Advancements in Aptamer Discovery Technologies.

Affinity reagents that specifically bind to their target molecules are invaluable tools in nearly every field of modern biomedicine. Nucleic acid-based aptamers offer many advantages in this domain, because they are chemically synthesized, stable, and economical. Despite these compelling features, aptamers are currently not widely used in comparison to antibodies. This is primarily because conventional aptamer-discovery techniques such as SELEX are time-consuming and labor-intensive and often fail to produce aptamers with comparable binding performance to antibodies. This Account describes a body of work from our laboratory in developing advanced methods for consistently producing high-performance aptamers with higher efficiency, fewer resources, and, most importantly, a greater probability of success. We describe our efforts in systematically transforming each major step of the aptamer discovery process: selection, analysis, and characterization. To improve selection, we have developed microfluidic devices (M-SELEX) that enable discovery of high-affinity aptamers after a minimal number of selection rounds by precisely controlling the target concentration and washing stringency. In terms of improving aptamer pool analysis, our group was the first to use high-throughput sequencing (HTS) for the discovery of new aptamers. We showed that tracking the enrichment trajectory of individual aptamer sequences enables the identification of high-performing aptamers without requiring full convergence of the selected aptamer pool. HTS is now widely used for aptamer discovery, and open-source software has become available to facilitate analysis. To improve binding characterization, we used HTS data to design custom aptamer arrays to measure the affinity and specificity of up to ∼10(4) DNA aptamers in parallel as a means to rapidly discover high-quality aptamers. Most recently, our efforts have culminated in the invention of the "particle display" (PD) screening system, which transforms solution-phase aptamers into "aptamer particles" that can be individually screened at high-throughput via fluorescence-activated cell sorting. Using PD, we have shown the feasibility of rapidly generating aptamers with exceptional affinities, even for proteins that have previously proven intractable to aptamer discovery. We are confident that these advanced aptamer-discovery methods will accelerate the discovery of aptamer reagents with excellent affinities and specificities, perhaps even exceeding those of the best monoclonal antibodies. Since aptamers are reproducible, renewable, stable, and can be distributed as sequence information, we anticipate that these affinity reagents will become even more valuable tools for both research and clinical applications.

Temporal Control of Aptamer Biosensors Using Covalent Self-Caging To Shift Equilibrium.

Aptamer-based sensors provide a versatile and effective platform for the detection of chemical and biological targets. These sensors have been optimized to function in multiple formats, however, a remaining limitation is the inability to achieve temporal control over their sensing function. To overcome this challenge, we took inspiration from nature's ability to temporally control the activity of enzymes and protein receptors through covalent self-caging. We applied this strategy to structure-switching aptamer sensors through the installation of a cleavable linker between the two DNA fragments that comprise the sensor. Analogous to self-caged proteins, installation of this linker shifts the equilibrium of the aptamer sensor to disfavor target binding. However, activity can be restored in a time-resolved manner by cleavage of the linker. To demonstrate this principle, we chose a photocleavable linker and found that installation of the linker eliminates target binding, even at high target concentrations. However, upon irradiation with 365 nm light, sensor activity is restored with response kinetics that mirror those of the linker cleavage reaction. A key benefit of our approach is generality, which is demonstrated by grafting the photocleavable linker onto a different aptamer sensor and showing that an analogous level of temporal control can be achieved for sensing of the new target molecule. These results demonstrate that nature's self-caging approach can be effectively applied to non-natural receptors to provide precise temporal control over function. We envision that this will be of especially high utility for deploying aptamer sensors in biological environments.

High-throughput enantiopurity analysis using enantiomeric DNA-based sensors.

