Functional-Group Specific Aptamers Indirectly Recognizing

Aug 10, 2012 - Aptamers are usually generated against a specific molecule. Their high selectivity makes them only suitable for studying specific targe...
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Functional-Group Specific Aptamers Indirectly Recognizing Compounds with Alkyl Amino Group Hongcheng Mei,‡,§ Tao Bing,‡,§ Xiaojuan Yang,‡,§ Cui Qi,‡,§ Tianjun Chang,‡,§ Xiangjun Liu,‡ Zehui Cao,‡ and Dihua Shangguan*,‡ ‡

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Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China § Graduate School of the Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Aptamers are usually generated against a specific molecule. Their high selectivity makes them only suitable for studying specific targets. Since it is nearly impossible to generate aptamers for every molecule, it can be of great interest to select aptamers recognizing a common feature of a group of molecules in many applications. In this paper, we describe the selection of aptamers for indirect recognition of alkyl amino groups. Because amino groups are small and positive charged, we introduced a protection group, p-nitrobenzene sulfonyl (p-nosyl) to convert them into a form suitable for aptamer selection. Taking Nε-p-nosyl-L-lysine (PSL) as a target, we obtained a group of aptamers using the SELEX technique. Two optimized aptamers, M6b-M14 and M13a exhibit strong affinity to PSL with the Kd values in the range of 2−5 μM. They also show strong affinity to other compounds containing p-nosyl-protected amino groups except those also possessing an α-carboxyl group. Both aptamers adopt an antiparallel Gquadruplex structure when binding to targets. An aptamer beacon based on M6b-M14 showed good selectivity toward the reaction mixture of p-nosyl-Cl and alkyl amino compounds, and could recognize lysine from amino acid mixtures indirectly, suggesting that aptamers against a common moiety of a certain type of molecules can potentially lead to many new applications. Through this study, we have demonstrated the ability to select aptamers for a specific part of an organic compound, and the chemical conversion approach may prove to be valuable for aptamer selection against molecules that are generally difficult for SELEX.

A

To assess this new approach, we tried to evolve aptamers binding alkyl amino groups. Compared to whole organic molecules that have been used to generate aptamers (e.g., ATP,19 L-tryptophan,20 cocaine,21 and L-tyrosinamide22), alkyl amino groups are usually considered too small to generate specific aptamers because it could not form multiple binding sites with aptamers. Additionally, amino groups exhibit positive charge at physiological pH condition, which may cause nonspecific binding to nucleic acids and result in the failure of aptamer selection. Although aptamers against the simplest molecule, ethanolamine, have been reported, these aptamers also bind to many other compounds.23,24 The binding motif of these aptamers is a type of G-quadruplexes,25 and the nonspecific electrostatic interaction has been found to play an important role in the binding of G-quadruplexes to compounds containing amino groups.26 To circumvent the above problems, we introduced a protective group to the amino group, and selected aptamers targeting the protected amino groups. pNitrobenzene sulfonyl (p-nosyl) is a common protective group

ptamers are single-stranded oligonucleotides, which can recognize target molecules with high affinity and specificity by folding into well-defined three-dimensional shapes.1,2 For many years, aptamers have attracted intense investigation because of their potential applications in areas such as affinity purification,3 biosensors,4−6 nanotechnology,7 DNA-based computing,8 drug discovery,9 and so on.10−12 Aptamers are in vitro selected from random pools of DNA or RNA molecules (containing about 1014 −10 15 different sequences) using SELEX (systematic evolution of ligands by exponential enrichment) technique via rounds of enrichment and amplification.13,14 The targets for aptamer selection have ranged from small molecules to living cells,15−17 and even tissues.18 Most aptamers are evolved against a specific target, their high selectivity makes them suitable for various applications only related to their specific target molecules. However, in many situations, it may be of great interest to have molecular probes for a certain type of molecules instead of just for one of the entire family. To achieve this goal using aptamer technology, we have explored aptamer selection against common features of a family of compounds and speculated that the selected aptamers can be used to distinguish one type of molecules from another. © 2012 American Chemical Society

Received: February 3, 2012 Accepted: August 10, 2012 Published: August 10, 2012 7323

