Exploring the Landscape of Aptamers: From Cross-Reactive to Selective to Specific, High-Affinity Receptors for Cocaine.

We reported over 20 years ago MNS-4.1, the first DNA aptamer with a micromolar affinity for cocaine. MNS-4.1 is based on a structural motif that is very common in any random pool of oligonucleotides, and it is actually a nonspecific hydrophobic receptor with wide cross-reactivity with alkaloids and steroids. Despite such weaknesses preventing broad applications, this aptamer became widely used in proof-of-concept demonstrations of new formats of biosensors. We now report a series of progressively improved DNA aptamers recognizing cocaine, with the final optimized receptors having low nanomolar affinity and over a thousand-fold selectivity over the initial cross-reactants. In the process of optimization, we tested different methods to eliminate cross-reactivities and improve affinity, eventually achieving properties that are comparable to those of the reported monoclonal antibody candidates for the therapy of overdose. Multiple aptamers that we now report share structural motifs with the previously reported receptor for serotonin. Further mutagenesis studies revealed a palindromic, highly adaptable, broadly cross-reactive hydrophobic motif that could be rebuilt through mutagenesis, expansion of linker regions, and selections into receptors with exceptional affinities and varying specificities.

Carbon-nanotube field-effect transistors for resolving single-molecule aptamer-ligand binding kinetics.

Small molecules such as neurotransmitters are critical for biochemical functions in living systems. While conventional ultraviolet-visible spectroscopy and mass spectrometry lack portability and are unsuitable for time-resolved measurements in situ, techniques such as amperometry and traditional field-effect detection require a large ensemble of molecules to reach detectable signal levels. Here we demonstrate the potential of carbon-nanotube-based single-molecule field-effect transistors (smFETs), which can detect the charge on a single molecule, as a new platform for recognizing and assaying small molecules. smFETs are formed by the covalent attachment of a probe molecule, in our case a DNA aptamer, to a carbon nanotube. Conformation changes on binding are manifest as discrete changes in the nanotube electrical conductance. By monitoring the kinetics of conformational changes in a binding aptamer, we show that smFETs can detect and quantify serotonin at the single-molecule level, providing unique insights into the dynamics of the aptamer-ligand system. In particular, we show the involvement of G-quadruplex formation and the disruption of the native hairpin structure in the conformational changes of the serotonin-aptamer complex. The smFET is a label-free approach to analysing molecular interactions at the single-molecule level with high temporal resolution, providing additional insights into complex biological processes.

Rapid isolation of anti-idiotype aptamers for quantification of human monoclonal antibodies against SARS-CoV-2 spike protein.

Therapeutic antibodies that block viral entry have already proven to be important, first line drugs for treatments of viral infections. In the case of SARS-CoV-2, combinations of multiple therapeutic antibodies may need to be rapidly identified and formulated in a way that blocks each new, predominant variant of the virus. For efficient introduction of any new antibody combination into patients, it is important to be able to monitor patient-specific pharmacokinetics of individual antibodies, which would include the time course of their specific capacity to block the viral spike proteins. Here, we present three examples of microfluidic-based rapid isolation of companion reagents useful for establishing combination antibody therapies. These reagents are specific three-dimensional imprints of variable regions of individual human monoclonal antibodies against the -spike protein of SARS-CoV-2 virus in the form of oligonucleotide-based ligands (aptamers). We implement these anti-idiotypic aptamers as bioreceptors in graphene-based field-effect transistor sensors to accomplish label free, rapid, and sensitive detection of matching antibodies within minutes. Through this work we have demonstrated the general applicability of anti-idiotype aptamers as capture reagents in quantification of active forms of monoclonal antibodies in complex biological mixtures.

Amplification of Signal on Cell Surfaces in Molecular Cascades.

We can formulate mixtures of oligonucleotide-antibody conjugates to act as molecular cascade-based automata that analyze pairs of cell surface markers (CD markers) on individual cells in a manner consistent with the implementation of Boolean logic-for example, by producing a fluorescent label only if two markers are present. While traditional methods to characterize cells are based on transducing signals from individual cell surface markers, these cascades can be used to combine into a single signal the presence of two or even more CDs. In our original design, oligonucleotide components irreversibly flowed from one antibody to another, driven by increased hybridizations, leading to the magnitude of the final signal on each cell being determined by the surface marker that was the least abundant. This is a significant limitation to the precise labeling of narrow subpopulations, and, in order to overcome it, we changed our design to accomplish signal amplification to a more abundant cell surface marker. We show the AMPLIFY function on two examples: (1) we amplify the fluorescent label from the CD19 marker onto a fivefold more abundant CD45, and (2) we amplify broadly distributed CD45RA to a more constant marker, CD3. We expect this new function to enable the increasingly complex Boolean analysis of cell surfaces.

