Hybrid Approach Combining Boronate Affinity Magnetic Nanoparticles

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

A Hybrid Approach Combining Boronate Affinity Magnetic Nanoparticles and Capillary Electrophoresis for Efficient Selection of Glycoprotein-Binding Aptamers Xinglin Li, Yunjie He, Yanyan Ma, Zijun Bie, Baorui Liu, and Zhen Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02907 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 4, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A Hybrid Approach Combining Boronate Affinity Magnetic Nanoparticles and Capillary Electrophoresis for Efficient Selection of Glycoprotein-Binding Aptamers Xinglin Li†, Yunjie He‡, Yanyan Ma†, Zijun Bie†, Baorui Liu‡, and Zhen Liu*,†

† State

Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and

Chemical Engineering, Nanjing University, Nanjing 210023, China ‡

Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing

University, Clinical Cancer Institute of Nanjing University, 321 Zhongshan Road, Nanjing 210008, China * Corresponding author: [email protected]

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Capillary electrophoresis (CE) and magnetic beads have been widely used for the selection of aptamers owing to their efficient separation ability. However, these methods alone are associated with some apparent drawbacks. CE suffers from small injection volumes and thereby only a limited amount of aptamer can be collected at each round. While the magnetic beads approach is often associated with tedious procedure and nonspecific binding. Herein we present a hybrid approach that combines the above two classical aptamer selection methods to overcome the drawbacks associated with these methods alone. In this hybrid method, one single round selection by boronate affinity magnetic nanoparticles (BA-MNPs) was first performed and then followed by a CE selection of a few rounds. The BA-MNPs-based selection eliminated non-binding sequences, enriching effective sequences in the nucleic acid library. While the CE selection, which was carried out in free solutions, eliminated steric hindrance effects in subsequent selection. Two typical glycoproteins, Ribonuclease B (RNase B) and alkaline phosphatase (ALP), were used as targets. This hybrid method allowed for efficient selection of glycoprotein-binding aptamers within 4 rounds (1 round of BA-MNPs-based selection and 3 rounds of CE selection) and the dissociation constants reached 10−8 M level. The hybrid selection approach exhibited several significant advantages, including speed, affinity, specificity and avoiding negative selection. Using one of the selected ALP-binding aptamers as an affinity ligand, feasibility for real application of the selected aptamers was demonstrated through constructing an improved enzyme activity assay. 2


Page 2 of 35

Page 3 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Aptamers are DNA (or

RNA) oligonucleotides that are able to bind different classes of

targets such as ions, small molecules, peptides, proteins or cells, with high affinity and specificity.1-3 Aptamers are often viewed as artificial antibodies and hold promise as substitutes of real antibodies in diagnosis and treatment of diseases.4 Nucleic acid aptamers are obtained through in vitro evolution-like protocols called systematic evolution of ligands by exponential enrichment (SELEX), which was first developed in early 1990’s for the selection of RNA5,6 and DNA7 aptamers. A huge number of methods have been developed to select aptamers, such as membrane filtration,5 affinity chromatography,6 magnetic beads,8-9 capillary electrophoresis (CE),10-12 surface plasmon resonance (SPR),

13

and

droplet microfluidics.14 Some new methods with significantly reduced rounds of SELEX have been continuously emerging, such as non-SELEX,15-17 high throughput sequencing (HTS)-based SELEX,18 on-chip SELEX,19 and fraction collection approach in CE-SELEX.20 Magnetic beads-based SELEX and CE-SELEX are two widely used selection methods. In magnetic beads-based methods,21,22 the target is immobilized on magnetic beads, bound sequences are isolated from unbound sequences through magnetic separation, and the bound sequences are released for PCR amplification. This method has some advantages, including easy-to-operate procedure and high success rate. However, it suffers from some apparent drawbacks, including time-consuming process and strong nonspecific

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

binding toward oligonucleotides. In CE-SELEX,10-12,20 bound and unbound sequences are separated by virtue of differential electrophoretic mobility and the bound sequences are collected for PCR amplification. CE-SELEX has several advantages, including fast selection speed, less nonspecific binding and possibility of determining the dissociation constants (Kd).23 However, it is also associated with some disadvantages. A major one is that only limited amounts of the aptamers can be collected at each round due to the small injection volume so that it is often hard to see the peaks for the target-aptamer complex.24 Clearly, new approaches that can combine the advantages of these methods and meanwhile overcome their drawbacks are of significant importance. Glycoproteins play essential roles in diverse biological processes, such as molecular recognition, inter- and intra-cell signaling, immune response, and so on.25 In addition, the occurrence of many diseases accompanies with change in the glycosylation state of related proteins, thus a lot of glycoproteins are of significance in clinic diagnositics.26 For instance, quite a lot of glycoproteins have been routinely used as disease biomarkers,27,28 such as -fetoprotein (AFP) and alkaline phosphatase (ALP). Therefore, efficient selection of glycoprotein-binding aptamers is of great importance. Boronate affinity materials have been widely used for the selective isolation and molecular recognition of cis-diol-containing compounds (particularly glycoproteins) with several significant advantages, including broad-spectrum selectivity, reversible covalent binding, pH-controlled capture/release, fast association/desorption kinetics, and good 4


