ANGEW CHEM丨Transition Metal-Functionalized DNA Double-Crossover Tiles: Enhanced Stability and Chirality Transfer to Metal Centers

Author: Sandeepa K. V.

DNA-only assemblies lack inherent functionality, due to the intrinsically passive nature of the DNA double helix. Hence, double crossover junction (DX) containing two DNA double helices linked together by two crossover junctions are alternate approach to provide the rigidity and addressability necessary to create extended tile systems. Herein, author reported design and optimization of the first metal-DNA crossover motif, via the incorporation of bis-2,9-diphenyl-1,10-phenanthroline (dpp) coordination motif that binds selectively Cu (I) metal. They implemented this strategy iteratively through three design generations (1-3), which optimize the stability of the assembly and the transfer of DNA chirality to the metal environment. 

Tile generation 1. The first iteration of DXM junction (1) involved the direct insertion of dpp ligands into the DAE tile. Ligands were directly attached at the crossover points, splitting the junction into upper and lower sections (Figure 1). This approach placed the metal coordination center directly in line with the crossover point without directly altering the existing base pairing interactions found in the DX motif. Junction 1 consists of five complementary DNA strands with two unmodified strands, R1 and B1, serving as the scaffolds that template the hybridization of the remaining three strands G1, C1 and Y1 (Scheme 1). The stepwise assembly of 1, concentration dependant studies and complexation with Cu (I) was monitored by non-denaturing polyacrylamide gel electrophoresis. 
 

Tile generation 2. Two versions of 2 were constructed: one with single stranded DNA tails (sticky ends, 2s) and the other with double stranded ends (blunt ends, 2b). 2 was designed such that there are two full helical turns between crossover points, as opposed to one turn in 1, in order to maintain thermal stability after removing hybridizing bases around the crossovers.

In native PAGE gels, 2b assembles as a single species. Addition of Cu (I) to the assembled product results in a single band with similar mobility to that of the sample without CuI. Consistent with the formation of a P-type CuI-dpp2 coordination complex they observed a new peak at 345 nm in the CD spectrum of 2b, confirming chirality transfer from the DNA to the metal environment. In contrast, 2s does not form as a single species, instead resulting in two distinct bands.

The thermal denaturation studies of 2b and its partialy-formed complement 2bopen showed a melting over a wide temperature range centered around 65 °C for 2bopen, and two melting transitions, at 55 °C and 65 °C for 2b. This likely corresponds to the melting of each dpp-modified strand hybridized to the red scaffold strand and unmetalated tile denatures in a non-cooperative manner. Metallization of 2bopen, observed a shift in the peak melting temperature from 65 to 70 °C signifying an increase in the cooperativity of the melting event. This is consistent with the dpp-modified strands being bound together through metal coordination. 2b observed the presence of two narrow melting transitions upon metalation as opposed to multiple broad transitions for the unmetalated tile.
 
Tile generation 3. Assemblies of 3 take advantage of asymmetry in the tile design, such that thermal annealing of the junction occurs in two steps. Two versions of this design, 3s and 3s' had complementary sticky ends, enabling the coassembly of 3s with 3s'. The resulting DX molecules are longer (28 nm for 3s) compared to 2. Native analytical PAGE showing the assembly of 3s and 3s’ and the resulting structures upon CuI addition. CD spectra of 3s and 3s’ before and after exposure to CuI showed the increase in signal at 345 nm signifies metal complexation.

As expected from the asymetric design, they observed two distinct melting transitions in thermal denaturation experiments for both 3s and 3s’. Upon addition of CuI to the tiles they observed a shift from two melting transitions to one single transition, suggesting that the coordination of CuI results in the linkage of the dpp-modified strands between duplexes. As a result, a shift from non-cooperative to cooperative melting transitions can be seen upon exposure of junctions to CuI.

 
Selectivity for CuI. With the successful incorporation of CuI, the possibility of coordinating other metals to the templated dpp ligand centers was investigated. Of the metal ions investigated (CuII, NiII, EuIII, CoII, PdII) only CuII produced CD spectra consistent with metal ion binding; however, CuII is not an optimal metal ion for the bis-dpp tetrahedral environment, as it prefers a five- or six- coordinate geometry. Interestingly, after 24 h a CD signal corresponding to CuI binding was observed to replace the signal corresponding to CuII binding.

 
DXM tile self-assembly. Adding CuI to 3s and 3s’, followed by heating, led to the formation of a significant population of Cu nanoparticles, resulting from the disproportionation of CuI to Cu0 and CuII in aqueous media. Separately annealed 3s and 3s’ were thus annealed together from 45 to 4 °C over 12 h, after which CuI was added. Both the pre- and post-CuI assemblies displayed distinct fibre-like structures by atomic force microscopy (AFM) in air. These structures were observed to be non-rigid, which can possibly be attributed to torsional freedom between the fused helices before and after metal coordination.
 
Reference: Alexander Rousina-Webb, Christophe Lachance-Brais, Felix J. Rizzuto, Mohammed Askari, and Hanadi Farouk Sleiman, Angew. Chem. Int. Ed., 2020, DOI:10.1002/anie.201913956.