UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas.

Neuroendocrine carcinomas, such as neuroendocrine prostate cancer and small-cell lung cancer, commonly have a poor prognosis and limited therapeutic options. We report that ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is elevated in tissues and plasma from patients with neuroendocrine carcinomas. Loss of UCHL1 decreases tumor growth and inhibits metastasis of these malignancies. UCHL1 maintains neuroendocrine differentiation and promotes cancer progression by regulating nucleoporin, POM121, and p53. UCHL1 binds, deubiquitinates, and stabilizes POM121 to regulate POM121-associated nuclear transport of E2F1 and c-MYC. Treatment with the UCHL1 inhibitor LDN-57444 slows tumor growth and metastasis across neuroendocrine carcinomas. The combination of UCHL1 inhibitors with cisplatin, the standard of care used for neuroendocrine carcinomas, significantly delays tumor growth in pre-clinical settings. Our study reveals mechanisms of UCHL1 function in regulating the progression of neuroendocrine carcinomas and identifies UCHL1 as a therapeutic target and potential molecular indicator for diagnosing and monitoring treatment responses in these malignancies.

Modular Aptamer Switches for the Continuous Optical Detection of Small-Molecule Analytes in Complex Media.

Aptamers are a promising class of affinity reagents because signal transduction mechanisms can be built into the reagent, so that they can directly produce a physically measurable output signal upon target binding. However, endowing the signal transduction functionality into an aptamer remains a trial-and-error process that can compromise its affinity or specificity and typically requires knowledge of the ligand binding domain or its structure. In this work, a design architecture that can convert an existing aptamer into a "reversible aptamer switch" whose kinetic and thermodynamic properties can be tuned without a priori knowledge of the ligand binding domain or its structure is described. Finally, by combining these aptamer switches with evanescent-field-based optical detection hardware that minimizes sample autofluorescence, this study demonstrates the first optical biosensor system that can continuously measure multiple biomarkers (dopamine and cortisol) in complex samples (artificial cerebrospinal fluid and undiluted plasma) with second and subsecond-scale time responses at physiologically relevant concentration ranges.

Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix.

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time is developed. As a proof of concept, real-time imaging of inter-cellular cyclic adenosine 3',5'-monophosphate (cAMP) signals in Dictyostelium discoideum amoeba cells is performed. A set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals is engineered. By combining multiple switches with different dynamic ranges, measure cAMP concentrations spanning three orders of magnitude in a single experiment can be measured. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements are achieved. Using different aptamer switches, this approach can be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

Extending the dynamic range of biomarker quantification through molecular equalization.

Precision medicine requires highly scalable methods of multiplexed biomarker quantification that can accurately describe patient physiology. Unfortunately, contemporary molecular detection methods are generally limited to a dynamic range of sensitivity spanning just 3-4 orders of magnitude, whereas the actual physiological dynamic range of the human plasma proteome spans more than 10 orders of magnitude. Current methods rely on sample splitting and differential dilution to compensate for this mismatch, but such measures greatly limit the reproducibility and scalability that can be achieved-in particular, the effects of non-linear dilution can greatly confound the analysis of multiplexed assays. We describe here a two-pronged strategy for equalizing the signal generated by each analyte in a multiplexed panel, thereby enabling simultaneous quantification of targets spanning a wide range of concentrations. We apply our 'EVROS' strategy to a proximity ligation assay and demonstrate simultaneous quantification of four analytes present at concentrations spanning from low femtomolar to mid-nanomolar levels. In this initial demonstration, we achieve a dynamic range spanning seven orders of magnitude in a single 5 µl sample of undiluted human serum, highlighting the opportunity to achieve sensitive, accurate detection of diverse analytes in a highly multiplexed fashion.

High-Throughput Strategy for Enhancing Aptamer Performance across Different Environmental Conditions.

