Label-Free Detection of Zeptomol miRNA via Peptide Nucleic Acid Hybridization Using Novel Cyclic Voltammetry Method

To harness the applicability of microribonucleic acid (miRNA) as a cancer biomarker, the detection sensitivity of serum miRNA needs to be improved. This study evaluated the detection sensitivity of miRNA hybridization using cyclic voltammograms (CVs) and microelectrode array chips modified with peptide nucleic acid (PNA) probes and 6-hydroxy-1-hexanethiol. We investigated the PNA probe modification pattern on array chips using fluorescently labeled cDNA. The pattern was not uniformly spread over the working electrode (WE) and had a one-dimensional swirl-like pattern. Accordingly, we established a new ion-channel sensor model wherein the WE is negatively biased through the conductive π–π stacks of the PNA/DNA duplexes. This paper discusses the mechanism underlying the voltage shift in the CV curves based on the electric double-layer capacitance. Additionally, the novel hybridization evaluation parameter ΔE is introduced. Compared to conventional evaluation using oxidation current changes, ΔE was more sensitive. Using ΔE and a new hybridization system for ultrasmall amounts of aqueous solutions (as low as 35 pL), 140 zeptomol label-free miRNA were detected without polymerase chain reaction (PCR) amplification at an adequate sensitivity. Herein, the differences in the target molar amount and molar concentration are elucidated from the viewpoint of hybridization sensitivity

Crystal structures of B-DNA with incorporated 2′-deoxy-2′-fluoro-arabino-furanosyl thymines: Implications of conformational preorganization for duplex stability

The fundamental conformational states of right-handed double helical DNA, the A- and B-forms, are associated with distinct puckers of the sugar moieties. The furanose conformation itself is affected by the steric and electronic nature of the ring substituents. For example, a strongly electronegative substituent at the C2′ position, such as in the 2′-deoxy-2′-fluororibofuranosyl analogue, will drive the conformational equilibrium towards the C3′-endo type (north). Conversely, the 2′-deoxy-2′-fluoroarabinofuranosyl modification with opposite stereochemistry at C2′ appears to have a preference for a C2′-endo type pucker (south). Incorporation of 2′-fluoroarabinofuranosyl thymines was previously shown to enhance the thermodynamic stability of B-DNA duplexes. We have determined the crystal structures of the B-DNA dodecamer duplexes [d(CGCGAASSCGCG)]2 and [d(CGCGAASTCGCG)]2 with incorporated 2′-deoxy-2′-fluoroarabinofuranosyl thymines S (south) at 1.55 Å resolution. In the crystal structures, all S residues adopt an O4′-endo conformation (east), well compatible with an overall B-form duplex geometry. In addition to the increased rigidity of S nucleosides, a clathrate-like ordered water structure around the 2′-fluorines may account for the observed larger thermodynamic stability of DNA duplexes containing 2′-deoxy-2′-fluoroarabino thymidine