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as templates for PCR amplification with FAM- and Biotinlabeled primers. The FAM-labeled ssDNAs were separated by alkali denaturation from streptavidin-coated sepharose beads, desalted with a NAP-5 column and used for next round of selection. In order to obtain aptamers with high specificity, glycine-immobilized beads were used for counter-selection during round 2−11, namely, the ssDNA pool was incubated with glycine-immobilized beads before mixing with PSLimmobilized beads. To further improve the specificity of aptamers, 1 mM Nε-p-nitrobenzoyl-L-lysine (PZL) was added to binding buffer during round 12−20 for competition. During the whole selection process, the selection pressure was enhanced gradually by increasing the volume of glycineimmobilized beads (from 0 to 60 μL) and the number of washing steps (n from 2 to 4 times), as well as decreasing the volume of PSL-immobilized beads (from 200 to 15 μL). After 20 rounds of selection, the selected ssDNA pool was PCRamplified with unlabeled primers and cloned into Escherichia coli using a TA cloning kit. Total 75 clones were sequenced by Beijing Genomics Institute (Beijing, China). Characterization of DNA Aptamers. Aptamer candidates were dissolved in binding buffer and denatured at 95 °C for 5 min, then rapidly cooled on ice, and kept at room temperature before use. For binding assay, 200 μL of 8 μM aptamer or control sequence were incubated with 8 μL of PSL-immobilized beads or control beads at room temperature for 30 min with gentle shaking. After it was washed twice with 200 μL of binding buffer, the bound sequences were eluted with 200 μL elution buffer after incubating at 95 °C for 5 min. The amounts of unbound, washed off, and eluted ssDNA were determined by UV absorbance at 260 nm. The equilibrium dissociation constant (Kd) of M6b-M14 and M13a were determined by static adsorption method19 and Isothermal Titration Microcalorimetry (ITC). For static adsorption assay, different concentrations of FAM-labeled aptamer were incubated with 10 μL of PSL-beads in buffer (10 mM Na2HPO4, 2 mM KH2PO4, 50 mM KCl, 5 mM MgCl2, 500 mM NaCl, pH 7.4) for 30 min. After centrifuging, the concentration of free aptamers was determined by measuring the fluorescence intensities of the supernatant with SpectraMax M5 at 538 nm with excitation at 485 nm. The amount of bound aptamers was calculated by subtracting the amount of the free aptamers from the total amount of aptamers. Kd was calculated by fitting the dependence of the amount of bound aptamers on the concentration of free aptamers to the equation (Y = BmaxX/(Kd + X) + NsX) using the Systat SigmaPlot version 11 (San Jose, USA). ITC assay was performed on a NANO ITC System (TA Instruments Inc., New Castle, DE, USA). Titrations were performed in buffer (10 mM Na2HPO4, 2 mM KH2PO4, 50 mM KCl, 5 mM MgCl2, 500 mM NaCl, pH 7.4). Injections of 5 μL of 1 mM PSL were added from a computer-controlled microsyringe at an interval of 500 s into M6b-M14 (17 μM)/ M13a (9.5 μM) solution (cell volume 1.0 mL) with stirring at 200 rpm at 25 °C. The experimental data were fitted to a theoretical titration curve using software supplied by TA, with ΔH (binding enthalpy kJ/mol), Ka (association constant), and n (number of binding sites per monomer), as adjustable parameters. The Kd was calculated by the equation Kd = 1/Ka. Competitive Binding Assay. The specificity of obtained aptamers to PSL was tested by competitive binding assay. With or without the presence of 3 mM organic molecules (competitors), 0.3 μM FAM-labeled aptamers was incubated

of amino groups and widely used in organic synthesis including peptide synthesis, and as the intermediate of some pharmaceuticals.27,28 The protection reaction can be rapidly accomplished by the reaction of p-nosyl-Cl with primary and secondary amino group under mild conditions, and the deprotection process is carried out by nucleophilic aromatic substitution with thiolate as nucleophile.29 In this paper, we describe the selection of aptamers against pnosyl protected alkyl amino group by utilizing Nε-p-nosyl-Llysine (PSL) as the target (Scheme 1). The obtained aptamers Scheme 1. Synthesis and Immobilization of PSL

were optimized and showed good selectivity to compounds with p-nosyl protected primary or secondary alkyl amino group except those also with an α-carboxyl group near the amino group. The effects of ionic strength, Mg2+ and pH on the binding ability and the specificity were investigated. The binding mechanism was also discussed. Further application for the recognition of compounds with alkyl amino groups, as well as detection of lysine in amino acid mixture showed that these aptamers have potential applications for a wide range of molecules.