Distinguishing between the two enantiomers of a molecule is a challenging task due to their nearly identical physical properties. Time-consuming chromatography methods are typically required for this task, which greatly limits the throughput of analysis. Here we describe a fluorescence-based method for the rapid and high-throughput analysis of both small-molecule enantiopurity and concentration. Our approach relies on selective molecular recognition of one enantiomer of the target molecule using a DNA aptamer, and the ability of aptamer-based biosensors to transduce the presence of a target molecule into a dose-dependent fluorescence signal. The key novel aspect of our approach is the implementation of enantiomeric DNA biosensors, which are synthesized from D- and L-DNA, but labeled with orthogonal fluorophores. According to the principle of reciprocal chiral substrate specificity, these biosensors will bind to opposite enantiomers of the target with equal affinity and selectivity, enabling simultaneous quantification of both enantiomers of the target. Using the previously reported DNA biosensor for L-tyrosinamide (L-Tym), we demonstrate the ability to rapidly and accurately measure both enantiopurity and concentration for mixtures of L- and D-Tym. We also apply our enantiomeric biosensors to the optimization of reaction conditions for the synthesis of D-Tym and provide mathematical modeling to suggest that DNA biosensors having only modest binding selectivity can also be used for fluorescence-based enantiopurity measurement. This research provides a generalizable method for high-throughput analysis of reaction mixtures, which is anticipated to significantly accelerate reaction optimization for the synthesis of high-value chiral small molecules.

Weak shock compaction on granular salt.

This study conducted integrated experiments and computational modeling to investigate the speeds of a developing shock within granular salt and analyzed the effect of various impact velocities up to 245 m/s. Experiments were conducted on table salt utilizing a novel setup with a considerable bore length for the sample, enabling visualization of a moving shock wave. Experimental analysis using particle image velocimetry enabled the characterization of shock velocity and particle velocity histories. Mesoscale simulations further enabled advanced analysis of the shock wave's substructure. In simulations, the shock front's precursor was shown to have a heterogeneous nature, which is usually modeled as uniform in continuum analyses. The presence of force chains results in a spread out of the shock precursor over a greater ramp distance. With increasing impact velocity, the shock front thickness reduces, and the precursor of the shock front becomes less heterogeneous. Furthermore, mesoscale modeling suggests the formation of force chains behind the shock front, even under the conditions of weak shock. This study presents novel mesoscale simulation results on salt corroborated with data from experiments, thereby characterizing the compaction front speeds in the weak shock regime.

RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors.

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 µM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.

Click-Particle Display for Base-Modified Aptamer Discovery.

Base-modified aptamers that incorporate non-natural chemical moieties can achieve greatly improved affinity and specificity relative to natural DNA or RNA aptamers. However, conventional methods for generating base-modified aptamers require considerable expertise and resources. In this work, we have accelerated and generalized the process of generating base-modified aptamers by combining a click-chemistry strategy with a fluorescence-activated cell sorting (FACS)-based screening methodology that measures the affinity and specificity of individual aptamers at a throughput of ∼107 per hour. Our "click-particle display (PD)" strategy offers many advantages. First, almost any chemical modification can be introduced with a commercially available polymerase. Second, click-PD can screen vast numbers of individual aptamers on the basis of quantitative on- and off-target binding measurements to simultaneously achieve high affinity and specificity. Finally, the increasing availability of FACS instrumentation in academia and industry allows for easy adoption of click-PD in a broader scientific community. Using click-PD, we generated a boronic acid-modified aptamer with ∼1 µM affinity for epinephrine, a target for which no aptamer has been reported to date. We subsequently generated a mannose-modified aptamer with nanomolar affinity for the lectin concanavalin A (Con A). The strong affinity of both aptamers is fundamentally dependent upon the presence of chemical modifications, and we show that their removal essentially eliminates aptamer binding. Importantly, our Con A aptamer exhibited exceptional specificity, with minimal binding to other structurally similar lectins. Finally, we show that our aptamer has remarkable biological activity. Indeed, this aptamer is the most potent inhibitor of Con A-mediated hemagglutination reported to date.

Strategies for Creating Structure-Switching Aptamers.

Aptamer biosensor that can switch its structure upon target binding offers a powerful strategy for molecular detection. However, the process of converting an aptamer into a "structure-switching" biosensor is challenging and often relies on trial-and-error without established design principles. In this Sensor Issues, we examine a variety of design approaches for incorporating structure-switching functionality into existing aptamers, and provide thermodynamic analyses to highlight the variables that most strongly influence their performance. Finally, we also describe emerging efforts for incorporating the structure-switching functionality directly into the aptamer selection process.

Convenient and scalable synthesis of fmoc-protected Peptide nucleic Acid backbone.

The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA.