Biology subjects

Eisenstein, M.

Publications and source records attributed to Eisenstein, M..

36 records · Page 2Linked to original sources

bioRxiv2021

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 suffers some notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background arising from the interaction of detection antibodies with the assay substrate or interferents in the sample matrix. Single-Molecule Colocalization Assay (SiMCA) overcomes this problem by employing total internal reflection fluorescence (TIRF) microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized fluorescent signals, we can essentially eliminate the confounding effects of background produced by non-specific binding of detection antibodies. We further employed a normalization strategy to account for the heterogeneous distribution of the capture antibodies, greatly improving the reproducibility of our measurements. In a series of experiments with TNF-, we show that SiMCA can achieve a three-fold lower limit of detection compared to conventional single-color assays using the same antibodies and exhibits consistent performance for assays performed in complex specimens such as chicken serum and human 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.

biophysics↗

bioRxiv2021

Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions

Seventy years following the discovery of peroxisomes, their proteome remains undefined. Uncovering the complete peroxisomal proteome, the peroxi-ome, is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover the peroxi-ome of the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known organellar proteome by [~]40% and paved the way for performing systematic, whole-organellar proteome assays. Coupled with targeted experiments this allowed us to discover new peroxisomal functions. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly-identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

cell biology↗

bioRxiv2021

Directed Evolution of Aptamer Discovery Technologies

ConspectusAlthough 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 lab 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 multi-parametric 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 matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of "non-nattural" 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. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, 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 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.

bioengineering↗

bioRxiv2021

A system for multiplexed selection of aptamers with exquisite specificity without counter-selection

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 counter-selection 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 counter-selection parameters. As a result, counter-selection 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 counter-selection. 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 counter-selection. 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. Significance statementAptamers offer the exciting potential to discriminate between structurally similar small molecules. However, generating such highly specific aptamers has been proven challenging using the conventional process of counter-selection. In this work, we describe a high-throughput screening platform that can characterize the specificity of millions of aptamers towards a group of structurally related molecules in a single experiment and generate exquisitely specific aptamers without any counter-selection. As exemplars, we generated aptamers with high affinity and specificity towards three structurally related kynurenine metabolites using our platform. Our platform can be readily adapted to other small molecule targets and should therefore accelerate the development of aptamer reagents with exquisite specificity.

bioengineering↗

bioRxiv2021

Real-time monitoring of drug pharmacokinetics within tumor tissue in live animals

The efficacy and safety of a chemotherapy regimen fundamentally depends on its pharmacokinetics. This is currently measured based on blood samples, but the abnormal vasculature and physiological heterogeneity of the tumor microenvironment can produce radically different drug pharmacokinetics relative to the systemic circulation. We have developed an implantable microelectrode array sensor that can collect such tissue-based pharmacokinetic data by simultaneously measuring intratumoral pharmacokinetics from multiple sites. We employ gold nanoporous microelectrodes that maintain robust sensor performance even after repeated tissue implantation and extended exposure to the tumor microenvironment. We demonstrate continuous in vivo monitoring of concentrations of the chemotherapy drug doxorubicin at multiple tumor sites in a rodent model, and demonstrate clear differences in pharmacokinetics relative to the circulation that could meaningfully affect drug efficacy and safety. This platform could prove valuable for preclinical in vivo characterization of cancer therapeutics, and may offer a foundation for future clinical applications.

bioengineering↗

bioRxiv2021

A Metric for Quantifying the Resolution of Molecular Assays

Many assay developers focus on limit of detection (LOD) as a primary performance metric, and LOD is indeed useful for assays designed to determine the presence or absence of an analyte. However, LOD is less useful for continuous assays designed to discriminate between concentrations of an analyte--e.g., glucose monitoring in diabetes, where clinical care is guided by particular concentration ranges and thresholds. In such scenarios, it would be valuable to quantify discriminatory resolution--i.e., whether an assay can differentiate 1 pM from 10 pM-- but no such standardized metric currently exists. Here, we propose a useful solution, termed resolution of molecular concentration (RMC). RMC offers a simple means for characterizing quantitative resolution and quickly comparing the quantitative performance of assays. By raising awareness of the limitations of current metrics for evaluating assay performance, we hope to empower the molecular diagnostics community to evaluate their methods in a more application-appropriate manner.