Page 4 of 35

Page 5 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


compatibility with mass spectrometry.29 A variety of new boronate affinity materials, such as macroporous monoliths,30-32 mesoporous materials,33,35 nanoparticles,36,37 molecularly imprinted polymers,38-40 have been developed rapidly for application in affinity separation,30-32 proteomics,41,42 metabolomics,43 and cell/tissue imaging.44 Boronate affinity materials also have already exhibited huge potential for aptamer selection. In a previous study by our group,45 boronate affinity monolithic capillaries have been demonstrated to be an efficient platform for the selection of glycoprotein-binding aptamers. The selection was finished by 6 rounds in two days and the dissociation constants reached the 10−8 M level. The advantages of boronate affinity monolithic column-based aptamer SELEX are obvious, including rapid selection speed, high specificity toward the target and facile capture/release of glycoproteins in a pH switchable fashion (high pH on, low pH off). Therefore, it is very necessary to further explore the feasibility and merits of other formats of boronate affinity materials for the selection of glycoprotein-binding aptamers. Herein, we report a hybrid approach that combines BA-MNPs-based selection with CE-based SELEX for efficient selection of glycoprotein-binding aptamers. The principle of the approach is shown in Figure 1. One round of BA-MNPs-based selection is first carried out. The target glycoprotein is immobilized onto BA-MNPs at an alkaline pH, and then a 5′-FAM-labeled ssDNA library is incubated with the BA-MNPs for a certain duration. After washing away nonspecifically bound ssDNA, the target-ssDNA complexes are collected through elution with an acidic solution. The complexes are submitted to PCR amplification 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and the obtained PCR products are sent for subsequent CE-SELEX. In the CE-SELEX, the PCR products of the previous round are incubated with the target glycoprotein for a certain period and then injected into the separation capillary for CE separation. The target-ssDNA complexes are collected into a PCR tube containing PCR mixture and then submitted to PCR amplification. The PCR products are evaluated by CE and then sent for a new round CE-SELEX until the binding affinity meets the requirement or will not increase any more. Finally, the obtained ssDNA with desired affinity is sent to cloning and sequencing. In other words, in this hybrid approach, one round of BA-MNPs-based selection is introduced into conventional CE-SELEX. Due to the involvement of one round of BA-MNPs-based selection, ssDNA that is specific to the target glycoprotein in the library is effectively enriched prior to the CE-based selection. As a result, the subsequent CE-selection becomes more efficient. On the other hand, due to the fact that CE-SELEX is carried out in free solution, steric hindrance effects due to target immobilization in the BA-MNPs-based selection can be eliminated. Thus, the hybrid approach can overcome the major drawbacks of conventional magnetic beads-based and CE-based selection methods. Two typical glycoproteins, RNase B and ALP, were used as targets. Aptamers with high specificity and affinity towards these two target proteins were selected within 4 rounds (1 round of BA-MNPs-based selection and 3 rounds of CE selection). The hybrid selection approach exhibited several merits, including rapid selection speed (only 2 days are needed), high affinity, high specificity, and avoiding negative selection. Feasibility of the selected 6


Page 6 of 35

Page 7 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aptamers for real application was demonstrated through an improved enzyme activity assay of ALP in human serum using one of the selected ALP-binding aptamers as the affinity ligand.

EXPERIMENTAL SECTION Reagents and Materials. RNase B, RNase A, and β-casein were obtained from Sigma-Aldrich (St. Louis, MO, USA). ALP (EC 3.1.3.1, specific activity 3000 U/mg) from calf intestinal mucosa, Glycine was purchased from Bio-Rad (Hercules, CA, USA). TaKaRa Taq™ enzyme and dNTP were from Takara Biotechnology (Dalian, China). The DNA library and the primers were purchased from Sangon (Shanghai, China). The DNA library was composed of 39 random nucleotides in the central and two constant primer regions at both ends. The sequence of the library and the primers were listed below: the DNA

library:

5′-FAM-CTTCTGCCCGCCTCCTTCC-(N39)

-GGAGACGAGATAGGCGGACACT-3′;

Primer

1:

5′-FAM

-CTTCTGCCCGCCTCCTTCC-3′; Primer 2: 5′-CTTCTGCCCGCCTCCTTCC-3′; Primer 3: 5′-AGTGTCCGCCTATCTCGTCTCC-3′. Other chemical reagents were of analytical grade. The DNA library and target proteins were dissolved in the loading buffer respectively (50 mM NH4HCO3 containing 50 mM NaCl, 1 mM MgCl2, pH 8.5 for RNase B selection and 50 mM glycine, 50 mM NaOH, containing 145 mM NaCl, pH 9.5 for ALP selection). Before selection, the DNA library (or PCR products) was heated at 94 °C for 10 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

min to denature the DNA library and then slowly cooled to room temperature. All the water used throughout this work was purified by a Milli-Q Advantage A10 system (Millipore, Milford, MA, USA). CE running buffer contained 25 mM Na2B4O7, 25 mM glycine and 5 mM KH2PO4 (pH 9.0) for RNase B and 25 mM Tris, 192 mM glycine and 25 mM NaCl (pH 8.5) for ALP, which was filtered through a hydrophilic membrane filter with pore size of 0.45 μm. Fused-silica capillaries of 75 μm ID × 365 μm OD were purchased from Yongnian Optical Fiber Factory (Hebei, China).

Instruments. CE-SELEX, CE evaluation and dissociation constant measurements were conducted on a P/ACE MDQ system (Beckman Coulter, Fullerton, CA, USA) which equipped with a photo diode array (PDA) detector and a laser-induced fluorescence (LIF) detector. The PDA detection wavelength was set at 214 nm. For LIF detection, the excitation wavelength was 488 nm whereas the emission wavelength was set at 520 nm. PCR amplification was performed on GeneAmp 9700 instrument (ABI, Shanghai, China).

