Stepping Library-Based Post-SELEX Strategy Approaching to the

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Stepping Library-Based Post-SELEX Strategy Approaching to the Minimized Aptamer in SPR Xiaoqin He,† Lei Guo,*,† Junlin He,‡ Hua Xu,† and Jianwei Xie† †

State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Academy of Military Medical Sciences and ‡State Key Laboratory of Toxicology and Medical Countermeasures, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China S Supporting Information *

ABSTRACT: When evolved from SELEX (systematic evolution of ligands by exponential enrichment), aptamers are generally about 70−130 nucleotides in length and needed to be effectively truncated for further diagnosis or therapeutic uses. Post-SELEX optimization is then aroused to simplify the aptamer sequence and improve the affinity property. In this work, we report a new post-SELEX strategy based on a stepping library for the first time. With a hypothesis that one nucleobase can influence the whole binding affinity through its adjacent base stacking and potential steric hydrogen bonding interaction, we designed a stepping library composed of all probable nucleotide truncation directions. We employed an aptamer 807−39nt toward EPO-α as a model, and surface plasmon resonance (SPR) as an efficient screening and evaluation method to optimize all label-free sequences in the library. We have successfully picked out In27 as the minimized aptamer from a mini library of only 35 sequences. Aptamer In27 has a sole loop, without the original stem portion of the initial aptamer, but retains the whole binding affinity. We have also defined the key nucleotide contribution by site mutagenesis with natural bases, and finally produced a degenerated sequence with higher or the same good affinities. Furthermore, we explored different binding behaviors between aptamer In27 and other recognition molecule such as agglutinin, monoclonal antibody, or receptor by competition or binding assays. Our work provides a new and efficient post-SELEX optimization strategy, as well as a minimized aptamer In27 with an explicit degenerated sequence and a defined binding behavior. That would enhance their great potential in future diagnosis and therapy.

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binding, or only nonprimer sequence contributes to the binding, but with reductant or even interfering nucleotides. It is the reason that only empirical structural truncation focused on special secondary structures is currently adopted by judging that the binding affinity may be largely generated from special secondary structure such like stem-loop, bulge, pseudoknot, three-way helix structure, and so on. New post-SELEX strategies are urgently needed to precisely optimize truncated aptamers.8 Currently, many attempts have been proposed to generate desired aptamers with same or ever higher binding affinity, but most of them followed an empirical trial-and-error approach.9 Briefly speaking, toward the evolved positive sequences in the enriched pool, random truncation, and site-directed mutagenesis were performed on homologous alignment, consensus secondary structure, and structure stability from the calculated lowest free energy (ΔG).10 For example, Shangguan et al. reported a panel of papers on truncation and mutation

ptamers are short single-chain DNA (ssDNA) or RNA oligonucleotides derived from an in vitro evolution and selection process called SELEX (systematic evolution of ligands by exponential enrichment),1,2 which can fold into unique three-dimensional structures and bind targets with high affinity and specificity. Known as “chemical antibodies”,3 aptamers outperform antibodies in nonimmunogenicity, automated chemical synthesis, better stability, and versatility in structural engineering and modifications. Aptamers are also a kind of promising reagents in biosensor, medical diagnosis, and disease treatment,4−6 and a few of aptamers have reached human clinical trials to date.7 Generally, an original aptamer selected by SELEX is composed of two fixed primers and a nonprimer sequence, which evolved from the random sequences in the pool, has a typical length of 70−130 nucleotides (nt). Among them, the recognition domain of an aptamer consists only a few nucleotides. A full-length aptamer is not necessary for direct clinical or laboratorial application, and further truncation is usually followed. Unfortunately, most key binding sites or domains of aptamers are not easy to be elucidated. Both primer and nonprimer sequences can form complicated intramolecular interactions and contribute simultaneously to the specific © 2017 American Chemical Society

Received: February 24, 2017 Accepted: May 15, 2017 Published: May 15, 2017 6559

DOI: 10.1021/acs.analchem.7b00700 Anal. Chem. 2017, 89, 6559−6566

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target protein. We wish that this new kind of stepping library promoted-truncation post-SELEX strategy can be further applied to other sequences in demand.

approaches of aptamers produced by SELEX. Precise comparison of predicated secondary structures of aptamers generated from same family or from different laboratories is proved to be a good beginning in post-SELEX.11−15 In one case, they truncated a DNA sgc8 aptamer by removing the initial loop from the end, then the first stem, the second loop, the third stem, and last obtained an aptamer sgc8c, which retained substantial affinity of sgc8. They suggested that a minimal structure of aptamers could be deduced from the comparison of similar secondary structure and Kd values among the same family sequences.11 We have also selected an antirecombinant human erythropoietin α (EPO-α) ssDNA aptamer 807 with a double stem-loop structure from a lectin-mediated affinity chromatographic SELEX procedure.16 We removed the primary nonpaired tailored segment from the end, then the first stem, and then the first loop step by step, and finally, we obtained a stem-loop structure, namely, aptamer 807−39nt, with a slightly superior affinity to the full-length aptamer 807.16 Given elimination of one, two, or more nucleotides, or one segment of a sequence can be randomly settled in each decision, a large uncertainty, as well as many useless sequences would be produced in this nucleotide trimming process. Sometimes in the traditional trial-and-error approach, it is difficult to obtain the optimized aptamer sequences with higher affinity and specificity in a limited range of sequences. A polarity reversal strategy was applied by Esposito et al. for generating a group of 14 analogues with 3′-3′ and 5′-5′ inversion of polarity sites toward thrombin aptamer, they can elucidate the binding mechanism, but did not find better binding affinity.17 Ferreira-Bravo et al. replaced the stem of DNA aptamer FA1 with a GC paired portion and found that the substitution had no obvious effect on the affinity. They truncated several segments from a tail portion of aptamer FA1, and found the random truncation cannot afford better binding activity.18 Nonaka et al. proposed a post-SELEX optimization using a genetic algorithm-based mutagenesis approach.19 They endowed higher probability of occurrence for high-affinity analogues in the next generation. With a combination of random site-mutation in one or two nucleotides, they finally obtained an improved aptamer with a 10-fold higher affinity than original VEGF aptamer from more than 50 mutated sequences of same length in three generations. Still, this advanced approach cannot address the key nucleotide for highaffinity binding, neither the conservative portion of the aptamer sequence. In this paper, based on a hypothesis that a single base in the nucleotide could affect the affinity binding by influencing its adjacent base stacking and potential steric hydrogen bonding interaction, and inspired by combinatorial chemistry, we create a mini-stepping library containing all possible directions for nucleotide truncation, to optimize a minimized aptamer without any redundant sequence. A SPR method was developed and employed to evaluate all the sequence analogues. With an anti-EPO-α DNA aptamer 807−39nt as an initial or model sequence, we successfully obtained a minimized aptamer In27 in a mini stepping library of only 35 sequences, which paved a new way to tackle the timeconsuming and arduous problem in post-SELEX optimization. We also reported a degenerated sequence of In27 by site mutagenesis to provide clear information on key nucleotides and conservative portion, which can be used for further understanding on the binding mechanism between aptamer and

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EXPERIMENTAL SECTION Instruments and Reagents. All affinity and kinetic measurements were performed on a Biacore T100 surface plasma resonance (SPR) instrument (GE Healthcare Life Sciences, U.S.A.). The circular dichroism (CD) spectra were measured on a MOS-450 multifunctional circular dichroism spectrometer (BioLogic Science Instruments, France). pH values were measured by a seven-compact pH meter (Millipore, U.S.A.). All oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China) with a high-performance liquid chromatographic purity. EPO-α (3.25 g/L, purity greater than 98.5%) was provided by SCIPROGEN Biopharmaceutical Co. (Shenzhen, China). Sulfosuccinimidyl-6-[biotin-amido]hexanoate (Sulfo-NHS-LC-Biotin) and 4′-hydroxyazobenzene2-carboxylic acid (HABA), and Zeba Spin desalting columns (Molecular weight cutoff: 7K MWCO) were purchased from Thermo Fisher Scientific (Thermo, U.S.A.). Sodium periodate (NaIO4) was purchased from J&K Scientific Ltd. (J&K, Beijing, China). Tween 20, avidin, biocytin hydrazide, carbodiimide (EDC), 2-(N-morpholino)ethanesulfonic acid (MES), and wheat germ agglutinin (WGA) were obtained from SigmaAldrich Co. (Sigma, U.S.A.). Antihuman EPO monoclonal antibody (EPO mAb, clone AE7A5), EPO soluble receptor (EPOR) was purchased from R&D Systems Co. (Minneapolis, MN, U.S.A.). Tris, HCl, NaCl, KCl, MgCl2, and other chemicals were obtained from China National Pharmaceutical Group Co. (Beijing, China). All reagents are of analytical grade or beyond. All solutions were prepared with ultrapure water with a resistivity of 18.2 MΩ·cm from a Milli-Q A10 water purification system (Millipore, U.S.A.). Before use, all solutions were sterilized by high pressure sterilizer (Zhongya Co., Shanghai, China). Construction of a Stepping Aptamer Analogue Library. The stepping library of ssDNA aptamer analogues consists of four groups with different stepping directions, that is, left contraction group, right contraction group, inner contraction group, and outer extension group (which also named as stem-nbp group). With aptamer 807−39nt as the initial and positive sequence, in the left contraction group, two nucleotides were gradually removed from the 3′-end in each time, and a group containing nine sequences of L37, L35, L33, L31, L29, L27, L25, L23, and L21 was obtained. In the right contraction group, two nucleotides were progressively removed from the 5′-end in each time, a group containing nine sequences of R37, R35, R33, R31, R29, R27, R25, R23, and R21 was composed. In the inner contraction group, one nucleotide was gradually removed from 5′-end as well as the other nucleotide from 3′-end in each time, which makes a group containing nine sequences of In37, In35, In33, In31, In29, In27, In25, In23, and In21. In the stem-nbp group, considering that the original secondary structure of 807−39nt is a loop structure with a 6-bp stem, the inherent stem was replaced by a d(GA)n segment in the 5′-end and a d(TC)n in the 3′-end, where n is 2, 3, 5, 6, 7, 10, 12, or 15, respectively, which makes a group containing eight sequences of stem-2bp (S2bp), stem-3bp (S3bp), stem-5bp (S5bp), stem-6bp (S6bp), stem-7bp (S7bp), stem-10bp (S10bp), stem-12bp (S12bp), and 6560

