Enzymatic Synthesis of Cu(II)-Responsive Deoxyribozymes through Polymerase Incorporation of Artificial Ligand-Type Nucleotides

Abstract

Metal-mediated artificial base pairs, consisting of ligand-type nucleotides and a bridging metal ion, have shown promise as functional units to develop stimuli-responsive DNA materials. Although a variety of metal-mediated base pairs have been constructed with artificial ligand-type nucleotides and various metal ions, the application of such metal-mediated base pairs has been relatively poorly explored mainly due to the cumbersome chemical synthesis of artificial DNA strands. Herein we report a facile enzymatic method to synthesize DNA strands containing a ligand-type hydroxypyridone (H) nucleotide, which forms a Cu II -mediated base pair (H−Cu II −H). A two-step primer extension reaction using two commercially available polymerases enabled the incorporation of a H nucleotide at an internal position of oligonucleotides. The polymerase synthesis was subsequently applied to the development of metal-responsive deoxyribozymes (DNAzymes), whose catalytic activity was regulated by the formation of a single H−Cu II − H base pair in its stem region. The DNAzyme activity was reversibly switched by the alternate addition and the removal of Cu II ions. Furthermore, metal-dependent orthogonal activation of a Cu II -responsive H-DNAzyme and a Hg II -responsive T-DNAzyme was experimentally demonstrated by utilizing both H−Cu II −H as well as widely explored T−Hg II −T base pairs. These results suggest that the incorporation of H−Cu II −H base pairs would facilitate the rational design of metal-responsive functional DNAs. Accordingly, the facile enzymatic synthesis of artificial ligand-bearing DNAs developed in this study would significantly expand the toolbox of DNA-based supramolecular chemistry and DNA nanotechnology.

Stable Hg(II)-mediated base pairs with a phenanthroline-derived nucleobase surrogate in antiparallel-stranded DNA

“…Similarly, other nucleic acid topologies and nucleic acid analogues have been probed with respect to the site-specific incorporation of metal ions in analogy to metal-mediated base-pair formation [28][29][30][31]. Several promising applications have already been established in this area, including the construction of structures with a modified electrical response [32,33], switchable devices [34], responsive devices [35,36], regulated primer extension [37], the specific detection of canonical nucleobases [38,39], the generation of DNA-templated metal nanoclusters [40], an expansion of genetic four-letter code [41], etc. Due to their accessibility from commercial resources, canonical nucleobases involved in metal-mediated base pairing have been most extensively studied, giving rise to C-Ag(I)-C and T-Hg(II)-T pairs [42].…”

Sharp Switching of DNAzyme Activity through the Formation of a CuII‐Mediated Carboxyimidazole Base Pair

“…Addition of 1 equiv of Cu II ions significantly stabilized the DNA duplex ( T m =57.7 °C, Δ T m =+35.2 °C), suggesting the formation of an Im C ‐Cu II ‐Im C base pair at neutral pH. The degree of Cu II ‐dependent duplex stabilization was much greater than that observed with H‐Cu II ‐H pairing (Δ T m =+13.7 °C [9] ). In fact, Im C ‐Cu II ‐Im C proved to be one of the most stabilizing artificial metallo‐base pairs ever reported [12d] .…”

“…Metallo‐base pairs have the ability to control DNA duplex stability and DNA hybridization in a metal‐responsive manner, [12] and were therefore expected to act as switches that control the structure and function of DNA. We have previously synthesized Cu II ‐responsive DNAzymes [9, 10] by incorporating a pair of hydroxypyridone (H) nucleobases, which form a Cu II ‐mediated H‐Cu II ‐H base pair, [13] into known DNAzyme structures. The activity of the modified DNAzymes (such as H‐DNAzyme [9] ) was controlled by adding and removing Cu II ions, but upgrading their on/off performance was an important issue.…”

Enzymatic synthesis of biphenyl-DNA oligonucleotides

“…PEX reaction with Therminator, Dpo4, or Kf exoand dBphTP on a template containing an abasic site 2. natural DNA synthesis after the installation of the dBph- pair with dNTPs and Therminator. Such a protocol has recently been applied for the synthesis of modified DNAzymes using metal base pairs [28] and DNA duplexes containing artificial silver base pairs. [66] In the former, a modified nucleotide was installed opposite natural dA and dT nucleotides and enzymatic synthesis was resumed with a different polymerase (Dpo4) while in the latter, a modified purine nucleotide was used to form an artificial silver base pair and natural DNA synthesis was resumed with Therminator.…”

“…[19,20] More recently, artificial metal base pairs have been proposed as alternative scaffolds for the construction of UBPs. [21][22][23][24][25][26][27][28] Such an expansion of the genetic alphabet has important practical repercussions since semisynthetic organisms capable of replicating DNA containing artificial nucleotides [29] and even transcribing modified mRNA into proteins [14] could be created and aptamers with enhanced binding properties were identified. [13,20,30,31] In this context, the zipper-like interstrand stacking motif ( Fig.…”