Advances in studies of aptamer-protein targets identification using

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Advances in studies of aptamer-protein targets identification using chromatographic approach Anna Drabik, Joanna Ner-Kluza, Przemyslaw Mielczarek, Laia Civit, Günter Mayer, and Jerzy Silberring J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00122 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 30, 2018

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Journal of Proteome Research

Advances in studies of aptamer-protein targets identification using chromatographic approach Anna Drabik*1, Joanna Ner-Kluza1, Przemyslaw Mielczarek1, Laia Civit2, Gunter Mayer2,3, Jerzy Silberring1 1

Faculty of Materials Science and Ceramics, AGH University of Science and Technology,

Krakow, Poland 2

Department of Chemical Biology, Life and Medical Sciences Institute, University of Bonn,

Germany 3

Center of Aptamer Research and Development, University of Bonn, Germany

AUTHOR INFORMATION Corresponding Author * Anna Drabik Ph.D. Department of Biochemistry and Neurobiology AGH University of Science and Technology Mickiewicza 30 Ave 30-059 Krakow, Poland Tel. +48-126175083

ACS Paragon Plus Environment

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E-mail: [email protected]

ABSTRACT Ever since the development of the process known as Systematic Evolution of Ligands by EXponential enrichment (SELEX) aptamers are widely used in variety of studies, including exploration of new diagnostic tools and discovery of new treatment methods. Aptamers abilities to bind to proteins with high affinity and specificity, often compared to antibodies, enable the search for potential cancer biomarkers and help to understand the mechanisms of cancerogenesis. The blind-spot of those investigations is usually the difficulty in selective extraction of targets attached to the aptamer. There are many studies describing the cell-SELEX for the prime choice of aptamers towards living cancer cells, or even whole tumors in the animal models. However, dilemma arises when a large number of proteins are being identified as potential targets, which is often the case. In this article, we present a new analytical approach designed to selectively target proteins bound to aptamers. During studies, we have focused on unambiguous identification of the molecular targets of aptamers characterized by the high specificity to the prostate cancer cells. We have compared four assay approaches using electrophoretic and chromatographic methods for “fishing-out” aptamer -protein targets, followed by mass spectrometry identification. We have established a new methodology, based on fluorescent-tagged oligonucleotides commonly used for the flow cytometry experiments or as optic aptasensors that allowed detection of specific aptamer-protein interactions by mass spectrometry. The use of atto488-labeled aptamers for tracking the formation of specific aptamer-target complexes provides the possibility to study putative protein counterparts without the necessity of applying enrichment techniques. Significantly, changes of the hydrophobic properties of atto488-labeled aptamer-protein complexes facilitate their separation by the reversed phase chromatography combined with fluorescence detection, followed by mass


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spectrometry-based protein identification. These comparative results of several methodological approaches confirmed universal applicability of this method to study aptamer-protein interactions with high sensitivity, showing superior properties over pull-down techniques.

KEYWORDS aptamer, target, protein, chromatography, electrophoresis, mass spectrometry

INTRODUCTION Aptamers are singly stranded oligonucleotides that possess unique properties to fold into a defined 3D structure, by which they specifically bind to various targets, from small molecules to whole cells and viruses1. Their biophysical binding characteristics are similar to those found for antibodies, however they do not induce immunogenicity and can be chemically modified to improve stability or enable labeling, as they are manufactured by chemical synthesis2. This allows for reproducible, cheap, and fast production3. Moreover, not only aptamers bind to cognate target proteins but also can inhibit associated biological functions4. Aptamers are selected from a large library of oligonucleotides that contains approximately 1015 different sequences, by a process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). This process consists of repetitive cycles of selection and amplification. During each cycle, oligonucleotides with specific affinity for a desired target are retained, recovered, and amplified5. After 10-15 cycles, this procedure leads to the enrichment of a library, predominantly consisting of putative target binding species, which can be ultimately called the aptamers6. Aptamers are widely used as tools for different approaches, including diagnostic and therapeutic applications7-9. Despite many advantages of the aptamer-based assays, some drawbacks can be observed, e.g. during cell-SELEX process, when intact cells are applied as a target


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and the number of identified potential aptamer-protein complexes is pronounced. The number of interactions is unknown, however the aptamer internalization process is usually observed after 45 minutes and before this event, the aptamer is bound exclusively to membrane proteins10,11. This might suggest that the set of proteins that interact with the aptamer may change with the time of incubation. Hence, not only membrane proteins are routinely used as diagnostic cancer biomarkers. It is estimated that c.a. 35% of biomarkers are membrane proteins12. Also, studies on intra-cellular proteins might be of a great value to reveal potential biomarkers of cancer disease13. As a consequence, it would be interesting to study and identify protein targets of aptamers that specifically recognize cancer cells and may enter their interior, what can be used for therapeutic purposes in a future14. Identification of such targets has proven difficult to elucidate, because of the less specific nature of a pull-down assay based on streptavidin-biotinylated aptamer interactions8-10. Authors often describe a group consisting of many proteins being potential targets for aptamers binding, and as a final result, one protein is selected based on the vaguely defined criteria13,14. It is problematic to assign, whether single protein molecule comprises of a unique aptamer target, or all of them actually bind oligonucleotides. Additional research needs to be carried out to verify this ambiguity.

