Barton Group Research

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:

Synthesis and Characterization of DNA-Binding Probes

The Barton group utilizes coordinatively saturated, substitution-inert, octahedral metal complexes to probe the structure and dynamics of double-helical DNA. Specifically, we design, synthesize, and study novel metal complexes capable of specific interactions with target sites in DNA duplexes. Two classes of complexes form the focus of our research efforts: Rh complexes, which photocleave the DNA backbone near the site of binding, and Ru complexes, which luminesce upon DNA binding. Compared with organic DNA-binding agents, these metal complexes offer a uniquely modular system, allowing combinations of recognition elements to be assembled on a single rigid, three-dimensional scaffold.

Crystal structures showing the two main binding modes of metal complexes to DNA. On the left, the complex inserts at a mismatch site, ejecting the mismatched bases (green) into the major groove. On the right, the complex intercalates into well-matched DNA.
By tuning the shape and functionality of these metal complexes, we have developed a series of Rh complexes capable of DNA sequence recognition through intercalation. More recent efforts have led to the discovery of a new class of metal complexes capable of recognizing single base mismatches. These complexes, dubbed metalloinsertors and exemplified by Rh(bpy)2(chrysi)3+ and Rh(bpy)2(phzi)3+, bind with high specificity and affinity to single base mismatches in DNA duplexes. Their mode of binding, elucidated through NMR studies and crystallography, constitutes the first observation of DNA insertion by a small molecule: the sterically expansive chrysi ligand inserts into the pi-stack of the DNA duplex from the minor groove and ejects the mispaired bases from the helix into the major groove.

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.

A Biological Role for DNA-Mediated Charge Transport

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.

BER enzymes MutY and EndoIII bound to DNA. By using DNA-mediated charge transport from the redox-active iron-sulfur cluster of one protein to the other, these enzymes may cooperate to scan large regions of the genome for defects in an efficient manner. (PDBIDs: 2KG2 and 1P59)
What role might DNA CT play in vivo?

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.

Electrochemistry on DNA Films

An illustration of a DNA monolayer on a gold surface. This motif is very useful for understanding how perturbations to the DNA base stack affect the efficiency of DNA charge transport.
Our electrochemistry research focuses on the development and utilization of electrochemical methods which take advantage of DNA CT chemistry. We are using this unique characteristic of DNA both to answer fundamental questions about the CT process and to develop a novel, highly sensitive platform for the detection of DNA-binding proteins and mRNAs.

A photograph of a multiplex chip. The chip can be used to measure up to 16 independent samples simultaneously.
Our electrochemical techniques exploit the exquisite sensitivity of DNA-mediated CT to perturbations in the π-stack formed by bases in the double helix. Thiol-modified DNA duplexes containing a redox-active probe are self-assembled into a monolayer on gold. By monitoring the DNA-mediated reduction of the redox probe, it is possible to sensitively probe the integrity of the π-stack in the region between the gold surface and the redox probe. We have used this technique to detect such perturbations as mismatches, lesions, as well as protein-DNA interactions including kinking and base-flipping. Current work to develop this technology into a viable protein and mRNA detection assay includes the incorporation of new, covalent redox probes and surface passivation strategies.

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.