A functional group-guided approach to aptamers for small molecules.

Aptameric receptors are important biosensor components, yet our ability to identify them depends on the target structures. We analyzed the contributions of individual functional groups on small molecules to binding within 27 target-aptamer pairs, identifying potential hindrances to receptor isolation-for example, negative cooperativity between sterically hindered functional groups. To increase the probability of aptamer isolation for important targets, such as leucine and voriconazole, for which multiple previous selection attempts failed, we designed tailored strategies focused on overcoming individual structural barriers to successful selections. This approach enables us to move beyond standardized protocols into functional group-guided searches, relying on sequences common to receptors for targets and their analogs to serve as anchors in regions of vast oligonucleotide spaces wherein useful reagents are likely to be found.

A Microfluidic Dual-Aptamer Sandwich Assay for Rapid and Cost-Effective Detection of Recombinant Proteins.

While monitoring expression of recombinant proteins is essential for obtaining high-quality biopharmaceutical and biotechnological products, existing assays for recombinant protein detection are laborious, time-consuming and expensive. This paper presents a microfluidic approach to rapid and cost-effective detection of tag-fused recombinant proteins via a dual-aptamer sandwich assay. Our approach addresses limitations in current methods for both dual-aptamer assays and generation of aptamers for such assays by first using microfluidic technology to isolate the aptamers rapidly and then employing these aptamers to implement a microfluidic dual-aptamer assay for tag-fused recombinant protein detection. The use of microfluidic technology enables the fast generation of aptamers and rapid detection of recombinant proteins with minimized consumption of reagents. In addition, compared with antibodies, aptamers as low-cost affinity reagents with an ability of reversible denaturation further decreases the cost of recombinant protein detection. For demonstration, an aptamer pair is isolated rapidly toward His-tagged IgE within two days, and then used in the microfluidic dual-aptamer assay for detecting His-tagged IgE in cell culture media within 10 min and with a limit of detection of 7.1 nM.

Oligonucleotide-Blocked Streptavidin for Biotinylation Analysis.

Binding between streptavidin, or its homologues, to biotin is one of the most widely exploited biological interactions in the biomedical sciences. Controlling the extent of biotinylation is important for meeting the requirements of the intended design and to preserve the native function of the biotin recipient. Within the protein world, a"trial-and-error" optimization approach toward biotinylation reaction conditions is often necessary due to widely varying properties of proteins. Therefore, product analysis is important. We show here that a oligonucleotide-blocked streptavidin, effectively "monovalent streptavidin", can tag biotin moieties individually and the tagged products visualized via a polyacrylamide gel shift assay to reveal the product distribution, i.e., [protein-(biotin)n] products where n = 1, 2, 3, etc. This is in contrast, and complementary, to current commercially available analytical reagents for biotinylation characterization, which use an absorbance or fluorescence signal to yield the mean number of biotin moieties.

Microfluidic isolation of aptamers with affinity towards multiple myeloma monoclonal immunoglobulins.

Multiple myeloma (MM) is a bone marrow cancer of resident plasma cells that affects 125,000 patients in the U.S. with about 30,000 new cases per year. Its signature is the clonal proliferation of a single plasma cell that secretes a patient specific monoclonal immunoglobulin (M-Ig). Targeting the M-Ig in patient serum could allow sensitive and noninvasive identification of minimal residual disease in multiple myeloma. Aptamers, which are single-stranded oligonucleotides with affinity and specificity to a target molecule, have recently been introduced as affinity reagents for recognition of MM M-Igs. Here we exploit microfluidic SELEX technology to enable rapid and efficient generation of aptamers against M-Ig proteins from MM patients. We first characterize the technology by isolating aptamers with affinity towards the monoclonal antibody rituximab as a model M-Ig and then apply the technology to isolating aptamers specifically targeting M-Igs obtained from serum samples of MM patients. We demonstrate that high-affinity DNA aptamers (KD < 50 nM) for M-Ig proteins from a patient sample could be isolated via microfluidic SELEX within approximately 12 h and using less than 100 micrograms of patient M-Ig. Such aptamers can potentially be used in personalized monitoring of minimal residual disease in MM patients.