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a

The sequence marked in grey is the sequence of initial aptamer 807-39nt.

stem-15bp (S15bp). The total number of sequences in this stepping library is 35. Monomer Biotinylation of EPO-α. EPO-α molecule can be biotinylated from its amino, carboxyl, or sialic acid groups. In one EPO-α molecule of 165 amino acids, there are 17 residues of arginine or lysine and one N-terminal amino group, and 17 residues of glutamate or aspartate and one C-terminal carboxyl group,20 while the terminal sialic acids occupied 18.7% (mol/mol) in the high glycosylation moiety, which contributed to 40% molecular mass.21 To maintain biotinylation labeling ratio around 1.0, here we added biotinylation agent and EPO-α protein as 1.5:1 (mol/mol) toward amino residual or terminal sialic acid (for preparation details, refer to Supporting Information). SPR Evaluation. All SPR experiments were performed with a streptavidin-coated chip (SA chip, GE Healthcare Life Sciences, U.S.A.) in a Tris-T buffer (20 mM Tris, 140 mM NaCl, 5 mM KCl, 5 mM MgCl2, and 0.005% Tween 20, pH was adjusted by HCl to pH 7.4−7.6). Prior to each SPR measurement, all buffers were filtered with a 0.22 μm filter membrane (Jinteng Co, Tianjin, China), degassed, and then diluted into an appropriate concentration of each oligonucleotide, the final buffer composition is as same as Tris-T. All oligonucleotide samples were heated at 95 °C for 5 min and cooled slowly to room temperature and then made ready for use. Biotinylated EPO-α (1 μM) was diluted in Tris-T buffer and injected into Biacore T100 at a flow rate of 10 μL/min for 60 s to achieve an immobilization level of 2000−2500 RU, under this condition aptamer 807−39nt at 500 nM has a binding response of about 30 RU. All multiple cycle kinetics (MCK) or single parameter comparison experiments were performed at the condition as flow rate: 30 μL/min, injection time: 120 s; dissociation time: 360 s; and regeneration: 5 mM NaOH at a flow rate of 30 μL/min for 15 s. Before each SPR binding experiment, the investigated ssDNA aptamer analogue was denatured at 95 °C for 5 min and slowly cooled to room temperature and then made ready for use. All experiments were replicated. When the biotinylated aptamer was used as an immobilizing agent, the conditions are 50 nM biotinylated aptamer for immobilization, flow rate: 10 μL/min, injection time: 300 s. In the competition or binding assay in the investigation of binding sites, 50 nM EPO-α was mixed with WGA, EPO mAb, or EPOR in a different series of concentrations as the analytes for SPR experiment. All the data were analyzed using the BIA evaluation 3.0 software (GE Healthcare Life Sciences, U.S.A.). The MCK data were nonlinearly fitted by a one-site binding model in a global format. For all the single parameter comparison experiments, the responses were normalized to the aptamer 807−39nt to avoid variation from different chip or regeneration. The response signal of 807−39nt was set at approximately 30 RU,

which was the response of the fresh chip when the immobilized signal of amino-terminal biotinylated EPO-α (NH2-Bio-EPOα) was about 2000 RU.