Aptamers selected under specific environmental conditions (e.g., pH, ion concentration, and temperature) often exhibit greatly reduced affinity when used in other contexts. This can be especially problematic for biomedical applications in which aptamers are exposed to sample matrices with distinctive chemical properties, such as blood, sweat, or urine. We present a high-throughput screening procedure for adapting existing aptamers for use in samples whose chemical composition differs considerably from the original selection conditions. Building on prior work from our group, we have utilized a modified DNA sequencer capable of screening up to 107 unique aptamer mutants for target binding under the desired assay conditions. As an exemplar, we screened all 11,628 single- and double-substitution mutants for a previously reported glucose aptamer that was originally selected in high-ionic strength buffer and exhibited relatively low affinity in physiological conditions. After a single round of screening, we identified aptamer mutants with ∼four-fold increased affinity in physiological conditions. Interestingly, we found that the impact of single-base substitutions was relatively modest but observed considerably greater binding improvements among the double mutants, highlighting the importance of cooperative effects between mutations. This approach should be generalizable to other aptamers and environmental conditions for a range of applications.

A massively parallel screening platform for converting aptamers into molecular switches.

Aptamer-based molecular switches that undergo a binding-induced conformational change have proven valuable for a wide range of applications, such as imaging metabolites in cells, targeted drug delivery, and real-time detection of biomolecules. Since conventional aptamer selection methods do not typically produce aptamers with inherent structure-switching functionality, the aptamers must be converted to molecular switches in a post-selection process. Efforts to engineer such aptamer switches often use rational design approaches based on in silico secondary structure predictions. Unfortunately, existing software cannot accurately model three-dimensional oligonucleotide structures or non-canonical base-pairing, limiting the ability to identify appropriate sequence elements for targeted modification. Here, we describe a massively parallel screening-based strategy that enables the conversion of virtually any aptamer into a molecular switch without requiring any prior knowledge of aptamer structure. Using this approach, we generate multiple switches from a previously published ATP aptamer as well as a newly-selected boronic acid base-modified aptamer for glucose, which respectively undergo signal-on and signal-off switching upon binding their molecular targets with second-scale kinetics. Notably, our glucose-responsive switch achieves ~30-fold greater sensitivity than a previously-reported natural DNA-based switch. We believe our approach could offer a generalizable strategy for producing target-specific switches from a wide range of aptamers.

Tailoring electrode surface charge to achieve discrimination and quantification of chemically similar small molecules with electrochemical aptamers.

Electrochemical biosensors based on structure-switching aptamers offer many advantages because they can operate directly in complex samples and offer the potential to integrate with miniaturized electronics. Unfortunately, these biosensors often suffer from cross-reactivity problems when measuring a target in samples containing other chemically similar molecules, such as precursors or metabolites. While some progress has been made in selecting highly specific aptamers, the discovery of these reagents remains slow and costly. In this work, we demonstrate a novel strategy to distinguish molecules with miniscule difference in chemical composition (such as a single hydroxyl group) - with cross reactive aptamer probes - by tuning the charge state of the surface on which the aptamer probes are immobilized. As an exemplar, we show that our strategy can distinguish between DOX and many structurally similar analytes, including its primary metabolite doxorubicinol (DOXol). We then demonstrate the ability to accurately quantify mixtures of these two molecules based on their differential response to sensors with different surface-charge properties. We believe this methodology is general and can be extended to a broad range of applications.

Binding and sequestration of poison frog alkaloids by a plasma globulin.

Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid-binding molecule in blood of poison frogs. Proteomic and biochemical studies establish this plasma protein to be a liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid-binding activity, ABG sequesters and regulates the bioavailability of 'free' plasma alkaloids in vitro. Unexpectedly, ABG is not related to saxiphilin, albumin, or other known vitamin carriers, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin-binding proteins. ABG represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points toward serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.

Improved immunoassay sensitivity and specificity using single-molecule colocalization.

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.

Combinatorial Polyacrylamide Hydrogels for Preventing Biofouling on Implantable Biosensors.

