Redmond Red as a Redox Probe for the DNA-Mediated Detection of Abasic Sites

Abstract

Redmond Red, a fluoropore containing a redox active phenoxazine core, has been explored as a new electrochemical probe for the detection of abasic sites in double stranded DNA. The electrochemical behavior of Redmond Red-modified DNA at gold surfaces exhibits stable, quasi-reversible voltammetry with a midpoint potential centered around -50 mV versus NHE. Importantly, with Redmond Red positioned opposite an abasic site within the DNA duplex, the electrochemical response is significantly enhanced compared to Redmond Red positioned across from a base. Redmond Red, reporting only if well stacked in the duplex, represents a sensitive probe to detect abasic sites electrochemically in a DNA-mediated reaction.Since its discovery, the ability of DNA to conduct charge has been exploited in a variety of arenas, including time resolved spectroscopy (1-3), conductivity measurements (4-7), and the development of biosensors (8). Our group, in particular, has explored the versatility of this intrinsic property of DNA by performing extensive electrochemical studies on surfaces modified with DNA duplexes (9-15). Typically in such systems, thiol-modified oligonucleotides are tethered to a gold electrode at the 5′ terminus by a Au-S bond and further modified at the opposite end with a redox probe, thus allowing the duplexes to act as an extension of the conducting medium. The reduction of the attached probe, as observed voltammetrically, serves as an indicator of the transport of charge via the DNA duplex. Importantly, it has been shown that DNA-mediated charge transport is remarkably sensitive to the integrity of the base stack; upon introduction of a single base mismatch located between the electrode and the redox probe, measured currents are dramatically diminished (9-10). This behavior not only provides conclusive evidence that the observed electrochemical reactions are indeed DNA-mediated but also allows naturally occurring perturbations, which may only subtly interrupt π-stack overlap, to be detected (11-15).Among these possibly destabilizing defects are abasic sites, which appear naturally and frequently among DNA sequences as the result of hydrolytic cleavage of the glycosidic bond (16-23).Specific enzymes are responsible for correcting these damaged sites prior to transcription and replication. When this repair pathway is delinquent, however, the persistence of these defects can impart deleterious effects on genetic encoding, leading to the development of cancer, among other diseases (24-26). Because they represent a potential threat, the reliable and specific detection of abasic sites is critical, although still very much under exploration. Development of an electrochemical system to detect these unwanted sites specifically could E-mail: jkbarton@caltech.edu * Author to whom correspondence should be addressed Supporting Information Available: Synthetic details, electrochemical experimental conditions, and characterization data (HPLC, MS, UV-vis, T m ) of modified DNA. This material is available free of charge via...

Solution, surface, and single molecule platforms for the study of DNA-mediated charge transport

“…From these examples we see that, in an added level of complexity to noncovalent probes, the covalent linkage now functions as the critical gatekeeper that can either promote or prevent the redox probe from achieving a CT-active conformation. In addition to MB, AQ, and TEMPO, we characterized the linkages and conditions necessary for the DNA-mediated reduction of a variety of other covalent redoxactive moieties including daunomycin, 106,118-120 disulfide bonds, 121 Redmond Red, 122 and Nile Blue. 123-125 …”

Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

“…Therefore, it is important to evaluate the interaction of DNA with various molecules (Pozo et al, 2005) and develop new strategies for sequence-specific detection of DNA samples. Different methods have been used to study such interactions, including fluorescence techniques (Zhang et al, 2010), mass spectroscopy (Sauer, 2006), quartz-crystal microbalance (Willner et al, 2002;Patolsky et al, 2000;Chen et al, 2007), surface plasmon resonance spectroscopy (Liu et al, 2005;Wang et al, 2004;Su et al, 2008), UV-vis spectroscopy (Ni et al, 2005;Shahabadi et al, 2008), Raman spectroscopy (Jiang et al, 2006;Coates et al, 1997;Xie et al, 2008), X-ray crystallography (Barton, 1985;Maciejewska et al, 2006), and electrochemical methods (White and Plaxco, 2010;Kumamoto et al, 2008;Gorodetsky et al, 2007Gorodetsky et al, , 2008Buzzeo and Barton, 2008;Zhang et al, 2008;Wang et al, 2008;Pournaghi-Azar et al, 2006). However, the electrochemical methods have attracted more attention than other methods because they are highly sensitive, easy to use, inexpensive, portable, and compatible with micro fabrication technologies Hejazi et al, 2007).…”

Sensing DNA through DNA Charge Transport

“…Abasic sites and destabilizing lesions, such as 8-oxoguanine, also significantly diminish DNA CT. 34,49 8-oxoG-A and 8-oxoG-C both destabilize the duplex structure, despite the different locations of their modification, which is sufficient to attenuate DNA CT. 34 It is because these base lesions are so easily detectable that the idea that nature might also use this chemistry for detection inside the cell became reasonable to consider. Indeed, a whole family of DNA repair enzymes has been found to contain [4Fe4S] clusters, redox cofactors featured commonly in proteins, and many experiments we have carried out support the idea that these repair proteins utilize DNA CT chemistry in their search for lesions within the cell.…”