A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.

Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors.

Quantification of the concentration of particular cellular metabolites reports on the actual utilization of metabolic pathways in physiological and pathological conditions. Metabolite concentration also constitutes the readout for screening cell factories in metabolic engineering. However, there are no direct approaches that allow for real-time assessment of the levels of intracellular metabolites in single cells. In recent years, the modular architecture of natural bacterial RNA riboswitches has inspired the design of genetically encoded synthetic RNA devices that convert the intracellular concentration of a metabolite into a quantitative fluorescent signal. These so-called RNA-based sensors are composed of a metabolite-binding RNA aptamer as the sensor domain, connected through an actuator segment to a signal-generating reporter domain. However, at present, the variety of available RNA-based sensors for intracellular metabolites is still very limited. Here, we go through natural mechanisms for metabolite sensing and regulation in cells across all kingdoms, focusing on those mediated by riboswitches. We review the design principles underlying currently developed RNA-based sensors and discuss the challenges that hindered the development of novel sensors and recent strategies to address them. We finish by introducing the current and potential applicability of synthetic RNA-based sensors for intracellular metabolites.

Assessment of Aptamer-Targeted Contrast Agents for Monitoring of Blood Clots in Computed Tomography and Fluoroscopy Imaging.

Objective: Random formation of thrombi is classified as a pathological process that may result in partial or complete obstruction of blood flow and limited perfusion. Further complications include pulmonary embolism, thrombosis-induced myocardial infraction, ischemic stroke, and others. Location and full delineation of the blood clot are considered to be two clinically relevant aspects that could streamline proper diagnosis and treatment follow-up. In this work, we prepared two types of X-ray attenuating contrast formulations, using fibrinogen aptamer as the clot-seeking moiety. Methods: Two novel aptamer-targeted formulations were designed. Iodine-modified bases were directly incorporated into a fibrinogen aptamer (iodo-FA). Isothermal titration calorimetry was used to confirm that these modifications did not negatively impact target binding. Iodo-FA was tested for its ability to produce concentration-dependent contrast enhancement in a phantom CT. It was subsequently tested in vitro with clotted human and swine blood. This allowed for translation into ex vivo testing, using fluoroscopy. FA was also used to functionalize gold nanoparticles (FA-AuNPs), and contrast capabilities were confirmed. This formulation was tested in vitro using clotted human blood in a CT scan. Results: Unmodified FA and iodo-FA demonstrated a nearly identical affinity toward fibrin, confirming that base modifications did not impact target binding. Iodo-FA and FA-AuNPs both demonstrated excellent concentration-dependent contrast enhancement capabilities (40.5 HU mM-1 and 563.6 HU µM-1, respectively), which were superior to the clinically available agent, iopamidol. In vitro CT testing revealed that iodo-FA is able to penetrate into the blood clots, producing contrast enhancement throughout, while FA-AuNPs only accumulated on the surface of the clot. Iodo-FA was thereby translated to ex vivo testing, confirming target-binding associated accumulation of the contrast material at the location of the clot within the dilation of the external carotid artery. This resulted in a 34% enhancement of the clot. Conclusions: Both iodo-FA and FA-AuNPs were confirmed to be effective contrast formulations in CT. Targeting of fibrin, a major structural constituent of thrombi, with these novel contrast agents would allow for higher contrast enhancement and better clot delineation in CT and fluoroscopy.

Disposable multiplexed electrochemical sensors based on electro-triggered selective immobilization of probes for simultaneous detection of DNA and proteins.

Electrically addressable covalent immobilization of probes on a multiplexed electrode for the simultaneous detection of multiple targets within the same sample is often regarded as a difficult milestone to be achieved. Herein, we demonstrated a reagentless disposable multiplexed electrochemical DNA and aptamer-based sensing platform for the simultaneous determination of various targets. The electrochemically triggered "click" chemistry was developed, and three biomarkers, including p53, thrombin, and VEGF165 were used as model analytes. The proposed sensor consisted of three independent screen-printed carbon electrodes (SPCE), with an alkyne-azide cycloaddition reaction that was activated selectively by means of electrical triggering, so that different DNA probes can be modified on the desired electrode units in sequence. In terms of simultaneous detection, the sensor was able to quantify the DNA target of p53 with a detection limit of 0.35 nM, whereas the limits of detection for protein quantification of thrombin and VEGF165 were 0.22 nM and 0.014 nM, respectively. The proposed sensor not only showed encouraging reproducibility and stability, but also performed well even in 50% serum samples. Therefore, the work described here offers a general strategy for developing a multiplexed sensor with promising potential to achieve rapid, simple and cost-effective analysis of biological samples.