Biofouling on the surface of implanted medical devices and biosensors severely hinders device functionality and drastically shortens device lifetime. Poly(ethylene glycol) and zwitterionic polymers are currently considered "gold-standard" device coatings to reduce biofouling. To discover novel anti-biofouling materials, a combinatorial library of polyacrylamide-based copolymer hydrogels is created, and their ability is screened to prevent fouling from serum and platelet-rich plasma in a high-throughput parallel assay. It is found that certain nonintuitive copolymer compositions exhibit superior anti-biofouling properties over current gold-standard materials, and machine learning is used to identify key molecular features underpinning their performance. For validation, the surfaces of electrochemical biosensors are coated with hydrogels and their anti-biofouling performance in vitro and in vivo in rodent models is evaluated. The copolymer hydrogels preserve device function and enable continuous measurements of a small-molecule drug in vivo better than gold-standard coatings. The novel methodology described enables the discovery of anti-biofouling materials that can extend the lifetime of real-time in vivo sensing devices.

A system for multiplexed selection of aptamers with exquisite specificity without counterselection.

Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counterselection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counterselection parameters. As a result, counterselection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel while also eliminating the need for counterselection. We employ a platform based on a modified benchtop sequencer to conduct a massively parallel aptamer screening process that enables the selection of highly specific aptamers against multiple structurally similar molecules in a single experiment, without any counterselection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes.

Directed Evolution of Aptamer Discovery Technologies.

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our laboratory to accelerate the discovery of high-quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multiparametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrixes such as serum. The third method is an alkyne-azide chemistry ("click chemistry")-based particle display (click-PD) that enables the generation and screening of "non-natural" aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Finally, we describe the non-natural aptamer array (N2A2) platform in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in high throughput, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized for affinity and specificity in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms.

Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize the biological function of such modifications have been greatly hampered by the lack of affinity reagents that can differentiate protein glycoforms with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases, which are capable of recognizing and differentiating between specific protein glycoforms. Using this approach, we were able to select base-modified aptamers that exhibit strong selectivity for specific glycoforms of two different proteins. These aptamers can discriminate between molecules that differ only in their glycan modifications, and can also be used to label glycoproteins on the surface of cultured cells. We believe our strategy should offer a generally-applicable approach for developing useful reagents for glycobiology research.

Accelerated Electron Transfer in Nanostructured Electrodes Improves the Sensitivity of Electrochemical Biosensors.

Electrochemical biosensors hold the exciting potential to integrate molecular detection with signal processing and wireless communication in a miniaturized, low-cost system. However, as electrochemical biosensors are miniaturized to the micrometer scale, their signal-to-noise ratio degrades and reduces their utility for molecular diagnostics. Studies have reported that nanostructured electrodes can improve electrochemical biosensor signals, but since the underlying mechanism remains poorly understood, it remains difficult to fully exploit this phenomenon to improve biosensor performance. In this work, electrochemical aptamer biosensors on nanoporous electrode are optimized to achieve improved sensitivity by tuning pore size, probe density, and electrochemical measurement parameters. Further, a novel mechanism in which electron transfer is physically accelerated within nanostructured electrodes due to reduced charge screening, resulting in enhanced sensitivity is proposed and experimentally validated. In concert with the increased surface areas achieved with this platform, this newly identified effect can yield an up to 24-fold increase in signal level and nearly fourfold lower limit of detection relative to planar electrodes with the same footprint. Importantly, this strategy can be generalized to virtually any electrochemical aptamer sensor, enabling sensitive detection in applications where miniaturization is a necessity, and should likewise prove broadly applicable for improving electrochemical biosensor performance in general.

A fluorescence sandwich immunoassay for the real-time continuous detection of glucose and insulin in live animals.

Biosensors that continuously measure circulating biomolecules in real time could provide insights into the health status of patients and their response to therapeutics. But biosensors for the continuous real-time monitoring of analytes in vivo have only reached nanomolar sensitivity and can measure only a handful of molecules, such as glucose and blood oxygen. Here we show that multiple analytes can be continuously and simultaneously measured with picomolar sensitivity and sub-second resolution via the integration of aptamers and antibodies into a bead-based fluorescence sandwich immunoassay implemented in a custom microfluidic chip. After an incubation time of 30 s, bead fluorescence is measured using a high-speed camera under spatially multiplexed two-colour laser illumination. We used the assay for continuous quantification of glucose and insulin concentrations in the blood of live diabetic rats to resolve inter-animal differences in the pharmacokinetic response to insulin as well as discriminate pharmacokinetic profiles from different insulin formulations. The assay can be readily modified to continuously and simultaneously measure other blood analytes in vivo.