bioengineering↗

bioRxiv2021

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 micron scale, their detection sensitivity degrades precipitously, thereby greatly reducing their utility in the context of molecular diagnostic applications. Studies have reported that nanostructured electrodes can greatly improve electrochemical biosensor sensitivity, but the underlying mechanism remains poorly understood, thus making it difficult to fully exploit this phenomenon to improve biosensor performance. In this work, we propose and experimentally validate a novel mechanism in which electron transfer is physically accelerated within nanostructured electrodes due to reduced charge screening, resulting in enhanced sensitivity. We show that this mechanism can be exploited to achieve up to 24-fold increase in signal and nearly four-fold lower limit-of-detection relative conventional planar electrodes. This accelerated electron transfer mechanism should prove broadly applicable for improving the performance of electrochemical biosensors.

bioengineering↗

bioRxiv2021

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 such modifications in the context of basic and clinical research are severely hampered by the lack of affinity reagents that can differentiate protein glycoforms. This lack of reagents is largely due to the challenges associated with generating affinity reagents that can bind to particular glycan epitopes 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 in an effort to isolate reagents that can differentiate between protein glycoforms. Using this approach, we were able to select multiple aptamers that exhibit strong selectivity for specific glycoforms of two different proteins, with the capacity to discriminate between molecules with identical tertiary structures that differ only in terms of their glycan modifications.

bioengineering↗

bioRxiv2020

Automated platform for high-throughput screening of base-modified aptamers for affinity and specificity

Aptamers incorporating chemically modified bases can achieve superior affinity and specificity compared to natural aptamers, but their characterization remains a labor-intensive, low-throughput task. Here we describe the non-natural aptamer array (N2A2) system, in which a minimally modified Illumina MiSeq instrument is used for the high-throughput generation and characterization of large libraries ([~]106) of base-modified DNA aptamer candidates on the basis of both target affinity and specificity. We first demonstrate the capability to screen multiple different base modifications to identify the optimal chemistry for high-affinity target binding. We next use N2A2 to generate aptamers that can maintain excellent specificity even in complex samples, with equally strong target affinity in both buffer and diluted human serum. Given that N2A2 requires only minor mechanical modifications to the MiSeq, we believe N2A2 offers a broadly accessible tool for generating high-quality affinity reagents for diverse applications.

bioengineering↗

bioRxiv2020

Rational Design of Aptamer Switches with Programmable pH Response

Aptamer switches that respond sensitively to local variations in pH could enable the development of molecular devices that greatly enhance diagnostic accuracy and therapeutic efficacy. Previous design strategies relied on inserting a known pH-sensitive DNA motif into the aptamer structure. Unfortunately, their performance was ultimately governed by the intrinsic pH-response of the inserted motif and could not be readily tuned to operate across arbitrary pH ranges. In this work, we present a general methodology for converting virtually any aptamer into a pH-responsive switch that can be modulated to undergo a strong change in affinity - in acidic, neutral, or alkaline conditions. This was achieved by inserting two orthogonal motifs that can be manipulated in parallel to tune sensitivity to different pH conditions without altering the core sequence of the aptamer itself. Using a single ATP aptamer, we achieve pH-dependent target binding and release under diverse pH conditions, producing as much as 1,000-fold pH-induced change in affinity. Importantly, we also demonstrate the novel capability to predictably engineer aptamer switches that only exhibit strong target affinity within a narrowly defined pH range. Our approach should offer a highly generalizable strategy for integrating pH-responsiveness into molecular devices.

