Investigating the Redox Abilities of DNA Aptamers Bound to Redox Cofactors for Possible Application in Biofuel Cells

Aptamers are single-stranded RNA or DNA sequences that can bind to a target with affinity and specificity. Deoxyribozymes or DNA enzymes represent another type of functional, single-stranded DNA that can catalyze chemical reactions. Both aptamers and deoxyribozymes could find potential use in a number of applications, including biofuel cells. These functional DNAs are identified through an iterative process termed in vitro selection. This process allows one to develop customized catalysts made of DNA that can function under the desired application conditions. Compared to proteins, DNA is cheaper to synthesize, easily modified for attachment to surfaces such as electrodes, and stable under various conditions. It is therefore desirable to exploit these properties of DNA for use in various applications. For example, a deoxyribozyme capable of peroxidase activity has been identified, and our lab has demonstrated the ability of this deoxyribozyme to function in a biofuel cell.

As a long term goal, we are interested in replacing protein enzymes with DNA catalysts in enzymatic biofuel cells because of challenges encountered, such as limited enzyme lifetime and low power densities. We are therefore investigating whether we could identify DNA sequences that may bind to redox cofactors in order to create an active site and mimic oxidoreductases. Oxidoreductase protein enzymes contain cofactors in their active sites that allow the enzymes to do redox chemistry. In enzymatic biofuel cells, the redox cofactors act as electron-transfer shuttles to transport electrons between the enzymes’ active sites and electrode surfaces. We reason that binding of DNA aptamers to the cofactors could provide a stable environment that allows the cofactor to be easily accessible to the electrode’s surface with improved electron transfer. We are focusing our efforts on the three commonly used redox cofactors:  flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD⁺) and pyrroloquinoline quinone (PQQ).

Using in vitro selection, we have isolated DNA aptamers that bind to PQQ. To assess the usefulness of these aptamer-cofactor complexes in biofuel cells, we conducted spectroscopic redox assays which showed that PQQ’s redox ability is not affected upon aptamer binding. We are currently using electrochemical techniques to further investigate the redox abilities of the PQQ-DNA complexes. Aptamers for FAD and NAD⁺ have previously been reported. We are also testing their redox abilities to compare how each cofactor is affected by aptamer binding. The results of these experiments as well as progress towards incorporating these aptamer-cofactor complexes in biofuel cells will be presented.