Design and Analysis of a Sample-and-Hold CMOS Electrochemical Sensor for Aptamer-based Therapeutic Drug Monitoring.

In this paper, we present the design and the analysis of an electrochemical circuit for measuring the concentrations of therapeutic drugs using structure-switching aptamers. Aptamers are single-stranded nucleic acids, whose sequence is selected to exhibit high affinity and specificity toward a molecular target, and change its conformation upon binding. This property, when coupled with a redox reporter and electrochemical detection, enables reagent-free biosensing with a sub-minute temporal resolution for in vivo therapeutic drug monitoring. Specifically, we design a chronoamperometry-based electrochemical circuit that measures the direct changes in the electron transfer (ET) kinetics of a methylene blue reporter conjugated at the distal-end of the aptamer. To overcome the high-frequency noise amplification issue when interfacing with a large-size (> 0.25 mm2) implantable electrode, we present a sample-and-hold (S/H) circuit technique in which the desired electrode potentials are held onto noiseless capacitors during the recording of the redox currents. This allows disconnecting the feedback amplifiers to avoid its noise injection while reducing the total power consumption. A prototype circuit implemented in 65-nm CMOS demonstrates a cell-capacitance-insensitive input-referred noise (IRN) current of 15.2 pArms at a 2.5-kHz filtering bandwidth. We tested our system in human whole blood samples and measured the changes in the ET kinetics from the redox-labeled aptamers at different kanamycin concentrations. By employing principal component analysis (PCA) to compensate for the sampling errors, we report a molecular noise floor (at SNR = 1) of 3.1 µM with sub 1-sec acquisition time at 0.22-mW power consumption.

Going beyond the Debye Length: Overcoming Charge Screening Limitations in Next-Generation Bioelectronic Sensors.

Electronic biosensors are a natural fit for field-deployable diagnostic devices because they can be miniaturized, mass produced, and integrated with circuitry. Unfortunately, progress in the development of such platforms has been hindered by the fact that mobile ions present in biological samples screen charges from the target molecule, greatly reducing sensor sensitivity. Under physiological conditions, the thickness of the resulting electric double layer is less than 1 nm, and it has generally been assumed that electronic detection beyond this distance is virtually impossible. However, a few recently described sensor design strategies seem to defy this conventional wisdom, exploiting the physics of electrical double layers in ways that traditional models do not capture. In the first strategy, charge screening is decreased by constraining the space in which double layers can form. The second strategy uses external stimuli to prevent double layers from reaching equilibrium, thereby effectively reducing charge screening. In this Perspective, we describe these relatively new concepts and offer theoretical insights into mechanisms that may enable electronic biosensing beyond the Debye length. If these concepts can be further developed and translated into practical electronic biosensors, we foresee exciting opportunities for the next generation of diagnostic technologies.

Engineering Aptamer Switches for Multifunctional Stimulus-Responsive Nanosystems.

Although RNA and DNA are best known for their capacity to encode biological information, it has become increasingly clear over the past few decades that these biomolecules are also capable of performing other complex functions, such as molecular recognition (e.g., aptamers) and catalysis (e.g., ribozymes). Building on these foundations, researchers have begun to exploit the predictable base-pairing properties of RNA and DNA in order to utilize nucleic acids as functional materials that can undergo a molecular "switching" process, performing complex functions such as signaling or controlled payload release in response to external stimuli including light, pH, ligand-binding and other microenvironmental cues. Although this field is still in its infancy, these efforts offer exciting potential for the development of biologically based "smart materials". Herein, ongoing progress in the use of nucleic acids as an externally controllable switching material is reviewed. The diverse range of mechanisms that can trigger a stimulus response, and strategies for engineering those functionalities into nucleic acid materials are explored. Finally, recent progress is discussed in incorporating aptamer switches into more complex synthetic nucleic acid-based nanostructures and functionalized smart materials.