EXPERIMENTAL SECTION Cells PC3 cells from American Type Culture Collection ATCC (USA) engineered to express luciferease were obtained from ProQinase (Germany). PC3luc cells were cultured in GlutaMax


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(Gibco) supplemented with 10% Fetal Bovine Serum FBS (Gibco) and 1% antibiotic/antifungal solution (Sigma-Aldrich) at 37°C in 5% CO2 humidified atmosphere.

Incubation of aptamers with the cells Aptamers stored at -20°C were thawed to the room temperature. Stock solution (25mM in DPBS buffer) of the oligonucleotide sequences were incubated for 5 min at 90°C, enabling achievement of the proper tree dimensional structures of aptamers during renaturation process (cooling down to the room temperature). Cells were incubated with the aptamer sequences A26, A33, and A33sc (Figure 1) (Microsynth AG) 25nM for 45 min at 37°C in binding buffer (5mM MgCl2, 1mM CaCl2, 0.1% ssDNA in DPBS (Sigma-Aldrich)). After binding, the excess of aptamers was removed with washing buffer (5mM MgCl2, 1mM CaCl2, 1mg/ml BSA in DPBS). The cells were carefully scraped off the flask surface, transferred into a Falcon tube, and centrifuged 3 min at 200xg. To retain the interaction between the proteins and the aptamers, cross-linking was performed, with the exception of experiment 1 (Strepatavidin-biotin pull-down assay). During cross-linking cells with bound aptamers were incubated with 1% formaldehyde (Sigma-Aldrich) in DPBS for 2 min at 4°C on a rotator15. The cells were immediately washed three times with DPBS buffer for 2 min to remove the remaining formaldehyde. All experiments were performed three times as three independent biological replicates with standard deviation less than 5%. The presence of the aptamers was confirmed using fluorescence microscopy.

Figure 1. A26; A33 and A33sc oligonucleotides sequences.


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Homogenization 108 PC3luc cells were homogenized in a Dounce homogenizer for 2 min at 4°C in DPBS buffer with proteinase inhibitors cocktail (Roche) at 60 strokes per min PC3luc cells after incubation with formaldehyde (linking formation), were homogenized in a Dounce homogenizer for 2 min at 4°C in the presence of 0.1% SDS in DPBS buffer at 60 strokes per min16. Homogenates were centrifuged for 45 min at 25000xg at 4°C. Protein concentration was measured using Bradford method (Sigma-Aldrich) according to the producer’s protocol.

Enrichment of biotinylated fractions Paramagnetic particles MagneSphere® (Promega Z5481 0.6 ml) were washed 3 times with 1 ml of DPBS buffer for 3 minutes on rotator at 4°C. The buffer was discarded using the magnetic separation device (two-position stand). After washing, protein extracts obtained from the cells were dissolved in DPBS buffer to the total volume of 500 µl and incubated with biotinylated variants of aptamers for 2 hours at 4°C with 0.6 ml of streptavidin - coated paramagnetic beads on rotator. Subsequently, cell homogenates were discarded using magnetic separation device, and paramagnetic particles were washed 3 times with 1 ml of DPBS buffer for 3 minutes on rotator at 4°C. For electrophoretic assays 25 µl of 8M urea was added and incubated for 15 min at 60°C. Supernatants were collected and 25 µl sample buffer (Bio-Rad) was applied to each sample, and incubated at 90°C for minutes. For chromatographic assay sample was obtained after incubation of paramagnetic particles for 15 min at 60°C with 25 µl of 8M urea.

SDS-PAGE


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One-dimensional electrophoresis was performed as follows. Twenty microliters of the Laemmli sample buffer (Bio-Rad) was mixed with the same volume of sample. Proteins were separated with the aid of the 10 % SDS-PAGE in Mini Protean system (Bio-Rad)16. The gels were stained with Coomassie Brilliant Blue G (CBB G) (Sigma-Aldrich) prior to the nanoLCMS/MS analysis. The separation was repeated three times for each biological replicate separately. In the following experiments, the gel was divided into two identical parts. One part of the gel was stained with SYPRO Ruby (Bio-Rad) prior MS-based identification of proteins, and the second part was electrotransferred onto PVDF membrane (Bio-Rad) with 100 mA current at 4°C, overnight, to confirm the presence of protein complexes with biotinylated forms of aptamers. Again, the electrophoretic separation and electrotransfer were run individually for each biological replicate. SDS-PAGE of atto488-labeled aptamers did not require any type of staining, since the protein targets could be tracked based upon the presence of an atto488 dye. Once more, the electrophoresis was performed in three independent repetitions for accurate statistical evaluation.

