Aptamer technology in clinical diagnostics
Aptamers are short single-chained oligo(deoxy)nucleotides that interact with target molecules with high affinity and specificity and can be utilised as cell–recognition tools. The EU-sponsored APTACELLSENS (An aptamer-based multiplex assay to recording molecular signatures of cancer cell fingerprints) proposal employed DNA aptamers with broad-spectrum interaction patterns for a multiplex assay to determine molecular fingerprints of the most prevalent cancers. The development of the assay makes profiling of cancer cells for diagnostic monitoring possible. The second objective of the project was the identification of the molecular targets of the employed aptamers using short interfering RNA (siRNA) methodology. Systematic evolution of ligands by exponential enrichment (SELEX) technology is a well-established and efficient technology for the generation of oligonucleotides with a high target affinity. SELEX was employed to identify the initial panel of aptamers. Next generation sequencing (NGS) was performed for the different selection cycles, and a series of initial aptamer candidates were obtained. Altogether 24 aptamers were further tested for binding to breast cancer cell line MCF7 and a prostate cancer cell line PC3 using radioactivity binding assay, quantitative PCR and flow cytometry. Aptamers showed selective targeting to cancer cells derived from solid tumours such as breast, prostate and lung cancer, while no targeting was observed to lymphoid cells. Additionally, confocal microscopy studies investigated aptamer recognition and internalisation to the cells. This was an important aspect addressing the future use of the aptamers for tumour targeting and therapy. The identification of the aptamer targets was carried out through the optimisation of a pull-down assay to identify the subsequent biomolecule for each particular aptamer. This methodology has been established by using fluorescently labelled aptamers and mass spectrometry analysis of the isolated proteins. Aptamers represent a new cutting edge technology with great potential. Data obtained by APTACELLSENS has established the viability of clinical application of aptamers for finding new biomarkers.
Keywords
Aptamer, APTACELLSENS, molecular fingerprints, cancer, SELEX
