Using Nature’s “Tricks” To Rationally Tune the Binding Properties of Biomolecular Receptors

Abstract

CONSPECTUS 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.

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates

“…Additionally, these results further exemplify that a single base change in a key target-recognition region significantly alters aptamer binding. 13 …”

Target Self‐Enhanced Selectivity in Metal‐Specific DNAzymes

“…Furthermore, since nonspecific binding is hard to avoid, using metal binding to trigger subsequent biological events, such as enzyme catalysis, might further increase selectivity. [2,3] In nature, metalloenzymes can sometimes use multiple cooperative metals, but it is quite challenging to harness this strategy in rational ligand design. [4,5] To test this idea, in vitro selection of DNAzymes from a random DNA library might be useful.…”

Re-evaluating the conventional wisdom about binding assays

“…Here, we aim to reevaluate the conceptual frameworks used when developing binding assays and to provide an intuitive understanding of molecular recognition, empowering researchers to make better use of contemporary technologies for quantitative analysis of biological systems. A number of excellent articles have already shown how to shift and manipulate binding curves from a physicochemical perspective [17]. Therefore, we endeavor to approach the topic from a stance that is agnostic to the specific assay chemistry or implementation.…”