Aptameric Probe Specifically Binding Protein Heterodimer Rather Than Monomers

Abstract Dimerization of proteins occurs frequently and plays integral roles in biological processes. However, no single molecular probe is available for in situ detection of protein dimers on cells and tissues because of the difficulty of isolating complete protein dimers for probe preparation and screening, which has greatly hampered the biomedical study of protein dimers. Herein, a G‐rich DNA aptamer (termed BG2) that only binds alkaline phosphatase (AP) heterodimers rather than monomers is reported. This aptamer is generated by the cell‐SELEX (systematic evolution of ligands by exponential enrichment) technique and proves to fold into a duplex stabilized antiparallel G‐quadruplex structure. Using BG2 as molecular probe, AP heterodimers are found to be expressed on several kinds of cancer cells. As an affinity ligand, BG2 could isolate AP heterodimers from cell lysate. BG2 is also demonstrated to be applicable for tumor imaging in mice xenografted with cells highly expressing AP heterodimers. AP isozymes are found in several tissues and blood throughout the body, but the function and tissue distribution of AP heterodimers are totally unknown; therefore, BG2 could serve as a molecular probe to uncover the mystery of AP heterodimers. The generation of aptameric probes by cell‐SELEX will open up a new situation for the study of protein dimers.

Based on this idea, we designed a restricted DNA library, 5'-ACGCTCGGATGCCACTACAGTYRRRRRRNNGGGNNNGGNNNGGNNGGNNNNNNNNGGNYYY YYYRTCTCATGGACGTGCTGGTGAC-3. In this library, a YRRRRRR and a YYYYYYR sequences (Y = C or T; R = A or G) were set within the random region next to the primer binding regions to increase the probability of forming a 7-bp stem; 5 G-tracts were placed in the central 30-bp regions to increase the probability of forming G-quadruplex; and other bases (N) were totally random. The SELEX was performed using a human cervical denocarcinoma cell line, HeLa, as target cells. The aptamer enrichment was monitored by flow cytometry. After three rounds of selection, the fluorescence intensity of cells was greatly increased; and almost no much fluorescence increase was observed after two further rounds of selection ( Figure S1a), suggesting that aptamers were enriched in three rounds. Compared with our previous aptamer selections those spent about 20 rounds, [9] the efficiency of SELEX was greatly improved by using the restricted library. The enriched pool also found to bind MCF-7 and LoVo cells ( Figure S1b and c).
The enriched DNA pool of the fifth round was sequenced by high throughput sequencing on an Illumina platform (Sangon Biotech Co. Ltd). About 15000 tests were sequenced, which contained 6628 unique sequences. Almost all sequences contained two complete complementary sequences near the primer regions, which could form the stem of hairpin structure, and 42% of them contained a conserved G-rich motif, GGGGTCGGTGTGGGTGGTTATGATTGG, suggesting that it may be the binding motif of aptamers.
The binding property of aptamer BG2. Many cell binding aptamers are selected at 4 o C or on ice; they usually showed weaker binding capability at 37 o C, which could hinder their applications in physiological conditions. [10] The binding assays showed that BG2 exhibited similar binding ability to LoVo cells at 4 o C,25 o C and 37 o C (Figure 2Sa), while the non-stem sequence, BG2c showed slightly weaker binding capability at 25 o C and 37 o C than at 4 o C ( Figure S2b), suggesting that the stem of BG2 stabilized its binding structure and made it work well at 37 o C. In addition, BG2 maintained good binding ability to LoVo cells after incubation at 37 o C in culture medium containing 10% FBS for 30 min, 1 hour and 3 hours ( Figure S2c). These results suggest that BG2 has the potential for in vivo usage.
The proteinase treatment experiment showed that treating cells with trypsin or proteinase K did not affect the binding of BG2 ( Figure S2d), suggesting that the molecular target of BG2 on cell surface may be a proteinase-resistant protein, a protein present in a membrane domain that are inaccessible to proteinase, or even not a protein.
Many antibodies and aptamers bind living cells but do not bind formalin-fixed cells or tissues. [11] The flow cytometry assay and confocal imaging showed that after fixed LoVo cells with 1% formaldehyde, BG2 still bound the fixed cells with similar affinity to living cells ( Figure S2e and f). This finding suggests that aptamer BG2 has the potential as a molecular probe for the analysis of live cells, fixed cells and tissue sections.
The AP antibodies. It should be noticed that there was cross-reaction between monoclonal antibodies against IAP, PLAP and GCAP because of the 85% homology between IAP and PLAP, and 98% homology between PLAP and GCAP. Four commercially available monoclonal antibodies were used in this study. Anti-PLAP (ab133602) and anti-IAP (ab186422) were used for western blot assay, in which anti-PLAP (ab133602) showed weak cross reaction to IAP; but anti-IAP (ab186422) bound both PLAP and IAP. Anti-PLAP (MA1-20245) and anti-IAP (GTX60746) were used for flow cytometric assay and cell imaging; both of them showed weak cross reaction between IAP and PLAP, but anti-IAP (GTX60746) also showed strong cross-reaction to PLAP when used in western blot assay. There was no commercially available anti-GCAP antibody, but the two anti-PLAP antibodies could bind GCAP.