Aptamers for DNA Damage and Repair

Abstract

DNA is damaged on a daily basis, which can lead to heritable mutations and the activation of proto-oncogenes. Therefore, DNA damage and repair are critical risk factors in cancer, aging and disease, and are the underlying bases of most frontline cancer therapies. Much of our current understanding of the mechanisms that maintain DNA integrity has been obtained using antibody-based assays. The oligonucleotide equivalents of antibodies, known as aptamers, have emerged as potential molecular recognition rivals. Aptamers possess several ideal properties including chemical stability, in vitro selection and lack of batch-to-batch variability. These properties have motivated the incorporation of aptamers into a wide variety of analytical, diagnostic, research and therapeutic applications. However, their use in DNA repair studies and DNA damage therapies is surprisingly un-tapped. This review presents an overview of the progress in selecting and applying aptamers for DNA damage and repair research.

Keywords: DNA damage; DNA repair; SELEX; aptamer; in vitro selection; mutation; therapeutics.

Figure 1

Our DNA is damaged by normal cell processes, contaminants in our food and environment, radiation and ultraviolet light. Damage may include strand-breaks, crosslinks (yellow line in DNA), or adducts (black and red stars). If not repaired, DNA damage can lead to cell death or heritable mutations and cancer.

Figure 2

When guanine is oxidized, forming 8-oxoguanine (8-oxo-G), the resulting preferential basepairing to A ultimately leads to a G to T tranversion mutation. Further oxidation results in the spiroiminodihydantoin (Sp) adduct diastereomers, for example, which are highly destabilizing to the DNA duplex.

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.

Figure 4

Conceptual figure highlighting potential applications of aptamers for DNA damage and repair. (A) Aptamers (green strands) could be incorporated into several platforms to create rapid analysis or point-of-care diagnostic kits to measured DNA lesion levels; (B) Aptamers combined with RNA tools such as “Spinach” could replace antibodies in cell imaging (allowing fluorescent imaging (bright dots) of damage/ repair proteins inside cells); (C) Highly specific aptamers (green strand) that inhibit repair proteins and polymerases (brown shape) could be used in cancer treatment and gene therapy.