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EXPERIMENTAL SECTION Procedure of Aptamers Selection. The 85 nt DNA library containing 45 central nucleotides of random sequence flanked by defined primer-binding sites was synthesized and purified by HPLC: 5′-ACGCTCGGATGCCACTACAG-N45CTCATGGACGTGCTGGTGAC-3′. The following labeled primers were used for amplification during the in vitro selection process: primer MP1F, 5′ FAM-ACGCTCGGATGCCACTACAG-3′; primer MP2B, 5′ Biotin-GTCACCAGCACGTCCATGAG-3′. For the first round of selection, 200 μL of PSL-modified beads and 16.5 nmol of DNA library were used. The PSL-modified beads were washed with 200 μL of binding buffer (137 mM NaCl, 0.5 mM MgCl2, 2.7 mM KCl, 2 mM KH2PO4, 10 mM Na2HPO4, pH 7.4) before selection. Each ssDNA pool dissolved in binding buffer was denatured at 95 °C for 5 min, cooled on ice for 15 min, and then kept at 25 °C prior to use. Iterative rounds of aptamer selection were performed as described previously15,20,30 with some modifications: PSL-modified beads were incubated with ssDNA pool in 200 μL of binding buffer at 25 °C for 30 min with gentle shaking, then the mixture was filtered through a pipet tip (200 μL) containing a frit, and washed with 200 μL × n of binding buffer. The bound DNA molecules were eluted by 200 μL of elution buffer (25 mM Tris-HCl, 10 mM EDTA, 3.5 M urea, pH 8.0) at 95 °C for 10 min and the fluorescence intensity of eluted DNA molecules was measured by SpectraMax M5 (Molecular Devices, USA) with excitation at 488 nm and emission at 530 nm (from round 2). The collected bound oligonucleotides were precipitated with ethanol and then used 7324

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with 10 μL of washed PSL-immobilized beads in 200 μL of binding buffer for 30 min at 25 °C with mild shaking. After filtered through a pipet tip (200 μL) containing a frit, the beads were washed with 400 μL of binding buffer. The bound sequences were eluted with elution buffer by heat treatment (95 °C, 5 min) as described above. The fluorescence intensity of the eluates were measured by SpectraMax M5 (Ex = 488 nm, Em = 530 nm, cutoff = 515 nm). The percentage of competition was calculated as

PSL were selected by the affinity chromatographic SELEX protocol as reported previously.20,30 To eliminate the nonspecific sequences binding to beads and the α-amino acid part of PSL, glycine-modified beads were used for counter selection during 2−11 rounds of selection. A sharp increase of enrichment of ssDNA molecules binding to PSL-modified beads was observed at round 8 and round 9 (Supporting Information Figure S-1). To generate aptamers binding to the whole p-nosylamide moiety, additional 9 rounds of selection were performed by adding 1 mM Nε-p-nitrobenzoyl-L-lysine (PZL) into the binding buffer to remove the sequences that only bind to the p-nitrobenzene moiety of both PZL and PSL. The maximum degree of enrichment was attained at round 18− 20. The enriched pool of the 20th round was cloned and 75 clones were sequenced. Characterization of Strong Binding Aptamers. The 75 DNA sequences (Supporting Information Table S-1) could be classified into five families according to their similarity. Seven sequences (M1, M2, M4, M6, M11, M13, and M67) from different families were synthesized and all of them were observed to bind to PSL-modified beads (data not shown). To identify the binding motif of these aptamers and optimize binding affinity, four aptamers (M4, M6, M13, and M67) were truncated based on comparison of their predicted secondary structures as described in previous report.15,20,30 As shown in Figure 1, all of the truncated sequences (M4a, M6a, M13a, and M67a) exhibited stronger binding ability to PSL-immobilized beads than their parent sequences. Since M6a and M13a have