Hybrid SELEX. In the hybrid method, one single round of BA-MNPs-based selection was first performed. For the selection of RNase B-binding aptamers, BA-MNPs (2 mg) were dispersed into 200 μL of 1 mg/mL RNase B dissolved in the loading buffer (50 mM NH4HCO3 containing 50 mM NaCl, 1 mM MgCl2, pH 8.5), then the mixture was shocked on a rotator at room temperature for 1 h and unbound RNase B was washed away by 8


Page 8 of 35

Page 9 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


applying 200 μL of the loading buffer for three times. Then the BA-MNPs were incubated with 100 μL of 1 μM DNA library dissolved in the loading buffer for 1 h. Subsequently the MNPs were washed in the same way to remove the unbound ssDNA for three times. Finally, the bound species (in the form of protein-ssDNA complexes) were eluted with 20 μL of 100 mM HAc solution and collected in PCR tubes (200 μL each). The bound species were submitted to PCR amplification, and then the amplified species were sent for subsequent SELEX by CE. ALP-binding aptamers were selected in the same procedure expect that the loading buffer was exchanged to 50 mM glycine, 50 mM NaOH, containing 145 mM NaCl, pH 9.5. After BA-MNPs-based selection, the selected ssDNA was PCR amplified and the amplified species were subjected to CE-SELEX. CE running buffer for the selection of RNase B-binding aptamers was the same as previously reported,45 i.e., 25 mM Na2B4O7 containing 25 mM glycine and 5 mM KH2PO4, pH 9.0. CE running buffer for the selection of ALP-binding aptamers was obtained through a brief optimization. Briefly, as the ALP and ssDNA library were not separated well in the running buffer for the selection of RNase B-binding aptamers, different concentrations of NaCl (0, 25, 50, 100 mM) and different pH values (pH 9.0, 8.5, 8.0, 7.5 and 7.0) of the running buffer were tried, but the separation was still unsatisfied. Then, the buffering agent Na2B4O7 was replaced with Tris and different concentrations of NaCl (25, 50, 100 mM) in the running buffer were tried. The buffer of 25 mM Tris containing 192 mM glycine and 25 mM NaCl (pH 8.5) gave the best 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

separation of ALP and the ssDNA library. A key step prior to CE-SELEX was to determine the aptamer collection time window under the CE conditions employed. Before experiments each day, the separation capillary was first flushed with 0.1 M NaOH at 20 psi for 20 min and CE separation buffer for 20 min. The capillary was rinsed with 0.1 M NaOH for 3 min and the CE running buffer for 3 min at 20 psi before each separation. The potential was 24 kV and the capillary was kept at 25 °C. Then the target protein (RNase B or ALP) and the FAM-labeled ssDNA were injected into the capillary to determine their migration time respectively. First, 1 mg/mL of target protein (CE running buffer of RNase B, 25 mM Na2B4O7, 25 mM glycine and 5 mM KH2PO4, pH 9.0 and CE running buffer of ALP, 25 mM Tris, 192 mM glycine and 25 mM NaCl, pH 8.5) was injected and separated with UV absorbance detection at 214 nm. Second, 1 μM FAM-labeled ssDNA was injected and separated with LIF detection at 488 nm excitation and 520 nm emission. The aptamer collection time window was set between the migration time of the target protein and FAM-labeled ssDNA. After that, the PCR products were incubated with target protein of appropriate concentration for 1 h and injected into the capillary by pressure of 5 psi for 5 s. CE-SELEX was monitored using LIF detection at 488 nm excitation and 520 nm emission. The bound species were collected into a PCR tube containing 47.5 μL of PCR mixture (0.2 mM dNTP, 0.025 U/L TaKaRa enzyme, 0.01 M primer 1 and 0. 1 nM primer 3) and submitted to PCR amplification. The PCR amplified species were evaluated by CE and then sent for a new round of CE selection until the binding affinity met the requirement or would 10


Page 10 of 35

Page 11 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


not increase any more. The RNase B-binding aptamers obtained at each round by the hybrid method were incubated with RNase B for 1 h and then evaluated by CE-LIF. Conventional CE-SELEX for the selection of RNase B-binding aptamers was performed by otherwise identical procedure as described above except that the BA-MNPs-based SELEX was absent. The aptamers obtained at each round were evaluated in the same way as described above.

Asymmetry PCR Amplification and ssDNA Formation. All eluates that contained the protein-ssDNA complexes were amplified by asymmetry PCR. Asymmetry PCR reagents were added to the eluates to get 0.2 mM dNTP, 0.025 U/L TaKaRa enzyme, 0.01 M primer 1 and 0.1 nM primer 3. Amplification conditions were: 5 min at 95 °C; 25 cycle of 30 s at 94 °C, 30 s at 60 °C , 30 s at 72 °C; 5 min at 72 °C. Amplified dsDNA was converted to ssDNA by heating them at 94 °C for 10 min and quickly cooled to -20 °C.

RESULTS AND DISCUSSION Characterization of BA-MNPs. As shown in Figures S1A and S1B, the average diameter of the BA-MNPs was determined by TEM to be ~100 nm, which is in good agreement with the diameter obtained previously.37 Figure S1C shows that the BA-MNPs dispersed well in water and were quickly attracted to the wall of the glass container by an external magnet. The selectivity of the BA-MNPs was critical to the aptamer selection in this study. As 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

shown in Figure S2, the BA-MNPs selectively captured adenosine (a typical cis-diol compound) but excluded deoxyadenosine (a typical non cis-diol compound), indicating successful functionalization of the MNPs with boronate acid. As shown in Figure S1D, both RNase B and ALP were captured by the BA-MNPs at alkaline pH’s (pH 8.5 for RNase B and pH 9.5 for ALP) but released at an acidic pH (2.7) while the DNA library dissolved in the loading buffer was not captured. Due to this desired selectivity, the BA-MNPs allowed for selective pre-immobilization of the target glycoproteins as well as selection of target-specific ssDNA.

