Simulative and Experimental Characterization of a pH-Dependent Clamp-like DNA Triple-Helix Nanoswitch

Abstract

Here we couple experimental and simulative techniques to characterize the structural/dynamical behavior of a pH-triggered switching mechanism based on the formation of a parallel DNA triple helix. Fluorescent data demonstrate the ability of this structure to reversibly switch between two states upon pH changes. Two accelerated, half microsecond, MD simulations of the system having protonated or unprotonated cytosines, mimicking the pH 5.0 and 8.0 conditions, highlight the importance of the Hoogsteen interactions in stabilizing the system, finely depicting the time-dependent disruption of the hydrogen bond network. Urea-unfolding experiments and MM/ GBSA calculations converge in indicating a stabilization energy at pH 5.0, 2-fold higher than that observed at pH 8.0. These results validate the pH-controlled behavior of the designed structure and suggest that simulative approaches can be successfully coupled with experimental data to characterize responsive DNA-based nanodevices. ■ INTRODUCTIONDNA nanotechnology allows us to design and engineer smart nanomaterials and nanodevices using synthetic DNA sequences. 1−6 For example, current methodologies and synthetic strategies, such as DNA tiles, origami, or supramolecular assembly, allowed the production of complex nanostructures of different shapes and dimensions. 7−11 The unparalleled versatility of these approaches allows precise positioning of molecule-responsive switching elements in specific locations of DNA nanostructures, leading to the construction of more complex functional nanodevices. 12−14 Similarly, enzyme−DNA nanostructures have been demonstrated to enhance enzyme catalytic activity and stability. 15 DNA motifs that rely on noncanonical DNA interactions, such as G-quadruplex, triplex, i-motif, hairpin, and aptamers, can be used to design such nanodevices due to their dynamic-responsive behavior toward chemical and environmental stimuli. 16,17 These responsive units often respond to specific chemical inputs through a bindinginduced conformational change mechanism that leads to a measurable output or function. The efficiency of this class of responsive nanodevices strongly depends on the designed structure-switching mechanism that controls their activity or functionality. Therefore, there is an urgent need to understand the energies involved in these responsive systems and the relationship between their structure and dynamics. 16 Among such functional DNA nanodevices, those based on the triple-helix motif are attracting interest for their strong and programmable pH dependence. 18−20 By rationally incorporating triplex-forming portions into DNA nanodevices, it is possible to trigger conformational changes and functions using pH as a chemical input. 21−24 Despite the fair amount of knowledge of the basic design principles and mechanism of action of triplex-based nanodevices, no reports describing the connection between their structural and dynamical properties are available. Toward this aim, simulative approaches represent valuable tools to shed ...

Engineering a responsive DNA triple helix into an octahedral DNA nanostructure for a reversible opening/closing switching mechanism: a computational and experimental integrated study

“…The root mean square deviation (RMSD), describing the evolution of the sampled conformations in terms of distance from the starting structure, has been monitored to follow the stability of the functional elements integrated on the cages at the two different pH values. At pH 5.0 the clamp-switch is stable for both the systems, being characterized by negligible deviations from the starting structure (data not shown), a result in line with the previous simulation of the isolated clamp-switch ( 32 ). At pH 8 a significant deviation from the starting structure is observed for the triple helix hosted on the LT-cage (Figure 2A , red line), but not on the T-cage (Figure 2A , black line).…”

“…The triplex-forming sequence, consisting of six bases, is connected to one of the W-C strands through a 25 bases loop specifically interacting at pH 5.0 through Hoogsteen hydrogen bonds with the double helical portion (Figure 1C , red and black strands, connected by black dots). When brought at pH 8.0, the clamp-switch domain should unfold (Figure 1D ) as demonstrated for the isolated switch in solution ( 32 ). To study the effect of the octahedral scaffold on the behaviour of these functional elements, we designed two different nanocage models.…”

“…Motivated by the above results here we propose a computational and experimental integrated approach to study the effect of engineering a functional responsive DNA-based element into a DNA nanostructure. We have selected a well-known and characterized pH-dependent clamp-switch based on the formation and dissociation of a parallel DNA triple helix ( 31 , 32 ), and we designed a novel octahedral cage decorated with two copies of this functional element. The effect of the nanostructure environment on the pH-dependent behaviour of the responsive element has been investigated through accelerated molecular dynamics (aMD) simulation, fluorescence and gel electrophoresis.…”

Light‐Induced Reversible Reconfiguration of DNA‐Based Constitutional Dynamic Networks: Application to Switchable Catalysis

“…[12] Fore xample,s tructural information includes the dictated formation of duplex nucleic acids and their separation by the strand displacement process,o rt he formation of triplex DNAstructures.Base-dictated functions of nucleic acids include catalytic properties (for example, DNAzymes), [13,14] sequence-specific binding properties of oligonucleotides (for example,a ptamers), [15] and enzymemimicking functions of catalyst-aptamer conjugates (for example,n ucleoapzymes). Forexample,the K + -ion-induced formation of G-quadruplexes and their separation by crown ethers, [17] the pH-stimulated formation and dissociation of i-motif or triplex structures, [18,19] and the metal-ion cooperative binding of duplex nucleic acids (for example,byT -Hg 2+ -T or C-Ag + -C bridges) and their ligand-induced separation (for example,by cysteine). Forexample,the K + -ion-induced formation of G-quadruplexes and their separation by crown ethers, [17] the pH-stimulated formation and dissociation of i-motif or triplex structures, [18,19] and the metal-ion cooperative binding of duplex nucleic acids (for example,byT -Hg 2+ -T or C-Ag + -C bridges) and their ligand-induced separation (for example,by cysteine).…”

“…[16] In addition, the switchable signal-triggered reconfiguration of nucleic acid structures is well established. Forexample,the K + -ion-induced formation of G-quadruplexes and their separation by crown ethers, [17] the pH-stimulated formation and dissociation of i-motif or triplex structures, [18,19] and the metal-ion cooperative binding of duplex nucleic acids (for example,byT -Hg 2+ -T or C-Ag + -C bridges) and their ligand-induced separation (for example,by cysteine). [20] Additionally,l ight was used as an auxiliary trigger for the switchable stabilization and separation of duplex nucleic acids in the presence of photoisomerizable units (for example,the stabilization of duplex nucleic acids by trans-azobenzene units and destabilization of the duplexes in the presence of cis-azobenzene).…”