Wearable aptamer-field-effect transistor sensing system for noninvasive cortisol monitoring.

Wearable technologies for personalized monitoring require sensors that track biomarkers often present at low levels. Cortisol­a key stress biomarker­is present in sweat at low nanomolar concentrations. Previous wearable sensing systems are limited to analytes in the micromolar-millimolar ranges. To overcome this and other limitations, we developed a flexible field-effect transistor (FET) biosensor array that exploits a previously unreported cortisol aptamer coupled to nanometer-thin-film In2O3 FETs. Cortisol levels were determined via molecular recognition by aptamers where binding was transduced to electrical signals on FETs. The physiological relevance of cortisol as a stress biomarker was demonstrated by tracking salivary cortisol levels in participants in a Trier Social Stress Test and establishing correlations between cortisol in diurnal saliva and sweat samples. These correlations motivated the development and on-body validation of an aptamer-FET array­based smartwatch equipped with a custom, multichannel, self-referencing, and autonomous source measurement unit enabling seamless, real-time cortisol sweat sensing.

Robust Heterochiral Strand Displacement Using Leakless Translators.

Molecular computing offers a powerful framework for in situ biosensing and signal processing at the nanoscale. However, for in vivo applications, the use of conventional DNA components can lead to false positive signals being generated due to degradation of circuit components by nuclease enzymes. Here, we use hybrid chiral molecules, consisting of both l- and d-nucleic acid domains, to implement leakless signal translators that enable d-nucleic acid signals to be detected by hybridization and then translated into a robust l-DNA signal for further analysis. We show that our system is robust to false positive signals even if the d-DNA components are degraded by nucleases, thanks to circuit-level robustness. This work thus broadens the scope and applicability of DNA-based molecular computers for practical, in vivo applications.

Hydrogel Microfilaments toward Intradermal Health Monitoring.

Digital health promises a paradigm shift for medicine where biomarkers in individuals are continuously monitored to improve diagnosis and treatment of disease. To that end, a technology for minimally invasive quantification of endogenous analytes in bodily fluids will be required. Here, we describe a strategy for designing and fabricating hydrogel microfilaments that can penetrate the skin while allowing for optical fluorescence sensing. The polyacrylamide formulation was selected to provide high elastic modulus in the dehydrated state and optical transparency in the hydrated state. The microfilaments can be covalently tethered to a fluorescent aptamer to enable functional sensing. The microfilament array can penetrate the skin with low pain and without breaking, contact the dermal interstitial fluid, and be easily removed from the skin. In the future, hydrogel microfilaments could be integrated with a wearable fluorometer to serve as a platform for continuous, minimally invasive monitoring of intradermal biomarkers.

Phenylalanine Monitoring via Aptamer-Field-Effect Transistor Sensors.

Determination of the amino acid phenylalanine is important for lifelong disease management in patients with phenylketonuria, a genetic disorder in which phenylalanine accumulates and persists at levels that alter brain development and cause permanent neurological damage and cognitive dysfunction. Recent approaches for treating phenylketonuria focus on injectable medications that efficiently break down phenylalanine but sometimes result in detrimentally low phenylalanine levels. We have identified new DNA aptamers for phenylalanine in two formats, initially as fluorescent sensors and then, incorporated with field-effect transistors (FETs). Aptamer-FET sensors detected phenylalanine over a wide range of concentrations (fM to mM). para-Chlorophenylalanine, which inhibits the enzyme that converts phenylalanine to tyrosine, was used to induce hyperphenylalaninemia during brain development in mice. Aptamer-FET sensors were specific for phenylalanine versus para-chlorophenylalanine and differentiated changes in mouse serum phenylalanine at levels expected in patients. Aptamer-FETs can be used to investigate models of hyperphenylalanemia in the presence of structurally related enzyme inhibitors, as well as naturally occurring amino acids. Nucleic acid-based receptors that discriminate phenylalanine analogs, some that differ by a single substituent, indicate a refined ability to identify aptamers with binding pockets tailored for high affinity and specificity. Aptamers of this type integrated into FETs enable rapid, electronic, label-free phenylalanine sensing.

Electrochemical Aptamer-Based Sensors for Improved Therapeutic Drug Monitoring and High-Precision, Feedback-Controlled Drug Delivery.