Selection of Experimental Conditions for the CE-SELEX. Appropriate separation conditions are essential for CE-based aptamer selection. Unbound ssDNA, free target protein and target-ssDNA complexes can be well separated under the conditions used. Also, a well-defined aptamer collection time window is critical for the selection. RNase B, which has a basic isoelectric point (pI = 8.9),39 and ssDNA, which exhibits apparent pI in the range of 4-5,46 were well separated by CE with the conditions previously used45 (See the Experimental Section for detailed conditions). As shown in Figure 2A, free RNase B exhibited multiple peaks due to its microheterogeneity of glycosylation, which appeared at 3.8-5.0 min; the free ssDNA exhibited one sharp peak and one broad peak, and it migrated to the detection window at 10.2-12.0 min; whereas the RNase B-DNA complex exhibited a sharp peak at 7.4 min. So the aptamer collection time window was determined to between 6 12


Page 12 of 35

Page 13 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and 8 min. As a comparison, the ALP used exhibits a pI value of 4.4-5.8,47-49 CE separation of ALP with its binding species is relatively difficult. Due to the high separation efficiency of CE, satisfactory CE separation was obtained through brief optimization of the separation conditions (See the Experimental Section for detailed conditions). As shown in Figure 2B, the free ALP exhibited two partially-resolved peaks at 3.3-3.7 min; free ssDNA exhibited a major and a minor peak located at 9.0-10.3 min; while the protein-DNA complexes exhibited four sharp peaks appeared at 6.4, 7.1, 7.9 and 8.3 min. So the aptamer-target collection time window was determined to be between 6 and 9 min.

Aptamer Selection by the Hybrid SELEX. RNase B-binding aptamers and ALP-binding aptamers were selected by the hybrid SELEX. The affinity of the selected aptamers towards the target glycoproteins at each round of CE-SELEX was evaluated using CE. Data fitting for the measurement of binding constants is exemplified with the aptamers selected at the second and third round of CE-SELEX (Figures S3 and S4). The bulk Kd values of the selected aptamers are shown in Figure 3. Two aspects of these results verified the effectiveness of the CE-SELEX. On the one hand, within the initial three round of CE-SELEX, the bulk Kd values decreased as the number of selection rounds increased. On the other hand, the deviation associated with the bulk Kd values also decreased as the number of selection rounds increased. For both RNase B-binding aptamers and ALP-binding aptamers, only three rounds of CE-selection were needed, which provided 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

bulk Kd value at the 10−8 M level. Further selection did not further decrease the Kd values. Thus, ssDNA selected at the third round of CE-SELEX or the fourth round of the hybrid SELEX was cloned and sequenced. The selectivity of the PCR products of finally selected ssDNA was evaluated by CE with laser-induced fluorescence (LIF) detection. As shown in Figure S5, three tiny peaks were observed for RNase A-ssDNA complexes while no apparent peaks were observed for -casein-ssDNA complexes. As shown in Figure S6, four tiny peaks were observed for RNase A-ssDNA complexes while one tiny peak was observed for -casein-ssDNA complexes. These results suggest good specificity of the selected ssDNA toward the target glycoproteins.

Characterization of the Selected Aptamers. After one round of BA-MNPs-based selection and three rounds of CE selection, the aptamers selected were sent to cloning and sequencing and three aptamers for each target glycoprotein were obtained. Their sequences and the experimental Kd values are listed in Table S1, while their predicted secondary structures are shown in Table S2. Since these clones were chosen randomly, they can be considered as a true statistical sampling of the selected pool as a whole. The Kd values for individual aptamers are in good agreement with the bulk Kd values for ssDNA selected at the third round of the CE-SELEX. These results indicate that the hybrid SELEX approach was effective. The affinity and selectivity of the selected aptamers towards the target 14


Page 14 of 35

Page 15 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


proteins as well as some competing proteins were evaluated by CE-LIF. Aptamer R1 for RNase B and aptamer A1 for ALP were chosen as representatives of the selected DNA sequences. As shown in Figure 4, aptamer R1 exhibited the highest affinity towards RNase B as compared with the two competing proteins and aptamer A1 exhibited the highest affinity towards ALP, while the DNA library had nearly the same binding affinity to the three proteins. These results also suggest that the aptamers selected by the hybrid SELEX method had good specificity to the target proteins.

Superiority of the Hybrid SELEX over CE-SELEX alone. The advantage of the hybrid SELEX over conventional CE-SELEX was investigated by comparing the selection of RNase B-binding aptamers by the two methods. As shown in Figure 5, the hybrid SELEX approach is clearly more advantageous than the conventional CE-SELEX. Even at the first round of CE selection, the amount of the selected ssDNA by the hybrid approach was about 11 times higher than that by the CE method. Moreover, the ssDNA obtained at each round by the hybrid approach exhibited a single sharp peak at fixed migration time, suggesting that the selected ssDNA had low microheterogeneity. This is attributed to the enrichment effect in the BA-MNPs-based selection. As a comparison, the ssDNA obtained at each round by the CE selection exhibited multiple relatively broad peaks at varying migration time, suggesting high microheterogeneity of the selected ssDNA.

