Abstract
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.
Highlights
Despite the field’s impressive achievements,[22−27] the performance of molecular nanodevices still pales in comparison with those of the naturally occurring biomolecular machines they seek to mimic
We have developed a class of DNA-based nanodevices containing up to four interacting binding sites that load and release a molecular cargo over narrow concentration ranges, the midpoint placements of which are controlled via both allosteric effectors and by environmental cues
This effect is generated by the presence of four interacting oxygen-binding sites that work together such that binding to one improves binding to the others. This produces a high-order, nonlinear dependence of occupancy on oxygen concentration resulting in a steeper, sigmoidal binding curve. The physics of such cooperativity can be understood using the allosteric model first formulated by Monod et al.,[39] which describes the cooperative receptor as populating an equilibrium between two conformational states, one with low affinity and the other with high
Summary
Perhaps the most challenging property of hemoglobin to mimic is the homotropic allosteric control (i.e., cooperativity) with which it binds and releases oxygen. Cooperativity arises when binding shifts this conformational equilibrium toward the higher affinity state with each successive binding event This produces a steeper binding curve (ligand concentration versus occupancy) and a narrower dynamic range than that observed for simple, single-site binding. Titrating this construct with a specific, 9-base target we obtain a binding curve with a Hill coefficient of 2.1 ± 0.1, which is within error of the 2.0 expected for an ideal two-site cooperative receptor (Figure 2B, right) This causes the useful dynamic range of the cooperative nanodevice to narrow to just 8.1(±0.9)-fold, rendering it much more sensitive to small changes in ligand concentration than would be a noncooperative receptor. Our nanodevice shows similar temperature dependence, with both its ligand affinity and cooperativity falling as the temperature rises (Figure 4J−L and Figure S5), presumably due to the increase of the entropic contribution cost of closing the loop during the first binding event
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