Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Abstract

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington's disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer.

Keywords: Drosophila melanogaster model; G-quadruplex; Huntington’s disease; aptamers; physicochemical characterization.

Figure 1

Representative 10% polyacrylamide gel electrophoresis under native conditions of MS3, at 3 μM concentration in the indicated buffer solutions, run at constant 80 V at r.t. for 75 min in TBE 1× buffer. The not-annealed (N.A.) V7t1 aptamer dissolved in HEPES/Na⁺ buffer (25 mM HEPES, 150 mM NaCl, pH = 7.4) at 4 μM concentration was used as reference oligonucleotide. Lane 1: MS3 in Na⁺ buffer; lane 2: MS3 in PBS; lane 3: MS3 in K⁺ buffer; lane 4: V7t1.

Figure 2

CD analysis of MS3 analogues: CD spectra of MS3-33 (a) and MS3-17 (c) in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM NaH₂PO₄/Na₂HPO₄, 1.8 mM KH₂PO₄/K₂HPO₄, pH = 7.4) at 5 °C before (black line), after melting/cooling (blue line), and at 100 °C (red line); CD spectra of MS3-33 (b) and MS3-17 (d) in the Na⁺ buffer (10 mM NaH₂PO₄/Na₂HPO₄, 90 mM NaCl solution, pH = 7.0) at 5 °C before (black line), after melting/cooling (blue line), and at 100 °C (red line).

Figure 3

CD melting experiments of MS3 analogues recorded at a scan rate of 0.5 °C min⁻¹: CD melting (black line) and cooling (red line) profiles of MS3-33 (a) and MS3-17 (b) in the Na⁺ buffer (10 mM NaH₂PO₄/Na₂HPO₄, 90 mM NaCl solution, pH = 7.0).

Figure 4

Experimental DSC profiles for MS3-33 (a) and MS3-17 (c) in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM NaH₂PO₄/Na₂HPO₄, 1.8 mM KH₂PO₄/K₂HPO₄, pH = 7.4); DSC profiles for MS3-33 (b) and MS3-17 (d) in the Na⁺ buffer (10 mM NaH₂PO₄/Na₂HPO₄, 90 mM NaCl solution, pH = 7.0). The red bar indicates the physiological temperature.

Figure 5

Representative 10% polyacrylamide gel electrophoresis under native conditions of MS3, MS3-33, and MS3-17 samples at 3 μM concentration in the selected PBS (lanes 2–4) and Na⁺ buffer (lanes 5–7) solutions, run at constant 80 V at r.t. for 75 min in TBE 1× buffer. The not-annealed (N.A.) V7t1 aptamer dissolved in HEPES/Na⁺ buffer (25 mM HEPES, 150 mM NaCl, pH = 7.4) at 4 μM concentration was used as a control oligonucleotide (lanes 1 and 8). Lane 1: V7t1; lane 2: MS3; lane 3: MS3-33; lane 4: MS3-17; lane 5: MS3; lane 6: MS3-33; lane 7: MS3-17; lane 8: V7t1.

Figure 6

Enzymatic resistance experiments performed on MS3-17 (a) and MS3-33 (b) incubated in 80% fetal bovine serum (FBS) as monitored by 20% denaturing polyacrylamide gel electrophoresis up to 48 h (time points: 0, 0.1, 0.5, 1, 2, 4, 6, 24, 30, and 48 h). For each compound, a representative 20% denaturing PAGE (8 M urea) is reported. Gels were run at a constant 200 V at r.t. for 3 h in TBE 1X as running buffer.

Figure 7

Uptake of FITC-conjugated MS3 and its truncated analogues in neuronal cells. SH-SY5Y cells were incubated with the three FITC-conjugated aptamers (green) for 24 h at different concentrations, as indicated. Then, cells were fixed, and nuclei were stained with DAPI (blue). Images were acquired with a confocal microscope. Scale bars, 6 μM. Mean fluorescence intensity of three independent experiments is shown, n > 50 cells; error bars, mean ± SD. *** p < 0.001, ** p < 0.01, Bonferroni test after significant ANOVA.

Figure 8

Time-course analysis of cell uptake of MS3 and its analogues. SH-SY5Y cells were incubated with a 4 µM solution of FITC-conjugated aptamers (green) at the indicated time points and fixed; then, nuclei were stained with DAPI (blue). Images were acquired with a confocal microscope. Higher magnification pictures in the insets. Scale bar, 6 µm. Mean fluorescence intensity of two independent experiments is shown, n > 50 cells; error bars, mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05, Bonferroni test after significant ANOVA. Mean fluorescence intensities at the plasma membrane (PM) or inside the cell (intracellular) were measured at the different times and expressed as percentage of total fluorescence; n > 30 cells.

Figure 9

Analysis of the biological stability in SH-SY5Y cells of MS3 and its analogues by time-course experiments. SH-SY5Y cells were incubated for 24 h with FITC-conjugated aptamers (green). After the removal of the residual aptamers by extensive washings, cells were incubated in a culture medium for the different indicated times. Nuclei were stained with DAPI. Images were acquired with a confocal microscope. Scale bar, 6 µm. Mean fluorescence intensity was measured at the different time points and expressed as an arbitrary unit (a.u.; left graph) or as the percentage of fluorescence detected at time 0 (set to 100%; right graph); error bars, mean ± SD; n > 40 cells. *** p < 0.001, Bonferroni test after significant ANOVA. Note that there is a statistically significant difference between the 72 and 96 h time points for MS3 but not for MS3-33 and MS3-17 (p-value < 0.001, Bonferroni test after significant ANOVA).

Figure 10

MS3-derived aptamers’ suppressed HD symptoms in a Drosophila model. Administration of MS3 and of its two analogues (MS3-17 and MS3-33) during adult period suppressed motor dysfunction in flies expressing HttFLQ128. Comparison was made between untreated and treated flies of the same age. (a) Climbing assay. The percentage of flies that, after the MS3 analogues’ (MS3-17 and MS3-33) chronic treatment throughout adulthood, could reach the 9 cm target was higher than both untreated and MS3-treated sibling flies at each point examined. (b) The average climbing height of MS3 analogues was significantly higher compared with MS3-treated flies also on day 12 after eclosion. (c) The survival curves of both treated groups declined slower in comparison with the curve of the untreated flies that quickly dropped on day 7, and the mean and maximum lifespan were significantly extended compared with untreated flies. **** p < 0.0001; *** p = 0.0005; ** p = 0.023, compared with untreated flies. Data represent mean ± SD.