Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs

Abstract

In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties. In the design of these covalent dimers, connecting linkers of different chemical nature, maintaining the same polarity along the strand or inverting it, have been introduced. These dimeric aptamers have been investigated using several biophysical techniques to disclose the conformational behavior, molecularity and thermal stability of the structures formed in different buffers. This in-depth biophysical characterization revealed the formation of stable G4 structures, however in some cases accompanied by alternative tridimensional arrangements. When tested for their VEGF₁₆₅ binding and antiproliferative activity in comparison with V7t1, these covalent dimers showed slightly lower binding ability to the target protein but similar if not slightly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells. These results provide useful information for the design of improved dimeric aptamers based on further optimization of the linker joining the two consecutive V7t1 sequences.

Keywords: G-quadruplexes; V7t1; VEGF165; aptamers; biophysical characterization; covalent dimers.

Figure 1

The 2% agarose gel electrophoresis under native conditions of V7t1 and its covalent dimeric analogues (here indicated as bisT7, bisHEG2, bisTEG2D) in both not-annealed (N.A.) (−) and annealed (A.) (+) form at 4 μM concentration in the selected HEPES/Na⁺ (a) and TRIS/K⁺ (b) buffer solutions. Gels were run at constant 60 V at r.t. for 2 h in Tris-Borate-EDTA (TBE) 1X as running buffer.

Figure 2

UV analysis on bisV7t1TEG2D at 2 µM concentration in the selected HEPES/Na⁺ buffer solution in both N.A. (a) and A. (b) form: overlapped UV-melting and UV-annealing profiles (red and light blue lines, respectively) recorded at 295 nm using a scan rate of 1 °C/min. n.d. = not determined.

Figure 3

Overlapped circular dichroism (CD) spectra of V7t1 (black line) and covalent V7t1 dimers (green, blue and red lines, respectively for bisV7t1T7, bisV7t1HEG2 and bisV7t1TEG2D), recorded at 15 °C and 2 µM concentration in the selected HEPES/Na⁺ (a, b) and TRIS/K⁺ (c) buffer solutions. All the investigated oligonucleotides were analyzed in both N.A. and A. form, reported respectively in panel a and b for HEPES/Na⁺ and as continuous and dashed lines in panel c for TRIS/K⁺ buffer solution.

Figure 4

N.A. bisV7t1T7 at 2 µM concentration in the selected HEPES/Na⁺ buffer: (a) CD-melting profile, recorded at 262 nm using a scan rate of 1 °C/min; (b,c) overlapped CD spectra recorded every 5 °C during the melting (b) and cooling processes (c). Arrows in panels b and c indicate the evolution of the CD signal over time. n.d. = not determined.

Figure 5

CD analysis on bisV7t1TEG2D at 2 µM concentration in the selected HEPES/Na⁺ buffer solution in both N.A. and A. form. (a) CD-melting and (b) CD-annealing profiles (red and light blue lines, respectively) of N.A. bisV7t1TEG2D recorded at 262 nm using a scan rate of 1 °C/min; (c) overlapped CD-melting and CD-annealing profiles (red and light blue lines, respectively) of A. bisV7t1TEG2D, both recorded at 262 nm (scan rate: 1 °C/min).

Figure 6

Native 7% electrophoretic mobility shift assay (EMSA) of A. (a) and N.A. (b) V7t1 and covalent V7t1 dimers incubated in the presence (+) or absence (−) of VEGF₁₆₅. GelGreen- and Coomassie-stained gels (left and right, respectively). 30 pmol of each aptamer were incubated with 40 pmol of the protein in a final volume of 9 μL in the selected HEPES/Na⁺ buffer, thus obtaining a final 1:1.3 oligo/protein ratio. Gels were run at constant 45 V for 2.3 h at r.t. in TAE 1X buffer.

Figure 7

Cell viability assay of V7t1, bisV7t1T7, bisV7t1HEG2, bisV7t1TEG2D and a control 24-mer on MCF-7 cells tested by MTT assays. Cell viability is reported as the % of live cells with respect to control untreated cells (100% cell viability). Reported results derive from 4 independent experiments. Statistical analysis was performed by using Student’s t-Test by comparing all samples to the control cells (* P < 0.05, ** P < 0.01 or *** P < 0.001) and ANOVA test by comparing all covalent V7t1 dimers to V7t1 (* P < 0.05).