Effects of Nucleic Acid Structural Heterogeneity on the Electrochemistry of Tethered Redox Molecules.

The cation condensation-induced collapse of electrode-bound nucleic acids and the resulting change in the electrochemical signal is a useful tool to predict the structure and redox probe location of heterogeneous structures of surface-tethered DNA probes─a common architecture employed in the development of electrochemical sensors. In this paper, we measure the faradaic current of an appended redox molecule at the 3' position of the nucleic acid using cyclic voltammetry before and after nucleic acid collapse for various nucleic acid architectures and heterogeneous mixtures on the same electrode surface. The voltammetric peak current change with collapse correlates with the proximity of the redox molecules from the surface. For stem-loop probes, the terminal methylene blue is initially held closer to the surface, such that inducing collapse, by reducing the dielectric permittivity of the interrogation solution, results in a ∼30% increase in current. However, when incorporating pseudoknot probes that hold methylene blue further away from the electrode surface, the current change is much larger (∼120%), indicating a larger conformation change. Upon a 50:50 ratio of the two, we observe a change in current that relates to the ratiometric distribution of the probe used to make the surfaces. Additionally, using cyclic voltammetry, we find that the change between diffusion-limited and diffusion-independent peak currents is dependent upon the distinct structural characteristics of DNA probes on the surface (stem-loop or pseudoknot), as well as the ratios of different DNA probes on the surface. Thus, we demonstrate that the heterogeneous nature of DNA probes governs the corresponding electrochemical signals, which can lead to a better understanding on how to predict the structures of functional nucleic acids on electrode surfaces and how this affects surface-to-surface variability and electrochemical response.

Nucleic Acid Identity, Structure, and Flexibility Affect the Electrochemical Signal of Tethered Redox Molecules upon Biopolymer Collapse.

We demonstrate that cation condensation can induce the collapse of surface-bound nucleic acids and that the electrochemical signal from a tethered redox molecule (methylene blue) upon collapse reports on nucleic acid identity, structure, and flexibility. Furthermore, the correlation of the electrochemical signal and structure is consistent with theoretical considerations of nucleic acid collapse. Changes in solution dielectric permittivity or the concentration of trivalent cations cause the structure of nucleic acids to become more compact due to an increase in attractive electrostatic interactions between the charged biopolymer backbone and multivalent ions in the solution. Consequently, the compaction of nucleic acids results in a change in the dynamics and location of the terminally appended redox marker, which is reflected in the faradaic current measured using cyclic voltammetry. In comparison to ssDNA, nucleic acid duplexes (dsDNA, DNA/peptide nucleic acid, and dsRNA) require nucleic-acid-composition-specific solution conditions for the collapse to occur. Moreover, the magnitude of current increase observed after the collapse is different for each nucleic structure, and we find here that these changes are dictated by physical parameters of the nucleic acids including the axial charge spacing and the periodicity of the helix. The work here aims to provide quantitative and predicative measures of the effects of the nucleic acid structure on the electrochemical signal produced from distal-end appended redox markers. This architecture is commonly employed in functional nucleic acid sensors and a better understanding of structure-to-signal correlations will enable the rational design of sensitive sensing architectures.

Perspective on the Future Role of Aptamers in Analytical Chemistry.

It has been almost 30 years since the invention of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology and the description of the first aptamers. In retrospect over the past 30 years, advances in aptamer development and application have demonstrated that aptamers are potentially useful reagents that can be employed in diverse areas within analytical chemistry, biotechnology, biomedicine, and molecular biology. While often touted as artificial antibodies with an ability to be selected for any target, aptamer development, unfortunately, lags behind development of analytical methodologies that employ aptamers, hindering deeper integration into the application of analytical tool development. This perspective covers recent advances in SELEX methodology for improving efficiency of the SELEX procedure and enhancing affinity and specificity of the selected aptamers, what we view as a critical barrier in the future role of aptamers in analytical chemistry. We discuss postselection modifications that can be used for enhancing performance of the selected aptamers in an analytical device by including understanding intermolecular interaction forces in the binding domain. While highlighting promising properties of aptamers that enable several analytical advances, we provide discussion on the challenges of penetration of aptamers in the analytical field.

Electrochemical Studies of Cation Condensation-Induced Collapse of Surface-Bound DNA.

In this paper, we demonstrate the ability to control and electrochemically monitor nucleic acid conformation by inducing collapse of short, surface-bound nucleotides (7-28 nucleotides). More specifically, we monitored changes in a 5'-electrode-bound DNA structure via changes in the faradaic current related to the reduction/oxidation of a 3'-terminal-appended redox molecule. Reversible DNA collapse was induced by cation condensation achieved by either reducing the dielectric permittivity of the interrogation solution or by the addition of multivalent cations such as the polyamine spermidine (3+). Additionally, we find that while the change in electrochemical signal associated with surface bound DNA collapse is dependent on nucleic acid length and surface packing density, the solution conditions (e.g., dielectric permittivity) required for collapse remain constant. As such, we find that collapse of the short DNA strands occurs when the effective charge of the DNA backbone is ∼73-89% neutralized by cations in solution/buffer, according to Manning's theory on cation condensation. This work provides new insight into the structure function relationship of surface-bound nucleic acids and how this is manifested in electrochemical signaling.