Post-SELEX Optimization and Characterization of a Prostate Cancer Cell-Specific Aptamer for Diagnosis

[Image: see text] The RNA aptamer A4 binds specifically to tumor prostate cells. A4 was modified (mA4) by adding deoxyribonucleotides to its ends to remove the reactive 2′ hydroxyl groups of RNA’s sugar at the ends of the aptamer and to make it more stable to widespread RNase contamination in laboratories. Thus, mA4 would be more suitable to use in the clinical settings of prostate cancer (PCa). We aimed to characterize this optimized oligonucleotide to verify its potential as a diagnostic tool. The sequences and structures of A4 and mA4 were compared through in silico approaches to corroborate their similarity. Then, the degradation of mA4 was measured in appropriate media and human plasma for in vitro tests. In addition, the binding abilities of A4 to prostate cells were contrasted with those of mA4. The effects of mA4 were assessed on the viability, proliferation, and migration of human prostate cell lines RWPE-1 and PC-3 in three-dimensional (3D) cell cultures. mA4 showed configurational motifs similar to those of A4, displayed a half-life in plasma substantially higher than A4, and exhibited a comparable binding capacity to that of A4 and unaltered viability, proliferation, and migration of prostatic cells. Therefore, mA4 maintains the crucial 3D structures of A4 that would allow binding to its target, as suggested by in silico and binding analyses. mA4 may be a good PCa reporter as it does not change cellular parameters of prostate cells when incubated with it. Its additional deoxyribonucleotides make mA4 inherently more chemically stable than A4, avoiding its degradation and favoring its storage and handling for clinical applications. These characteristics support the potential of mA4 to be used in diagnostic systems for PCa.