitro evolution of functional DNA using capillary electrophoresis, J. Am. Chem. Soc. 126 (2004) 20–21. [12] R.K. Mosing, S.D. Mendonsa, M.T. Bowser, Capillary electrophoresis-SELEX selection of aptamers with affinity for HIV-1 reverse transcriptase, Anal. Chem. 77 (2005) 6107–6112.
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Low pH capillary electrophoresis application to improve capillary electrophoresis-systematic evolution of ligands by exponential enrichment Qian Li a , Xinying Zhao b , Hongyang Liu a , Feng Qu a,∗ a b
School of Life Science, Beijing Institute of Technology, Beijing 100081, China Beijing Centre for Physical and Chemical Analysis, Beijing 100089, China
a r t i c l e
i n f o
Article history: Received 22 April 2014 Received in revised form 21 August 2014 Accepted 22 August 2014 Available online 27 August 2014 Keywords: Protein Aptamers selection Low pH capillary electrophoresis Low pH CE-SELEX
a b s t r a c t In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) as a CE-SELEX variant is proposed. Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein are incubated with a FAM labeled ssDNA library, respectively. Incubation mixture is separated in low pH CE (pH 2.6), where positively charged protein, protein–ssDNA complex and negatively charged ssDNA library migrate oppositely without EOF driven. Analysis of protein–ssDNA complex under positive voltage and unbound ssDNA library under negative voltage by CE–UV are applied for interactive evaluation. By increasing injection time, larger amount protein–ssDNA complex can be collected conveniently at the cathode end whereas ssDNA migrates to anode. Finally, stability of protein–ssDNA complex in low pH CE separation is discussed.
1. Introduction Aptamers are single-stranded DNA(ssDNA) or RNA, which are selected from a random synthesized ssDNA library through the process of systematic evolution of ligands by exponential enrichment (SELEX) [1,2]. Aptamers bind targets with high affinity and specificity, which is comparable to interactive between antigen and antibody. Moreover, aptamers have a wider range of targets from small molecules to large proteins and composite targets of cell and bacteria, so they have great potential in medical and pharmacy [3,4] as well as in biosensor analysis as recognition molecules [5,6]. Since Ellington [1] and Tuerk [2] reported SELEX in 1990, many SELEX variants have been developed, a recent review has outlined various modified SELEX methods [7]. Usually, aptamers selection from a random ssDNA library requires multiple cycles (4–20 rounds) of partitioning, separation and PCR amplification steps. High efficiency separation of targetssDNA complex from unbound ssDNA library is an important step in SELEX. Many separation methods have been applied in SELEX, such as affinity chromatography, membrane filtration etc. Recent years, some advantageous separation methods such as polyacrylamide gel electrophoresis [8], magnetic beads on microchip [9,10] and
∗ Corresponding author. Tel.: +0086 10 68918015; fax: +0086 10 68915956. E-mail address: [email protected] (F. Qu). http://dx.doi.org/10.1016/j.chroma.2014.08.073 0021-9673/© 2014 Elsevier B.V. All rights reserved.
© 2014 Elsevier B.V. All rights reserved.
capillary electrophoresis (CE) [11–26] have been applied to improve separation in SELEX. Unlike others, CE is a fast and high resolution separation method, allows protein–ssDNA complex separation in free solution without immobilization of target to solid support and other solid media involvement. Some aptamers against important functional proteins have been selected in only 1–4 rounds by CE-SELEX, which greatly reduce selection time and selection cost. Aptamers of HIV-1 reverse transcriptase [12], farnesyltransferase [13], signal transduction protein[14], bovine catalase [15], neuropeptide Y [17], human IgE [18], MutS [19,20] and h-Ras [21], ricin toxin [22], protein kinase C [23], leptin [24], Ara h 1 protein [25], rhVEGF165 [26] have been reported based on CE-SELEX. Above aptamers selection by CE applies conventional electrolyte solution (pH 7–8) in electrophoresis, both protein–ssDNA complex and unbound ssDNA library migrate to the cathode end under electroosmotic flow (EOF) driven. In order to collect a complete protein–ssDNA complex fraction at cathode capillary outlet end without unbound ssDNA library in it, complex collection time must be strictly controlled. In addition, due to very small injection volume in CE, only a small portion of protein–ssDNA complex is injected, which certainly reduces the possibility of finding sequences with high binding affinity, so injection and collection have to be repeated several times to get more protein–ssDNA complex. Even though enrichment of aptamers can be achieved due to exponentially amplified by PCR [27], the question “ssDNA sequences diversity is lost in CE system” mentioned by Li et al. [8]
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Table 1 Capillary electrophoresis analysis conditions. Items
Protein–ssDNA complex separation and collection under CE–UV
Protein–ssDNA complex identification under CE–LIF
Instruments Capillary length
Agilent 7100 CE 33.5 cm/25 cm (effective)
Running electrolyte
Low pH solution 50 mM NaH2 PO4 /H3 PO4 (pH 2.6) 50 mbar, 10 s 15 ◦ C 10 kV UV 195 nm/254 nm
Beckman P/ACE MDQ 50.2 cm/40 cm (effective) Common solution 50 mM Na2 B4 O7 /H3 BO3 (pH 8.7) 0.5 psi, 5 s 25 ◦ C 20 Kv LIF ex /em 488/520 nm
Injection Temperature Voltage Detection
has to be faced. Therefore, convenient protein–ssDNA complex collection and expansion of injection amount of CE are beneficial to CE-SELEX. In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) is proposed. In order to improve CE-SELEX, low pH CE is applied to interactive evaluation of protein and ssDNA and convenient larger amount protein–ssDNA complex collection. With transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein, under low pH solution (pH 2.6), protein–ssDNA complex and ssDNA library migrate in reverse direction without EOF driven. Binding of protein and ssDNA library is illustrated by protein–ssDNA peak analysis under positive voltage and unbound ssDNA library analysis under negative voltage, respectively. Moreover, protein–ssDNA complex is conveniently collected at the cathode end without unbound ssDNA library involving. Furthermore, larger amount protein–ssDNA complex collection is completed just by increasing injection time. 2. Experimental 2.1. Instruments Agilent 7100 Capillary Electrophoresis equipped with a DAD detector (Agilent Technology, USA) was applied for protein and ssDNA library mixture separation and protein–ssDNA complex collection. Beckman P/ACE MDQ (Beckman-coulter, Fullerton, CA, USA) equipped with a LIF detector was applied for collected protein–ssDNA complex identification, by which FAM labeled ssDNA was detectable. 2.2. Reagents and materials Random synthesized ssDNA library was 6-carboxyfluorescein (FAM) labeled sequences 5 -FAM-AGC AGC ACA GAG GTC AGA TG(N40 )-CCT ATG CGT GCT ACC GTG AA-3 (ssDNA library), which was supplied by Sangon Biological Engineering Technology and Services (Shanghai, China). Transferring (Trf) was purchased from Sigma, bovine serum albumin (BSA) from Amresco, cytochrome c (Cyt c) from Roche. Na2 B4 O7 ·10H2 O, H3 BO3 , NaH2 PO4 , H3 PO4 were of analytical grade, purchased from Beijing Reagent Plant. 75 m fused silica capillary was provided by Sino Sumtech (Handan, Hebei, China). 2.3. Capillary electrophoresis conditions Table 1 showed capillary electrophoresis analysis conditions using Agilent 7100 Capillary Electrophoresis for complex analysis and collection while Beckman P/ACE MDQ for collected protein–ssDNA complex identification.ssDNA library was heated to 94 ◦ C for 5 min, then cooled down to 0 ◦ C quickly. Protein was incubated with ssDNA library on ice for 30 min, the incubation mixture
was injected for analysis and protein–ssDNA complex collection. protein–ssDNA complex was separated and collected under low pH (50 mM NaH2 PO4 /H3 PO4 pH 2.6) solution by CE–UV. The collected protein–ssDNA complex was identified using a common solution (50 mM Na2 B4 O7 /H3 BO3 pH 8.7) by CE–LIF. Three steps to capillary washing was done before each run. In protein–ssDNA complex separation and collection experiment, capillary was rinsed with methanol, water and low pH electrolyte solution (pH 2.6) each for 3 min. In protein–ssDNA complex identification experiments, capillary was rinsed with 0.1 mol/L NaOH, water and common solution (pH 8.7) each for 3 min. 3. Results and discussions 3.1. Principle of protein–ssDNA complex separation and collection in low pH CE For aptamers selection, one of the critical steps is to isolate protein–ssDNA complex from unbound ssDNA library. In conventional CE-SELEX, protein and ssDNA library incubation mixture is injected at the anode capillary inlet end, three components migrate in the order of protein, protein–ssDNA complex and ssDNA library from anode to cathode driven by EOF [11–26]. When EOF is inhibited in low pH solution (pH < 3), three compounds migrate in a different direction depending on their charge/mass ratio. Positively charged protein and protein–ssDNA complex move to the cathode meanwhile negatively charged ssDNA library moves to the anode. Fig. 1 demonstrates the migration and detection of protein, protein–ssDNA complex and ssDNA library under positive or negative separation voltage, where protein–ssDNA complex collection is always completed at cathode end. As we known, coated capillary is usually applied to inhibit EOF and protein adsorption. However, some proteins adsorption on coated capillary is inevitable. In our experiment, protein and protein–ssDNA complex cannot be identified using coated capillary, so coating media binding protein and protein–ssDNA has to be considered. However, in low pH solution (pH < 3), EOF and protein adsorption are inhibited simply without additives involvement. 3.2. Analysis of protein and ssDNA library incubation mixture Three protein targets Trf (5.6–5.8), BSA (pI 4.7–4.8) and Cyt c (pI 10.2) were incubated with a FAM labeled ssDNA library, respectively, incubation reaction was always in 10 L final volume, then incubation mixtures were analyzed under positive and negative voltage. 3.2.1. Positive voltage analysis Fig. 2 showed incubation mixture analysis under positive voltage. Trf and Trf–ssDNA complex reached baseline separation (Fig. 2A). With ssDNA library concentration increasing, Trf peak decreased accompanied with Trf–ssDNA complex peak increased. Both 195 nm and 254 nm detection showed the change of Trf and Trf–ssDNA complex clearly, which confirmed the interactive of Trf and ssDNA library as well as the formation of Trf–ssDNA complex. Fig. 2B showed the similar results of BSA and ssDNA library. In contrast, with ssDNA library concentration increasing, Cyt c peak decreased significantly, but only a very small peak after Cyt c was detected at 195 nm and 254 nm (Fig. 2C). Results in Fig. 2 indicated Trf, BSA, Cyt c and Trf–ssDNA, BSA–ssDNA complex held positive net charges, and migrated toward cathode and were detectable by UV. Peak area of Trf–ssDNA complex was obviously larger than BSA–ssDNA complex, which illustrated more Trf–ssDNA complex was produced and Trf bound more ssDNA than BSA. So Trf had stronger interactive with this ssDNA library than BSA. For strong basic protein Cyt c, there was no
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Fig. 1. Diagram of protein, protein–ssDNA complex and ssDNA library separation and collection in low pH CE. (A) positive voltage; (B) negative voltage.