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RESULTS AND DISCUSSION Development of SPR Screening and Evaluation Method. Various methods have been developed for screening and evaluation of aptamers in the post-SELEX optimization, including SPR, biolayer interferometry, isothermal titration calorimetry, electrophoretic mobility shift assay (EMSA), microscale thermophoresis, backscattering interferometry, fluorescence anisotropy, and so on. Among them, SPR is a kind of label-free and real-time technique; it can provide affinity data as well as kinetic information for high-throughput assays. SPR has been widely used in the investigations on protein− protein, protein−DNA or RNA, DNA hybridization, and cell receptor−ligand interactions.22,23 Here we optimized the key parameters to develop a robust and sensitive SPR screening and evaluation method. We compared two sites of EPO-α for biotinylation on SA chip, one was the amino-terminal of EPO-α (i.e., NH2-BioEPO-α), and the other was its sialic acid terminal one (sialylBio-EPO-α), with a linker of 10−14 bond length to efficiently avoid the steric hindrance effect. Both biotinylated EPO-α show good recognition toward aptamer 807−39nt. Sialyl-Bio-EPO-α offered about 1.5-fold response of the same series of aptamers than NH2-Bio-EPO-α even in the half immobilization signal (Figure S1), indicating more active binding domains were exposed to aptamer, but both of them provided similar Ka, Kd, and KD results. However, sialyl-Bio-EPO-α offered less stability under multiple cycles of regeneration, here we preferred to use the immobilized NH2-Bio-EPO-α as the ligand to perform all evaluation experiments. Other key parameters such as purity of biotinylated EPO-α, the elimination of nonspecific interaction, good regeneration condition, and run-to-run stability of protein ligand to guarantee the efficiency in SPR evaluation were depicted in Supporting Information and Figures S2 and S3. Construction and Evaluation of a Combinational Stepping Mini-Library. To address the tedious or low efficiency problem in the empirical trial-and-error approach, here we suggest a construction of small combinational stepping library, which is enlightened by the molecular block or module concept of combination chemistry and the nature of nucleic acid structure. We hypothesize that each nucleobase in the aptamer probably influences the whole binding by altering the adjoining base stacking interaction and possible spatial neighboring hydrogen bonding interaction. Since base stacking and hydrogen bonding are the basic driving forces influence the local conformation variability as well as responsible for the stabilization of three-dimensional structure,24−26 shortening or extending the sequence by each or either two nucleotides with merely changed nucleobases could influence the base stacking and hydrogen bonding interaction and thus alter the binding 6561

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Figure 1. Binding curve of single-sequence comparison in each cluster and kinetic results of positive sequences measured by MCK.

property. Considering all the possible nucleotide truncation directions, we constructed a stepping library of four clusters toward original aptamer, that is, left contraction, right contraction, inner contraction, and outer extension (Scheme 1), in which the initial sequence was altered by varying two consecutive nucleotides each time. We regarded that all kinds of potential base-stacking and steric hydrogen bonding variations originated from truncation process are well included in this kind of successive trimming library. We used SPR to evaluate all the clusters one by one. First, we performed the single runs of each analogue at 500 nM in the binding buffer plus Tween-20, that is, Tris-T buffer, to pick out all the analogues of positive binding responses larger than zero

and compared the positive signals among each cluster with aptamer 807−39nt. Then we performed MCK determination toward each positive analogue, compared the Ka, Kd, and KD differences inside the cluster and deduced the influence of sequence variation on binding interaction. Our expectation is to find a minimized aptamer holding the same or even better binding affinity, with shortest length containing no redundant segment, which we considered as the analogue exerting full potential for specific binding. Here we choose aptamer 807−39nt as the initial aptamer to be optimized. We have successfully selected this aptamer with high specificity for EPO-α after rough truncation.16 Aptamer 807−39nt is a G-rich sequence with a stem-loop second 6562

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loop, which exactly exhibits as In27, we altered the stem nucleotides as a sequential d(AG)n:d(CT)n (n = 2, 3, 5, 7, 10, 15), to observe the variation of kinetics and binding affinity. We found that the KD value was decreased from S2bp to S7bp and then kept almost constant until S15bp. It is indicated that when the stem forms half of a duplex (S5bp), it can support the loop for binding very well. S6bp or S7bp is the most favorite one with the KD values of about 40 ± 2 nM (S6bp) or 30 ± 5 nM (S7bp). Apparently, a stem of duplex (S10bp) or one and a half duplex (S15bp) cannot contribute more to the binding affinity. While in the case of a stem of 2bp or 3bp, we observed an obvious 90% loss of affinity (390 ± 50 nM for S2bp, and 440 ± 40 nM for S3bp), suggests that some key nucleotides for recognition should exist around both the ends of In27, and the addition of several nucleotides without a fined shape will shield the key base sites for full binding. In all kinetic data, all the Snbp sequences have kept similar dissociation rates but varied association rates. S2bp and S3bp have lower associate rates as well as lower affinities, also indicates that shorter stem has some adverse effect on binding by hindering the easy folding of the sole loop. Site Mutagenesis on a Possible G-Quadruplex Structure of In27. For the optimized aptamer In27 with only the loop portion from the original aptamer 807−39nt, we supposed it might form some kind of three-dimensional structure, such as G-quadruplex in virtue of its G-rich nature.30 The CD spectra (Figure 2A) showed that the aptamer 807−

