In the Barton group, we examine the chemical and physical properties of DNA and the biological implications of those properties. Our research has shown that DNA is more than just the library of the cell, existing only to hold genetic information; on the contrary, DNA is a molecule rich in complexity and full of surprises.
Previously, the Barton group has shown that the overlapping π system of stacked DNA bases can mediate the transfer of electrical charge (electrons and holes) over long distances. This DNA-mediated charge transfer (DNA CT) occurs in a variety of sequence contexts and between various types of charge donors and acceptors, shows a shallow distance dependence, and is exquisitely sensitive to perturbations to the base stack such as chemical damage and base pair mismatches. (For recent reviews, please see Genereux and Barton, 2010; Genereux, Boal, and Barton, 2010; and Genereux and Barton, 2009.)
Our current research is focused in three areas:
Metal complexes that recognize specific sites in DNA have great potential to be used in biological applications including early cancer diagnostics and chemotherapeutics.
Among the Rh complexes, Rh(L)2(chrysi)3+ have been successfully employed to detect single nucleotide polymorphisms (SNP). They have also been shown to preferentially inhibit the proliferation of mismatch repair (MMR)-deficient cells over MMR-proficient ones. Currently, we are designing new bifunctional conjugates of Rh complexes to use as highly selective drug delivery systems.
Luminescent Ru complexes have been used extensively in mechanistic studies of cellular uptake. More efforts are being directed toward the development of Ru-based luminescent reporters of MMR-deficient cancer cells.
DNA CT studies focus on examining the mechanistic and kinetic properties of electron and hole propagation along DNA. Photooxidants containing transition metals such as ruthenium, rhodium, and iridium, tethered to DNA, have proven to be invaluable tools in these DNA studies. Modified nucleobases, which are incorporated into the DNA base stack and form oxidative products upon reaction, yield information on charge occupancy along the strand. The DNA base pair stack facilitates CT over long distances but is remarkably sensitive to perturbations in base-base interactions, which occur at sites containing single base mismatches and lesions. Since CT efficiency is dependent on DNA structure and dynamics, measurements of CT efficiency can act as a general reporter of these characteristics. Interestingly, damaged bases in DNA are known to lead to errors in DNA replication thus ultimately altering cellular function and possibly leading to diseases such as cancer.
One mechanism by which damaged bases are naturally repaired at an efficient rate in the cell involves the base excision repair (BER) pathway. While the excision step in repair has been well characterized, precisely how these damaged bases are located in the genome by BER enzymes remains unknown. It is of interest to determine whether BER enzymes depend on the CT property of DNA or instead use some other type of processive mechanism to recognize lesions. The CT properties of BER enzymes such as Endonuclease III and MutY, both of which contain a redox-active [4Fe-4S]2+ cluster, are currently under investigation. Based on our model that exploits the CT property of DNA to locate lesions efficiently, glycosylases are expected to spend more time bound to damage-containing DNA. The techniques we use to study BER protein/DNA interactions include single-molecule atomic force microscopy, electrochemistry to measure redox activity, biochemistry to monitor cooperativity and function within cells, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy.
In order to gain knowledge of how BER proteins may communicate with one another and with DNA, we are exploring the activity of wild-type and mutant proteins in vivo. We are able to create site-specific mutations in proteins, purify, and then test reactivity in cellular environments. Other important transcription factors that may utilize DNA CT, such as SoxR and p53, are being investigated in cells and in vitro. Furthermore, mitochondria, which power the cell, contain their own DNA susceptible to oxidative damage. We are working to recognize mtDNA damage hotspots and elucidate signaling mechanisms under conditions of oxidative stress to probe how mitochondria may take advantage of DNA CT.
An additional focus of the subgroup is the development of multiplexed gold electrode chips to be used to expand our protein and mRNA detection assays into an array-based format. Such arrays will make it possible to simultaneously detect multiple transcription factors and mRNAs on a single chip with minimal sample preparation or purification. The future of this technology shows great promise for use in clinical settings for the detection of transcription factors or mRNAs which are markers of cancers and other diseases.