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Superiority of the Hybrid SELEX over Magnetic Beads-based Selection alone. The advantage of the hybrid SELEX over magnetic beads-based selection alone can be rationalized from several aspects. First, as shown in Figure S5, the RNase B-binding aptamers selected by the hybrid SELEX exhibited low cross-reactivity towards RNase A. RNase A and RNase B share an identical polypeptide core. The only difference between the two analogues is that RNase B contains one N-linked glycan chain but RNase A does not. During the BA-MNPs-based selection, the target RNase B was immobilized on the surface of BA-MNPs through boronate affinity interaction between the BA-MNPs and the only glycan chain. Thus, most of the ssDNA selected by this step could bind with not only RNase B but also RNase A. If subsequent CE-SELEX had not been performed and only BA-MNPs-based selection had been performed (with more rounds), the ssDNA selected would not have been able to differentiate RNase B from RNase A. Instead, in the hybrid method, subsequent CE-SELEX, which was carried out in free solutions, was performed after the BA-MNPs-based selection, thereby ssDNA that could bind with RNase A were largely excluded. Second, traditional magnetic beads-based SELEX often requires 8-12 rounds of selection,50 whereas only 4 rounds were needed in the hybrid SELEX. Third, magnetic beads are usually associated with non-specific binding towards ssDNA. To reduce or eliminate the effect of non-specific binding, negative selection is usually required by magnetic beads-based selection.8-9,51,52 As a comparison, negative selection is not necessary in the hybrid SELEX, due to the desirable selectivity of the BA-MNPs and the selection in 16


Page 16 of 35

Page 17 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


free solution in the CE-SELEX.

Comparison with boronate affinity monolithic capillary-based selection. Compared to the boronate affinity monolithic capillary based SELEX we proposed previously,45 the hybrid selection method showed several merits. First, the hybrid approach is more efficient. It took only 4 rounds of selection but the boronate affinity monolithic capillary-based SELEX needed 6 rounds. Second, the hybrid approach is free of non-specific binding of ssDNA due to nanoconfinement effect. It has been revealed that nanoscale pores in monolithic columns are associated with significant confinement effect.53 ssDNA may be retarded on the monolithic capillary due to non-specific nanoconfinement effect. Third, the hybrid approach may provide aptamers with high specificity towards the target. In the boronate affinity monolithic capillary-based SELEX, the target was immobilized on the monolith at each round of selection and thus the binding of ssDNA with some regions of the target could be hampered due to steric hindrance.

Aptamer-based Enzyme Activity Assay of ALP. Conventional enzyme activity assay directly adds the substrate into the sample under investigation. Biological samples, particularly human plasma and serum, usually contain plentiful species that may interfere with the detection of product of the enzymatic reaction. For instance, it has been revealed that the coexistence of glucose and amino acids apparently inhibits the enzyme activity of 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

alkaline phosphatase (ALP).54-55 An antibody specific to ALP has been used to fish out the enzyme to eliminate the interference of the sample matrix,56-57 but antibodies suffer from some drawbacks such as poor stability, difficulty in immobilization and high cost. In a previous study, selective removal of interfering species from the sample matrix by a molecularly imprinted polymer has been demonstrated to be an important means to get reliable analytical results.58 To demonstrate the feasibility of the aptamers selected for real-word applications, the ALP-binding aptamer selected herein was employed as an affinity ligand to specifically capture ALP for constructing new enzyme activity assay. Among the ALP-binding aptamers selected, aptamer A1 was chosen as the affinity ligand due to its highest affinity. The aptamer was first modified at its 3′ end with a thiol-containing moiety. The thiol-modified aptamer was then immobilized onto gold layer-modified inner liners (see the Experimental section). The obtained inner liners were mounted onto a 96-well microplate (Figure S7). Human serum samples were added to the wells for enzyme activity assay. Because the ALP-binding aptamer on the inner liners allowed us to selectively capture ALP from the human serum samples, the interference of the sample matrix was effectively eliminated. A calibration curve was first established, which showed good linearity within 0-200 U/L (R2 = 0.99) (Figure S8). Then the aptamer-modified wells were used for the assay of ALP in a human serum sample. For example, the ALP concentration in the serum sample from a healthy individual was determined to be 77.9 ± 6.9 U/L by the standard addition method (Figure S9). Considering 18


Page 18 of 35

Page 19 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the 2-fold dilution prior to the assay, the ALP concentration in normal human serum was calculated to be 155.8 ± 13.8 U/L, which is consistent with the ALP level for adults (40-190 U/L).59 In order to further verify our method, the ALP concentrations in the serum from healthy individuals (n = 5) and patients with osteosarcoma (n = 5) were measured. The results as well as those by the commercial ALP detection kit are listed in Table 1. Overall, the results by our method are comparable to the results by the commercial kit. However, most of the results by our method are slightly lower than those by the kit. This is probably due to the elimination of the interference of the sample matrix by the aptamer-based method. Besides, the results by the aptamer-based enzyme activity assay clearly showed that the ALP level in patients with osteosarcoma was apparently than that in healthy individuals, which is in agreement with previously reports.60,61

CONCLUSION We

have

established

a

hybrid

SELEX

method

for

efficient

selection

of

glycoprotein-binding aptamers through combining magnetic beads-based selection and CE-based SELEX. As an advanced functional material, the BA-MNPs allowed for facile capture/release of glycoproteins in a pH switchable fashion, and thus the BA-MNPs-based selection eliminated non-binding sequences, enriching effective sequences in the nucleic acid library. The subsequent CE-based selection, which was carried out in free solutions, avoided steric hindrance effects in subsequent selection. Due to these merits, the proposed 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

method effectively overcame the major drawbacks of the conventional magnetic beads-SELEX and CE-SELEX. The efficiency of the hybrid selection approach was experimentally confirmed through selecting aptamers specifically bound to two typical glycoproteins, RNase B and ALP. The selected aptamers showed high affinity and high specificity. Using one of the selected ALP-binding aptamers as affinity ligand, the improved enzyme activity assay of ALP within human serum well demonstrated the feasibility of the selected aptamers for application to real complex biosamples. Therefore, the hybrid SELEX approach can be a promising tool for efficient selection of high-performance aptamers.

