p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short singlestranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice.p53 | RNA aptamer | contrast screening | SELEX | tumor N ucleic acid aptamers, as single-stranded DNA or RNA oligonucleotides that are able to bind various targets with high specificity, were first isolated from a pool of random sequences with a process called systematic evolution of ligand exponential enrichment (SELEX) in 1990 by two laboratories (1, 2). Over the years, an array of methods have been invented to facilitate SELEX screening, and specific aptamers binding to partners ranging from small molecules to large proteins have been isolated. However, an RNA aptamer that can distinguish a protein with a single amino acid mutation from the wild-type (WT) protein remains absent (3-11).Protein with a single amino acid substitution is the cause of a plethora of human diseases (12)(13)(14). A well-known example is sickle-cell anemia, which is caused by a point mutation in the β-globin chain of hemoglobin (15). Also, point mutations in multiple tumor suppressor proteins cause cancer (16-18). The protein p53 is a tumor suppressor and functions as a transcription factor to regulate the expression of genes involved in DNA repair, cell cycle, and apoptosis. A mutation within one allele of this gene can result in inactivation of the remaining WT allele in a dominant-negative manner, and mutations from six mutation hot spots located in the DNA-binding surface of p53 are frequently found in almost all cancer types (19). Actually, more than half of human cancer cases relate to mutations in p53, and the single amino acid substitution p53R175H is one of the mutations at the p53R175 hot spot (20,21). R175H mutation abolishes the p53 WT functions in both MEF cells and thymocytes (22). p53R175H possesses a marked anti-apoptotic gain-of-function in lung cancer cells (23). Also, p53R175H cooperates better than any other mutant ...