Apta-histochemistry? Enhanced Tissue Analysis with Aptamers
Immunohistochemistry is widely used for the detection of cellular markers for tumor diagnosis. Labeled primary antibodies or primary antibodies and labeled secondary antibodies are used to bind and image a cell surface marker of interest within the tissue sample. While antibody affinity reagents have been validated for use in an ever-growing number of blood-based and tissue-based diagnostic tests, aptamers offer some unique advantages (1). Breast Cancer Tumor Image courtesy of Joseph Szulczewski, David Inman, Kevin Elicein, and Patricia Keely; National Cancer Institute: Carbone Cancer Center at the University of Wisconsin
Aptamer Advantages in Immunochemistry
Aptamers are DNA- or RNA-based ligands capable of selectively binding practically any molecular target. The aptamer selection process starts with an initial library containing over 1015 oligonucleotides. Each stage of the aptamer selection process involves binding, elution, and amplification to identify selective, high-affinity aptamers for the target of interest (3). Aptamers are chemically synthesized, simplifying production, reducing production time, improving consistency from batch to batch, and reducing regulatory concerns when compared with traditional antibody production (1). Aptamers are highly selective and easily conjugated to a wide range of tags for imaging without affecting selectivity or affinity (3). Small aptamer size, approximately one tenth the size of an antibody, enables better staining in dense tissue and staining of co-localized proteins, a case where steric hindrance can be an issue for large antibodies (1). The use of Cell-SELEX for aptamer discovery enables aptamer selection to tumor cells prior to identification of a unique cell surface marker, accelerating the development of new affinity reagents for diagnostics and therapeutics. (Learn more about Cell-SELEX)
Selective Staining of Metastatic Breast and Prostate Tissue
Using Cell-SELEX, researchers have identified an aptamer with high selectivity for metastatic breast and prostaste cancer tissue. The aptamer, Apt63, was found to bind the beta subunit of F1F0 ATP synthase (ATP5B). Arrays composed of frozen and FFPE (formalin-fixed, paraffin-embedded) tissue samples were tested with labeled aptamer. Plasma membrane staining with aptamer Apt63 correlated with tumor stage and metastasis, with minimal staining of non-metastatic cancer tissue and no staining of normal breast tissue. The aptamer was also found to be cytotoxic to metastatic breast and prostate cancer cells (4).
Staining of Lung Adenocarcinoma Tissue
Researchers in Russia selected three aptamers to postoperative lung adenocarcinoma tissue for analysis of tumor cells, blood vessels, and connective tissue. Aptamer LC-18 was selective for lung adenocarcinoma cells and elastic fibers of transformed tumor arteries. Aptamer LC-224 was selective for connective tissue and blood vessels positive for lung adenocarcinoma. Aptamer LC-17 bound both positive cells and connective tissues. All three aptamers showed no binding to normal lung tissue. The team established a protocol for staining formalin-fixed (not paraffin-embedded) tissue with a single aptamer and simultaneous staining of fresh frozen tissue with three aptamers. Use of these aptamers enabled tumor characterization and identification of malignant vessels and connective tissues. Successful staining of fresh tissue supports the use of these aptamers for visualization of intraoperative tumors and targeted therapy (5).
Staining of Amyloid Plaques
Researchers at Massachusetts General Hospital and Harvard Medical School discovered an RNA aptamer selective for amyloid beta (Aβ) that binds amyloid plaques both ex vivo and in vivo. Frozen human tissue samples from subjects with Alzheimer’s disease were sectioned on a cryostat at a thickness of 10 mm and fixed for 10 minutes in 4% paraformaldehyde followed by overnight incubation at 4ºC with biotinylated aptamer. A streptavidin-dye conjugate was utilized for visualization with a fluorescence microscope. In vivo detection of amyloid plaques was also observed with labeled aptamer in APP/PS1 transgenic mice (3). Though the aptamer had no therapeutic effect on the amyloid plaques, it showed significant diagnostic potential.
Custom Aptamer Selection for Tissue Analysis
A growing number of researchers are exploring aptamer-based methods for enhanced tissue analysis. The wide range of options for aptamer selection and labeling offer the potential to utilize aptamer affinity reagents in almost any tissue imaging or cell imaging application.
Contact Base Pair today for more information on selection of aptamers for specific cells or cell surface markers.
- Bauer, M., et al. The application of aptamers for immunohistochemistry. Nucleic Acid Therapeutics. 2016. 26(3):120-126.
- Ellington, A.D. and J.W. Szostak. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990. 346:818–822.
- Farrar, C.T., et al. RNA aptamer probes as optical imaging agents for the detection of amyloid plaques. PLOSone. 26 Feb 2014. https://doi.org/10.1371/journal.pone.0089901.
- Speransky, S., et al. A novel RNA aptamer identifies plasma membrane ATP synthase beta subunit as an early marker and therapeutic target in aggressive cancer. Breast Cancer Research and Treatment. 2019. 176:271-289.
- Zamay, G.S., et al. DNA aptamers for the characterization of histological structure of lung adenocarcinoma. Molecular Therapy Nucleic Acids. 2017. 6:150-162.