Western blot SAV analysis The presence of specific protein targets was confirmed by the detection of biotinylated forms of aptamers (Microsynth AG) by streptavidin conjugated with alkaline phosphatase (Vector Laboratories). The blotted membranes were treated with casein solution (Vector Laboratories) as a blocking agent, for 1 h at room temperature, washed three times with Tris- buffered saline TBS (Sigma-Aldrich), and subsequently incubated with alkaline phosphatase conjugated streptavidin for 1 h in TBS supplemented with 0.1% Tween 20 TBST (Sigma-Aldrich). Finally, after washing with TBST buffer, the blots were visualized by alkaline phosphatase substrate kit II (Vector


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Laboratories). Both PVDF membranes and SDS-PAGE scans were acquired using the GelDoc XR+ (Bio-Rad).

KIF27 and XRCC5 immunodetection InvitrogenTM KIF27 (PA5-49134) and XRCC5 antibody (PA5-35087) were used to confirm the presence of Kinesin-like protein KIF27 and X-ray repair cross-complementing protein 5 in examined samples (triplicate). The blotted membranes were treated with casein solution as blocking agent for 1 h at room temperature, washed three times with TBS buffer, and subsequently incubated with KIF27 and XRCC5 antibody (Invitrogen) for 1 h with TBST in 1:1000 dilution. Subsequently, the membrane was washed three times with TBST buffer and incubated with alkaline phosphatase conjugated streptavidin for 1 h with TBST. Finally, the blots were visualized by the alkaline phosphatase substrate kit II. PVDF membranes were analyzed using the GelDoc XR+.

HPLC separations Chromatographic separation of protein extracts gained from PC3luc cells after incubation with atto488-labeled aptamers A26; A33 and A33sc were performed using the Prominence system (Shimadzu) equipped with the SPD-M20A UV-VIS detector and the RF-20A fluorescence detector. The cells were washed three times with DPBS buffer to remove remaining aptamer, and were homogenized in a Dounce homogenizer for 2 min at 4°C in DPBS buffer with proteinase inhibitors cocktail (Roche) at 60 strokes per min. PC3luc cells after incubation with formaldehyde (linking formation), were homogenized in a Dounce homogenizer for 2 min at 4°C in the presence of 0.1% SDS in DPBS buffer at 60 strokes per min16. Homogenates were


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centrifuged for 45 min at 25000xg at 4°C. Protein concentration was measured using Bradford method (Sigma-Aldrich) according to the producer’s protocol. C8 5µm 150x4.6mm Kinetex Reversed Phase column (Phenomenex) was used. The gradient consisting of eluent A (100% H2O 0.1% FA) and B (100% ACN 0.1% FA) was maintained, at a 600 µl/min flow-rate. A gradient was produced from 2 to 70 % B in 50 min, 85 % B was kept for 5 min, and again reduced to 2 % until 60 min.

Protein MS-based identification Gel bands excised with a scalpel were chopped into 1mm cubes, rinsed with water, and transferred into the LoBind Eppendorf® tubes. CBB or SYPRO Ruby stains were removed with 100 µL of 100 mM NH4HCO3 after 5 min of incubation at 40°C with shaking, and an equal volume of ACN was added. The supernatant was discarded after 5 min of incubation at 40°C with shaking. Then, the gel pieces were treated with 100 µL of 100% ACN and reswollen in 25 µL of 50 mM dithiothreitol DTT (Sigma-Aldrich) in 50 mM NH4HCO3, followed by 10 min incubation at 90°C with mixing at 500 RPM. DTT solution was removed. Subsequently, the reduced proteins were carbamidomethylated by the addition of 25 µL of 100 mM iodoacetamide (IAA; Sigma-Aldrich) during incubation for 10 min at 90°C with mixing at 500 RPM. Finally, 25 µL of 10 ng/µL trypsin Gold (Promega) in 50 mM NH4HCO3 was added, and the mixture was incubated overnight at 37°C with mixing at 500 RPM. After completion of digestion, the supernatants were transferred into the fresh LoBind Eppendorf® tubes, followed by addition 50 µL of 50 mM NH4HCO3, and after 10 min of incubation at 40°C with shaking, an equal volume of 100% ACN was added and again incubated for 10 min at 40°C with shaking. Subsequent extraction of peptides was repeated twice with 50 µL 5% formic acid (v/v) in 100% ACN, and


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the combined supernatants were evaporated to dryness in a vacuum centrifuge (Labconco). Prior to the nanoLC-MS/MS analysis, the peptides were resuspended in 20 µL of 0.1% formic acid (Sigma-Aldrich).