AUTHOR INFORMATION * Corresponding Author Tel.: +86 25 8968 5639; fax: +86 25 8968 5639. E-mail address: [email protected] (Z. Liu).

ACKNOWLEDGEMENTS We acknowledge the financial support of the National Science Fund for Distinguished Young Scholars (No. 21425520) from the National Natural Science Foundation of China and the Key Grant of 973 Program (No. 2013CB911202) from the Ministry of Science and Technology of China. 20


Page 20 of 35

Page 21 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ASSOCIATED CONTENT Supporting Information. Experimental details, characterization of BA-MNPs, binding isotherms, CE analysis, photographs of the aptamer-modified 96-well microplate and enzyme activity assay are given in the supporting information.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

REFERENCES (1) Famulok, M.; Mayer, G.; Blind, M. Acc. Chem. Res. 2000, 33, 591-599. (2) Gold, L.; Brody, E.; Heilig, J.; Singer, B. Chem. Biol. 2002, 9, 1259-1264. (3) Ellington, A. D.; Conrad, R. Biotechnol. Annu. Rev. 1995, 1, 185-214. (4) Rusconi, C. P.; Scardino, E.; Layzer, J.; Pitoc, G. A.; Ortel, T. L.; Monroe, D.; Sullenger, B. A. Nature 2002, 419, 90-94. (5) Tuerk, C.; Gold, L. Science 1990, 249, 505-510. (6) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818-822. (7) Ellington, A. D.; Szostak, J. W. Nature 1992, 355, 850-852. (8) Huang, C. J.; Lin, H. I.; Shiesh, S. C.; Lee, G. B. Biosens. Bioelectron. 2012, 35, 50-55. (9) Song, Y. L.; Zhu, Z.; An, Y.; Zhang, W. T.; Zhang, H. M.; Liu, D.; Yu, C. D.; Duan, W.; Yang, C. J. Anal. Chem. 2013, 85, 4141-4149. (10) Mendonsa, S. D.; Bowser, M. T. Anal. Chem. 2004, 76, 5387-5392. (11) Mendonsa, S. D.; Bowser, M. T. J. Am. Chem. Soc. 2005, 127, 9382-9383. (12) Mallikaratchy, P.; Stahelin, R. V.; Cao, Z. H.; Cho, W.; Tan, W. H. Chem. Commun. 2006, 3229-3231. (13) Misono, T. S.; Kumar, P. K. R. Anal. Biochem. 2005, 342, 312-317. (14) Zhang, W. Y.; Zhang, W. H.; Liu, Z. Y.; Li, C.; Zhu, Z.; Yang, C. J. Anal. Chem. 2012, 84, 350-355 (15) Berezovski, M.; Drabovich, A.; Krylova, S. M.; Musheev, M.; Okhonin, V.; Petrov, A.; Krylov, 22


