DNA-Templated Aptamer Probe for Identification of Target Proteins

Mar 7, 2017 - BP was first added to recruit the binding protein via aptamer recognition, and subsequently CP was added to let the cross-linker close t...
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A DNA-templated Aptamer Probe for Identification of Target Proteins Wenjing Bi, Xue Bai, Fan Gao, Congcong Lu, Ye Wang, Guijin Zhai, Shanshan Tian, Enguo Fan, YuKui Zhang, and Kai Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04895 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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A DNA-templated Aptamer Probe for Identification of Target Proteins Wenjing Bi,† Xue Bai,† Fan Gao,‡ Congcong Lu,‡ Ye Wang,‡ Guijin Zhai,† Shanshan Tian,† Enguo Fan,§,∥ Yukui Zhang,‡,⊥ Kai Zhang*,† †

Tianjin Key Laboratory of Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China ‡ Department of Chemistry, Nankai University, Tianjin 300071, China § Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany ∥

Department of Microbiology and Parasitology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/School of Basic Medicine, Peking Union Medical College, Beijing 100005, China

⊥

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

ABSTRACT: Using aptamers as molecular probes for biomarker discovery has attracted a great deal of attention in recent years. However, it is still a big challenge to accurately identify those protein markers that are targeted by aptamers under physiological conditions due to weak and non-covalent aptamer-protein interactions. Herein, we developed an aptamer based dual-probe using DNA-templated chemistry and photo-crosslinking technique for the identification of target proteins that are recognized by aptamers. In this system, the aptamer was modified by a single strand DNA as binding probe (BP), and another complementary DNA with a photo-active group and reporter group was modified as capture probe (CP). BP was first added to recruit the binding protein via aptamer recognition, and subsequently CP was added to let the crosslinker close to the target via DNA self-assembly, and then a covalent bond between CP and its binding protein was achieved via photo-crosslinking reaction. The captured protein can be detected or affinity enrichment using the tag, finally identified by MS. By use of lysozyme as a model substrate, we demonstrated that this multiple functionalized probe can be utilized for a successful labeling and enrichment of target protein even under complicated and real environment. Thus a novel method to precisely identify the aptamer-targeted proteins has been developed and it has a potential application for discovery of aptamer-based biomarkers.

Aptamers are single-stranded DNA or RNA molecules evolved from random oligonucleotide libraries by repetitive binding of the oligonucleotides to target molecules, a wellknown method named SELEX (systematic evolution of ligands by exponential enrichment).1,2 Aptamers can recognize not only small-molecules,3 but also macro-biomolecules4 such as proteins,5,6 cells7-9 and viruses10. Because of their high specificity and selectivity to targets, aptamers are usually called “chemical antibodies”.1,2 Owing to their properties of small size, good stability, easy modification and target versatility, aptamers can be used as an alternative to antibodies in many applications.4 Accumulated evidence suggests that aptamers can recognize molecular signatures like membrane proteins that are exposed on cell-surface to indicate the specific physiological states of cells9,11 and to distinguish certain types of cancer cells.12,13 Therefore aptamers are highly promising to be used in the detection of biomarkers of certain diseases and indeed an aptamer-based “fishing” method has been developed for the identification of biomarkers.14,15 However, it is still a big challenge to accurately identify the targets of aptamers due to

the unstable, weak and non-covalent interactions between aptamers and their targets.16 Photoaffinity crosslinking technique has become a powerful tool to investigate those weak molecular interactions because it can covalently link interaction partners like protein and DNA, small molecules or another protein. Famulok et al. reported a general strategy called ABAL (aptamer based affinity labeling),17 in which a photo reactive group was introduced into an aptamer to trap its targets covalently. However, the complicated synthetic steps might limit its wide range application and the insertion of the crosslinker inside aptamers may change the structure of the probe that eventually might affect the recognition of aptamers to target proteins. DNA-encoded chemical libraries (DEL) is a technology to site-specifically incorporate desired chemical groups for chemical synthesis of large number of compounds via selfassembly of complementary double-stranded DNA.18 Because this DNA templated technique was easier to implement the desired groups on bioactive substances but with a minimized impact, it has been developed as a useful tool in the applications of protein identification including drug target

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proteins,19 transcription factors20 and the readers of histone marks.21 In the present work, combining DNA-templated chemistry with photo-crosslinking technology, we developed an aptamer-based dual-probe system to analyze the target proteins. Using lysozyme (LYS) as a model substrate, we designed and prepared binding and capture probes to enable a specific aptamer recognition and a flexible photo-crosslinkingbonding for target protein. Our data demonstrated that this strategy allows an effective labeling and enrichment of the aptamer-targeting protein and holds a great potential for a more broad and general application in the identification of target proteins recognized by aptamers.