structure, and has been proved its good applicability in biosensing and bioaffinity separation.16,27−29 Our MCK measurement of 807−39nt in this SPR assay provide a KD value of 54 ± 2 nM toward EPO-α, consistent with previous KD determination results in EMSA (39 ± 27 nM)16 or capillary electrophoresis with laser-induced fluorescence detection (86 ± 13 nM).28 SPR revealed more kinetics information, shown as a slightly fast association (Ka = (1.7 ± 0.14) × 104 M−1 s−1) and slow dissociation rate (Kd = (9.1 ± 0.28) × 10−4 s−1) between aptamer 807−39nt and EPO-α. As depicted in Figure 1, first, in the left contraction cluster (L37, L35, L33, L31, L29, L27, L25, L23, L21), only L37, L35, and L33 kept some binding activity around 360 nM, although diminished much comparing with aptamer 807−39nt. The other six sequences lack of --(GG)--(GGGG)--(GG)---(GGG) segment completely lost their binding affinity. It indicates that the G-rich segment could play an important role in the binding. When the right arm of the stem varied from 6 to 2 nt in length (from L37, L35 to L33), the KD values varied a little, indicates that the affinity contribution from the right arm is not so restrained by nucleotide length. In the right contraction cluster (R37, R35, R33, R31, R29, R27, R25, R23, R21), R37, R35, R33, and R31 provided some but different affinities toward EPO-α. When we cut 2 nt at the first and second time from the 5′ end, the binding affinity had a significant loss (R37, 560 ± 170 nM; R35, 1300 ± 150 nM); while we further cut 2 nt at the third and fourth time, the affinity was greatly recovered (R33, 140 ± 40 nM; R31, 130 ± 30 nM). It suggested that some important nucleotides for recognition were exposed in this right truncation procedure. Further cut was not favorable, which made the affinity totally lost. It indicated that only a G-rich segment composed of a full four consecutive Gn (n = 2, 3, 4) showed favorable recognition. A big surprise came out from the inner contraction cluster (In37, In35, In33, In31, In29, In27, In25, In23, In21). Seven out of nine sequences in this cluster showed binding affinity with a fall-rise order except In25. The KD values were gradually increased from 807 to 39nt to In33 (750 ± 30 nM), and then progressively declined to In27 (54 ± 2 nM), while total activity was resumed. We demonstrated this difference in their kinetic behaviors. From In37 to In27 in the descending order, the dissociation rate did not vary much, ∼1.5 × 10−3 s−1, but the association rate decreased at first, and the lowest rate was (2.0 ± 0.08) × 103 (In33), then rapidly increased to (3.1 ± 0.28) × 104 (In27), which is 15-fold greater than the lowest one. In27 is the very sequence which keeps the total affinity of the initial aptamer 807−39nt. It is interesting to note that in the inner cluster, In37 and other descending sequences until to In29 contain a shortened stem from 5 bp to 1 bp, and In27 is the exact loop without any stem. It is indicated that the loop itself acts as the recognition portion to the binding, and the stem with suitable length can keep the activity of this loop, but short stem length hindered the recognition much. Obviously, In27 still contains the full G-rich segment. In the case of In25, while the loop was shortened by one nucleotide at each end, its affinity was deteriorated 40-fold than In27. Combined with results of the right contract cluster, it is suggested that the nucleotides in the both ends of the loop are very important to the binding affinity. The results of the outer extension cluster (S2bp, S3bp, S5bp, S7bp, S10bp, S12bp, S15bp) confirmed that the stem here mainly acted as a holder for loop stabilization, but shorter stem length hampered the specific recognition. Toward the original

Figure 2. (A) CD spectra of 807−39nt and In27 in the binding buffer (Tris buffer). The characterized wavelengths for 807−39nt and In27 are 260(−), 290(+), and 240(−), 260(+), 290(+), respectively. Both concentrations of 807−39nt and In27 are 1 O.D. All the aptamers were heated at 95 °C, 5 min, then slowly cooled to room temperature, and then ready for CD measurement. D1 and D2 indicate measures were made at the first and second day. (B) Supposed G-quadruplex conformation of aptamer In27.

