The diagnostic potentiality of the RNA aptamer against progesterone receptor isolated by crush and soak (CRUSOAK)-SELEX

• Published in: Mikrochimica acta (1966) Vol. 191; no. 6; p. 346
• Main Authors: Presela, Ravinderan, Prabu, Siva Sankar, Ch’ng, Ewe Seng, Tang, Thean-Hock, Citartan, Marimuthu
• Format: Journal Article
• Language: English
• Published: Vienna Springer Vienna 01.06.2024; Springer Nature B.V
• Subjects: Analytical Chemistry; Antibodies; Aptamers, Nucleotide - chemistry; Binding; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Crushing; Deoxyribonucleic acid; Diagnostic systems; DNA; Female; Humans; Microengineering; Nanochemistry; Nanotechnology; Original Paper; Receptors; Receptors, Progesterone - metabolism; Ribonucleic acid; RNA; SELEX Aptamer Technique - methods; RNA aptamer; Histostaining; Progesterone receptor; CRUSOAK-SELEX
• ISSN: 0026-3672; 1436-5073
• DOI: 10.1007/s00604-024-06423-z

Aptamers are a class of molecular recognition elements that exhibit high binding affinity and specificity against their respective targets. In view of the many advantages aptamers harbor over their counterpart antibodies, we were impelled to isolate an RNA aptamer against progesterone receptor, particularly its DNA binding domain. A total of eight SELEX cycles were executed against the recombinant Progesterone Receptor DNA-binding domain (PR DBD). The RNA–protein complex in the gel shift assay was subjected to crush and soak method to elute the binders prior to conventional sequencing, the step of which was based upon to coin the term CRUSOAK-SELEX. The sequencing revealed three different classes of sequences, with one class termed, PRapt-3, showing the strongest binding against PR DBD. The dissociation constant of PRapt-3 RNA aptamer was estimated at 380 nM ± 35 nM. PRapt-3 was successfully used to develop aptamer-based diagnostic assays such as ELASA, aptamer-based dot blot, and aptamer-based western blot. The prominent highlight is the performance of the aptamer in aptacytostaining, which was unachievable with antibodies. Compared to its counterpart antibodies, PRapt-3 has a better penetration capacity in aptahistostaining using the formalin-fixed paraffin-embedded (FFPE) breast cancer cells and tissue blocks. This study represents the first ever demonstration of an aptamer against progesterone receptor and its diagnostic capacity. Graphical Abstract