Page 22 of 35

Page 23 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


S. N. J. Am. Chem. Soc. 2005, 127, 3165-3171. (16) Berezovski, M.; Musheev, M.; Drabovich, A.; Krylov, S. N. J. Am. Chem. Soc. 2006, 128, 1410-1411. (17) Berezovski, M. V.; Musheev, M. U.; Drabovich, A. P.; Jitkova, J. V.; Krylov, S. N. Nat. Protoc. 2006, 1, 1359-1369. (18) Hoon, S.; Zhou, B.; Janda, K. D.; Brenner, S.; Scolnick, J. Biotechniques 2011, 51, 413-416. (19) Lou, X. H.; Qian, J. R.; Xiao, Y.; Viel, L.; Gerdon, A. E.; Lagally, E. T.; Atzberger, P.; Tarasow, T. M.; Heeger, A. J.; Soh, H. T. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 2989-2994. (20) Luo, Z. F.; Zhou, H. M.; Jiang, H.; Ou, H. C.; Li, X.; Zhang, L. Y. Analyst 2015, 140, 2664-2670. (21) Tang, J. J.; Xie, J. W.; Shao, N. S.; Yan, Y. Electrophoresis 2006, 27,1303-1311. (22) Gopinath, S. C. B. Anal. Bioanal. Chem. 2007, 387, 171-182. (23) Drabovich, A.; Berezovski, M.; Krylov, S. N. J. Am. Chem. Soc. 2005, 127, 11224-11225. (24) Tok, J.; Lai, J.; Leung, T.; Li, S. F. Y. Electrophoresis 2010, 31, 2055-2062. (25) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370-2376. (26) Ludwig, J. A.; Weinstein, J. N. Nat. Rev. Cancer 2005, 5, 845-856. (27) Germann, U. A. Eur. J. Cancer 1996, 32A, 927-944. (28) Cordon-Cardo, C.; O’Brien, J. P.; Boccia, J.; Casals, D.; Bertino, J. R.; Melamed, M. R. J. Histochem. Cytochem. 1990, 38, 1277-1287.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(29) Li, D. J.; Chen, Y.; Liu. Z. Chem. Soc. Rev. 2015, 44, 8079-8123 (30) Ren, L. B.; Liu, Z.; Liu, Y. C.; Dou, P.; Chen, H. Y. Angew. Chem. Int. Ed. 2009, 48, 6704-6707. (31) Li, H. Y.; Liu, Y. C.; Liu, J.; Liu, Z. Chem. Commun. 2011, 47, 8169-8172. (32) Li, D. J.; Li, Y.; Li, X. L.; Bie, Z. J.; Pang, X. H.; Zhang, Q.; Liu, Z. J. Chromatogr. A. 2015, 1384, 88-96. (33) Xu, Y. W.; Wu, Z. X.; Zhang, L. J.; Lu, H. J.; Yang, P. Y.; Webley, P. A.; Zhao, D. Y. Anal. Chem. 2009, 81, 503-508. (34) Liu, L. T.; Zhang, Y.; Zhang, L.; Yan, G. Q.; Yao, J.; Yang, P. Y.; Lu, H. J. Anal. Chim. Acta 2012, 753, 64-72. (35) Tan, J.; Wang, H. F.; Yan, X. P. Anal. Chem. 2009, 81, 5273-5280. (36) Liang, L.; Liu, Z. Chem. Commun. 2011, 47, 2255-2257. (37) Bie, Z. J.; Chen, Y.; Ye, J.; Wang, S. S.; Liu, Z. Angew. Chem. Int. Ed. 2015, 54, 10211-10215. (38) Wulff, G. Pure Appl. Chem. 1982, 54, 2093-2102. (39) Li, L.; Lu, Y.; Bie, Z. J.; Chen, H. Y.; Liu, Z. Angew. Chem. Int. Ed. 2013, 52, 7451-7454. (40) Wang, S. S.; Ye, J.; Bie, Z. J.; Liu, Z. Chem. Sci. 2014, 5, 1135-1140. (41) Zhang, L. J.; Xu, Y. W.; Yao, H. L.; Xie, L. Q.; Yao, J.; Lu, H. J.; Yang, P. Y. Chem. Eur. J. 2009, 15, 10158-10166. (42) Wang, Y. L.; Liu, M. B.; Xie, L. Q.; Fang, C. Y.; Xiong, H. M. Lu, H. J. Anal. Chem. 2014, 86, 2057-2064.

24


Page 24 of 35

Page 25 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(43) Jiang, H. P.; Qi, C. B.; Chu, J. M.; Yuan, B. F.; Feng, Y. Q. Sci. Rep. 2015, 5, 7785-7793. (44) Yin, D. Y.; Wang, S. S.; He, Y. J.; Liu, J.; Zhou, M.; Ouyang, J.; Liu, B. R.; Chen, H. Y.; Liu, Z. Chem. Commun. 2015, 51, 17696-17699. (45) Nie, H. Y.; Chen, Y.; Lü, C. C.; Liu, Z. Anal. Chem. 2013, 85, 8277-8283. (46) Liang, L.; Dou, P.; Dong, M. M.; Ke, X. K.; Bian, N. S.; Liu, Z. Anal. Chim. Acta 2009, 650, 106-110. (47) Latner, A. L.; Parsons, M.; Skillen, A. W. Enzymologia 1970, 40, 1-6. (48) Lazdunski, M.; Brouillard, J.; Ovellet, L. Canadian J. Chem. 1965, 43, 2222-2235. (49) Besman, M.; Coleman, J. E. J. Biol. Chem. 1985, 260, 11190-11193. (50) Bowser, M. T. Analyst 2005, 130, 128-130. (51) Qian, J. R.; Lou, X. H.; Zhang, Y. T.; Xiao, Y.; Soh, H. T. Anal. Chem. 2009, 81, 5490-5495. (52) Chen, X. J.; Huang, Y. K.; Duan, N.; Wu, S. J.; Ma, X. Y.; Xia, Y.; Zhu, C. Q.; Jiang, Y.; Wang, Z. P. Anal. Bioanal. Chem. 2013, 405, 6573-6581. (53) Li, Q. J.; Tu, X. Y.; Ye, J.; Bie, Z. J.; Bi, X. D.; Liu, Z.; Chem. Sci. 2014, 5, 4065-4069. (54) Pollak, A.; Coradello, H.; Leban, J.; Maxa, E.; Sternberg, M.; Widhalm, K.; Lubec, G. Clin. Chim. Acta 1983, 133, 15-24. (55) Hoylaerts, M. F.; Manes, T.; Millan, J. L. Clin. Chem. 1992, 38, 2493-2500. (56) Gordon, K.; Balyasnikova, I. V.; Nesterovitch, A. B.; Schwartz, D. E.; Sturrock, E. D.; Danilov, S. M. Tissue Antigens 2010, 75, 136-150. (57) Stevens, J.; Danilov, S.; Fanburg, B. L.; Lanzillo, J. J. J. Immunol. Methods 1990, 132,

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

263-273. (58) Bi, X. D.; Liu, Z. Anal. Chem. 2014, 86, 12382-12389. (59) Hausamen, T. U.; Helger, R.; Rick, W.; Gross, W. Clin. Chim. Acta 1967, 15, 241-245. (60) Min, D. L.; Lin, F.; Shen, Z.; Zheng, S.; Tan, L. N.; Yu, W. X.; Yao, Y. Asia-Pac. J. Clin. Oncol. 2013, 9, 71-79. (61) Pochanugool, L.; Subhadharaphandou, T.; Dhanachai, M.; Hathirat, P.; Sangthawan, D.; Pirabul, R.; Onsanit, S.; Pornpipatpong, N. Clin. Orthop. Relat. R. 1997, 345, 206-214.

