Aptamers are single-stranded DNA or RNA oligonucleotides that can be used to recognize ligands ranging in size from small organic molecules to large molecules such as proteins. Aptamers are usually identified by a procedure called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). When coupled with a sensing mechanism, signaling aptamers can be created that can be used as biosensors to selectively detect ligands in complex mixtures of molecules. The goal of this Exploratory Research Grant application is to test a novel, alternative approach to SELEX in which the ability of oligonucleotides to form intramolecular triplexes is used as the basis to create aptamers that can recognize 8-oxo-dG and 8-oxo-dA, nucleosides that result from oxidative damage of cellular DNA. The project has three specific aims.
Specific Aim 1 will create aptamer libraries that can potentially form intramolecular triplexes in the presence of 8-oxo-dG or 8-oxo-dA. Aptamers hybridized with a biotinylated capture probe will be incubated with 8-oxo-dG or 8-oxo-dA;ligand induced triplex formation will displace the probe;ligand-free aptamers will be captured on streptavidin beads;and ligand- bound aptamers will be isolated and sequenced.
Specific Aim 2 will convert aptamers identified in Specific Aim 1 to signaling aptamers by derivatizing these molecules with a fluorophore/quencher pair to create a molecular beacon that is able to produce a fluorescent signal in the presence of the 8-oxo-dG or 8-oxo-dA ligand.
Specific Aim 3 will test the abilities of the signaling aptamers developed in Specific Aim 2 to detect 8- oxo-dG and 8-oxo-dA in DNA derived from human cells that have been subjected to oxidative stress. If successful, these studies could lead to the development of detection and diagnostic agents that can be used in a simple assay to monitor oxidative damage to cellular DNA.
The goal of this application is to determine if novel, triplex-based aptamers can be developed to detect oxidized bases in DNA. The formation of these damaged bases, resulting from either oxidative stress or normal metabolic processes, can have important biological consequences and may be involved in processes that affect human health, including aging and male infertility. Simple methods to detect these lesions could provide useful diagnostic or prognostic tools for evaluating disease processes in a clinical setting.