The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation

Abstract

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 ...

Aptamers as targeted therapeutics: current potential and challenges

“…When non-formulated aptamers are administered into the blood stream, even using stabilizing backbone modifications, small aptamers are subject to rapid excretion through renal filtration. To overcome renal filtration and extend circulation time, aptamers are generally formulated with a bulky moiety, such as high molecular mass PEG 69 , cholesterol 79, 80 , proteins 81, 82 , liposomes 83 , organic or inorganic nanomaterials 10, 84 , or are multimerized 85–87 to create a multivalent molecule above the cutoff threshold for the renal glomerulus (30–50 kDa). PEG is a well-studied, hydrophilic biomaterial which decreases aggregation and increases solubility of the conjugates.…”

“…This complexity confers comparable binding affinity and specificity, and even superior advantages over antibodies (Box 1, Table 1). Aptamers are capable of distinguishing between closely related molecules, such as conformational isomers 7 , targets containing different functional groups 8, 9 , or even an amino acid mutation 10 .…”

Future strategies for the discovery of therapeutic aptamers

“…These biomarkers may be drug-specific targets that can directly affect the response of a patient’s disease tissue to a therapeutic regimen. Aptamers targeting point-mutated protein [4] and misfolded proteins [5] might be serve as a personalized treatment that affects therapeutic responses.…”

MDM2 oligomers: antagonizers of the guardian of the genome

“…Although delivery of nucleic acids remains an obstacle, aptamers offer the ability to specifically target deleterious point mutations characteristic of tumors, such as the p53 mutation R175H. 131 Moreover, delivery of p53R175H aptamers via nanoparticles showed impressive efficacy in a mouse xenograft tumor model. Similarly to the potential for PRIMA-1 MET synergism with MDM2 inhibitors, aptamers could also be useful adjuvants to expand the effective tumor profile of MDM2 inhibitors.…”