Nano Chromatography Combined with Tandem Mass Spectrometry nanoLC-MS/MS analysis The nanoLC-MS/MS analysis used to separate protein digests was performed using the Proxeon nanocapillary chromatography system (Bruker Daltonics). Separations were performed using a PepMap column (15 cm long, 75 µm ID, C18, 3 µm particle size, 100 Å pore size, Thermo-Scientific) The gradient was formed using 0.1% HCOOH in H2O (solvent A) and 0.1% HCOOH in ACN (solvent B), at a flow rate of 300 nL/min. The system was controlled by the Hystar software (Bruker Daltonics). A gradient was produced from 2 to 50% B in 30 min and up to 90% B at 35 min, then kept until 45 min, and again reduced to 2% until 55 min. The chromatographic system was directly coupled to the Amazon ETD (Bruker Daltonics) mass spectrometer16,17. The instrument operated in a positive-ion mode. During analysis, the two most intense peaks (threshold above 500 000) in the range of 450−1800 m/z were automatically fragmented using CID in the data-dependent acquisition mode. Charge state parameters included the preferable charge states 2+ and 3+. Accumulation time to achieve the number of ions entering the trap was less than 1 ms. Trap drive was set to 35. Two technical replicates of each sample were run.

Data Processing The acquired mass spectra were analyzed using the Bruker Data Analysis 4.0 software (Bruker Daltonics) and were identified using the Mascot 2.4.1 algorithm (Matrix Science) against the


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Swiss-Prot/TrEMBL sequence database ver. 2012_03 (535248 sequences; 189901164 residues)16. Search parameters were set as follows: taxonomy: human; modification: carbamidomethyl (fixed) or methionine dioxidation (variable); up to 1 missed cleavage; peptide charges: +1, + 2, and +3; mass tolerance: 0.8 Da for precursor mass and 0.6 Da for fragment mass. Proteins with at least two fragmented, unique peptides detected were considered, and an additional criterion was an ion score higher than 40, which is above the level of false positives (p ≤ 0.05).

RESULTS AND DISCUSSION In this paper, we describe the sensitive methodology established for the identification of protein targets of two aptamers selected via a cell-SELEX in the mouse cancer model, A26 and A33 that specifically recognize PC3luc cells, a prostate cancer cell line11. Previous studies for the elucidation of target counterparts of these aptamers were based on the pull down affinity assays using biotinylation combined with mass spectrometry analysis, although the major drawback are non-specifically bound proteins that can be found in the pulled down extract. To minimize this problem, four approaches were tested and their effectivity compared, including the conventional biotin-streptavidin system and the novel approach consisting of fluorescently labeled aptamers (Figure 2).


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Figure 2. Scheme of the workflow. 1.

Streptavidin-biotin pull-down assay

PC3luc cells were cultured, followed by 45 min incubation with biotinylated forms of aptamers A26; A33 and A33sc (scrambled; control sequence). These conditions were selected in order to achieve the optimum time frames to validate the aptamer binding proteins localized both on the cell membrane and inside the cell10. The cells were extensively washed and homogenized in a Dounce homogenizer, followed by incubation with streptavidin-coated paramagnetic beads in order to isolate the aptamer-protein complex. Enriched fractions were eluted and separated using SDS-PAGE. Several protein bands were identified in the PC3luc protein extracts after incubation with A26 and A33 that were not observed in A33sc control sample. However, the number of non-specific interactions present on the gel was predominant, represented by bands in the lane A33sc. Bands representing the potential aptamer-binding targets were excised both from A26 and A33 gel lanes along with A33sc unstained corresponding gel fragments (Figure 3).


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Figure 3. SDS-PAGE of protein extracts of PC3luc cells incubated with biotinylated aptamers, followed by enrichment with streptavidin coated magnetic beads. Numbers 1-6 represent the excised bands. Proteins within the gel pieces were reduced, alkylated, digested with trypsin, and identified using nanoliquid chromatography combined with tandem mass spectrometry (nanoLC-MS/MS), according to our previously described protocol

16, 17

. The results from three biological and two

technical replicates are presented in Table 1.

