Modular Catalysis: Aptamer Enhancement of Enzyme Kinetics in a Nanoparticle Reactor.

Enzyme activity requires sequential binding and chemical transformation of substrates. While directed evolution and random mutagenesis are common methods for improving catalytic activity, these methods do not allow for independent control of KM and kcat. To achieve such control, we envisioned that the colocalization of aptamers and enzymes that act on the same molecule could increase catalytic efficiency through preconcentration of substrate. We explored this concept with cocaine esterase and anticocaine aptamers having varying KD values, both encapsulated in MS2 virus-like particles. Rate enhancements were observed with magnitudes dependent on both aptamer:enzyme stoichiometry and aptamer KD, peaking when aptamer KD and enzyme KM were roughly equivalent. This beneficial effect was lost when either aptamer binding was too tight or the aptamers were not constrained to be close to the catalyst. This work demonstrates a modular way to enhance catalysis by independently controlling substrate capture and release to the processing enzyme.

Systematically Modulating Aptamer Affinity and Specificity by Guanosine-to-Inosine Substitution.

Aptamers are widely used in small molecule detection applications due to their specificity, stability, and cost effectiveness. One key challenge in utilizing aptamers in sensors is matching the binding affinity of the aptamer to the desired concentration range for analyte detection. The most common methods for modulating affinity have inherent limitations, such as the likelihood of drastic changes in aptamer folding. Here, we propose that substituting guanosine for inosine at specific locations in the aptamer sequence provides a less perturbative approach to modulating affinity. Inosine is a naturally occurring nucleotide that results from hydrolytic deamination of adenosine, and like guanine, it base pairs with cytosine. Using the well-studied cocaine binding aptamer, we systematically replaced guanosine with inosine and were able to generate sequences having a range of binding affinities from 230 nM to 80 µM. Interestingly, we found that these substitutions could also modulate the specificity of the aptamers, leading to a range of binding affinities for structurally related analytes. Analysis of folding stability via melting temperature shows that, as expected, aptamer structure is impacted by guanosine-to-inosine substitutions. The ability to tune binding affinity and specificity through guanosine-to-inosine substitution provides a convenient and reliable approach for rapidly generating aptamers for diverse biosensing applications.

Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging.

Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson-Crick-Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2'-O-methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme-nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.

Combating small molecule environmental contaminants: detection and sequestration using functional nucleic acids.

Small molecule contaminants pose a significant threat to the environment and human health. While regulations are in place for allowed limits in many countries, detection and remediation of contaminants in more resource-limited settings and everyday environmental sources remains a challenge. Functional nucleic acids, including aptamers and DNA enzymes, have emerged as powerful options for addressing this challenge due to their ability to non-covalently interact with small molecule targets. The goal of this perspective is to outline recent efforts toward the selection of aptamers for small molecules and describe their subsequent implementation for environmental applications. Finally, we provide an outlook that addresses barriers that hinder these technologies from being widely adopted in field friendly settings and propose a path forward toward addressing these challenges.