EXPERIMENTAL SECTION Materials and reagents. Tris, sodium choride (NaCl, >99.5%) and magnesium chloride (MgCl2, >98%) were purchased from Sigma Aldrich. Succinimidyl-ester diazirine (SDA), monomeric avidin beads and HPLC solvents were purchased from Thermo Fisher Scientific Ltd. LYS, aptamer with a sequence of 5’-ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG ATG TCG CTG TAG -3’ and all DNA oligonucleotides were obtained from Sangon biotech (Shanghai) Co., Ltd. Synthesis and characterization of aptamer probe. The LYSaptamer sequence was selected as report.22 DNA sequences were designed with the help of m-fold webserver (http://unafold.rna.albany.edu/?q=mfold/download-mfold), the favorable secondary structure of aptamer was shown in Figure S1. Binding probe containing a LYS aptamer and a single stranded DNA was obtained by a custom synthesis directly. Capture probe was synthesized via a typical coupling reaction for amide bond formation between amino modified DNA and succinimidyl-ester diazirine (SDA) (Figure S2). Amino modified DNA was dissolved in NaHCO3 buffer (200 mM, pH= 8.5) then centrifuged for 5 minutes at 10,000 g. SDA was added in a molar ratio of DNA (the last reaction concentration was 100 µM): SDA = 1: 20, then the mixture was shaken at 25 ºC for 2 hours. DNA was precipitated using ice cold ethanol. Supernatant was removed and the pellet was washed 3 times using ethanol. The washed pellet was redissolved in 1×binding buffer and stored at the -20 ºC before use. All synthesized DNA probes were characterized by MALDI−TOF MS with a Bruker Autoflex III TOF/TOF mass spectrometer. Default settings (acquisition of mass spectra in the linear negative ion mode within an acceleration voltage of 20 kV) were applied for the detections. The software was Flexanalysis for the analysis of spectral processing and peak detection. The MALDI-TOF spectral data are shown in Figure S3. Fluorescence labeling of LYS using dual DNA probe. The aptamer was heated to 95 ℃ for 5 min then cooled to room temperature before use. Binding probe and LYS were pre-incubated in 1 × binding buffer at 4 ℃ overnight. Then the FAM (5carboxy-fluorescein)-CP was added to the complexes and the mixture was incubated at room temperature for another one hour to form DNA duplex. After UV irradiation for 8 min on ice, the sample was heated to 95 ℃ for 10 min and analyzed by 15% SDS-PAGE. The proteins were visualized by scanning the gel on a bio-rad imager. The fluorescence intensity was determined by Image J tool. Affinity enrichment of LYS using dual DNA probe. Binding probe, LYS and Hela cell lysates were pre-incubated in reaction medium at 4 ℃ overnight. After biotin-CP was added, the mixture was shaken at desired temperature for another one hour before UV irradiation on ice for 8 min. The mixture was incubated with monomeric avidin beads at 4 ℃ for 4 hours. The beads were then

