Through a systematic bioinformatics analysis on the reads by Cygwin (Redhat), sequences were extracted and listed. CEA, CA50 and CA72-4 respectively. Combined with high-throughput sequencing, we identified 6 aptamers which specifically target for these three biomarkers of gastrointestinal cancer. Intriguingly, the predicted secondary structures Puromycin Aminonucleoside of RNA aptamers from each antigen showed significant structural similarity, suggesting the structural recognition between the aptamers and the antigens. Moreover, we determined the dissociation constants of all the aptamers to their corresponding antigens by fluorescence spectroscopy, which further demonstrated high affinities between the aptamers and the antigens. In addition, immunostaining of gastric adenocarcinoma cell line AGS using CEA Aptamer probe showed positive fluorescent signal which proves the potential of the aptamer as a detection tool for gastric cancer. Furthermore, substantially decreased cell viability and growth were observed when human colorectal cell line LS-174T was transfected with each individual aptamers. Taking together, these novel RNA aptamers targeting gastrointestinal cancer biomarker CEA, CA50 and CA72-4 will aid further development and standardization of clinical diagnostic method with better sensitivity and specificity, and potentially future therapeutics development of gastric cancer. Introduction According to the statistics from World Health Organization 2015, Gastric cancer is the third most common cause of cancer-related death in the world [1]. Nevertheless, gastric cancer remains difficult to cure due to the lack of early detection biomarkers and most patients are diagnosed at late stage or with advanced CD253 disease. For the last decades, detection of biomarkers or tumor markers has been widely used in clinical management which assist the screening, diagnosis, prediction of prognosis and recurrence, and post-treatment monitoring. Common biomarkers for gastrointestinal cancer can be largely classified as carcinoembryonic antigen (CEA), and tumor associated antigens, such as cancer antigen 19C9 (CA19-9), cancer antigen 50 (CA50) and cancer antigen 72C4 (CA72-4) [2]. CEA, with a molecular Puromycin Aminonucleoside weight of 180C200 kD, is a cell surface glycoprotein that plays a role in cell adhesion and intracellular signaling [3]. Cancer antigen 50 (CA50), with a molecular weight of ~210 kD, is defined by the monoclonal antibody C 50 developed against a colorectal cancer (CRC) cell line COLO-205. It has elevated levels in serum and can be observed in a variety of malignancies, especially gastrointestinal cancers [4]. Puromycin Aminonucleoside Cancer antigen 72C4 (CA72-4), with a molecular weight of 220C400 kD, is a mucin-like high molecular weight tumor associated antigen, and it is considered as the first choice of tumor marker for gastric carcinoma because of a superior sensitivity than CEA and CA19-9 [1]. Currently, enzyme immunoassay (EIA) and radio-immunoassay (RIA) are commonly employed for the detection of tumor markers [2]. Although the interaction between antibodies and antigens are highly Puromycin Aminonucleoside specific, the biggest drawback and conundrum of immunoassays are harmonization and standardization of the assays for clinical applications [5]. Different manufactures of the commercial detection kits of tumor markers may design different binding sites/ epitopes for the immunoassays which leading to fluctuations on quality and challenge on the standardization. Moreover, variation in the induction of immune response in biological system also contributes to the uncertainty of the quality and the production suffers from lot-to-lot variation. In addition, a minimal perturbation of conformational epitopes on native proteins will cause a failure in probing against the corresponding antigen by a monoclonal antibody. Technically, the relatively low stability and short shelf life of immunoassays also post challenge for frequent calibrations and fluctuations of the results. All these limitations lead to an urgent need for the development.