The electrochemical aptamer-based (E-AB) sensing platform appears to be a convenient (rapid, single-step, and calibration-free) and modular approach to measure concentrations of specific molecules (irrespective of their chemical reactivity) directly in blood and even in situ in the living body. Given these attributes, the platform may thus provide significant opportunities to render therapeutic drug monitoring (the clinical practice in which dosing is adjusted in response to plasma drug measurements) as frequent and convenient as the measurement of blood sugar has become for diabetics. The ability to measure arbitrary molecules in the body in real time could even enable closed-loop feedback control over plasma drug levels in a manner analogous to the recently commercialized controlled blood sugar systems. As initial exploration of this, we describe here the selection of an aptamer against vancomycin, a narrow therapeutic window antibiotic for which therapeutic monitoring is a critical part of the standard of care, and its adaptation into an electrochemical aptamer-based (E-AB) sensor. Using this sensor, we then demonstrate: (i) rapid (seconds) and convenient (single-step and calibration-free) measurement of plasma vancomycin in finger-prick-scale samples of whole blood, (ii) high-precision measurement of subject-specific vancomycin pharmacokinetics (in a rat animal model), and (iii) high-precision, closed-loop feedback control over plasma levels of the drug (in a rat animal model). The ability to not only track (with continuous-glucose-monitor-like measurement frequency and convenience) but also actively control plasma drug levels provides an unprecedented route toward improving therapeutic drug monitoring and, more generally, the personalized, high-precision delivery of pharmacological interventions.

Microcompartments for Protection and Isolation of Nanoscale DNA Computing Elements.

Physical isolation of molecular computing elements holds the potential for increasing system complexity by enabling the reuse of standardized components and by protecting the components from environmental degradation. However, once elements have been compartmentalized, methods for communicating into these compartments are needed. We report the compartmentalization of steroid-responsive DNA aptamers within giant unilamellar vesicles (GUVs) that are permeable to steroid inputs. Monodisperse GUVs are loaded with aptamers using a microfluidic platform. We demonstrate the target-specific activation of individual aptamers within the GUVs and then load two noninterfering aptamers into the same GUV and demonstrate specific responses to all possible combinations of the two input steroids. Crucially, GUVs prevent the degradation of DNA components by nucleases, providing a potential mechanism for deploying nucleic acid components in vivo. Importantly, our compartments also prevent nonspecific cross-talk between complementary strands, thereby providing a method for parallel execution of cross-reacting molecular logic components. Thus, we provide a mechanism for spatially organizing molecular computing elements, which will increase system modularity by allowing standardized components to be reused.

Aptamer-field-effect transistors overcome Debye length limitations for small-molecule sensing.

Detection of analytes by means of field-effect transistors bearing ligand-specific receptors is fundamentally limited by the shielding created by the electrical double layer (the "Debye length" limitation). We detected small molecules under physiological high-ionic strength conditions by modifying printed ultrathin metal-oxide field-effect transistor arrays with deoxyribonucleotide aptamers selected to bind their targets adaptively. Target-induced conformational changes of negatively charged aptamer phosphodiester backbones in close proximity to semiconductor channels gated conductance in physiological buffers, resulting in highly sensitive detection. Sensing of charged and electroneutral targets (serotonin, dopamine, glucose, and sphingosine-1-phosphate) was enabled by specifically isolated aptameric stem-loop receptors.

Insulin Hexamer-Caged Gadolinium Ion as MRI Contrast-o-phore.

High-relaxivity protein-complexes of GdIII are being pursued as MRI contrast agents in hope that they can be used at much lower doses that would minimize toxic-side effects of GdIII release from traditional contrast agents. We construct here a new type of protein-based MRI contrast agent, a proteinaceous cage based on a stable insulin hexamer in which GdIII is captured inside a water filled cavity. The macromolecular structure and the large number of "free" GdIII coordination sites available for water binding lead to exceptionally high relaxivities per one GdIII ion. The GdIII slowly diffuses out of this cage, but this diffusion can be prevented by addition of ligands that bind to the hexamer. The ligands that trigger structural changes in the hexamer, SCN- , Cl- and phenols, modulate relaxivities through an outside-in signaling that is allosterically transduced through the protein cage. Contrast-o-phores based on protein-caged metal ions have potential to become clinical contrast agents with environmentally-sensitive properties.

New therapeutic approaches and novel alternatives for organophosphate toxicity.