bioengineering↗

bioRxiv2020

Measuring aptamer folding energy using a molecular clamp

Folding energy offers a useful metric for characterizing the stability and function of aptamers. However, experimentally measuring the folding energy is challenging and there is currently no general technique to measure this parameter directly. In this work, we present a simple approach for measuring aptamer folding energy. First, the aptamer is stretched under equilibrium conditions with a double-stranded DNA "molecular clamp" that is coupled to the aptamer ends. We then measure the total internal energy of stressed DNA molecules using time-lapse gel electrophoresis and compare the folding and unfolding behavior of molecular clamp-stressed molecules that incorporate either the aptamer or unstructured random single-stranded DNA in order to derive the aptamer folding energy. Using this approach, we measured a folding energy of 10.40 kJ/mol for the HD22 thrombin aptamer, which is consistent with other predictions and estimates. We also analyzed a simple hairpin structure, generating a folding energy result of 9.05 kJ/mol, consistent with the value predicted by computational models (9.24 kJ/mol). We believe our strategy offers an accessible and generalizable approach for obtaining such measurements with virtually any aptamer.

biophysics↗

bioRxiv2020

Continuous detection of glucose and insulin in live animals

Real-time biosensors that can continuously measure circulating biomolecules in vivo would provide valuable insights into a patients health status and their response to therapeutics even when there is considerable variability in pharmacokinetics and pharmacodynamics across patient populations. Unfortunately, current real-time biosensors are limited to a handful of analytes (e.g. glucose and blood oxygen) and are limited in sensitivity (high nanomolar). In this work, we describe a general approach for continuously and simultaneously measuring multiple analytes with picomolar sensitivity and sub-second temporal resolution. As exemplars, we report the simultaneous detection of glucose and insulin at picomolar concentrations in live diabetic rats. Using our system, we demonstrate the capacity to resolve inter-individual differences in the pharmacokinetic responses to insulin and discriminate profiles from different insulin formulations at a high temporal resolution. Critically, our approach is general and could be readily modified to continuously and simultaneously measure other circulating analytes in vivo by swapping the affinity reagents, thus making it a versatile tool for biomedical research.

bioengineering↗

bioRxiv2019

Independent Control of the Thermodynamic and Kinetic Properties of Aptamer Switches

Molecular switches that change their conformation upon target binding offer powerful capabilities for biotechnology and synthetic biology. In particular, aptamers have proven useful as molecular switches because they offer excellent binding properties, undergo reversible folding, and can be readily engineered into a wide range of nanostructures. Unfortunately, the thermodynamic and kinetic properties of the aptamer switches developed to date are intrinsically coupled, such that high temporal resolution (i.e., switching time) can only be achieved at the cost of lower sensitivity or high background. Here, we describe a general design strategy that decouples the thermodynamic and kinetic behavior of aptamer switches to achieve independent control of sensitivity and temporal resolution. We used this strategy to generate an array of aptamer switches with effective dissociation constants (KD) ranging from 10 M to 40 mM and binding kinetics ranging from 170 ms to 3 s--all generated from the same parent ATP aptamer. Our strategy is broadly applicable to other aptamers, enabling the efficient development of switches with characteristics suitable for diverse range of biotechnology applications.

bioengineering↗

bioRxiv2019

Gateway mechanism confers ultrasensitivity on motor’s function

Biological switches are wide spread in many biological systems. Among them, the switch of the bacterial flagellar motor has generated much interest because it affects a mechanical process rather than a chemical reaction, it controls the direction of rotation of a rotary motor rather than being an on/off switch, and it is exceptionally ultrasensitive. Yet, the molecular mechanism underlying its function has remained unknown. Here we resolved unique features of this mechanism: On the one hand, it is tightly regulated by multiple means, involving three binding sites and two different covalent modifications, with the binding specificity being dictated by the type of covalent modification and by a strict binding sequence. On the other hand, it endows the motor with flexibility as it involves an intermediate stage of brief switches that provides a "go/no go" situation, in which the motor can either proceed to a stable rotation in the new direction or shift back to the original direction. This intermediate stage appears to be a means of the cell to produce angular deflection of swimming while maintaining directional persistence. Furthermore, we show by mathematical modeling that such a switching mechanism can provide ultrasensitivity. This unique combination of tight regulation, flexibility, and ultrasensitivity makes this switching mechanism of special interest.