Cost-effective monitoring of microRNA-205 applied as a biomarker using G-quadruplex DNAzyme and 1,1'-oxalyldiimidazole chemiluminescence.

Trace levels of microRNA-205, known as a biomarker of lung cancer, in human serum was quantified for the first-time using G-quadruplex DNAzyme linked to detection complementary probe and 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL). First, capture complementary probes immobilized on the surface of paramagnetic bead selectively bound with microRNA-205 existing in human serum. Then, with the addition of detection complementary probe linked to hemin aptamer, a complex linked to hemin aptamer was formed with the completion of hybridization between microRNA-205 and two complementary probes. With the addition of hemin in the solution, finally, a complex linked to G-quadruplex DNAzyme was formed from the interaction of hemin aptamer and hemin. Resorufin, luminescent dye, was formed from the reaction of Amplex Red and H2O2 in the presence of the complex linked to DNAzyme acting as a horseradish peroxidase (HRP)-mimicking enzyme. The concentration of resorufin formed from the reaction was dependent on the concentration of microRNA-205 in human serum. Thus, the brightness of resorufin emitted in ODI-CL reaction was enhanced with the increase of microRNA-205. The limit of detection (LOD) of the biosensor with ODI-CL detection, capable of sensing microRNA-205 (dynamic range: 0.4-62.5 nM), was as low as 0.13 nM. It was confirmed that the biosensor can quantify trace levels of microRNA-205 with statistically acceptable accuracy, precision, and recovery.

Aptamer-Based TB Antigen Tests for the Rapid Diagnosis of Pulmonary Tuberculosis: Potential Utility in Screening for Tuberculosis.

Pulmonary tuberculosis is the most common manifestation of tuberculosis, and to this day, sputum smear microscopy remains the most widely used diagnostic test in resource-limited settings despite its suboptimal sensitivity. Here we report the development of two DNA aptamer-based diagnostic tests, namely aptamer linked immobilized sorbent assay (Aptamer ALISA) and electrochemical sensor (ECS), for the direct detection of a TB biomarker HspX in sputum. First we compared the performance of Aptamer ALISA with anti-HspX polyclonal antibody-based enzyme linked immunosorbent assay (Antibody ELISA) in a blinded study of 314 sputum specimens. Aptamer ALISA displayed a high sensitivity of 94.1% (95% CI 86.8-98%) as compared to 68.2% sensitivity (95% CI 57.2-77.9%) of Antibody ELISA ( p-value < 0.05) using culture as the reference standard without compromising test specificity of 100%. Out of nine smear-negative culture-positive samples, six were positive by Aptamer ALISA and only two were detected by Antibody ELISA. ALISA detected as positive 80 of 85 culture-positive TB as compared to 57 of 81 diagnosed as TB by X-ray ( p-value < 0.0001). These findings demonstrate the superiority of the aptamer-based test over smear microscopy, antibody-based ELISA, and chest X-ray for TB detection ( p-value < 0.0001 for all). Further, we have developed a ∼30 min point-of-care ECS test that discriminates between tuberculous and nontuberculous sputum with a sensitivity of ∼92.3% and specificity of 91.2%. The tests developed in the current study cost ∼$1-3/test and have potential utility in active case finding in high-risk groups and screening for pulmonary TB among presumptive TB subjects.

Rapid aptasensor capable of simply detect tumor markers based on conjugated polyelectrolytes.

