Abstract
The biosensor community has long focused on achieving the lowest possible detection limits, with specificity (the ability to differentiate between closely similar target molecules) and sensitivity (the ability to differentiate between closely similar target concentrations) largely being relegated to secondary considerations and solved by the inclusion of cumbersome washing and dilution steps or via careful control experimental conditions. Nature, in contrast, cannot afford the luxury of washing and dilution steps, nor can she arbitrarily change the conditions (temperature, pH, ionic strength) under which binding occurs in the homeostatically maintained environment within the cell. This forces evolution to focus at least as much effort on achieving optimal sensitivity and specificity as on achieving low detection limits, leading to the "invention" of a number of mechanisms, such as allostery and cooperativity, by which the useful dynamic range of receptors can be tuned, extended, narrowed, or otherwise optimized by design, rather than by sample manipulation. As the use of biomolecular receptors in artificial technologies matures (i.e., moves away from multistep, laboratory-bound processes and toward, for example, systems supporting continuous in vivo measurement) and these technologies begin to mimic the reagentless single-step convenience of naturally occurring chemoperception systems, the ability to artificially design receptors of enhanced sensitivity and specificity will likely also grow in importance. Thus motivated, we have begun to explore the adaptation of nature's solutions to these problems to the biomolecular receptors often employed in artificial biotechnologies. Using the population-shift mechanism, for example, we have generated nested sets of receptors and allosteric inhibitors that greatly expanded the normally limited (less than 100-fold) useful dynamic range of unmodified molecular and aptamer beacons, enabling the single-step (e.g., dilution-free) measurement of target concentrations across up to 6 orders of magnitude. Using this same approach to rationally introduce sequestration or cooperativity into these receptors, we have likewise narrowed their dynamic range to as little as 1.5-fold, vastly improving the sensitivity with which they respond to small changes in the concentration of their target ligands. Given the ease with which we have been able to introduce these mechanisms into a wide range of DNA-based receptors and the rapidity with which the field of biomolecular design is maturing, we are optimistic that the use of these and similar naturally occurring regulatory mechanisms will provide viable solutions to a range of increasingly important analytical problems.
Highlights
Nature employs proteins and nucleic acids for high affinity, high specificity recognition of an enormous range of molecular targets
Hybridization is, likewise, generalizable to the highspecificity, high-affinity detection of any nucleic acid sequence. These observations have motivated decades of research aimed at harnessing the power of biological recognition in such technologies as sensors, “smart” responsive adhesives and materials, synthetic biology, and molecular computing
Despite the many positive attributes of and successful development of technologies based on biological recognition, the physics of single-site binding limits the utility of bioreceptors in many applications
Summary
Nature employs proteins and nucleic acids for high affinity, high specificity recognition of an enormous range of molecular targets. We converted the system from heterotropic allostery, in which the activator and target differ, to homotropic allostery, in which the two are identical Under these circumstances hybridization of the first copy of the target weakens the stem and improves the affinity with which the second copy binds, leading to cooperative behavior and improved responsiveness: the tailed beacon binds its target molecule with a Hill coefficient of 1.54 ± 0.10, corresponding to a (17 ± 3)-fold dynamic range (Figure 9, middle right). Using a relatively high stability stem, this system achieves a Hill coefficient of 1.94 ± 0.17 and a dynamic range of only (9.6 ± 1.6)-fold (Figure 9, bottom right), achieving a degree of cooperativity within experimental error of an ideal two-site receptor They serve as illustrations of the principles involved, the approaches that we have taken to the design of cooperative molecular beacons are likewise not transferable to structurally more complex receptors. Used this mechanism to produce cooperative two-site mercury(II), cocaine, and doxorubicin binding receptors achieving Hill coefficients of up to 1.98 ± 0.04 and dynamic ranges of as little as 9.2-fold (Figure 10).[53]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