microbiology↗

bioRxiv2019

Click-PD: A Quantitative Method 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 [~]10^7 per hour. Our \"click-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 based on quantitative on- and off-target binding measurements to simultaneously achieve high affinity and specificity. Finally, it requires minimal specialized equipment or reagents besides a FACS instrument which is now widely-available. Using click-PD, we generated a boronic acid-modified aptamer with [~]1 M 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 (ConA). 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 ConA aptamer exhibited exceptional specificity, with minimal binding to other structurally-similar lectins. Finally, we show that our aptamer remarkable biological activity. Indeed, to the best of our knowledge, this aptamer is the most potent inhibitor of Con A-mediated hemagglutination reported to date.

bioengineering↗

bioRxiv2019

High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences

Nanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultra-short DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNV) out of reach. To overcome these limitations, we report a nanopore-based sequencing strategy in which short target sequences are first circularized and then amplified via rolling-circle amplification to produce long stretches of concatemeric repeats. These can be sequenced on the Oxford Nanopore Technologys (ONT) MinION platform, and the resulting repeat sequences aligned to produce a highly-accurate consensus that reduces the high error-rate present in the individual repeats. Using this approach, we demonstrate for the first time the ability to obtain unbiased and accurate nanopore data for target DNA sequences of < 100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences and even enables quantitative detection of specific variants present at ratios of < 10%. Our method is simple, cost-effective, and only requires well-established processes. It therefore expands the utility of nanopore sequencing for molecular diagnostics and other applications, especially in resource-limited settings. One Sentence SummaryWe introduce a simple method of accurately sequencing ultra-short (<100bp) target DNA on a nanopore sequencing platform.

bioengineering↗

bioRxiv2018

A direct selection strategy for isolating aptamers with pH-sensitive binding activity

An aptamer reagent that can switch its binding affinity in a pH-responsive manner would be highly valuable for many biomedical applications including imaging and drug delivery. Unfortunately, the discovery of such aptamers is difficult and only a few have been reported to date. Here we report the first experimental strategy for generating pH-responsive aptamers through direct selection. As an exemplar, we report streptavidin-binding aptamers that retain nanomolar affinity at pH 7.4 but exhibit a ~100-fold decrease in affinity at pH 5.2. These aptamers were generated by incorporating a known streptavidin-binding DNA motif into an aptamer library and performing FACS-based screening at multiple pH conditions. Upon structural analysis, we found that one aptamers affinity-switching behavior is driven by a non-canonical G-A base-pair that controls its folding in a highly pH-dependent manner. We believe our strategy could be readily extended to other aptamer-target systems because it does not require a priori structural knowledge of the aptamer or the target.

synthetic biology↗

bioRxiv2017

Conformational states during vinculin unlocking differentially regulate focal adhesion properties

Focal adhesions (FAs) are multi-protein complexes that connect the actin cytoskeleton to the extracellular matrix, via integrin receptors. The growth, stability and adhesive functionality of these structures are tightly regulated by mechanical stress, yet, despite the extensive characterization of the integrin adhesome, the mechanisms underlying FA mechanosensitivity are still poorly understood. One of the key candidates for regulating FA-associated mechanosensing is vinculin, a prominent FA component, which was proposed to possess either closed (\"auto-inhibited\") or open (active) conformations. However, a direct demonstration of the nature of conformational transition between the two states is still absent. In this study we combined multiple structural and biological approaches to probe the transition from auto-inhibited to active conformation, and determine its effects on FA structure and dynamics. We further show here that the closed to open transition requires two sequential steps that can differentially regulate FA growth and stability.

biochemistry↗