39nt itself formed an antiparallel G-quadruplex conformation with a feature of peaks at 260 nm (−) and 290 nm (+).31 While for aptamer In27, the peaks initially appeared at 240 nm (−), 260 nm (+, shoulder peak), and 290 nm (+) with a slight wavelength shift, and slowly transferred to the characteristic peaks of about 260 nm (−) and 290 nm (+), indicating a conformation transition to an antiparallel G-quadruplex without a stem support. A possible G-quadruplex was depicted in Figure 2B. We followed a general domain-directed principle to design the site mutation clusters, and tried to finally find the degenerated sequence and deeply understand the key nucleotide contribution to the binding between aptamer In27 and EPO-α. We named intervals among consecutive GG as the domain clusters, including 1A2A (D1), 5TCTGTTTT13T(D2), 18 19 T T(D3), 22TT24T(D4), and 27G(D5). We basically followed an exhaustive site-mutation approach for the 1A2A sites in D1, 6563

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partial activity when the first dA was totally depleted, supporting our hypothesis that removal of adjacent nucleotide could affect the whole binding. However, the second nucleotide, dA, is a conservative one. It cannot be altered at all. Site-mutation cluster D2 contains eight sequences, 6T, 6A, 6G, 6X, 8T, 6T8T, 10X, and 10X11X. Only 6T remained the binding affinity very well, which 6A and 6G kept half of the response of In27 at the same concentration. Other single or combined alteration in the sixth or eighth nucleotide such as 6X, 8T, or 6T8T provided faster association approaching to a steady-state response and more rapidly decreased dissociation responses, indicating key recognition in these two nucleotide sites. Furthermore, if one or two dT in the consecutive five dT segment was deleted (11X, 1011XX), the binding affinity of In27 was totally lost (Figure 3B). It is suggested that D2 domain consisting of 11 nucleotides is an important and conservative domain, which guarantees the whole binding affinity between In27 and EPO-α. Both site-mutation clusters D3 and D4 have one altered sequence in each cluster. Given that d(T)n is a common loop linker in the G-quadruplex, only one dT-to-dA in 18T19T or 22 TT24T segment was prepared to compare the binding affinity. We also found a fast but low association/dissociation response to a steady state (Figure S4), indicating the small loops such as TT in D3 and TTT in D4 domain are also important for the binding recognition. In the site-mutation cluster of D5, all three altered sequences at the final nucleotide in In27 showed less binding affinity. It seems that dG at the 3′-end is quite specific; it cannot be removed or changed to other nucleotides (Figure S5). Second, for the regarded G-quadruplex backbone, based on alteration of dT to the sole dC in the sixth site to simplify the sequence composition without any affinity sacrifice, we made a dG-to-dT alteration in those three or four consecutive dG nucleotides, that is, 14GGG17G, 25GG27G. Results of Gquadruplex backbone cluster of nine sequences in Figure S6 showed that the dG-to-dT mutation deteriorated the binding affinity. Moreover, we supposed that the 14GGG17G containing four dG nucleotides might have some inherent mobility (17T of ca. 5 RU vs 14T, 15T, 16T of no response; 14T15T of ca. 10 RU vs 16T17T, 14T17T of less than 2 RU), while 25G was more probably involved in the G-quadruplex formation in comparison with 27G (25T of less than 2 RU response vs 27T of ca. 5 RU). Degenerated Cluster and Its Valuable Sequences. Afterward, we created a degenerated cluster with all efficient site mutations based on the aforementioned SPR evaluation results. 1T/C/G in D1 cluster and 6T in D2 cluster were picked out and combined to form 1T, 1C, 1G, 6T, 1T6T, 1C6T, and 1G6T, total seven sequences. At the same concentration, 1T, 1T6T, 6T, and 1C have higher responses compared to In27, and 1C6T has a moderate response between aptamer In27 and 807−39nt, while the other two sequences, 1G and 1G6T, have slightly lower but still comparable response to aptamer 807−39nt. We then deduced a degenerated sequence as d(1NAGGT6YTGT10TTTTGGGGTT20GGTTTGGG), where N represents dA, dT, dC, or dG, and Y represents dC or dT, respectively. The kinetic evaluation showed that, the KD values of the degenerated sequences were between 20 to 88 nM (Table 2, Figure S7). Sequence 1T obtained a highest affinity of 20 ± 2 nM with a 2-fold Ka value and same Kd value comparing with In27. In the case of only natural base mutation without any

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TCT8G in D2, 27G in D5, and in each domain cluster, we did not introduce any dC to avoid new G-C pairing, and only altered dT to dA or shorten the successive dT nucleotides. Those five clusters contain 20 sequences (Table 1). As shown Table 1. Site-Mutation Clustersa

a

The altered nucleobase is shown in gray background.