Table 1. Proteins identified in the excised SDS-PAGE bands after enrichment on the streptavidin magnetic beads. Detailed MS results are included in supplementary materials. SDS-PAGE band number 1

Proteins identified simultaneously in A26 and A33, and not present in A33sc FLNA Filamin-A FAS Fatty acid synthase PRP8 Pre-mRNA-processing-splicing factor NU205 Nuclear pore complex PPRC1 Peroxisome proliferator-activated DYH6 Dynein heavy chain 6 COMA1 Collagen alpha-1 SHPRH E3 ubiquitin-protein ligase SHPRH CO7A1 Collagen alpha-1 CNTN3 Contactin-3 UBR2 E3 ubiquitin-protein ligase UBR2

Protein Id P21333 P49327 Q6P2Q9 Q92621 Q5VV67 Q9C0G6 Q8NFW1 Q149N8 Q02388 Q9P232 Q8IWV8


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F178A Protein FAM178A DYH3 Dynein heavy chain 3 HSP90B Heat shock protein 90B HSP90A Heat shock protein 90A ACTN1 Alpha-actinin1 EF2 Elongation factor 2 NUCL Nucleolin XRCC5 X-ray repair cross-complementing protein 5 CIP2A Protein CIP2A SHPRH E3 ubiquitin-protein ligase SHPRH SHRM3 Protein Shroom 3 XRCC6 X-ray repair cross-complementing protein 6 F1711 Protein FAM171A1 HS90B Heat shock protein 90B TFR1 Transferrin receptor protein NUCL Nucleolin C2TA MHC class II transactivator XRCC5 X-ray repair cross-complementing protein 5 ZEP2 Transcription factor GRP78 78kDa glucose-regulated protein GRP75 Stress-70 protein HSP7C Heat shock protein cognate 71 kDa MOES Moesin LMNA Prelamin-A/C HSP7C Heat shock protein cognate 71 kDa HNRPR Heterogeneous nuclear ribonucleoprotein R H12 Histone1.2 H4 Histone H4 H2A1B Histone H2A CH10 10kDa Heat shock protein PROF1 Profilin-1 RS27A Ubiquitin-40S ribosomal protein H12 Histone1.2 ANXA5 Annexin A5 H10 Histone 10 ROA1 Heterogeneous nuclear ribonucleoprotein A1 1433Z 14-3-3 protein zeta ADT2 ADP/ATP translocase 2 RL13 60s ribosomal protein H2A1B Histone H2A H4 Histone H4 RA1L2 Heterogeneous nuclear ribonucleoprotein A1-like 2

2

3

4

5

6

2.

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Q8IX21 Q8TD57 P08238 P07900 P12814 P13639 P19338 P13010 Q8TCG1 Q149N8 Q8TF72 P12956 Q5VUB5 P08238 P02786 P19338 P33076 P13010 P31629 P11021 P48721 P11142 P26038 P02545 P11142 O43390 P16403 P62805 P04908 P61604 P07737 P62979 P16403 P08758 P07305 P09651 P63104 P05141 P26373 P04908 P62805 Q32P51

SAV pull-down assay fixed with formaldehyde

For additional verification, we have cross-linked the aptamers to their targets in the presence of 1% formaldehyde in DPBS buffer to induce covalent interactions of oligonucleotides with lysine groups and to maintain the aptamer-protein complex intact under denaturing environment15. Protein extracts from PC3luc cells after incubation with biotinylated aptamers, were bound with SAV-MB, washed with washing buffer containing 0.1% SDS, and then separated by SDSPAGE. One half of the gel was blotted onto a PVDF membrane to detect the presence of biotinylated complexes with streptavidin alkaline phosphatase conjugate. The bands that


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corresponded to the PVDF detected proteins were excised from the second half of the gel, stained with SYPRO Ruby (where noticeably reduced amount of nonspecific interactions was present), followed by MS-based identification (Figure 4). The list of identified proteins from three biological and two technical replicates is presented in Table 2.

Figure 4. PVDF and SYPRO Ruby SDS-PAGE of protein extracts from PC3luc cells incubated with biotinylated aptamers, followed by enrichment with streptavidin coated magnetic beads. Numbers 1-5 represent the excised bands from A26 and A33 samples, likewise corresponding A33sc unstained gel fragments. Table 2. Proteins identified in the excised SDS-PAGE bands selected based upon immunodetection of biotinylated complexes. SDS-PAGE band number 1

Proteins identified simultaneously in A26 and A33, and not present in A33sc PLEC Plectin NU205 Nuclear pore complex DYH1 Dynein heavy chain 1 AHNK2 Protein AHNAK2 COMA1 Collagen alpha-1 CO7A1 Collagen alpha-1(VII) UBR2 E3 ubiquitin-protein ligase UBR2 F178A Protein FAM178A F186A Protein FAM186A MUC16 Mucin-16

Protein Id Q15149 Q92621 Q9P2D7 Q81VF2 Q8NFW1 Q02388 Q8IWV8 Q8IX21 A6NE01 Q8WXI7


15


2

3

4 5

EF2 Elongation factor 2 NUCL Nucleolin XRCC5 X-ray repair cross-complementing protein 5 XRCC6 X-ray repair cross-complementing protein 6 SHPRH E3 ubiquitin-protein ligase SHPRH F1711 Protein FAM171A1 XRCC5 X-ray repair cross-complementing protein 5 SI1L1 Signal-induced proliferation-associated protein CLAP1 CLIP associated protein MOES Moesin H4 Histone H4 ADT2 ADP/ATP translocase 2

P13639 P19338 P13010 P12956 Q149N8 Q5UVB5 P13010 O43166 Q7Z460 P26038 P62805 P05141

ANXA5 Annexin A5 ROA1 Heterogeneous nuclear ribonucleoprotein A1 RL13 60s ribosomal protein

P08758 P09651 P26373

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Although PC3luc cells were washed under harsh conditions with the use of 0.1% SDS, the substantial number of identified proteins suggests the possibility of existence of nonspecific interactions or several proteins that bind the aptamers. Therefore, we have performed a yet another experiment using the fluorescent, atto488-labeled forms of aptamers A26, A33, and A33sc.

