Circular and linear: a tale of aptamer selection for the activation of SIRT1 to induce death in cancer cells

Abstract

It is a challenge to select the right target to treat conditions without affecting non-diseased cells. Cancer belongs to the top 10 causes of death in the world and it remains difficult to treat. Amongst cancer emerging targets, silent information regulator 1 (SIRT1) - a histone deacetylase - has shown many roles in cancer, ageing and metabolism. Here we report novel SIRT1 ligands that bind and modulate the activity of SIRT1 within cells and enhance its enzymatic activity. We developed a modified aptamer capable of binding to and forming a complex with SIRT1. Our ligands are aptamers, they can be made of DNA or RNA oligonucleotides, their binding domain can recognise a target with very high affinity and specificity. We used the systematic evolution of ligands by exponential enrichment (SELEX) technique to develop circular and linear aptamers selectively binding to SIRT1. Cellular consequences of the interaction were monitored by fluorescence microscopy, cell viability assay, stability and enzymatic assays. Our results indicate that from our pool of aptamers, circular AC3 penetrates cancerous cells and is recruited to modulate the SIRT1 activity. This modulation of SIRT1 resulted in anticancer activity on different cancer cell lines. Furthermore, this modified aptamer showed no toxicity on one non-cancerous cell line and was stable in human plasma. We have demonstrated that aptamers are efficient tools for localisation of internal cell targets, and in this particular case, anticancer activity through modulation of SIRT1.

Fig. 1. Workflow representation of the selection process. In the case of the circular library, the oligonucleotides were phosphorylated and ligated. In the case of the linear library, the oligonucleotides were unmodified. First stage of the cycle starts with the SIRT1 interacting with either library. Unbound sequences were eluted using different NaCl ionic concentration. The bound sequences were then amplified by PCR. In the case of circular library, the sequences were phosphorylated and ligated before entering the second round. The linear library sequences were used from the purification step after the PCR one. Once eight cycles (circular) and twelve (linear) were performed, the sequences were cloned, analysed and studied for biological activity.

Fig. 2. Cell viability results at 72 hours of incubation with aptamers at 2.5 μM final concentration. The results represent the mean ± SEM of 2 independent experiments. The p-value for AC3 treatment relative to TBHP show for A549: p < 0.00005; Hepg2: p < 0.0005; MCF7: p < 0.0001; MDA-MB468: p < 0.005; U2OS: p < 0.0005; Caco-2: p < 0.001 and Beas2B: p < 0.05.

Fig. 3. Stability of AC3 in human plasma up to 24 hours monitored by HPLC-UV. (A) Comparison of human plasma elution time with AC3. (B) Degradation of AC3 in human plasma as a function of time.

Fig. 4. Fluorescence microscopy analysis of AC3 binding to SIRT1 enzyme and nuclei localisation. AC3-FAM (green-yellow), nuclei (DAPI, blue) and SIRT1 (Texas Red, red). Overlap of images can produced alterations in the colour results such as yellow produced by overlap of red and green, magenta produced by red and blue and cyan produced by green and blue. (A) A549, scale 100 μm. (B) HepG2 (augmentation in Fig. 20, ESI. (C) Caco2, scale 200 μm). (D) U2OS, scale 200 μm. (E) MCF7, scale 20 μm. (F) MCF7 zoomed. (G) MDAMB-468, scale 100 μm. (H) Beas2B, scale 200 μm. Fluorescence intensity was measured by LUMIStar luminometer microplate readers as: green ex. 495 nm, em. 509 nm; blue ex. 359 nm, em. 461 nm and red ex. 596 nm, em. 615 nm. The results represent the mean ± SEM of two different experiments performed twice. Images were taken at a magnification of 100× in a Cytation 3.