Using RNA Aptamers and the Proximity Ligation Assay for the Detection of Cell Surface Antigens

Abstract

The detection and typing of tumor cells based on differentially or similarly expressed antigens (biomarkers) have proven to be increasingly important for the diagnosis and treatment of various cancers. Sensitive techniques for the detection of cell surface antigens are therefore crucial for the early and accurate detection of cancer. Although techniques such as ELISA and tissue staining have proven their worth, these techniques often either require substantial amounts of starting material or are prone to high background and false negatives. The proximity ligation assay (PLA) has proven to be an exquisitely sensitive technique with very low background. Two probes that bind adjacent to one another on a protein target can be ligated, yielding a unique amplicon that can be sensitively detected by real-time PCR. We have now adapted PLA to cell surface protein targets using modified RNA aptamers, and have shown that aptamer-based cell surface PLA can successfully detect and differentiate between cells that differentially express a tumor antigen, the prostate specific membrane antigen (PSMA).

Applications of Aptamers as Sensors

“…To adapt the RNA aptamers to DNA ligation, the aptamers were hybridized via sequence-specific tails to DNA oligomers, which in turn were ligated with a DNA splint. This use of reagents that can form a single, defined ligation junction may have led to less variance than would have been the case with comparable, multiply labeled antibodies (Supplemental Figure 5a) (124). Upon cell binding and splint-initiated ligation, the anti-PSMA aptamer probe constructs detected as few as 10 LNCaP prostate tumor cells on their own or in the presence of 100,000 noncognate HeLa cells.…”

“…Another advantage of PLA is that proximity recognition need not be limited to individual molecules; it can also involve the surfaces such as bacterial spores, vegetative cells, and cancer cell lines (122)(123)(124). RNA aptamers previously selected against the homodimer prostate-specific membrane antigen (PSMA) were used for the in situ detection of this protein on the cell surface.…”

The future of aptamers in medicine

“…That study showed that the selected aptamers can be used as molecular probes for effective diagnosis and subcategorisation of AML. Aptamer-based cell-surface proximity ligation assay (PLA) has also been used to successfully detect and differentiate between cells that differentially express the prostate-specific membrane antigen (PSMA) 26. In PLA, two probes that bind adjacent to one another on a target analyte are ligated, yielding a unique amplicon that can be sensitively detected by real-time PCR 26.…”

“…Aptamer-based cell-surface proximity ligation assay (PLA) has also been used to successfully detect and differentiate between cells that differentially express the prostate-specific membrane antigen (PSMA) 26. In PLA, two probes that bind adjacent to one another on a target analyte are ligated, yielding a unique amplicon that can be sensitively detected by real-time PCR 26. This aptamer-based cell-surface PLA assay, which is specific for PSMA, has a great potential in the early diagnosis and typing of prostate cancer.…”

Diagnosis of prostate cancer using anti-PSMA aptamer A10-3.2-oriented lipid nanobubbles

“…Therefore, aptamers have been applied extensively in studies on molecular imaging and gene therapy. [14][15][16][17][18] Simultaneously, the aptamer as an element of the targeted probe can avoid immunogenicity and its high purity can also guarantee its non-toxicity and safety compared with other ligands. [19][20][21] Early aptamers including A10 and A9 were screened from the synthesized RNA aptamer library, and can both effectively bind to PSMA.…”