3.

Atto488 labeled aptamers electrophoresis-based assay

The protein extracts were separated using electrophoretic method (Figure 5). The list of identified proteins from three biological and two technical replicates is presented in Table 3. Electrophoretic-based method again allowed for obtaining the results with some additionally present co-existing proteins that were identified in the excised gel bands even though they were not specific aptamer-binding targets, meaning they were also present in scrambled sequence A33sc.


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Figure 5. SDS-PAGE of protein extracts from PC3luc cells after incubation with atto488aptamers. Numbers 1-5 represent the excised bands. Table 3. Proteins identified in the excised SDS-PAGE bands visualized by the presence of atto488 dye. SDS-PAGE band number 1

2

3

4 5

4.

Proteins identified simultaneously in A26 and A33, and not present in A33sc PLEC Plectin NU205 Nuclear pore complex AHNK2 Protein AHNAK2 COMA1 Collagen alpha-1 VP13C Vacuolar protein sorting associated UBR2 E3 ubiquitin-protein ligase UBR2 F186A Protein FAM186A KI67 Proliferation marker protein Ki-67 MUC16 Mucin-16 XRCC5 X-ray repair cross-complementing protein 5 KIF27 Kinesin-like protein KIF27 F1711 Protein FAM171A1 XRCC6 X-ray repair cross-complementing protein 6 SI1L1 Signal-induced proliferation-associated protein CLAP1 CLIP associated protein H4 Histone H4 HNRPR Heterogeneous nuclear ribonucleoprotein R H10 Histone H10 RL13 60s ribosomal protein

Protein Id Q15149 Q92621 Q8IVF2 Q8NFW1 Q709C8 Q8IWV8 A6NE01 P46013 Q8WXI7 P13010 Q86VH2 Q5VUB5 P12956 O43166 Q7Z460 P62805 O43390 P07305 P26373

Atto488 labeled aptamers chromatography-based assay


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During chromatographic separation of PC3luc protein extracts labeled with atto488-aptamers, we have noticed the relevant changes in hydrophobic interactions between un-bound proteins and proteins targeted with aptamers. As a result, we found the aptamer-bound fraction eluting with unquestionably longer retention time (26th min of separation) in comparison to the nonproteinaceous bound fluorescent oligonucleotides and eluted in unretained fraction (11th min of the gradient). The reversed-phase C8 column was used, together with two types of detectors: fluorescence and UV-VIS (Figure 6).

Figure 6. Chromatograms of PC3luc protein extracts after incubation with atto488-aptamers; blue arrows indicate collected fractions; a) atto488-A26, b) atto488-A33 and c) atto488-A33sc oligonucleotide sequence.


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The bound protein fractions were collected and evaporated to dryness. Proteins were reduced, alkylated, digested with trypsin and analyzed with the nanoLC-MS/MS. Results are shown in Table 4. The compounds identified in fractions 0, 0’, and 0’’with retention times between 11-16 minutes were non-protein contaminants. This phenomenon might suggest the non-specific type of interactions that is typical for all oligonucleotide sequences. Fractions representing fluorescence and retention times longer than 25 minutes are representative for proteins with altered hydrophobic properties, and prolonged interactions with stationary phase, what can be explained by the presence of atto488-labeled aptamer attached to the polypeptide chain. Proteins identified in collected fractions after incubation with A26 and A33 were distinct from those, incubated with A33sc control sequence.

Table 4. Proteins identified in HPLC fractions using the fluorescence detector. Aptamer A26

HPLC fraction 1 2 3 4

A33

1 2 3 4 5 6

A33sc

1

2

3

Identified proteins KIF27 Kinesin-like protein XRCC6 X-ray repair cross-complementing protein 6 KI67 Proliferation marker protein Ki-67 CAD23 Cadherin 23 PPARD Peroxisome proliferator-activated receptor delta KIF27 Kinesin-like protein KIF27 KIF14 Kinesin-like protein KIF14 XRCC5 X-ray repair cross-complementing protein 5 F1711 Protein FAM171A1 F186A Protein FAM186A CC120 Coiled-coil domain contacting protein 120 MACF1 Microtubule-actin cross-linking factor 1 MUC16 Mucin-16 SI1L1 Signal-induced proliferation-associated protein F186A Protein FAM186A AHNK2 Protein AHNAK2 VP13C Vacuolar protein sorting associated UBR2 E3 ubiquitin-protein ligase UBR2 CO7A1 Collagen alpha-1 (VII) CLAP1 CLIP associated protein

