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. 2016 May 19:6:25799.
doi: 10.1038/srep25799.

Bioactivity of 2'-deoxyinosine-incorporated aptamer AS1411

Xinmeng Fan  1 , Lidan Sun  2 , Yun Wu  1 , Lihe Zhang  1 , Zhenjun Yang  1
Affiliations

Bioactivity of 2'-deoxyinosine-incorporated aptamer AS1411

Xinmeng Fan et al. Sci Rep. .

Abstract

Aptamers can be chemically modified to enhance nuclease resistance and increase target affinity. In this study, we performed chemical modification of 2'-deoxyinosine in AS1411, an anti-proliferative G-rich oligodeoxynucleotide aptamer, which binds selectively to the nucleolin protein. Its function was augmented when 2'-deoxyinosine was incorporated at positions 12, 13, 15, and 24 of AS1411, respectively. In addition, double incorporation of 2'-deoxyinosine at positions 12 and 24 (FAN-1224dI), 13 and 24 (FAN-1324dI), and 15 and 24 (FAN-1524dI) promoted G-quartet formation, as well as inhibition of DNA replication and tumor cell growth, and induced S-phase cell cycle arrest. In further animal experiments, FAN-1224dI, FAN-1324dI and FAN-1524dI resulted in enhanced treatment effects than AS1411 alone. These results suggested that the position and number of modification substituents in AS1411 are critical parameters to improve the diagnostic and therapeutic function of the aptamer. Structural investigations of the FAN-1524dI/nucleolin complex structure, using molecular dynamics simulation, revealed the critical interactions involving nucleolin and 2'-dI incorporated AS1411 compared with AS1411 alone. These findings augment understanding of the role of 2'-deoxyinosine moieties in interactive binding processes.

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Figures

Figure 1
Figure 1
(A) Secondary structure sketch map of AS1411. (B) Chemical structures of 2′-deoxynucleosides.
Figure 2
Figure 2. CD spectra of 2′-dI modified AS1411.
CD data is obtained with a 5 μM concentration in the presence of in 10mM sodium phosphate buffer, pH 7.0, containing 0.1 M KCl. All aptamers are boiled for 5 min, and anneal at 60 °C for 50 h.
Figure 3
Figure 3. DNA synthesis in untreated 2 kind of cells (PBS as control) and cells treated with AS1411 (control oligonucleotide) or FAN-1224dI/FAN-1324dI/FAN-1524dI (active oligonucleotide).
Cells were treated a final concentration of 18 μM for 72 h and then expose to 50 μM EdU for 2 h at 37 °C. DNA was stained with 5 μg/mL Hoechst 33342 (50 μl per well) for 30 min and imaged under a fluorescent microscope. (A) MCF-7 cells. (B) MDA-MB-231 cells.
Figure 4
Figure 4
CCK-8 assays showing the growth of MCF-7 cells (A) and 293 cells (B) treated with FAN-1224dI, FAN-1324dI, FAN-1524dI or PBS as a control. oligonucleotides (or PBS as control) are added directly to the culture medium to give a final concentration of 7.5 μM (day 1). On days 2-4 further oligonucleotide equivalent to half the initial dose is added. Cells are assayed using the cell counting kit-8 (CCK-8) (Dojindo Laboratorie, Japan) on 3, 5, 7 days after treatment. The OD450 nm value is proportional tothe number of viable cells in the sample.
Figure 5
Figure 5. Immunofluorescence images of MCF-7 cells incubate with anti-nucleolin antibody at 4 °C overnight and 550 nM FAM-FAN-1324dI for 30 min at 37 °C.
Permeabilize cells with 0.5% (v/v) Triton X-100, cells are then stained with 5 μg/mL Hoechst 33342 (50 μL per well) for 30 min, Confocal images are obtained using NIS-Elements and Bitplane imaris software. Antibody in red and FAM-FAN-1324dI in green, nucleolus are shown in blue.
Figure 6
Figure 6. FAN-1224dI, FAN-1324dI and FAN-1524dI suppresses growth of MCF-7 xenografts.
Figure 7
Figure 7
(A) RMSD curves for the whole complex structure during the MD simulation. (B) The overall structure and structure comparation of FAN-1524dI-RBD12 and AS1411-RBD12. (a) The average structure of FAN-1524dI-RBD12 from MD. The protein and FAN-1524dI are colored blue and green, respectively. 2′-dI at position 15 is colored purple; (b) Superimposition of FAN-1524dI-RBD12 (blue) and AS1411-RBD12 (yellow) structures. The red arrow depicts the protein rotation; (c) The complex structure used for constructing the initial FAN-1524dI-RBD12 for MD and the protein and AS1411 are colored yellow and green, respectively.
Figure 8
Figure 8
Binding mode analysis of FAN-1524dI-RBD12 (A) and AS1411-RBD12 (B). In both panels, residues (blue) and nucleotide (green) making favorable interactions are represented as solid sticks with black labels. Green dots represent hydrogen bonds.
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