relative competing ability = (F0 − Fb)/F0 × 100%

where Fb is the fluorescence intensity of the eluate with organic molecules (competitor) and F0 is the fluorescence intensity of the eluate without organic molecules. Each experiment was repeated three times. Influence of Buffer Composition and pH on Aptamer Binding. To investigate the influence of buffer composition and pH on aptamer binding, the binding assay was performed in buffers with different compositions or pH values other than those of the binding buffer. For the influence of Na+, K+, or Mg2+, different concentration of NaCl, KCl, and MgCl2 was added in phosphate buffer (2 mM KH2PO4 and 10 mM Na2HPO4, pH 7.4); for the influence of organic solvent, different proportion of DMF or ethanol was added to phosphate buffer containing additional 0.5 M NaCl; for the influence of pH, 2 M NaOH or 2 M HCl were added to the buffer (10 mM Na2HPO4, 2 mM KH2PO4,150 mM NaCl) to adjust the pH to 3−11. In addition, Tris-HCl buffer (10 mM Tris-HCl, 500 mM NaCl, pH 7.4) was also used for investigation. Aptamer Beacon Design and Amino Compounds Recognition. Aptamer beacon was constructed using a fluorophore labeled aptamer (0.05 μM): M6b-M14-F (5′FAM-ATCATGGTGGGTATCGGCACTCGTTGGTTGAT3′) and a quencher labeled complementary sequence (0.15 μM): M14-Q9, 5′-CACCATGAT-BHQ1-3′. The working solution contained 100 mM Na2HPO4, 20 mM KH2PO4, 500 mM NaCl, at pH 7.4. The determination process of amino compounds were as following: 10 μL of 1 M p-nosyl chloride DMF solution was added to 100 μL of 10 mM amino compounds in 0.1 M Na2CO3/NaHCO3 buffer (pH 10), and reacted at 0 °C for 30 min with shaking, then 5 μL of the reaction mixture was added to 100 μL of aptamer beacon system (0.05 μM M6b-M14-F, 0.15 μM M14-Q9) and incubated for 3 h at room temperature. The fluorescence spectra from 500 to 600 nm were recorded by SpectraMax M5 with excitation at 488 nm and cutoff at 515 nm.

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RESULTS AND DISCUSSION In Vitro Selection of DNA Aptamers against PSL. The p-nosyl protection was chosen because it might increase the surface area of alkyl amino groups for interacting with aptamers and also eliminate the positive charge of amino group, which would make the aptamer selection less challenging. For evolving aptamers against p-nosyl protected alkyl amino group, we chose Nε-p-nosyl-L-lysine (PSL) as the target. The synthesis and immobilization of PSL on epoxy-actived Sepharose 6BTM beads was shown in Scheme 1 (see Supporting Information for details). The α-amino group of PSL was designed for the immobilization of PSL on solid support, and the α-carboxyl group was designed for neutralizing the positive charge of α-amino group that could result in nonspecific binding of DNAs to solid support. DNA aptamers binding to

Figure 1. Predicted secondary structure of aptamers (top) and the binding of different aptamers to PSL-immobilized beads (bottom). EP: the enriched pool. Bound percentage = nb/n0 × 100%, wherein nb is the amount of ssDNA bound on beads and n0 is the amount of total ssDNA used for each binding assay. 7325

