We demonstrate here the rational design of purely entropic domains as a versatile approach to achieve control of the input/output response of synthetic molecular receptors. To do so and to highlight the versatility and generality of this approach, we have rationally re-engineered two model DNA-based receptors: a clamp-like DNA-based switch that recognizes a specific DNA sequence and an ATPbinding aptamer. We show that, by varying the length of the linker domain that connects the two recognition portions of these receptors, it is possible to finely control their affinity for their specific ligand. Through mathematical modeling and thermodynamic characterization, we also demonstrate for both systems that entropy changes associated with changes in linker length are responsible for affinity modulation and that the linker we have designed behaves as a disordered random-coil polymer. The approach also allows us to regulate the ligand concentration range at which the receptors respond and show optimal specificity. Given these attributes, the use of purely entropic domains appears as a versatile and general approach to finely control the activity of synthetic receptors in a highly predictable and controlled fashion.

The rational regulation of the pK a of an ionizable group in a synthetic device could be achieved by controlling the entropy of the linker connecting the hydrogen bond forming domains. We demonstrate this by designing a set of pH-responsive synthetic DNA-based nanoswitches that share the same hydrogen bond forming domains but differ in the length of the linker. The observed acidic constant (pK a) of these pH-dependent nanoswitches is linearly dependent on the entropic cost associated with loop formation and is gradually shifted to more basic pH values when the length of the linker domain is reduced. Through mathematical modeling and thermodynamic characterization we demonstrate that the modulation of the observed pK a is due to a purely entropic contribution. This approach represents a very versatile strategy to rationally modulate the pK a of synthetic devices in a highly predictable and accurate way.

We demonstrate here the use of 2-(4-chlorophenyl)-2-cyanopropanoic acid (CPA) and nitroacetic acid (NAA) as convenient chemical fuels to drive the dissipative operation of DNA-based nanodevices. Addition of either of the...

Here we report the rational design of a synthetic molecular nanodevice that is directly inspired from hemoglobin, a highly evolved protein whose oxygen-carrying activity is finely regulated by a sophisticated network of control mechanisms. Inspired by the impressive performance of hemoglobin we have designed and engineered in vitro a synthetic DNA-based nanodevice containing up to four interacting binding sites that, like hemoglobin, can load and release a cargo over narrow concentration ranges, and whose affinity can be finely controlled via both allosteric effectors and environmental cues like pH and temperature. As the first example of a synthetic DNA nanodevice that undergoes a complex network of nature-inspired control mechanisms, this represents an important step toward the use of similar nanodevices for diagnostic and drug-delivery applications.

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 ...

Cooperativity enhances the responsiveness of biomolecular receptors to small changes in the concentration of their target ligand, albeit with a concomitant reduction in affinity. The binding midpoint of a two-site receptor with a Hill coefficient of 1.9, for example, must be at least 19 times higher than the dissociation constant of the higher affinity of its two binding sites. This trade-off can be overcome, however, by the extra binding energy provided by the addition of more binding sites, which can be used to achieve highly cooperative receptors that still retain high affinity. Exploring this experimentally, we have employed an “intrinsic disorder” mechanism to design two cooperative, three-binding-site receptors starting from a single-site—and thus noncooperative—doxorubicin-binding aptamer. The first receptor follows a binding energy landscape that partitions the energy provided by the additional binding event to favor affinity, achieving a Hill coefficient of 1.9 but affinity within a factor of 2 of the parent aptamer. The binding energy landscape of the second receptor, in contrast, partitions more of this energy toward cooperativity, achieving a Hill coefficient of 2.3, but at the cost of 4-fold poorer affinity than that of the parent aptamer. The switch between these two behaviors is driven primarily by the affinity of the receptors’ second binding event, which serves as an allosteric “gatekeeper” defining the extent to which the system is weighted toward higher cooperativity or higher affinity.

The emerging field of RNA nanotechnology harnesses the versatility of RNA molecules to generate nature-inspired systems with programmable structure and functionality. Such methodology has therefore gained appeal in the fields of biosensing and diagnostics, where specific molecular recognition and advanced input/output processing are demanded. The use of RNA modules and components allows for achieving diversity in structure and function, for processing information with molecular precision, and for programming dynamic operations on the grounds of predictable non-covalent interactions. When RNA nanotechnology meets bioanalytical chemistry, sensing of target molecules can be performed by harnessing programmable interactions of RNA modules, advanced field-ready biosensors can be manufactured by interfacing RNA-based devices with supporting portable platforms, and RNA sensors can be engineered to be genetically encoded allowing for real-time imaging of biomolecules in living cells. In this article, we report recent advances in RNA-based sensing technologies and discuss current trends in RNA nanotechnologyenabled biomedical diagnostics. In particular, we describe programmable sensors that leverage modular designs comprising dynamic aptamer-based units, synthetic RNA nanodevices able to Page 1 of 24 Analytical & Bioanalytical Chemistry perform target-responsive regulation of gene expression, and paper-based sensors incorporating artificial RNA networks.

Electrochemical Aptamer-Based (EAB) sensors, comprised of an electrode bound DNA aptamer with a redox reporter on the distal end, offer the promise of high-frequency, real-time molecular measurements in complex sample...