Programmable pH-Triggered DNA Nanoswitches

Abstract

ABSTRACT:Here we have rationally designed a tunable DNA-based nanoswitch whose closing/opening can be triggered over specific different pH windows. This nanoswitch forms an intramolecular triplex DNA structure through pH-sensitive parallel Hoogsteen interactions. We demonstrate that by simply changing the relative content of TAT/CGC triplets in the switch we can rationally tune its pH-dependence over more than 5 pH units. By using a combination of such nanowitches with different pH sensitivity, we also demonstrate how we can engineer a pH nanosensor that can precisely monitor pH variations over 5.5 units of pH. With their fast response time (<200 msec) and high reversibility, these pH-triggered nanoswitches appear particularly suitable for applications ranging from the real-time monitoring of pH changes in-vivo to the development of pH sensitive smart nanomaterial.Nature often employs finely pH-regulated biomolecules to modulate and tune a number of biological activities 1 ranging from enzyme catalysis 2 to protein folding 3 , membrane function 4 and apoptosis 5 . For these reasons, developing probes, switches or nanomaterials that are able to respond to specific pH changes should prove of utility for several applications in the fields of in-vivo imaging, clinical diagnostics, and drug-delivery [6][7][8] .By taking advantage of the high versatility and designability of DNA chemistry 9-19 several groups have recently developed pH-triggered DNA-based probes or nanomachines [20][21][22][23][24][25][26][27][28][29][30] . Such probes typically exploit DNA secondary structures that display pH-dependence due to the presence of specific protonation sites. These structures include I-motif [21][22][23]26,29,31 , intermolecular triplex DNA 25,28,32 , DNA tweezers 20 and, more recently, the Amotif 33 . Despite the promising and advantageous characteristics of some of these DNA-based nanomachines, which include fast response times and sustained efficiency over several cycles, a drawback inevitably affects their performances: they all respond (with an exception 33a ) over a fixed pH window that typically spans 1.5 to 2 pH units 26,34,33b . These nanomachines, therefore, cannot be adapted to provide a useful output outside these fixed pH-windows.Here we describe a method to rationally design and program pH-triggered DNA-based nanoswitches whose pH-dependence can be finely tuned and modulated over more than 5 units of pH. We created our switches by taking advantage of the wellcharacterized pH sensitivity of the parallel Hoogsteen (T,C)-motif in triplex DNA [34][35][36] . To do so we have designed a DNAbased triplex pH-triggered nanoswitch that consists in a double intramolecular hairpin stabilized with both Watson-Crick (W-C) and parallel Hoogsteen interactions (Fig. 1). More specifically, one hairpin of the triplex nanoswitch is formed by the W-C hybridization of two 10-base complementary portions separated by a 5 base loop. This duplex DNA is then able to form a triplex structure via the formation of a second hairpin through...

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

“…In response to this limitation, we demonstrate here that simulative approaches can help in the optimization of DNA-based nanodevices controlled by pH changes that have been recently proposed for both diagnostic and drug-delivery applications. 20,23,24 Moreover, the simulation approaches we have employed here might help in the characterization of response time and prediction of the effect of environmental conditions which obviously represent crucial factors in the performance of such DNAbased tools. Finally, we note that, while we have focused here on DNA-based switches controlled by pH, the applicability of simulation approaches in the field of DNA nanotechnology could be expanded to other noncanonical DNA structures (i.e., i-motif, G-quadruplex, etc.…”

Artificial molecular and nanostructures for advanced nanomachinery

“…DNA switches have been designed that open and close depending on pH. 79 These switches operate based on Hoogsteen base pairing (an alternative type to Watson-Crick) and can be tuned to respond to a wide pH range of 6.5 to 10.2, depending on the amount of TAT/CGC triplets present in the sequence. The switches demonstrated an extremely fast response time (o100 ms) and could potentially be incorporated into larger nanomachines to trigger response to external stimuli.…”

DNA Nanobiosensors: An Outlook on Signal Readout Strategies

“…Such stem-loop configurations can also be used to monitor global environmental changes such as changes in temperature by using a fluorophore-quencher pair on the ends of the strands. Biosensors for other stimuli such as pH changes are based on structures that involve triplex [17] (Figure 1(c)), i-motif formation [18] (Figure 1(d)), or poly-dA helix formation (Figure 1(e)) [19], all of which are 2 Journal of Nanomaterials sequence-specific and occur under acidic conditions. Gquadruplex formation is another sequence-specific conformational change that forms under specific ionic conditions such as the presence of K + or Na + [20] (Figure 1(f)).…”

“…A similar example used a graphene surface instead of gold and worked on the basis of pHdependent triplex formation [31]. In addition, solution-based triplex-forming nanoswitches have also been developed for pH detection [17]. This switch was designed so that the fluorophore-quencher pair remains closer when the switch forms a triplex, acting as an indicator of the pH range.…”