DNA-Mediated Charge Transport for Long-Range Sensing and Protein Detection

The structural core of DNA, a continuous stack of aromatic heterocycles—the base pairs—that extends down the helical axis, gives rise to the fascinating electronic properties of this molecule that is so critical for life. This π-stacked structure facilitates a unique form of charge conduction, termed DNA-mediated charge transport (DNA CT). Experiments with diverse platforms, in solution, on surfaces, and with single molecules, collectively provide a broad and consistent perspective on the essential characteristics of this chemistry. Notably, DNA CT can proceed over long molecular distances, but is remarkably sensitive to perturbations in base pair stacking. These characteristics suggest that DNA CT may be used for long-range sensing both in nature and in nanoelectronic applications. Here, measurements of DNA CT with surface and single molecule platforms are used to (i) determine how ground state DNA CT varies over regimes of increasing distance and (ii) apply this chemistry to the electrical detection of DNA-binding proteins. First, the design and fabrication of multiplexed, DNA-modified electrodes on silicon chips is reported. These lithographically patterned chips with 16 individually addressable gold electrodes allow for the measurement of DNA CT with four different types of DNA, side by side on the same surface, with four-fold redundancy. Discrimination of DNA with a single base mismatch and detection of sequence-specific restriction enzyme activity are both achieved with these chips. Scaling of these devices to microelectrode dimensions is also demonstrated. Importantly, these chips show greater reproducibility and consistency than commercially available rod electrodes. This greater signal quality, combined with the capacity to examine different samples side by side, opens the door for more complex applications of this platform. The fully developed, multiplexed chips are first used to compare DNA CT over short and long distance regimes. DNA is evaluated in this context because the efficacy of a long-range sensor, in either nature or nanoelectronics, is determined largely by its capacity to facilitate CT in a manner that is minimally affected by the CT distance. DNA CT over 34 nm in 100-mer monolayers is found to yield electrochemical signals that are comparable in size to shorter 17-mer DNA. Signal attenuation from a single base-pair mismatch in the 100-mer is also comparable to that for 17-mers, and confirms that CT in these 100-mer films is DNA-mediated. Efficient cleavage by a restriction enzyme indicates that the 100-mer DNA adopts a native, upright conformation. The alkanethiol linker used to anchor the DNA to the electrode is found to limit the electron-transfer rate for both DNA lengths. Thus the impact of increasing the CT distance on DNA CT is too small to be resolved by this platform, even over 34 nm. These measurements put DNA among the longest and most conductive molecular wires reported to date. Next, DNA CT with multiplexed chips is extended to the electrochemical detection of methyltransferases, proteins that are attractive targets because of their prominent role in the initial stages of many types of cancer. Electrochemical detection of binding and activity by these proteins is achieved by two different methods. First, DNA-binding and base-flipping by these proteins disrupts the DNA π-stack and may be used for direct “signal OFF” detection. Using this method, the concentration- and cofactor- dependence of SssI methyltransferase, the bacterial analog of human methyltransferases, are examined. Second, methylation-conferred protection of DNA against cutting by a restriction enzyme may be used for “signal ON” detection of methyltransferase activity. With this approach, the use of both unmethylated and hemimethylated DNA substrates is demonstrated for the sensitive detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity. Importantly, the electrochemical format of these assays requires minimal equipment, is low cost, and may be easily applied to high throughput studies, making it an accessible option for a variety of research and clinical settings. Alongside work with this surface, electrochemical platform, a single molecule, carbon nanotube-DNA (CNT-DNA) platform is also used to evaluate DNA CT over increasing distances and to detect protein binding. CNT-DNA devices consist of a single molecule of DNA that is made to bridge a gap cut in a CNT covalently, such that current flow through the device is DNA-mediated. Upon introduction of DNA bridges of varying length (15-mer, 60-mer, and 100-mer), the device resistance is minimally affected, echoing the result of long distance electrochemistry experiments. These devices are also used to detect SssI methyltransferase binding by the direct “signal OFF” method used with multiplexed chips; DNA-binding and base-flipping disrupts DNA CT and shuts off current flow through the device. CNT-DNA devices are used to electronically measure the sequence-specific, cofactor-dependent, and reversible binding of SssI. DNA methylation catalyzed by SssI is also detected based on its alteration of the protein-binding affinity of the device. This detection approach, which relies on DNA as both a recognition element and electrical transducer, represents a unique strategy for the specific, single molecule detection of protein binding and activity.