In this paper, a very simple, easily-operated and universal platform is proposed for tumor marker detection. In this strategy, tumor marker-specific aptamer, which can quench the fluorescence of polyfluorene-based cationic conjugated polyelectrolytes (PFN+), are used as recognizing probes. Upon addition of tumor marker, the aptamer can be assembled into the tumor marker-aptamer complex, resulting in fluorescence recovery of PFN+ and the detection of the targets. The most widely-used tumor markers, carcinoembryonic antigen (CEA) and fetoprotein (AFP) have been chosen as the model analytes for this work. The sensing method is capable of rapidly detect target protein within 5 min without complex handling procedure and expensive instruments. Compared with previous studies, the assay presented here is really simple and avoids either conjugated polyelectrolytes (CPEs) modification or oligonucleotide labeling. This method also shows a wide detection range of 3 orders of magnitude and the detection limit is 0.316 ng/mL for CEA and 1.76 ng/mL for AFP. Furthermore, the approach requires only a convenient"mix-and-detect" procedure and offers a universal platform for the sensitive detection of any target molecule of choice according to the selected aptamer.

Using two-site binding models to analyze microscale thermophoresis data.

The emergence of microscale thermophoresis (MST) as a technique for determining the dissociation constants for bimolecular interactions has enabled these quantities to be measured in systems that were previously difficult or impracticable. However, most models for analyses of these data featured the assumption of a simple 1:1 binding interaction. The only model widely used for multiple binding sites was the Hill equation. Here, we describe two new MST analytic models that assume a 1:2 binding scheme: the first features two microscopic binding constants (KD(1) and KD(2)), while the other assumes symmetry in the bivalent molecule, culminating in a model with a single macroscopic dissociation constant (KD,M) and a single factor (α) that accounts for apparent cooperativity in the binding. We also discuss the general applicability of the Hill equation for MST data. The performances of the algorithms on both real and simulated data are assessed, and implementation of the algorithms in the MST analysis program PALMIST is discussed.

Assessment of Variability in the SOMAscan Assay.

SOMAscan is an aptamer-based proteomics assay capable of measuring 1,305 human protein analytes in serum, plasma, and other biological matrices with high sensitivity and specificity. In this work, we present a comprehensive meta-analysis of performance based on multiple serum and plasma runs using the current 1.3 k assay, as well as the previous 1.1 k version. We discuss normalization procedures and examine different strategies to minimize intra- and interplate nuisance effects. We implement a meta-analysis based on calibrator samples to characterize the coefficient of variation and signal-over-background intensity of each protein analyte. By incorporating coefficient of variation estimates into a theoretical model of statistical variability, we also provide a framework to enable rigorous statistical tests of significance in intervention studies and clinical trials, as well as quality control within and across laboratories. Furthermore, we investigate the stability of healthy subject baselines and determine the set of analytes that exhibit biologically stable baselines after technical variability is factored in. This work is accompanied by an interactive web-based tool, an initiative with the potential to become the cornerstone of a regularly updated, high quality repository with data sharing, reproducibility, and reusability as ultimate goals.

Molecular simulations and Markov state modeling reveal the structural diversity and dynamics of a theophylline-binding RNA aptamer in its unbound state.

RNA aptamers are oligonucleotides that bind with high specificity and affinity to target ligands. In the absence of bound ligand, secondary structures of RNA aptamers are generally stable, but single-stranded and loop regions, including ligand binding sites, lack defined structures and exist as ensembles of conformations. For example, the well-characterized theophylline-binding aptamer forms a highly stable binding site when bound to theophylline, but the binding site is unstable and disordered when theophylline is absent. Experimental methods have not revealed at atomic resolution the conformations that the theophylline aptamer explores in its unbound state. Consequently, in the present study we applied 21 microseconds of molecular dynamics simulations to structurally characterize the ensemble of conformations that the aptamer adopts in the absence of theophylline. Moreover, we apply Markov state modeling to predict the kinetics of transitions between unbound conformational states. Our simulation results agree with experimental observations that the theophylline binding site is found in many distinct binding-incompetent states and show that these states lack a binding pocket that can accommodate theophylline. The binding-incompetent states interconvert with binding-competent states through structural rearrangement of the binding site on the nanosecond to microsecond timescale. Moreover, we have simulated the complete theophylline binding pathway. Our binding simulations supplement prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline form an initial complex, most notably, a major rearrangement of the C27 base from a buried to solvent-exposed orientation. Theophylline appears to bind by a combination of conformational selection and induced fit mechanisms. Finally, our modeling indicates that when Mg2+ ions are present the population of binding-competent aptamer states increases more than twofold. This population change, rather than direct interactions between Mg2+ and theophylline, accounts for altered theophylline binding kinetics.