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washed with 1 × phosphate-buffered saline (PBS, pH 7.4) three times to reduce nonspecific binding. The aptamer-protein hybrid was further eluted by incubating for 1 h with elution buffer (the biotin competes with the biotin-CP and releases the aptamerprotein hybrid from the monomeric avidin beads) at room temperature. The eluates were concentrated to a complete dryness with a SpeedVac before moderate loading buffer was added. Then the sample was heated at 95 ºC for 10 minutes and analyzed by 15% SDS−PAGE. The target was subsequently identified by LCMS/MS (Figure S4). Extraction of LYS from egg white. The egg white was separated from fresh eggs and diluted to 50% (v/v) with phosphate buffer (pH 7.4, 20 mM). The diluted egg solution was homogenized in an ice-bath for 30 min, and then centrifuged at 4 ℃ at 10,000 g for 10 min. The supernatant was used as a LYS source. The proteins in supernatant was examined using 15% SDS-PAGE and visualized by silver staining. The protein concentration was measured using BCA method after diluting the supernatant 25 times. Protein identification by HPLC-MS/MS. The corresponding gel bands were excised and subjected to in-gel digestion as described previously.23 After desalting, the tryptic digest was reconstituted in 7 µL HPLC buffer A (0.1% (v/v) formic acid in water), and 5 µL was injected into a Nano-LC system (EASY-nLC 1000, Thermo Fisher Scientific, Waltham, MA). Each sample was separated by a C18 column (50 µm inner-diameter × 15 cm, 2 µm C18) with a 50 min HPLC-gradient at a flow rate of 200 nL/min (linear gradient from 2 to 35% HPLC buffer B (0.1% formic acid in acetonitrile) in 40 min, and then to 90% buffer B in 10 min). The HPLC elute was electrosprayed directly into an Orbitrap QExactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The source was operated at 1.8 kV. The mass spectrometric analysis was carried out in a data-dependent mode with an automatic switch between a full MS scan and an MS/MS scan in the obitrap. For full MS survey scan, automatic gain control (AGC) target was 1e6, scan range was from 350 to 1750 with the resolution of 70,000. The 10 most intense peaks with charge state 2 and above were selected for fragmentation by higher-energy collision dissociation (HCD) with normalized collision energy of 27%. The MS2 spectra were acquired with 17,500 resolution. The exclusion duration for the data-dependant scan was 10 sec, and the exclusion window was set at 2.2 Da. The resulting MS/MS data were searched against UniProt human database (downloaded Nov 13, 2016) for Hela lysates or UniPort gallus database downloaded Nov 13, 2016) for LYS using Proteome Discoverer software (v1.4) with an overall false discovery rate (FDR) for peptides of less than 1%. Peptide sequences were searched using trypsin specificity and allowing a maximum of two missed cleavages. Carbamidomethylation on Cys was specified as fixed modification. Oxidation of methionine and acetylation on protein N-terminal were fixed as variable modifications. Mass tolerances for precursor ions were set at ±10 ppm for precursor ions and ±0.02 Da for MS/MS.

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Figure 1. Schematic representation of aptamer probe for LYS detection based on DNA templated chemistry and photo crosslinking technology. Step 1: incubation of aptamer with LYS. Step 2: the addition of CP probe to initiate BP/CP hybridization. Step 3: UV-irradiation to initiate photo-crosslinking. Step 4: the labeling with a fluorophore or biotin for in-gel imaging or pull-down. Step 5: MS analysis.

RESULTS AND DISCUSSION Analytical strategy and the preparation of probes A brief description of the strategy is shown in Figure 1. We synthesized two DNA probes, a “binding probe” (BP) and a “capture probe” (CP). BP includes a native, modification-free hairpin lysozyme aptamer for target recognition and a single-stranded DNA. CP contains a complementary DNA sequence and a modified photo-reactive diazirine group as well as a tag for in-gel imaging or pull-down. The synthesis and characterization of the probes are shown in Figure S2 and Figure S3. The analysis is a five step process: Firstly, BP was incubated with target protein at 4 ℃ overnight. Secondly, CP was added to the mixture to let the diarizine groups close enough to the target via DNA self-assembly. Thirdly, a covalent bond formation between CP probe and its target protein was achieved via photo-crosslinking reaction under 365 nm. The captured protein can be detected or affinity pulldown using FAM or biotin tag. Finally, target proteins were identified by MS. Using lysozyme that has important functions in antiinflammatory, anti-viral, and anti-tumor activities24 as a typical model, we characterized the feasibility, specificity and sensitivity of the probes, respectively.

lane 6: BP/CP without LYS; lane 7: CP only; arrows indicate positions of conjugate.

Optimization of probe and experimental conditions For the aptamer probe, altering the chain length of CP can conveniently adjust the spatial position of the crosslinker toward the targets, which eventually can influence the labeling efficiency. To obtain an optimal position of the cross-linker, we have designed four different overhang lengths of CP (n=0, 3, 6, 9). The value of ‘n’ indicates the number of overhang bases within CP that does not hybridize with BP after CP/BP hybridization. To evaluate the labeling efficiency, the fluorescence intensity was measured (Fig. 3) and the results from three independent experiments showed that CP of n=3 gave the highest labeling yield (Figure 3A). The results further suggest that the arm of CP can provide a better spatial position for the crosslinker to access binding partners. However, considering a longer crosslinking radius not only leads to a decreased labeling efficiency, but also brings in nonspecific reaction, thus we chose n=3 as the optimal length in the following experiments. Next, we investigated the interaction between probe and LYS using series of varied concentrations of probe incubated with a defined concentration of LYS. As shown in Figure 3B, while increase the concentration of the probes lead to an improved labeling yield of LYS, the by-product (BP/CP) is also increased especially at high probe concentrations, which may be due to ligand-independent intermolecular crosslinking reactions. To avoid false-positive results derived from high probe concentrations, we finally chose 20 µM as the optimal probe concentration.

