Diagnostic Applications of Morpholinos and Label-Free Electrochemical Detection of Nucleic Acids.

Diagnostic applications of morpholinos take advantage of their unique properties including backbone charge neutrality, a weak impact of ionic strength on their hybridization behavior, and their resistance to enzymatic degradation. This chapter overviews how these properties have advanced transduction and other capabilities useful for the analysis of nucleic acids. In many cases, the benefits stem from electrostatic mechanisms; for example, use of low ionic strengths improves sensitivity of detection while decreasing background signals because only the nucleic acid analyte is charged. While most literature reports focus on in vitro assays in buffer, morpholinos have been also used for biodistribution measurements of species such as fungal rRNA and miRNA. After reviewing the diagnostic applications of morpholinos, the chapter describes preparation of morpholino monolayers on metal supports for electrochemical diagnostics and the procedure for performing label-free detection of DNA from changes in surface capacitance.

Capacitive monitoring of morpholino-DNA surface hybridization: experimental and theoretical analysis.

Impedance and cyclic voltammetry methods, complemented by Poisson-Boltzmann (PB) modeling, are used to study hybridization of DNA analyte strands to monolayers of morpholino oligomers (MOs) immobilized by one end to mercaptopropanol-passivated gold electrodes. MOs, like peptide nucleic acids (PNAs), are uncharged molecules that recognize nucleic acids following conventional base-pairing rules. The capacitive response to hybridization, determined from real-time impedance measurements, is analyzed with emphasis on understanding the underlying structural changes and on providing a foundation for label-free diagnostics. The capacitive response is correlated with the instantaneous surface molecular populations by labeling DNA and MO strands with ferrocene tags and using cyclic voltammetry to monitor their respective coverages in real-time. This approach allows analysis of hybridization-induced changes in interfacial capacitance as a function of duplex coverage, the DC bias used for readout, buffer molarity, and probe coverage. The results indicate that unhybridized MO layers exist in a compact state on the solid support. For hybridized layers, the intrinsic signal per hybridization event is strongly enhanced at low ionic strengths but, interestingly, does not depend on the readout bias in the sampled range negative of the capacitive minimum. A PB model incorporating an effective medium description of the hybridizing films is used to establish how hybridization-derived changes in dielectric composition and charge distribution at the surface translate into experimentally observed variations in interfacial capacitance.

Morpholino monolayers: preparation and label-free DNA analysis by surface hybridization.

Surface hybridization, a reaction in which nucleic acid molecules in solution react with nucleic acid partners immobilized on a surface, is widely practiced in life science research. In these applications the immobilized partner, or "probe", is typically single-stranded DNA. Because DNA is strongly charged, high salt conditions are required to enable binding between analyte nucleic acids ("targets") in solution and the DNA probes. High salt, however, compromises prospects for label-free monitoring or control of the hybridization reaction through surface electric fields; it also stabilizes secondary structure in target species that can interfere with probe-target recognition. In this work, initial steps toward addressing these challenges are taken by introducing morpholinos, a class of uncharged DNA analogues, for surface-hybridization applications. Monolayers of morpholino probes on gold supports can be fabricated with methods similar to those employed with DNA and are shown to hybridize efficiently and sequence-specifically with target strands. Hybridization-induced changes in the interfacial charge organization are analyzed with electrochemical methods and compared for morpholino and DNA probe monolayers. Molecular mechanisms connecting surface hybridization state to the interfacial capacitance are identified and interpreted through comparison to numerical Poisson-Boltzmann calculations. Interestingly, positive as well as negative capacitive responses (contrast inversion) to hybridization are possible, depending on surface populations of mobile ions as controlled by the applied potential. Quantitative comparison of surface capacitance with target coverage (targets/area) reveals a nearly linear relationship and demonstrates sensitivities (limits of quantification) in the picogram per square millimeter range.

Charging behavior of single-stranded DNA polyelectrolyte brushes.

DNA monolayers are widely used in fundamental and applied genomics and are versatile experimental models for elucidating the behavior of charged polymers at interfaces. The physical behavior of these systems is to a large extent governed by their internal ionic microenvironment, which is investigated here for layers of end-tethered, single-stranded DNA oligonucleotides (DNA brushes). Retention of counterions by the DNA brush manifests as lowered susceptibility of the interfacial capacitance to external salt conditions. A physical model based on concepts adapted from polymer science was used to further elucidate the connection between monolayer organization and its charging behavior. The data indicate a reorganization of the monolayer with changes in ionic strength and strand coverage that is consistent with that expected for a polyelectrolyte brush. A method for electrochemical quantification of strand coverage, based on shift of reduction potential for redox counterions associated with the DNA monolayer, is also described. These results provide guidance for development of label-free electrochemical diagnostics employing DNA monolayers and formulate a description of monolayer behavior within a polymer science framework.