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M6b-M14 and M13a. These results imply that the bulge-stem II-loop region of M6b and the loop of M13a adopt an antiparallel G-quadruplex structure in the presence of high concentration of K+ or Na+. The double-G tract in stem I of M6b probably do not take part in the G-quadruplex formation because the strongest binding sequence, M6b-M14, does not possess this G tract. Influence of Buffer Composition and pH on Aptamer Binding. It is well-known that the driving force of aptamer binding includes hydrogen bonds, electrostatic and stacking interactions, hydrophobic effect and shape complementarity.2,34 Many factors, such as pH, the composition of buffer, can change the microenvironment and conformation of aptamer and target, and consequently affect their binding.35,36 Ionic strength is an important factor in affecting the folding of nucleic acids and changing the interaction between nucleic acids and their ligands by screening the charges of nucleic acids and ligands.34−36 Na+ and K+ were used here to adjust ionic strength of the solution respectively. As shown in Figure 3, both aptamers M6b-M14 and M13a had very low affinity to PSL modified beads in phosphate buffer (2 mM KH2PO4 and 10 mM Na2HPO4, pH 7.4). With the stepwise increase of Na+, the affinity of M6b-M14 and M13a increased gradually up to a plateau at 600 mM of Na+. Increasing the concentration of K+ had a similar effect on M13a, but produced much smaller increase of the affinity of M6b-M14 than that of M13a. These results indicate that high ionic strength favors the binding of M13a and M6b-M14 to PSL, and different monovalent metallic ions have different effects on the binding of M6b-M14. By comparing the effect of Na+ and K+ on the binding ability and CD spectra of both aptamers, a good correlation was revealed between the antiparallel G-quadruplex formation and the binding ability. The CD signals of antiparallel G-quadruplex of both aptamers became stronger with the increase of the concentration of Na+ and K+ (Figure 2), which is consistent with the increase of their binding affinity (Figure 3). The stronger increase effect of K+ on the affinity of M13a than that of M6b-M14 also accords with the trend of the effect of K+ on the CD signals of antiparallel G-quadruplex of both aptamers (Figure 2c, d). This set of results suggest that these two aptamers adopt antiparallel G-quadruplex structure to bind to PSL. The differentiation in the binding behavior and the CD signal between M13a and M6b-M14 under the above conditions implies the differences of these two aptamers in the antiparallel G-quadruplex structure and the binding mode. Mg2+ has been reported to stabilize the secondary and tertiary structure of aptamer and to facilitate the formation of a ligand−aptamer complex in some cases.36,37 Increasing Mg2+ concentration in the range of 1−10 mM greatly increased the affinity of M13a, and weakly increased the affinity of M6b-M14 (Figure 3a, c). Further increasing Mg2+ concentration to 15 mM greatly decreased the affinity of M13a. The different effects of Mg2+ on the binding affinity of M13a and M6b-M14 are similar as those of K+, also implying the differences of these two aptamers in the binding mode. The pH value is connected with protonation or deprotonation of aptamer and target, which may affect the affinity by increasing or decreasing the electrostatic interaction between aptamer and its target.34,36 The low and high pH values were found unfavorable to the binding of M6b-M14 and M13a to PSL (Figure 3b,d). The optimized pH range for both aptamers’ binding is 6−8, which is identical to the condition for aptamer selection.

stronger binding ability than others, they were further optimized by truncation and mutation assay. The secondary structure prediction31 showed that M6a adopted a bulged hairpin structure (Figure 1). The truncation and mutation assay (see Supporting Information for details) suggests that stem I with modest length plays important role in the aptamers’ binding ability, the four G tracts in loop and stem II region are critical for the aptamer binding, but stem II is not necessary for binding (Supporting Information Figure S-2). Two truncated and mutated sequences (M6b and M6b-M14) were found to have similar or better binding ability (Figure 1). The predicted secondary structure of aptamer M13a is a hairpin structure with a one-base bulge on the stem (Figure 1). Truncation or mutation of M13a caused decrease or almost complete loss of binding ability (Supporting Information Figure S-3), suggesting that it is an optimal aptamer sequence. Both M6b and M13a possess four double-G tracts in the bulge-stem II-loop or loop region, which is characteristic of DNAs that form G-quadruplex structures. G-quadruplex structures are often found to be the binding motifs of many aptamers.17,20,25,30 To illuminate whether G-quadruplex structure was formed by these aptamers, a CD spectrum analysis was carried out (Figure 2) (see Supporting Information

Figure 2. CD spectra of M6b-M14 (a, b) and M13a (c, d) in the absence or presence of Na+ or K+ and PSL. Aptamers (3 μM) was dissolved in 20 mM Tris-HCl buffer (pH 7.4), the final concentration of PSL is 10 μM.

for details). CD signals can provide information concerning the structure of G-quadruplex (i.e., antiparallel, parallel, hybrid-type structure): the antiparallel structure has a positive elliptic maximum at 295 nm and a negative minimum at 260 nm; while the parallel structure have a positive maximum at 264 nm and a negative minimum at 240 nm; and the mixed-hybrid type have a positive at 295 nm plus a positive shoulder near 265 nm and a negative minimum at 235−240 nm.32,33 As shown in Figure 2, both sequences M6b-M14 and M13a showed a negative band near 240 nm and a broad band in the range of 260−285 nm in 20 mM Tris-HCl buffer (pH 7.4) without K+ and Na+, suggesting that no G-quadruplexes formed. However, in the presence of 500 mM Na+ or K+, the CD spectra of M6b-M14 and M13a showed a new positive band around 295 nm and a decreased signal near 260 nm, suggesting the formation of antiparallel G-quadruplex. Further addition of PSL to the highsalt buffer system did not significantly change the CD feature of 7326

