AbstractBackgroundChromosomal heterogeneity leads to the abnormal expression and mutation of tumor‐specific genes. Drugs targeting oncogenes have been extensively developed. However, given the random mutation of tumor suppressor genes, the development of its targeted drugs is difficult.MethodsOur early research revealed that artificial circular single‐stranded DNA (CSSD) can restore multiple tumor suppressor genes to inhibit tumor malignant progression by adsorbing miRNA. Here, we improved CSSD to a fully closed single‐stranded DNA with G quadruplex DNA secondary structure (G4‐CSSD), which made G4‐CSSD with higher acquisition rate and decreased degradation. The Cancer Genome Atlas (TCGA) and Human Protein Atlas database were used to predict tumour suppressor genes in colon cancer. Cellular and animal experiments were performed to validate the role of G4‐CSSD in cancer cell progression.ResultsIn colon cancer, we observed the simultaneous low expressions of chloride channel accessory 1 (CLCA1), UDP‐GlcNAc:betaGal beta‐1,3‐N‐acetylglucosaminyltransferase 6 (B3GNT6) and UDP glucuronosyltransferase family 2 member A3 (UGT2A3), which indicated an favourable prognosis. After repressing miR‐590‐3p with G4‐CSSD590, the upregulation of CLCA1, B3GNT6 and UGT2A3 inhibited the proliferation and metastasis of colon cancer cells.ConclusionsThis study may provide basis for new treatment methods for colon cancer by restoration of tumor suppressor genes.