Organophosphate compounds (OPCs) are commonly used as pesticides and were developed as nerve agents for chemical warfare. Exposure to OPCs results in toxicity due to their covalent binding and inhibition of acetylcholinesterase (AChE). Treatment for toxicity due to OPC exposure has been largely focused on the reactivation of AChE by oxime-based compounds via direct nucleophilic attack on the phosphorous center. However, due to the disadvantages to existing oxime-based reactivators for treatment of OPC poisoning, we considered non-oxime mechanisms of reactivation. A high throughput screen of compound libraries was performed to discover previously unidentified reactivation compounds, followed by studies on their analogs. In the process, we discovered multiple non-oxime classes of compounds, the most robust of which we have already reported [1]. Herein, we report other classes of compounds we identified in our screen that are efficient at reactivation. During biochemical characterization, we also found some compounds with other activities that may inspire novel therapeutic approaches to OPC toxicity. Specifically, we found compounds that [1] increase the rate of substrate hydrolysis by AChE and, [2] protect the enzyme from inhibition by OPC. Further, we discovered that a subset of reactivator compounds recover activity from both AChE and the related enzyme butyrylcholinesterase (BuChE). We now report these compounds, their activities and discuss how each relates to therapeutic approaches that would provide alternatives to traditional oxime-based reactivation.

INTEGRATED MICROFLUIDIC SELEX USING FREE SOLUTION ELECTROKINETICS.

Systematic evolution of ligands by exponential enrichment (SELEX) offers a powerful method to isolate affinity oligonucleotides known as aptamers, which can then be used in a wide range of applications from drug delivery to biosensing. However, conventional SELEX methods rely on labor intensive and time consuming benchtop operations. A simplified microfluidic approach is presented which allows integration of the affinity selection and amplification stages of SELEX for the isolation of target-binding oligonucleotides by combining bead-based biochemical reactions with free solution electrokinetic oligonucleotide transfer. Free solution electrokinetics allows coupling of affinity selection and amplification for closed loop oligonucleotide enrichment without the need for offline processes, flow handling components or gel components, while bead based selection and amplification allow efficient manipulation of reagents and reaction products thereby realizing on-chip loop closure and integration of the entire SELEX process. Thus the approach is capable of multi-round enrichment of oligonucleotides using simple transfer processes while maintaining a high level of device integration, as demonstrated by the isolation of an aptamer pool against a protein target (IgA) with significantly higher binding affinity than the starting library in approximately 4 hours of processing time.

High-Affinity Nucleic-Acid-Based Receptors for Steroids.

Artificial receptors for hydrophobic molecules usually have moderate affinities and limited selectivities. We describe three new classes of high affinity hydrophobic receptors for nonaromatic steroids based on deoxyribonucleotides, obtained through five high stringency selections coupled with tailored counter-selections. The isolation of multiple classes of high affinity steroid receptors demonstrates the surprising breadth of moderately sized hydrophobic binding motifs (<40 nucleotides) available to natural nucleic acids. Studies of interactions with analogs indicate that two classes, four-way junctions and 4XGN motifs, comprise receptors with shapes that prevent binding of specific steroid conjugates used in counter-selections. Furthermore, they strongly prefer nonhydroxylated steroid cores, which is typical for hydrophobic receptors. The third new class accommodates hydroxyl groups in high-affinity, high-selectivity binding pockets, thus reversing the preferences of the first two classes. The high-affinity binding of aptamers to targets efficiently inhibits double-helix formation in the presence of the complementary oligonucleotides. The high affinity of some of these receptors and tailored elimination of binding through counter-selections ensures that these new aptamers will enable clinical chemistry applications.

An Integrated Microfluidic SELEX Approach Using Combined Electrokinetic and Hydrodynamic Manipulation.

This article presents a microfluidic approach for the integration of the process of aptamer selection via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs bead-based biochemical reactions in which affinity-selected target-binding oligonucleotides are electrokinetically transferred for amplification, while the amplification product is transferred back for affinity selection via pressure-driven fluid flow. The hybrid approach simplifies the device design and operation procedures by reduced pressure-driven flow control requirements and avoids the potentially deleterious exposure of targets to electric fields prior to and during affinity selection. In addition, bead-based reactions are used to achieve the on-chip coupling of affinity selection and amplification of target-binding oligonucleotides, thereby realizing on-chip loop closure and integration of the entire SELEX process without requiring offline procedures. The microfluidic approach is thus capable of closed-loop, multiround aptamer enrichment as demonstrated by selection of DNA aptamers against the protein immunoglobulin E with high affinity ( KD = 12 nM) in a rapid manner (4 rounds in approximately 10 h).