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Figure 3. (a and b) Effects of [Na+], [K+], [Mg2+], pH, DMF, and ethanol on the binding of M6b-M14. (c and d) Effects of [Na+], [K+], [Mg2+], pH, DMF, and ethanol on the binding of M13a.

the aptamer. As shown in Figure 4, the target molecule PSL exhibited the strongest binding ability to both aptamers. The

Hydrophobic effect has been reported to play important role on aptamer binding.20,34 The addition of organic solvent to the binding buffer may affect the interaction between aptamers and their targets. As shown in Figure 3b, M6b-M14 showed good tolerance to DMF and ethanol, despite the fact that its binding ability decreased gradually with the increase of DMF and ethanol. About 40% binding ability of M6b-M14 remained in the binding buffer containing 50% DMF or 50% ethanol. M13a exhibited similar tolerance to ethanol as M6b-M14 but showed less tolerance to DMF, 5% DMF caused a 60% decrease of binding ability of M13a (Figure 3d). Equilibrium Dissociation Constants (Kd) of M6b-M14 and M13a. On the basis of the above results, a buffer consisted of 10 mM Na2HPO4, 2 mM KH2PO4, 500 mM NaCl, pH 7.4 was used as the optimal binding condition for the binding assay of M6b-M14 and M13a. Under this condition, the equilibrium dissociation constants (Kd) of M6b-M14 and M13a binding versus PSL immobilized on beads were determined to be 2.27 ± 0.80 and 2.54 ± 1.41 μM by the static adsorption method (Supporting Information Figure S-4). Since the Kd values measured by static adsorption method is versus the immobilized PSL, we further measured the Kd values of both aptamers binding to free PSL by ITC method. The measured Kd values were 3.98 ± 0.72 and 4.59 ± 1.56 μM (Supporting Information Figure S-5), which agreed well with the Kd values versus immobilized PSL, implying that these aptamers mainly bind to the p-nitrobenzenesulfonamide moiety of PSL and not to the linking moiety of immobilized PSL. Specificity of M6b-M14 and M13a. The specificity of M6b-M14 and M13a to p-nosyl-protected amino group was investigated by competitive binding assay (see Experimental Section for details), in which the strong relative competing ability means a strong binding ability of the tested molecule to

Figure 4. Competitive binding assay of PSL and different molecules to M6b-M14 (a) and M13a (b) . Relative competing ability= (F0 − Fb)/ F0 × 100%, Fb is the fluorescence intensity of the eluate with organic molecules (competitor), and F0 is the fluorescence intensity of the eluate without organic molecules. Each experiment was repeated three times.

compounds containing p-nosyl protected primary and secondary alkyl amino group, p-nosyl aminoethanol (PSAE), p-nosylN,N-bis(2-hydroxyethyl)amine (HEPN), and p-nosyl aminocaproic acid (PSAC) showed strong binding ability to both aptamers. However, PZL, p-nosyl-OH (PSA), and L-Lys showed very weak binding ability. These results suggest that these aptamers do not merely bind to the nitrobenzene moiety 7327