apparent Cyt c -ssDNA complex peak, the interactive of Cyt c and ssDNA library could not be confirmed in this way. 3.2.2. Negative voltage analysis Fig. 3 showed incubation mixture analysis under negative voltage. Negatively charged ssDNA library migrated toward anode and was detectable. With Trf, BSA and Cyt c concentration increasing, unbound ssDNA library peak reduced clearly (Fig. 3A–C). 195 nm and 254 nm detection gave the same results. When Trf and BSA concentration increased in the range of 1–5 M, ssDNA library peak decreased obviously. However, when Cyt c concentration increased to 5 M, the decrease of ssDNA library was negligible, which corresponded to the result of Fig. 2C, where a very small complex peak was visible. Protein concentration increase caused unbound ssDNA reduction, which also illustrated the interactive of protein and ssDNA library. 3.2.3. Interactive comparison Fig. 4A showed Trf–ssDNA and BSA–ssDNA complex peak area increased with ssDNA library concentration increasing. Trf–ssDNA peak area larger than BSA–ssDNA illustrated the stronger interactive of Trf with ssDNA library than BSA. Fig. 4B showed unbound ssDNA peak area decreased with Trf, BSA and Cyt c concentration increasing, the decrease trend also indicated their interactive strength was in the order of Trf > BSA> Cyt c. In contrast, the
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In order to confirm protein–ssDNA complex was collected at the outlet end (under positive voltage) and inlet end (under negative voltage), both collection fractions were analyzed by sensitive LIF detection (conditions see Table 1). Fig. 5 showed the results of collected complex identification. Both Trf–ssDNA 100
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evidence based on protein peak area decrease was not as strong as that of complex peak increase or unbound ssDNA peak decrease, even though protein decrease trend (based on slopes value) followed the same order of Trf > BSA> Cyt c (Fig. 4C). Fig. 4 illustrated that under positive or negative voltage analysis, both complex increase and unbound ssDNA decrease could be used for interactive evaluation and comparison. Since the interaction between protein and ssDNA library is an equilibrium reaction, their concentration ratio will determine specific and nonspecific interaction strength. With positive voltage analysis or negative voltage analysis, the total binding strength between protein target and designed ssDNA library can be illustrated, but specific or nonspecific interaction cannot be identified. To improve specific binding, other follow-up work is always needed, for example, (1) to optimized protein and ssDNA library ratio; (2) with other protein target as negative control; (3) increase selection round, etc.
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Fig. 2. Analysis of three proteins and protein–ssDNA complexes in incubation mixture with ssDNA library concentration increasing under positive voltage with 195 nm and 214 nm UV detection.
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Fig. 3. Analysis of unbound ssDNA library in incubation mixture with three proteins concentration increasing under negative voltage with 195 nm and 214 nm UV detection.
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Fig. 4. Interactive comparison of Trf, BSA and Cyt c with ssDNA library.
and BSA–ssDNA complex fractions gave clear FAM–ssDNA signal, whereas Cyt c–ssDNA complex fraction only gave a negligible peak. CE–LIF detection results confirmed the formation of Trf–ssDNA and BSA–ssDNA complex and their successful collection without unbound ssDNA. 3.4. Protein–ssDNA complex stability in low pH solution In conventional CE, protein and ssDNA library mixture was incubated and separated in the higher pH solution (pH 7–8). Here, in low pH CE, protein and ssDNA library mixture was incubated in pure H2 O but separated in low pH solution. In order to validate the stability of protein–ssDNA complex in 15 min low pH separation, three
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proteins and ssDNA library mixtures incubated in low pH solution for 30 min, respectively, were compared with those incubated in H2 O. Since affinity binding of protein and ssDNA depended mainly on ssDNA secondary structure, low pH solution could result in part protonation of protein and ssDNA base, but it did not impede Trf–ssDNA and BSA–ssDNA complex formation. Trf–ssDNA complex and BSA–ssDNA complex formed in low pH incubation were showed in Fig. 6, which indicated the stability of Trf–ssDNA and BSA–ssDNA in low pH solution, even though smaller peaks of Trf–ssDNA and BSA–ssDNA complex and a little worse resolution of separation were shown comparing with those in H2 O incubation. Therefore, low pH CE separation process less than 15 min would
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Fig. 6. Comparison of three protein–ssDNA complexes formed in pH 2.6 solution and H2 O.
not damage Trf–ssDNA and BSA–ssDNA complex formed in H2 O. Strong basic Cyt c and its complex peaks, however, could not be identified after low pH incubation.
3.5. Collection amount expansion of protein–ssDNA complex In conventional CE, to get high separation efficiency, injection amount of the incubation mixture was very limited. For example, common 5 s injection volume at 50 mbar in Agilent 7100 corresponded to 66 nL, accounted for 6% of total capillary volume. In this work, to collect more protein–ssDNA complex, injection amount was enlarged by increasing injection time. For a 33.5 cm capillary length (effective 25 cm) in Agilent 7100, the maximum injection time 30 s was tried, which corresponded to injection volume 396 nL, accounted for 36% of total capillary volume. Fig. 7 showed peak area (PA) increase of Trf and Trf–ssDNA when injection time increased from 5 s to 25 s, the increase of PA of Trf and Trf–ssDNA was linear with injection time (inj-t), PATrf = 261Xinj-t − 817.4 (R2 = 0.9990) and PATrf-ssDNA = 844.32Xinj-t − 1524.8 (R2 = 0.9992). Comparing with common 5 s injection, 25 s injection obtained 7.26 times more Trf–ssDNA complex. With longer injection time, the electric current became unstable. Above results showed that larger amount of protein–ssDNA complex could be collected just by simply increasing injection time.