Feasibility of the aptamer-probe labeling To evaluate the feasibility of this method, we designed a group of parallel in vitro experiments to examine the labeling of the probe. BP probe was firstly incubated with proteins and subsequently with CP-FAM, and then followed by photo-crosslinking reaction. We observed four fluorescence bands in the SDS-PAGE image, as shown in Figure 2. CP, BP/CP, CP/LYS and BP/CP/LYS can be identified from band 7, 6, 4 and 1, respectively. Figure 2 also demonstrates that the BP/CP/LYS can be successfully labeled only when BP and CP are added, as well as UV irradiation. There is no obvious labeling product in negative controls (lane 2-7: no irradiation, no BP, no CP, and BP without LYS), indicating that all the conditions such as DNA hybridization, specific aptamer recognition and adequate UV irradiation are indispensable for a valid protein labeling and the observed labeling products are probe-specific rather than artifact. These results adequately demonstrate the feasibility of this proposed assay.

Figure 2. In vitro labeling of LYS by the aptamer probe. Lane 1: BP/CP/LYS/hv; lane 2: BP/CP/LYS without irradiation; lane 3: CP/LYS without irradiation; lane 4: CP/LYS/hv; lane 5: no CP;

Figure 3. (A) The optimization of relative position of photocross-linker towards the target by changing the overhang length within CP. SDS-PAGE analysis of labeling experiments using CPs containing four different overhang lengths. LYS = 20 µM. BP/CP-FAM = 20 µM. Error bars are calculated based on three independent experiments. (B) The effects of various concentrations of BP/CP probes to labeling yields. LYS = 20 µM.

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Figure 4. (A) The effects of various concentrations of binding buffer on non-specific binding. BSA = 20 µM and CP-FAM = 20 µM. (B) The effects of various concentrations of binding buffer on specific binding. LYS = 20 µM, BP/CP-FAM = 20 µM. Moreover, as the salt concentration of buffer is well known to have an important effect on protein-DNA interactions,19 we further investigated the effect of salt concentration on labeling yield to optimize the experimental conditions aiming at a maximum yield of crosslinking products. We first examined the nonspecific photo-crosslinking between a CP and BSA (bovine serum albumin), a typical protein known to have an extensive non-specific binding with diverse small molecules and ligands. As shown in Figure 4A, a remarkable decrease of BSA-derived crosslinking products is detected with an increased salt concentration. In Figure 4B, the influence of salt concentration on specific binding is shown and it is obvious that the labeling yield first increases and then decreases with the increase of salt concentration, which demonstrates the influence of salt ions on aptamer-protein interaction.17 Finally, we chose 1× binding buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5mM MgCl2) as the optimal reaction medium.

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To evaluate the affinity enrichment ability of the aptamer probe in a complicated background, we prepared another CP probe modified by a biotin tag. After purification, targets were detected by SDS-PAGE. As shown in Figure 6, a protein can be isolated from samples and the molecular weight of the band matches to the expected molecular weight of LYS. Further MS analysis (Table S2 and Figure 4) confirmed that the identified protein band indeed represents LYS. To further evaluate the ability of the probes for target protein enrichment, we used high sensitive mass spectrometry to compare the identified proteins before and after enrichment. LYS was mixed with 10 times amount of proteins from Hela cell lysates, then the mixture was subjected to enrichment via the aptamer probe containing a biotin tag. The samples before and after enrichment were subjected to in-solution digestion and HPLC-MS/MS analysis (Supporting Information). As shown in Table S3, the number of identified proteins is decreased significantly from 863 to 125 after enrichment. More importantly, the number and score of the identified peptides demonstrated that the abundance of the identified proteins decreased significantly after enrichment (For example, the number of identified peptides for protein CPS1 decreased from 48 to 1 after enrichment), while the score of LYS was increased from 86.64 to 368.96 after enrichment. In summary, although part of residual proteins can still be found in high sensitive mass spectrometry but with a significantly decreased signal, majority of the background proteins can be eliminated through enrichment. Thus these results further confirmed the good enrichment ability of the aptamer probe. Therefore, it can be concluded that this aptamer probe can be used to efficiently fish and identify the aptamer targeted proteins in combination with proteomics approach.