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or the L-Lys moiety of PSL. Instead, the sulfonamide moiety is primarily responsible for the binding. Two p-aminobenzenesulfonyl amide compounds, sulfanilamide (SA) and sulfamethoxazole (SMZ) did not show any competing ability. Benzene sulfonyl aminoethanol (BSAE) and p-methyl-benzene sulfonyl aminoethanol (MSAE) showed much weaker competing ability than PSAE. These results suggest that the nitro moiety takes part in the aptamer binding. The relative weaker competing ability of PSAC than PSAE and PSL may be owing to the electrostatic repulsion between the negatively charged carboxyl group of PSAC and the sugar−phosphate backbone of DNA at pH 7.4. Compared with PSAC, the competing ability of two pnosyl α-amino acids, p-nosyl aminoacetic acid (PSAA), and N(p-nosyl)-L-phenylalanine (NPSP) was much lower, which implies that the α-carboxyl group strongly interferes with these aptamers’ binding to the sulfonamide moiety. The specificity investigation suggests that the whole p-nosyl-amide moiety is mainly involved in the aptamer−target interaction, and these aptamers have the potential to be used for recognition of compounds containing alkyl amino group protected by p-nosyl group. Recognition of p-Nosyl Protected Amino Compounds by M6b-M14 Aptamer Beacon. The specificity assay showed that M6b-M14 and M13a had strong binding ability to p-nosyl protected amino compounds except those with an αcarboxyl group. To demonstrate that these aptamers could be used for recognition of amino compounds, we designed an aptamer beacon6 that consisted of a FAM labeled M6b-M14 (M6b-M14-F) and a BHQ labeled complementary sequence (M14−5C9-BHQ) (Figure 5a). The response of aptamer beacon to PSL is shown in Figure 5b. The fluorescence intensity of aptamer beacon increased with the rise of PSL level in the range of 0.1−100.0 μM, a linear response was obtained in the concentration range of 0.5−10 μM (R = 0.9946) (Supporting Information Figure S-6). The specificity assay showed that this aptamer beacon had the same specificity as M6b-M14, since it only responded to compounds with the pnosyl protected primary and secondary alkyl amino group (PSL, PSAC, HEPN, and PSAE), and did not respond to other compounds (Figure 5c). This aptamer beacon was further tested for the response to real samples containing amino compounds. Reaction mixtures of amino compounds and p-nosyl-Cl were added separately to the working solution of aptamer beacon. Only the reaction mixtures of L-lysine, ethanolamine and diethanolamine increased the fluorescence intensity of the aptamer beacon, while the other tested compounds, such as ethanol, glycine and L-phenylalanine did not change the fluorescence of the aptamer beacon (Figure 5d), suggesting that the side-products, as well as the excess p-nosyl-Cl and p-nosyl-OH did not interfere the response of aptamer beacon. Since aptamer M6b-M14 does not bind to the p-nosyl protected α-amino acids, the aptamer beacon was also tested against L-lysine from amino acid mixture. As shown in Figure 5d, after adding p-nosyl-Cl, the amino acid mixture (AAM, including glycine, L-histidine, Lcysteine, L-phenylalanine, L-tryptophan, L-arginine, L-glutamic acid, and L-valine) without lysine only slightly increased the fluorescence intensity of the aptamer beacon, while the amino acid mixture containing L-lysine (AAM+lysine) greatly increased the fluorescence intensity, which implies that this aptamer has the potential in recognition and isolation of peptides containing lysine. Because p-nosyl-Cl can react with amino group rapidly in mild condition (Supporting Information

Figure 5. (a) Illustration of the working principle of aptamer beacon; (b) the fluorescence spectra of aptamer beacon in the prescence of different concentration of PSL; (c) the increase of fluorescence intensity of aptamer beacon in the prescence of different organic molecules; (d) the fluorescence spectra of aptamer beacon after adding the reaction solution of different compounds and p-nosyl-Cl.

Figure S-7), the above results show that aptamers against pnosyl protected alky amino groups could have many applications for compounds containing alkyl amino groups, such as separation, identification, and detection of these compounds.

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CONCLUSIONS To generate aptamers against alkyl amino groups, we used pnosyl protected L-lysine (PSL) as the selection target, and evolved a group of aptamers by affinity chromatography-based SELEX technique. Two optimized aptamers, M6b-M14 and M13a show strong affinity to PSL with the Kds at the level of 2−5 μM in optimal buffer condition. The mutation assay, CD spectral assay and the binding assay under different buffer conditions suggest that both M6b-M14 and M13a adopt an antiparallel G-quadruplex structure when binding to targets. The specificity assay indicates that M6b-M14 and M13a bind to p-nosyl protected alkyl amino groups, except those with an αcarboxyl group. An aptamer beacon based on M6b-M14 shows good selectivity to p-nosyl protected alkyl amino compounds. This aptamer beacon system can directly respond to the 7328

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Analytical Chemistry

Article

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reaction solution of alkyl amino compounds and p-nosyl-Cl, and indirectly detect lysine from amino acid mixtures. The above results have demonstrated that (1) aptamers recognizing a certain type of molecules can be generated by SELEX technique by targeting common features of this family of molecules. (2) For small groups that usually can not be used as target for aptamer selection, the introduction of a protection group is an effective method to obtain their aptamers. (3) Aptamers against a common moiety of a certain type of molecules have the potential to be used as new tools for the separation, detection or identification of molecules of this type.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel./Fax: 86-10-62528509. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from Grant 973 Program (2011CB935800 and 2011CB911000), NSF of China (21075124 and 20805049) and 863 Program (2008AA02Z206).

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dx.doi.org/10.1021/ac300281u | Anal. Chem. 2012, 84, 7323−7329