Selection of DNA aptamers for extra cellular domain of human epidermal growth factor receptor 2 to detect HER2 positive carcinomas.

Background: Human epidermal growth factor receptor 2 (Her2, an orphan receptor of ErbB family) is considered as an important biomarker as it plays a key role in the development and progression of aggressive types of breast, ovarian, stomach and gastric cancer. In the present study, we developed novel DNA aptamers against the extra-cellular domain (ECD) of Her2 protein for detection of Her2-positive carcinomas.

Methods: We cloned and expressed Her2-ECD protein in E. coli system. After purification, the protein was used as a bait for screening of specific DNA aptamer candidate from a pool of 10 random oligonucleotides through in vitro Systematic Evaluation of Ligands by Exponential Enrichment (SELEX) process. The aptamer-protein binding kinetics was elucidated by isothermal calorimetry. The specificity of FAM-labelled ECD_Apt1 towards Her2-positive cell lines was estimated by FACS and immunofluorescence assay. The specificity of the candidate was also verified with the tissue samples of breast cancer patients by immunohistochemistry process.

Results: Among four selected candidates, ECD_Apt1 (having minimum ∆G = -3.24) showed the highest binding affinity (K  = 6.33 ± 0.86 nM) to Her2-ECD protein. The aptamer-protein sandwich assay showed a linear rise in chemiluminescence (at 490 nm wavelength) in the dynamic range of 100-700 nM ECD_Apt1 with a detection limit of 12.5 ± 2.5 ng/mL. Biotinylated ECD_Apt1 showed stronger cytoplasmic staining in Her2-positive breast cancer cell lines (SKBR3) compared to Her2-negative cells (MDA MB 231, MCF7). In paraffin-embedded breast cancer tissue sections, it showed specific and selective localization in the cytoplasmic niche of malignant duct cancer cells without any cross-reactivity to fibroblasts, inflammatory cells and adipocytes.

Conclusions: Binding assays, cytochemical and histochemical studies support ECD_Apt1 as a potential theranostic agent for Her2-positive carcinomas. ECD_Apt1 could be an effective low-cost alternative to conventional anti-Her2 antibody in solid phase immunoassays for cancer diagnosis and related applications.