Abstract
We described the selection of a novel nucleic acid antibody-like prostate cancer (PCa) that specifically binds to the single-stranded DNA molecule from a 277-nt fragment that may have been partially paired and bound to the PCA3 RNA conformational structure. PCA3-277 aptamer ligands were obtained, and the best binding molecule, named CG3, was synthesized for validation. Aiming to prove its diagnostic utility, we used an apta-qPCR assay with CG3-aptamer conjugated to magnetic beads to capture PCA3 transcripts, which were amplified 97-fold and 7-fold higher than conventional qPCR in blood and tissue, respectively. Histopathologic analysis of 161 prostate biopsies arranged in a TMA and marked with biotin-labeled CG3-aptamer showed moderate staining in both cytoplasm and nucleus of PCa samples; in contrast, benign prostatic hyperplasia (BPH) samples presented strong nuclear staining (78% of the cases). No staining was observed in stromal cells. In addition, using an apta-qPCR, we demonstrated that CG3-aptamer specifically recognizes the conformational PCA3-277 molecule and at least three other transcript variants, indicating that long non-coding RNA (lncRNA) is processed after transcription. We suggest that CG3-aptamer may be a useful PCa diagnostic tool. In addition, this molecule may be used in drug design and drug delivery for PCa therapy.
Highlights
SELEX (Systematic Evolution of Ligands by EXponentional enrichment) is the technology used for in vitro selection of aptamers[3,4], evolving ligands against single molecules to complex target mixtures, or even whole organisms
We successfully generated a genomic library amplified by PCR after eight rounds of in vitro selection against the single-stranded DNA molecule from a 277-nt fragment that may have partially been paired and bound to the prostate cancer antigen 3 (PCA3) RNA conformational structure
Researchers have used the properties of DNA to understand RNA folding[24] and previous measurements of flexible single-stranded nucleic acids[25,26,27], they have not reached a consensus about conformations
Summary
Our in silico analysis of the PCA3 transcript generated a remarkable tertiary structure with a highly significant free energy (Δ G > − 975.60 kcal/mol), showing an extensive base pairing within the molecule that led to a very constrained structure with many hairpins and loops (Fig. 1). Sequences were classified according to the presence of five different motifs identified (CCAU, CCCA, UCCA, UGCC and UGUC) during the 1st, 3th and 8th selection rounds. To demonstrate whether PCA3-277 is a linear or conformational structure, we performed binding assays with denatured and native RNA molecules by dot-blot (solid-phase), immunohistochemistry, and by magnetic capture in solution. The binding of aptamers (CG3 and BC4) to the denatured PCA3 RNA, heated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