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4. Conclusion Low pH CE for SELEX provides a CE-SELEX variant (LpH-CESELEX), which can be used for fast interactive evaluation of protein and ssDNA library and convenient larger amount protein–ssDNA complex collection, improve protein–ssDNA complex separation and collection for CE-SELEX. Comparing with conventional CESELEX, LpH-CE-SELEX has the following advantages: (1) both positive and negative voltage separation can be applied for interactive evaluation before aptamers selection to reduce risk and blindness of aptamers selection; (2) protein–ssDNA complex collection amount can be amplified easily by increasing injection time; (3) reverse migration of protein–ssDNA complex and unbound ssDNA makes complete protein–ssDNA complex collection at cathode without unbound ssDNA involvement, there is no need to exactly control collection time; (4) low pH collection does not affect the recovery of ssDNA in subsequent PCR due to larger amount pH 7.4 HCl–Tris buffer is used in PCR. Acknowledgements This work is supported by the National Natural Science Foundation of China (no. 21175011, 21375008), National Basic Research Program of China (973 program, no. 2012CB910603). References [1] A.D. Ellington, J.W. Szostak, In vitro selection of RNA molecules that binds specific ligands, Nature 346 (1990) 818–822. [2] C. Tuerk, L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 249 (1990) 505–510. [3] P. Sundaram, H. Kurniawan, M.E. Byrne, J. Wower, Therapeutic RNA aptamer in clinical trials, Eur. J. Pharm. Sci. 48 (2013) 259–271. ´ Aptamers molecules [4] F. Radom, P.M. Jurek, M.P. Mazurek, J. Otlewski, F. Jelen, of great potential, Biotechnol. Adv. 31 (2013) 1260–1274. [5] M. Citartan, S.C.B. Gopinath, J. Tominaga, S-C. Tan, T-H. Tang, Assays for aptamer-based platforms, Biosens. Bioelectron. 34 (2012) 1–11. [6] J. Huang, X. Yang, X. He, K. Wang, J. Liu, H. Shi, Q. Wang, Q. Guo, D. He, Design and bioanalytical applications of DNA hairpin-based fluorescent probes, Trends Anal. Chem. 53 (2014) 11–20. [7] G. Aquino-Jarquin, J.D. Toscano-Garibay, RNA aptamer evolution: two decades of SELEction, Int. J. Mol. Sci. 12 (2011) 9155–9171. [8] Y. Liu, C. Wang, F. Li, S. Shen, D.L.J. Tyrrell, X.C. Le, X-F. Li, DNase-mediated single-cycle selection of aptamers for proteins blotted on a membrane, Anal. Chem. 84 (2012) 7603–7606. [9] J. Qian, X. Lou, Y. Zhang, Y. Xiao, H.T. Soh, Generation of highly specific aptamers via micromagnetic selection, Anal. Chem. 81 (2009) 5490–5495. [10] X. Lou, J. Qian, Y. Xiao, L. Viel, A.E. Gerdon, E.T. Lagally, P. Atzberger, T.M. Tarasow, A.J. Heeger, H.T. Soh, Micromagnetic selection of aptamers in microfluidic channels, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 2989–2994. [11] S.D. Mendonsa, M.T. Bowser, In vitro evolution of functional DNA using capillary electrophoresis, J. Am. Chem. Soc. 126 (2004) 20–21. [12] R.K. Mosing, S.D. Mendonsa, M.T. Bowser, Capillary electrophoresis-SELEX selection of aptamers with affinity for HIV-1 reverse transcriptase, Anal. Chem. 77 (2005) 6107–6112.
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