As the number of aptamer modifications increases, these challenges will no longer hinder the therapeutic use of aptamers in general

As the number of aptamer modifications increases, these challenges will no longer hinder the therapeutic use of aptamers in general. DNA repair studies and DNA damage therapies is usually surprisingly un-tapped. This review presents an overview of the progress in selecting and applying aptamers for DNA damage and repair research. as the target. However, the authors were interested in isolating ligand-induced binding aptamers (i.e., those that only bind to the target in certain conditions). Therefore, the selections included a range of neomycin concentrations. As a result, the emerging aptamers could only bind to the target, Fpg, in the presence of the antibiotic, neomycin. Regardless, TBA-354 the best aptamer displayed high affinity to this repair protein, with a reported is usually described; however, homologues of all these proteins are found in eukaryotes. This process is initiated by MutS, a protein that recognizes and binds to mispaired nucleotides. MutS then works together with MutL to direct the excision of the newly synthesized DNA strand by MutH [48]. This is followed by removal of the mismatch and subsequent re-synthesis by DNA polymerases [49]. The Krylov group has been using non-equilibrium capillary electrophoresis TBA-354 of equilibrium mixtures (NECEEM) SELEX [50] to identify many aptamers to various repair proteins. In 2006, NECEEM was first used to select aptamers to MutS from gene is the most frequently mutated codon in human cancers. As a result, many aptamers have been generated to mutant KRAS proteins and peptides [58,59]. Rabbit polyclonal to AKR7L In the most recent example, an RNA aptamer was generated that specifically bound to a mutant KRAS protein with a point mutation in codon 12 (KRASV12). Excitingly, binding to the wild-type KRAS was more than 50 fold lower than the mutant [33]. A second example is the gene which is considered the guardian of the genome. is lost or mutated in about half of human cancer cases [60,61]. The single amino acid substitution p53R175H is one mutation which abolishes p53 function. In 2015, Chen et al. were able to isolate an RNA aptamer that binds to the p53 mutant p53R175H. Remarkably, this RNA aptamer (p53R175H-APT) also displayed a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays [62]. 5. Selection Challenges and Considerations The SELEX process involves iterative rounds of in vitro binding, partitioning and amplification (Figure 3) [63,64,65]. Despite the simplicity, a major advantage of the process is the flexibility in the enrichment strategy, binding conditions and nucleic acids design and type [66,67]. Due to this flexibility, aptamers have been selected to a wide range of targets, including whole cells, viruses, proteins and small molecules [68]. For reviews on the many modifications and improvements to the SELEX procedure over the past 25 years, see [69,70,71]. Here, conditions specific to DNA damage and repair targets are highlighted. Open in a separate window Figure 3 Conceptual representation of classic Systematic Evolution of Ligands by EXponential enrichment (SELEX) and important modifications. Classic SELEX consists of iterative rounds of binding, partitioning and PCR amplification. Single-stranded DNA or RNA libraries are incubated with the target-of-interest (blue circles). A partitioning step removes non-specific sequences (light grey strands). PCR amplification is then used to make multiple copies of the selected sequences (dark grey). Modifications to the classic SELEX process to isolate aptamers for DNA damage and repair targets include: the use of capture-SELEX for small molecules allowing them to be selected without immobilization; altered binding conditions to improve binding to strand breaks and improving activity in vivo; rigorous counter selection to ensure binding specificity; and the use of NECEEM for difficult protein targets. 5.1. DNA Adducts The nucleobases of DNA have molecular weights ranging from approximately 110C150 g/mol. Nucleosides range from 240C285 g/mol, and nucleotides are around 500 g/mol. As a result, the selection targets for DNA damage aptamer libraries are very small, and therefore pose some of the same challenges as small molecule SELEX. Several reviews and methods highlight the conceptual and technical challenges in isolating aptamers to targets of less than 1000 g/mol [11,72]. This explains, in part, the relatively small number of different DNA adduct aptamers as compared to repair proteins, and is consistent with the general trend of fewer small molecule aptamers as compared to aptamers to large targets such as proteins and even cells [68]. The biggest potential break-through in addressing the challenges associated with small molecule aptamer selection was the development of Capture-SELEX, which yields structure-switching aptamers [73]. This method circumvents the needs to immobilize small molecules on a TBA-354 solid-support and further introduces a selection pressure for the selected aptamers to.