title: 3D Cell-SELEX: Development of RNA aptamers as molecular probes for PC-3 tumor cell line.
abstract: Human prostate cancer (PCa) is a highly heterogeneous and multifactorial disease. Current clinical biomarkers are not sufficiently accurate, thus being unable to predict the clinical outcome. Therefore, searching for new biomarkers aiming to improve diagnosis, prognosis and therapy is still required. In this study, we performed 3D Cell-SELEX against PC-3 prostate cancer cell line, a novel strategy to select specific nucleic acid ligands against spheroid cells in 3D cell culture. This original system combines Cell-SELEX, a process that exploits the cellular structure to generate specific ligands, and 3D cell culture, an approach that mimics the tissue microenvironment in vitro. In the first round of 3D Cell-SELEX, a negative selection against RWPE-1, non-tumor cell line, was performed to subtract non-tumor specific aptamers. The supernatant was used in eight additional rounds of selection, which were performed against PC-3 cell line. After nine selection cycles, eight PC-3 specific RNA aptamers were selected and sequenced. The aptamers presented sizes between 20 and 50 nucleotides-long, with low free energy (∆G<-13.6), which contributed for their spontaneous folding and high stability. Furthermore, our results showed the aptamer A4 as a specific ligand to prostate tumor cells, with dissociation constant in the nanomolar scale. Therefore, the novel 3D Cell-SELEX procedure improved the selection of PCa cell-surface ligands and the aptamer A4 has shown potential for the identification of prostate tumor cells, suggesting the application of this molecule in further screening assays for PCa.
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia (UFU), 38400-902 Uberlândia, MG, Brazil. Electronic address: alingosouza@yahoo.com.br.
authors.forename: Aline G
authors.initials: AG
authors.lastname: Souza
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia (UFU), 38400-902 Uberlândia, MG, Brazil; Laboratory of Cancer Molecular Genetics, Faculty of Medical Sciences, University of Campinas, SP, Brazil.
authors.forename: Karina
authors.initials: K
authors.lastname: Marangoni
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia (UFU), 38400-902 Uberlândia, MG, Brazil.
authors.forename: Patrícia T
authors.initials: PT
authors.lastname: Fujimura
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia (UFU), 38400-902 Uberlândia, MG, Brazil.
authors.forename: Patrícia T
authors.initials: PT
authors.lastname: Alves
authors.affiliation: Center of Molecular Biology and Genetic Engineering, University of Campinas, SP, Brazil.
authors.forename: Márcio J
authors.initials: MJ
authors.lastname: Silva
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia (UFU), 38400-902 Uberlândia, MG, Brazil.
authors.forename: Victor Alexandre F
authors.initials: VA
authors.lastname: Bastos
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia (UFU), 38400-902 Uberlândia, MG, Brazil; University of California-Davis, Department of Medical Microbiology and Immunology, Davis, CA, USA.
authors.forename: Luiz R
authors.initials: LR
authors.lastname: Goulart
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia (UFU), 38400-902 Uberlândia, MG, Brazil.
authors.forename: Vivian A
authors.initials: VA
authors.lastname: Goulart
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D045744:Cell Line, Tumor; D006801:Humans; D008024:Ligands; D008297:Male; D015335:Molecular Probes / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011471:Prostatic Neoplasms / Q000235:genetics*; D059016:Tumor Microenvironment / Q000235:genetics
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D015335:Molecular Probes
keywords: 3D cell culture; Cell-SELEX; Prostate cancer; RNA aptamers