26


Page 26 of 35

Page 27 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Captions Figure 1. Schematic illustration of the hybrid SELEX approach.

Figure 2. CE electropherograms of the selection of RNase B-binding aptamers (A) and ALP-binding aptamers (B). i) FAM labeled DNA library, ii) glycoprotein-ssDNA complex at the third round of hybrid SELEX and iii) glycoprotein-ssDNA complex at the fourth round of hybrid SELEX, iv) glycoprotein standard (detected by UV absorbance). Conditions for (A): separation buffer, 25 mM Na2B4O7, 25 mM glycine and 5 mM KH2PO4, pH 9.0; RNase B, 1 mg/mL; ssDNA 1 μM; complex, 1 mg/mL RNase B and the corresponding PCR products, incubated for 1 hour. Conditions for (B): separation buffer, 25 mM Tris, 192 mM glycine and 25 mM NaCl, pH 8.5. ALP, 1 mg/mL; DNA library, 1 μM; complex, 1 mg/mL ALP and the corresponding PCR products, incubated for 1 hour. All the curves have been artificially shifted up to fit them on the graph.

Figure 3. Bulk Kd values for the ssDNA selected at different CE-SELEX cycle in the hybrid selection approach. A) RNase B-binding aptamers, B) ALP-binding aptamers.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Comparison of the affinity of the selected aptamers and the ssDNA library towards different proteins. (A) RNase B-binding aptamer R1; (B) ALP-binding aptamer A1; (C) the ssDNA library. 3 L FAM-labeled aptamer or DNA library was incubated with each protein at different concentrations for 1 h and then analyzed by CE-LIF. Separation buffer: 25 mM Na2B4O7, 25 mM glycine and 5 mM KH2PO4 (pH 9.0) for RNase B and 25 mM Tris, 192 mM glycine and 25 mM NaCl (pH 8.5) for ALP and the ssDNA library.

Figure 5. Comparison of the efficiency of (A) the hybrid approach and (B) CE-based SELEX alone for the selection of RNase B-binding aptamers. Sample: i) FAM-labeled ssDNA library; ii) RNase B-ssDNA complex at the first round SELEX; iii) RNase B-ssDNA complex at the second round SELEX; iv) RNase B-ssDNA complex at the third round ofSELEX. Separation buffer: 25 mM Na2B4O7, 25 mM glycine and 5 mM KH2PO4, pH 9.0. All the curves have been artificially shifted up to fit them on the graph. The peaks in B slightly shifted towards the longer times, which was probably due to slight change in the pH of the running buffer.

28


Page 28 of 35

Page 29 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

29



A 1.0

0.10

Intensity / RFU

0.08

iv

0.6

0.06

iii

0.4

0.04 complex

ii

0.2

0.02 ssDNA

i

0.0 0

Absorbance / AU

RNase B

0.8

0.00 2

4

6

8

10

12

Time / min 0.10

2.4

0.08

ALP

iv

1.8

0.06

1.2

iii

0.6

ii

0.04

complex

0.02 ssDNA

i

0.0

0.00 0

2

4

6

8

10

Time / min

Figure 2

30


12

Absorbance / AU

B 3.0 Intensity / RFU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 35

Page 31 of 35

A

0.8

RNase B aptamers

Kd (μM)

0.6

0.49±0.13

0.4 0.21±0.08

0.2 0.080±0.032

0.098±0.028

3

4

0.0 1

2

Cycle number of CE-SELEX

B

0.4

ALP aptamers 0.3

Kd (μM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.25±0.08

0.2

0.086±0.021

0.1

0.073±0.025 0.051±0.010

0.0 1

2

3

4

Cycle number of CE-SELEX

Figure 3

31



Fraction of bound DNA

A 0.8

RNase B β -casein RNase A

0.6

0.4

0.2

0.0 0.0

0.2

0.4

0.6

0.8

1.0

Concentration of protein / μ M

Fraction of bound DNA

B 0.8

ALP β -casein RNase B

0.6

0.4

0.2

0.0 0.0

0.1

0.2

0.3

0.4


C 0.8 Fraction of bound DNA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

RNase B β -casein ALP

0.6

0.4

0.2

0.0 0.0

0.2

0.4

0.6

0.8


Figure 4

32


1.0

Page 33 of 35

A

1.0

Hybrid SELEX

Intensity / RFU

0.8

0.6

iv

0.4

iii

0.2

ii i

0.0 0

2

4

6

8

10

12

8

10

12

Time / min

B 1.0 CE-SELEX

0.8

Intensity / RFU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


iv

0.6

iii

0.4

ii

0.2

i

0.0 0

2

4

6

Time / min

Figure 5

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 35

Table 1. Comparison of the ALP concentrations (U/L) measured by the current method with that by the conventional enzyme activity assay. Patients with osteosarcoma

Healthy individuals

Current

Conventional

method

enzyme activity assay

1

274.4 ± 24.4

2

No.

No.

Current method

295.1

1

120.9 ± 10.5

292.6 ± 27.3

301.4

2

123.7 ± 10.9

3

308.5 ± 26.6

304.4

3

140.6 ± 11.8

4

263.1 ± 20.8

291.9

4

106.1 ± 11.2

5

314.7 ± 28.4

331.7

5

112.0 ± 11.3

34


Page 35 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC graphic

35


Hybrid Approach Combining Boronate Affinity Magnetic Nanoparticles

Recommend Documents