Protein Id Q86VH2 P12956 P46013 Q9H251 Q03181 Q86VH2 Q15058 P13010 Q5VUB5 A6NE01 Q96HB5 Q9UPN3 Q8WXI7 O43166 A6NE01 Q8IVF2 Q709C8 Q8IWV8 Q02388 Q7Z460

The aim underlying this study was to explore two aptamers identified by cell-SELEX that have been proven to selectively bind to cancerous cells and to develop novel, more specific target identification protocol 18,19. Based on the comparison of different strategies we have revealed the


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method for identification of aptamer-bound proteins characterized with the lowest number of non-specific interactions (Figure 7). These methods involved: (1) the classic streptavidin-biotin electrophoretic approach; (2) assay based on changing the nature of aptamer-protein interaction by fixing in formaldehyde solution, and (3) fluorescence labelled conjugates in SDS-PAGE, and finally the (4) HPLC-based approach of atto488-labelled complexes.

Figure 7. Diagram presenting the number of proteins identified in subsequent methodological approaches.

Based on the obtained data, it seems that chromatographic approach is superior to electrophoretic techniques, both in terms of specificity and simplicity. By the comparison of proteins identified in PC3luc cell extracts incubated with A26, A33, and A33sc aptamers, we have successfully selected specific targets that were present in A26 and A33-bound fractions and, at the same time, were absent in A33sc protein extracts. The identified proteins KI67, CAD23, and PPARD were present in PC3luc cells incubated with A26 aptamer, and CC120 and MACF1 were found for A33 aptamer. Moreover, we have identified two other proteins: X-ray repair cross-complementing protein complex, and Kinesin-like protein KIF27 as binding targets


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characteristic for both aptamers. The presence of XRCC5 and KIF27 proteins in fluorescent aptamers

bound

fractions

were

additionally

verified

using

western

blotting

with

immunodetection on PVDF membrane (Figure 8). Kinesin-like protein KIF27 and XRCC5/6 Xray repair cross-complementing protein 5/6 have been previously described as potential cancer biomarkers20, 21. Alterations in the expression of kinesins might represent a novel mechanism of promoting the progression and metastasis, as a result of higher proliferation rate and increased cell migration20. KIF27 has been involved in cell projection process that is associated with many biological functions, such as cell motility, cancer cell invasion, endocytosis, exocytosis, and cytokinesis20. This may also explain the role of KIF27 in the internalization of aptamers to the interior of the cancerous cell. The XRCC5/6 dimer was identified in many cancerous cell types, also in PC3 prostate cancer cell exosomes22. Upregulation of XRCC5/6 dimer accompanies tumorigenesis and progression21. Elevated XRCC5/6 concentration observed in cells may have causative or permissive effect, however it brings the novel “blind spot” of cancerogenesis that can be exploited therapeutically.

Figure 8. Immunodetection of KIF27 and XRCC5 protein.


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Presented results and a new, more sensitive methodology may be advantageous for the purpose of study of cancer biomarkers and diagnosis, monitoring and treatment of diseases. It is also important to conclude, for future considerations, that there is an obvious difference between cellSELEX identification of the aptamers, acting at the organ level, and identification of the binding counterparts at the molecular level. This fact may help in identification of protein-targets important not only because they might be potential biomarkers, but also they hold the key to understand biological processes in cancer cells.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Grant “META” No. 5/EuroNanoMed/2012, in addition to from the Polish Ministry of Science and Higher Education 2013/09/B/NZ4/02531, and 2016/21/B/NZ6/01307. ASSOCIATED CONTENT The following supporting information is available free of charge at ACS website http://pubs.acs.org List of supplementary components: 1. Table S1 – List of identified proteins in workflow 1 (Streptavidin-biotin pull-down assay) 2. Table S2 – List of identified proteins in workflow 2 (Streptavidin-biotin pull-down assay fixed with formaldehyde)


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3. Table S3 – List of identified proteins in workflow 3 (Atto488 labeled aptamers electrophoresis-based assay) 4. Table S4 – List of identified proteins in workflow 4 (Atto488 labeled aptamers chromatography-based assay)

