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Biomarkers and Early Detection: Molecular Diagnostics

Nucleic acid aptamers as diagnostic tools for prostate cancer.

University of Texas - Austin, Austin, TX

Abstract

B19

Prostate cancer is the second leading cause of death amongst males in America. Diagnosis and prognosis of prostate cancer is heavily based upon cell morphology and the levels of prostate specific antigen (PSA), two factors that are prone to false negatives and misdiagnosis. The identification of molecular alternatives to the detection of all tumors in general as well as cell specific markers to specific tumor types can be very useful. Nucleic acid aptamers serve as excellent detection tools due to their affinity for the target they are selected against as well their versatile nature that allows them to be coupled to molecules such as siRNA, DNA tags, quantum dots, peptides etc. We have now directly selected aptamers against prostate cancer biomarker molecules on the surfaces of cultured prostate cancer cell lines, LNCaP and PC3. These cell lines differ in the expression of the tumor marker prostate specific membrane antigen (PSMA), a very promising cell surface biomarker. LNCaP cells over-express this surface marker while PC3 have very little PSMA on their surface. Two aptamers, LN101 and LN104, selected against LNCaP cells and two aptamers, PC301 and PC304, selected against PC3 specifically were generated from negative selection while four aptamers, LP702, LP705, LP719 and LP720 were generated through positive selections. The sensitive nature of these aptamers as cancer diagnostic tools was demonstrated by FACs, confocal microscopy and a novel technique called the Proximity Ligation Assay (PLA). The principle of PLA involves the use of two affinity tags on the surface of the target, which when in proximity to one another can ligate in the presence of a connector nucleotide (splint) and the amplicon generated is detected via real-time PCR. In order to adapt aptamers to PLA, we constructed a five-piece PLA system where aptamers were annealed to specific 3'and 5' DNA probes via specific RNA linkers or extensions that had been chosen based on their ability to leave the aptamer structure intact. This construct was used in target specific detection of cognate cancer cells in a heterogeneous mixture of cells, the sensitivity of detection being as efficient as 10 LNCaP and 10 PC3 cells in a mixture of 10⁵ HeLA cells, with little to no cross-reactivity. In conclusion, aptamer-mediated PLA is extremely sensitive and target specific presenting itself as a potential molecular sensor for early cancer detection.