Figure 3. SPR binding curve of aptamers in the D1 (A) or D2 (B) site mutation cluster toward immobilized EPO-α. Aptamer In27 here used as the positive control for comparison. The concentration of each aptamer is 500 nM.

in Figure 3A, in site-mutation cluster D1, sequences of 1T, 1C, and 1G had comparable affinity to In27, while the other four sequences of 1X (X means deletion of nucleotide in this site), 2T, 1T2T, and 2G lost their affinities at a large extent. It is indicated that the first nucleotide from 5′-end in this In27 sequence has a large flexibility, which can be altered from dA to all other three kinds of nucleotides. Analogue of 1X still kept 6564

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then the mixture of EPO-α protein at a fixed concentration of 50 nM and its specific agglutinin, that is, WGA at varied concentrations were injected. The response trends of aptamer 807−39nt and In27 were similar, and only slight difference was found in signal response (Figures 5 and S9). For In27, the

Table 2. Kinetic and Affinity Results of Sequences in the Degenerated Cluster Seq In27 1T 1C 1G 6T 1T6T 1C6T 1G6T

Ka (M−1 s−1) (3.1 (7.6 (4.9 (2.6 (3.3 (3.6 (5.6 (2.9

± ± ± ± ± ± ± ±

Kd (s−1) 4

0.28)×10 0.38)×104 0.40)×104 0.25)×104 0.64)×104 0.71)×104 0.15)×104 0.77)×104

(1.6 (1.5 (2.0 (1.5 (1.9 (2.1 (3.1 (2.5

± ± ± ± ± ± ± ±

0.07)×10−3 0.20)×10−3 0.04)×10−3 0.05)×10−3 0.09)×10−3 0.04)×10−3 0.46)×10−3 0.19)×10−3

KD (nM) 52 20 40 57 50 58 56 88

± ± ± ± ± ± ± ±

4 2 2 3 3 0.2 8 2

chemical modification, 1T offers the largest binding probability among all digging-out sequences from aptamer In27. What is the difference in recognition ability between aptamer In27 or original aptamer 807−39nt? We separately immobilized these two biotinylated aptamers (3′-Bio-39nt and 3′-Biod(T)12-In27) to examine their affinities toward EPO-α. Here a 12mer poly(dT) used as a spacer at the 3′ end of In27 to overcome the steric hindrance and increase the accessibility of a target protein to the aptamer. Aptamer In27 offered sufficient response to EPO-α, proved to be a good capturing and recognition module. It has a slightly lower response than 807− 39nt at the same concentration, which might be attributed to the stem stabilization effect of aptamer 807−39nt on the sole loop. The obtained KD values are similar to the results when EPO-α was immobilized as ligand (Figure 4).

Figure 5. Change of response signal when 50 nM EPO-α protein mixture with different concentrations of WGA, EPO mAb, or EPOR in the SA chip on SPR when immobilized 3′-Bio-d(T)12-In27.

response was gradually increased with the increased WGA concentration from 5 to 200 nM (Figure S10A), indicating that aptamer and WGA have different binding sites toward EPO-α, and WGA can be used as a signal enhancer in further biosensing experiments. We are not surprised at this confirmed sandwich type structure, because our aptamer 807−39nt was originally selected from an agglutinin-directed affinity chromatographic SELEX.16 The EPO mAb experiments gave us a similar result with WGA but of unique feature. Addition of EPO mAb at a low concentration below about 1-fold of EPO-α did not influence the binding very much, but at higher concentration, for example, 2- or 5-fold, the signal response was enhanced to 2- to 6-fold. It is indicated that the primary binding site of EPO mAb is different from aptamer. Considering that EPO mAb and EPO has a strong affinity with KD of pM (Figure S11), the overall sandwiched binding behavior is only observed at nM level, which is closed to aptamer’s binding affinity, it seems that the binding is dominated by the aptamer, not the EPO mAb in this assay format. At last, we investigated the binding sites between aptamer and EPOR (Figure S10C). When the EPOR concentration was increased from 16 to 256 nM, the signal response was decreased, when the concentration ratio was about 1:1 (EPOα/EPOR = 50 nM:64 nM), the signal response was decreased to half of the signal without EPOR. It is indicated that the free EPOR in the solution competes with the immobilized aptamers for the recognition site on the EPO-α. EPOR could inhibit the binding activity of aptamers, and vice versa. It offered us a positive clue that aptamer 807−39nt and In27 can be further used as the inhibitor to EPO-EPOR interaction. Since many kinds of tumor cells overexpressed EPOR and EPO to adapt the hypoxia microenvironment, it is very important when the aptamers can act as useful inhibitors. We have previously observed that aptamer 807−39nt has a positive inhibition effect on the nude-mouse transplanted tumor model.33 Therefore, we wish that aptamer In27, its degenerated sequence, as well as other derived or modified variants, can exert a same or stronger effect.

