Selection and Validation of Aptamers Against Osteosarcoma By Cell-Selex Method

Abstract

Osteosarcoma is known as the most common malignant mesenchymal stem cell development by endochondral ossification. Treatment resulting from an early diagnosis of such aggressive pediatric bone cancer can greatly increase the survival rate of the patient. For this purpose, aptamers comprise a cheaper and stable alternative product compared to antibodies. The ability of these short nucleotide sequences to be synthetically designed and used either for diagnostic or therapeutic purposes by targeting the relevant cell receptors has been attracting the interest of many researchers.

Aptamers are single-chain DNA or RNA oligonucleotides that bind to a variety of targets, from small organic molecules to proteins. Single-chain oligonucleotide libraries can theoretically form three-dimensional structures that can be combined with various types of target molecules. Aptamers can be selected through SELEX (Systematic Evolution of Ligands by Exponential Enrichment) if the target is known or cell-SELEX if the target is unknown. Cell-SELEX enables aptamers selection using whole cells as a target. Advantages of aptamers include repeatable denaturation and renaturation, easy synthesis and preservation of chemical modifications of the targeted aptamers throughout the selection cycles.

In this thesis, a 80-100 nt aptamer (ssDNA) library was designed to select by cell-SELEX, aptamers that specifically recognize the osteosarcoma cell line (MG-63) with high selectivity and sensitivity using osteoblast cells (control normal cells) for counter-selection. The work also include designing the vector system for proliferation of aptamers, sequence and homology analysis of the potential aptamers followed by flow cytometry and confocal microscopy.

We have successfully generated an aptamer against MG-63 cells for the first time, after 10 cycles of enrichment using cell-SELEX. Out of 36 potential sequences, 14 sequences were full-length, and out of these, three aptamer sequences were analyzed by flow cytometry and one aptamer was selected for further analysis. This aptamer was found to be specific against MG-63 cells as compared to osteoblasts and other cancer cell lines, exhibiting a nanomolar range Kd value, namely 11.9±0.9 nM. Internalization of the aptamer was observed by confocal microscopy when MG-63 cells were incubated with the aptamer at 37C, but not at 4C. This dual characteristic of the selected aptamer highlights its potential to be used for treatment at 37C (in case it is found to interfere with any relevant receptor or cascade inhibiting cancer proliferation) and for diagnostics at 4C (if coupled for instance with a fluorescence molecule).

Overall, the aptamer herein selected and characterized against osteosarcoma is a powerful and valuable tool for its detection and treatment, given the high selectivity and sensitivity of the aptamer.