Labeling and enrichment of targets in a complex environment To explore the specificity and selectivity of the probe, we performed the experiments in the presence of BSA. As shown in Figure 5A, it is obvious that the probe is able to label LYS with a high selectivity. Similar labeling experiment of LYS in a more complicated background, i.e. the whole Hela cell lysate, was also conducted (Figure 5 B) and again a specific labeling was achieved. All together, the aptamer probe has an outstanding specificity to its target even in a complex system.

Figure 5. The selectivity of the aptamer probe for LYS labeling in the presence of (A) BSA and (B) the whole cell lysate. The target protein is marked by the red arrow. ‘mis’ means a random DNA sequence that unable identify LYS.

Figure 6. Affinity pull-down of lysozyme by the aptamer probe and monomeric avidin beads. Lane1: marker. Lane2: lysozyme input. Lane 3: lysozyme enriched from sample shown in lane 2. Lane 4: LYS mixed with Hela cell lysates. Lane 5: LYS enriched from samples shown in lane 4. The red arrow indicates the target.

The application of the aptamer probe in egg white for LYS detection To further examine the application of this probe in real biological samples, we used this probe to detect LYS from chicken egg white containing LYS.25 Egg white sample was incubated with the aptamer probe as described in Experimental section. As shown in Figure 7A, LYS was detected as a very faint band (lane 3) only when the total amount of crude chicken egg white is about 15 µg, while after FAM labeling, an obvious band was observed even with low amount of loaded proteins (60 ng, lane 2, Figure 7B), which should be the BP/CP/LYS complex by comparing the band with lane 2 and 3 according to their molecular weight. This detected protein band (15 µg, lane 3 in Figure 7B) was further excised from the gel and analyzed by MS (details shown in Table S4), which confirmed that it is indeed lysozyme. Except for target protein, other high abundance of proteins almost can not be observed in the FAM labeling experiment, which demonstrated that this method is highly specific for target protein. Of course, it should be noted that the redundant CP-BP as a background might have a potential effect on the detection of target proteins. If one

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want to specifically remove CP-BP from the samples, a centrifugal filtration can be used before SDS-PAGE or in-solution digestion taking advantage of the smaller molecular weight of CP-BP compared with target proteins. In conclusion, the results clearly demonstrated that the LYS in egg white sample can be specifically labelled using the dual probe thus indicating that the aptamerprobe might be promising in the identification of aptamer targeting proteins in real biological samples.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China with Grants (21275077), National Basic Research Program of China (Grants 2013CB910903, 2016YFC0903000, and 2012CB910601), and Tianjin Municipal Science and Technology Commission (No. 14JCYBJC24000).

REFERENCES

Figure 7. Detection of LYS in crude egg white. (A) Silver staining of protein samples used for labeling experiment; (B) In-gel imaging assay. Lane 1: marker. Lane 2-3: labeling results of LYS under different amount of egg white proteins. (Lane 2: 60 ng, lane 3: 15 µg). The red arrows indicate the position of LYS.

CONCLUSION By combining photo crosslinking method and DNA-templated chemistry, a dual-probe method was developed and successfully applied for the detection of protein that is recognized by aptamer. Owing to UV irradiated photo-crosslinking, the probe can invert the weak and transient intermolecular interactions into covalent ones, which enables the further exact analysis of the targeted proteins. By conjugating the crosslinker to another single-stranded DNA, great influence of additional chemical groups on aptamer structure characteristics can be skilfully avoided. In addition, because of its double probe features, the probe can be flexibly designed to obtain an optimal crosslinking position with minimum changes to whole probe and furthermore this probe can be applied to analyze any other individual protein/aptamer interactions easily. Using LYS as an example, the optimized probe showed a high selectivity and specificity to target protein, even in real biological background, such as egg white sample, suggesting this strategy is suitable for aptamer targeted proteins identification, which should hold a great potential and can be considered as a novel tool particularly when combined with quantitative proteomics technique for aptamer-targeted biomarker detections.

ASSOCIATED CONTENT Supporting Information Available: Additional experimental results, favorable secondary structure of aptamer, synthesis and characterization of aptamer dual-probe, The MALDI-TOF MS Characterization of the DNA-based probes, the typical MS/MS spectra of tryptic peptide of LYS, the list of LYS aptamer and DNA sequences, the list of identified tryptic peptides of LYS and identified proteins before and after enrichment. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

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