ACKNOWLEDGMENT We would like to gratefully acknowledge SHIM-POL (Poland) Company for facilitating the fluorescence detector for the purpose of this research. We would like to kindly thank ProQinase GmbH (Germany) for providing PC3luc cell lines enabling the realization of the project, which was partially supported by the grant “META” No. 5/EuroNanoMed/2012. Presented studies were also supported by the grants from the Polish Ministry of Science and Higher Education 2013/09/B/NZ4/02531 and 2016/21/B/NZ6/01307. REFERENCES (1) Wilson, D. S.; Szostak, J. W. In vitro selection of functional nucleic acids. Annu Rev Biochem. 1999, 68, 611-647. (2) Mayer, G. The chemical biology of aptamers. Angew Chem Int Ed Engl. 2009, 48, 26722689. (3) Wolter, O.; Mayer, G. Aptamers as Valuable Molecular Tools in Neurosciences. JNeurosci. 2017, 37, 2517-2523. (4) Famulok, M.; Hartig, J. S.; Mayer, G. Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. Chem Rev. 2007, 107, 3715-3743. (5) Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990, 249, 505-510. (6) Phillips, J. A.; Lopez-Colon, D.; Zhu, Z.; Xu, Y.; Tan, W. Applications of aptamers in cancer cell biology. Analytica chimica Acta. 2008, 621, 101-108. (7) Keefe, A. D.; Pai, S.; Ellington, A. Aptamers as therapeutics. Nat Rev Drug Discov. 2010, 9, 537-550.


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(8) Berezovski, M. V.; Lechmann, M.; Musheev, M. U.; Mak, T. W.; Krylov, S. N. Aptamerfacilitated biomarker discovery (AptaBiD). J Am Chem Soc. 2008, 130, 9137-9143. (9) Mallikaratchy P. Evolution of Complex Target SELEX to Identify Aptamers against Mammalian Cell-Surface Antigens. Molecules. 2017, 22, 1-17. (10 ) Cibiel, A.; Quang, N. N.; Gombert, K.; Theze, B.; Garofalakis, A.; Duconge, F. From ugly duckling to swan: unexpected identification from cell-SELEX of an anti-Annexin A2 aptamer targeting tumors. PLoS One. 2014, 9, e87002. (11) Théodorou, I.; Quang, N. N.; Gombert, K.; Thézé, B.; Le-landais, B.; Ducongé, F. In Vitro and In Vivo Imaging of Fluorescent Aptamers. Methods Mol Biol. 2016, 1380, 135-150. (12) Polanski, M.; Anderson, N. L. A list of candidate cancer biomarkers for targeted proteomics. Biomark insights. 2007, 1, 1-48. (13) Chang, Y. M.; Donovan, M. J.; Tan, W. Using aptamers for cancer biomarker discovery. J Nucl Acids. 2013, 2013, 817350. (14) Cibiel, A.; Dupont, D. M.; Ducongé, F. Methods To Identify Aptamers against Cell Surface Biomarkers. Pharmaceuticals. 2011, 4, 1216-1235. (15) Hasegawa, H.; Savory, N.; Abe, K.; Ikebukuro, K. Methods for Improving Aptamer Binding Affinity. Molecules. 2016, 21, 421. (16) Drabik, A.; Ner-Kluza, J.; Bodzon-Kulakowska, A.; Suder P. MYTHBUSTERS: a universal procedure for sample preparation for mass spectrometry. Eur J Mass Spectrom. 2016, 22, 269273. (17) Drabik, A.; Bodzon-Kulakowska, A.; Suder, P. Application of the ETD/PTR reactions in top-down proteomics as a faster alternative to bottom-up nanoLC-MS/MS protein identification. J Mass Spectrom. 2012, 10,1347-1352. (18) Van Simaeys, D.; Turek, D.; Champanhac, C.; Vaizer, J.; Se-fah, K.; Zhen, J.; Sutphen, R.; Tan, W. Identification of cell membrane protein stress-induced phosphoprotein 1 as a potential ovarian cancer biomarker using aptamers selected by cell systematic evolution of ligands by exponential enrichment. Anal Chem. 2014, 86, 4521-4527. (19) Cerchia, L.; Hamm, J.; Libri, D.; Tavitian, B.; de Franciscis, V. Nucleic acid aptamers in cancer medicine. FEBS Letters. 2002, 528, 12-16. (20) Rath, O.; Kozielski, F. Kinesins and cancer. Nat Rev Cancer. 2012, 12, 527-539. (21) Pearl, L.H.; Schierz, A.C.; Ward, S.E.; Al-Lazikani, B.; Pearl, F.M. Therapeutic opportunities within the DNA damage response. Nat Rev Cancer. 2015, 15, 166-1680. (22) Worst, T.S.; von Hardenberg, J.; Gross, J.C.; Erben, P.; Schnoelzer, M.; Hausser, I.; Bugert, P.; Michel, M.S.; Boutros, M. Database-augmented Mass Spectrometry Analysis of Exosomes Identifies Claudin 3 as a Putative Prostate Cancer Biomarker. Mol Cell Proteomics. 2017, 16, 998-1008.


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Table of Contents


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Advances in studies of aptamer-protein targets identification using

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