"Illusionary" polymerase activity triggered by metal ions: use for molecular logic-gate operations.

In recent years, an intense interest has grown in the interactions of nucleic acids with metal ions. Examples of such novel interactions include the specific binding of aptamers with metal ions and selective incorporation of metal ions as cofactors to promote the catalytic activities of nucleic acid enzymes (deoxyribozymes or ribozymes). Furthermore, certain metal ions, such as Hg, Ag, Cu, Ni, and Co, specifically bind to nucleosides or ligandosides to form metal-ion-mediated base pairs. This nonnatural base paring is stabilized by coordination of the metal ions to the nucleosides in a manner that is different from natural hydrogen bonding between complementary nucleosides. The novel interaction of nucleic acids with metal ions has recently been utilized for the construction of molecular-scale logic gates, which are essential for the development of molecular-scale computers and other computational devices. Most representatively, deoxyribozymes have been employed to build logic gates based on the fact that their catalytic activities can be regulated by the presence of specific metal ions. However, these kinds of logic gates typically rely on relatively complicated designs for gate switching and frequently require the involvement of RNA or chimeric DNA as an operational substrate. In addition to leading to high construction costs, this phenomenon ultimately requires complex operational features for controlling the system as a result of the susceptibility of RNA molecules to degradation. From this perspective, it would be highly desirable to develop molecular-scale logic gates that operate in a more simple and cost-effective manner. The results of the investigation described below have led to a new, simple strategy for construction of molecular-scale logic gates that are based on the “illusionary” polymerase activity at the mismatched site triggered by metal ions. As illustrated in Scheme 1, the underlying principle for operation of this system relies on specific interactions between metal ions (Hg or Ag) and the respective mismatched base pairs (thymine–thymine (T– T) or cytosine–cytosine (C–C)). Forward (F) and reverse (R) primers were designed to form T–T (Scheme 1a) or C–C (Scheme 1b) mismatches with template DNA at its 3’ end. The mismatched primers cannot be extended in the absence of the respective metal ions, because the terminal mismatching stalls action of the polymerase enzyme at the 3’ end, thus preventing the elongation reaction promoted by the polymerase. However, in the presence of Hg or Ag ions, the terminal T or C base at the 3’ end of the primer can form an nonnatural but stable T-Hg-T or C-Ag-C base pair with template DNA. This stabilization induces the polymerase activity and, as a consequence, amplification products are formed (Scheme 1). This activity is termed illusionary polymerase activity herein because it is derived from the illusion of DNA polymerase that the metal-ion-mediated base pair is perfectly matched. Figure 1 shows gel electrophoresis images of the products obtained from PCR mixtures containing F/R primers with terminal mismatched T (Figure 1a) or C base (Figure 1 b) at the 3’ end. As envisioned, use of the T–T and C–C mismatched primers results in formation of gel bands that correspond to amplification products only when the respective Hg and Ag ions are present (lane 3 in Figure 1a,b). In contrast, employment of perfectly matched primers results in the generation of amplification products regardless of whether or not the respective metal ions are present (lane 1, 4 in Figure 1a,b). Importantly, the fact that no significant difference is seen between the band intensities for systems with and without the metal ions indicates that the metal ions do not have an adverse effect on the polymerase reaction. To further support the proposal that the metal ions induce polymerase activity, melting curve analyses were performed for the extension products obtained from the T–T and C–C Scheme 1. Illustration of polymerase activity triggered by metal ions. a) Extension of T–T mismatched primer in the presence of Hg ions. b) Extension of C–C mismatched primer in the presence of Ag ions.