Figure 4. Kinetic and affinity characterization of EPO-α protein to immobilized aptamers in the SA chip: (A) 3′-Bio-39nt, (B) 3′-Biod(T)12-In27. Each aptamer with a concentration of 50 nM was immobilized onto the SA chip for 300 s, which showed a conjugation response of 1500 RU and 1650 RU for 3′-Bio-39nt and 3′-Bio-d(T)12In27, respectively.

Aptamer In27 has a great potential as a sensing module because its minimized sequence for recognition, and many more variants can be constructed around this core sequence. For example, we designed a dimer of In27 with a spacer of d(T)12 since it is generally considered that a bivalent aptamer can improve the affinity.18,32 Results revealed that the binding affinity was slightly decreased (KD, 170 nM) toward an immobilized EPO-α protein. With consideration of stem stabilization effect, a d(A)n oligonucleotide was added to hybridize this dimer, and a recovered binding affinity was observed as expected (Figure S8). When n increased from 12 to 15, the KD values were decreased from 140 to 86 nM, which indicates some inherent steric hindrance of the dimer is efficiently overcome by a convenient hybridization way. Investigation on the Binding Sites of Aptamer, Agglutinin, EPO mAb, EPO receptor, Toward EPO-α. Hereby we used aptamers 807−39nt and In27 to mutually corroborate the binding sites by a competitive binding assay. First, the biotinylated aptamer was immobilized on the SA chip, 6565

DOI: 10.1021/acs.analchem.7b00700 Anal. Chem. 2017, 89, 6559−6566

Analytical Chemistry

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CONCLUSION

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ASSOCIATED CONTENT

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b00700. Monomer biotinylation of EPO-α. Optimization of a SPR screening and evaluation method. The scheme of EPO-α biotinylation (Scheme S1). MCK curves of aptamer 807−39nt using immobilized NH2-Bio-EPO-α or sialyl-Bio-EPO-α (Figure S1). MCK curves of aptamer 807−39nt in Tris or in Tris-T buffer (Figure S2). The baseline response variation of different regeneration conditions (Figure S3). SPR binding curves of aptamers in the D3 and D4 (Figure S4), D5 (Figure S5) site mutation cluster, the GQ backbone cluster (Figure S6), and the degenerated cluster (Figure S7) toward immobilized EPO-α. The kinetic and affinity data for aptamer In27-dimer, dimer-(dA)12 hybridization, or dimer-(dA)15 hybridization and EPO-α, respectively (Figure S8). The change of signal response when 50 nM EPO-α protein mixture with different concentrations of WGA, EPO mAb or EPOR in the SA chip by SPR when immobilized 3′-Bio-39nt (Figure S9). The competition or binding assay of WGA, EPO mAb or EPOR toward EPO-α when using 3′-Bio-(dT)12-In27 as immobilized ligand by SPR (Figure S10). MCK analysis on EPO mAb or EPOR on immobilized EPO-α (Figure S11; PDF).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 86-10-66931650. Fax: 86-1068225893. ORCID

Lei Guo: 0000-0003-4780-712X Notes

The authors declare no competing financial interest.

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In conclusion, we have developed an efficient post-SELEX strategy to truncate the sequence of aptamer for better binding affinity. With a stepping mini-library approach, we picked out aptamer In27 as a minimizer of all aptamers toward target protein EPO-α from only 35 analogues. Benefited from the SPR method for label-free evaluation, we then deduced a degenerated sequence of In27 based on site mutagenesis and its probable G-quadruplex conformation. We have also successfully applied In27 for its target protein recognition, dimer design and optimization, and binding sites investigation, to show the great potential and convenience of aptamer In27. We are looking forward to the further wide applications of the stepping librarybased post-SELEX strategy, and aptamer In27 in the corresponding aptameric field.

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Article

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (No. 21175152). We thank Dr. Zhongcai Gao for his kind support for the SPR evaluation experiments in the earlier stage. 6566

DOI: 10.1021/acs.analchem.7b00700 Anal. Chem. 2017, 89, 6559−6566