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications.

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G-C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165 Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165.

A universal fluorescent aptasensor based on AccuBlue dye for the detection of pathogenic bacteria.

We report a universal fluorescent aptasensor based on the AccuBlue dye, which is impermeant to cell membranes, for the detection of pathogenic bacteria. The sensor consists of AccuBlue, an aptamer strand, and its complementary strand (cDNA) that partially hybridizes to the aptamer strand. We have fabricated two models by changing the sequence of the reaction between the elements in the system. One is the "signal on" model in which the aptamer is first bound to the target, followed by the addition of cDNA and AccuBlue, at which time the cDNA hybridizes with the free unreacted aptamer and forms a double-stranded DNA (dsDNA) duplex. Such hybridization causes AccuBlue to insert into the dsDNA and exhibit significantly increased fluorescence intensity because of the specific intercalation of the AccuBlue into dsDNA rather than single-stranded DNA (ssDNA). The other model, "signal off," involves hybridization of the aptamer with cDNA first, resulting in high fluorescence intensity on the addition of AccuBlue. When the target is added, the aptamer binds the target, causing the cDNA to detach from the dsDNA duplex and resulting in low fluorescence as a result of the liberation of AccuBlue. Because this design is based purely on DNA hybridization, and AccuBlue is impermeant to cell membranes, it could potentially be adapted to a wide variety of analytes.

Multifunctional electrochemical aptasensor for aptamer clones screening, virus quantitation in blood and viability assessment.

A novel attempt was made to develop a disposable multifunctional sensor for analysis of vaccinia virus (VACV), a promising oncolytic agent that can replicate in and kill tumor cells. Briefly, we developed aptamers specific to VACV that were negatively selected against human serum as well as human and mouse blood to be further utilized for viral analysis directly in serum and blood. In addition, the aptamers were negatively selected against heat-inactivated VACV to enable them to distinguish between viable and nonviable virus particles. The selected aptamers were integrated onto an electrochemical aptasensor to perform multiple functions, including quantification of VACV, viability assessment of the virus, and estimation of the binding affinity between the virus and the developed aptamers. The aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and a VACV-specific aptamer onto a gold nanoparticles modified screen-printed carbon electrode (GNPs-SPCE). Square wave voltammetry was employed to quantify VACV in serum and blood within the range of 150-900 PFU, with a detection limit of 60 PFU in 30 µL. According to the electrochemical affinity measurements, three virus specific aptamer clones, V-2, V-5, and V-9 exhibited the highest affinity to VACV. Furthermore, flow cytometry was employed to estimate the dissociation constants of the clones which were found to be 26.3, 40.9, and 24.7 nM, respectively. Finally, the developed aptasensor was able to distinguish between the intact virus and the heat-inactivated virus thanks to the tailored selectivity of the aptamers that was achieved via negative selection.

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis.

The binding affinity of 17ß-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17ß-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 µM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.

Aptamer selection by high-throughput sequencing and informatic analysis.

Traditional methods for selecting aptamers require multiple rounds of selection and optimization in order to identify aptamers that bind with high affinity to their targets. Here we describe an assay that requires only one round of positive selection followed by high-throughput DNA sequencing and informatic analysis in order to select high-affinity aptamers. The assay is flexible, requires less hands on time, and can be used by laboratories with minimal expertise in aptamer biology to quickly select high-affinity aptamers to a target of interest. This assay has been utilized to successfully identify aptamers that bind to thrombin with dissociation constants in the nanomolar range.

An aptamer-based assay for thrombin via structure switch based on gold nanoparticles and magnetic nanoparticles.

An aptamer-based assay for thrombin with high specificity and sensitivity was presented. In the protocol, the aptamer for thrombin was immobilized on magnetic nanoparticle, and its complementary oligonucleotide was labeled with gold nanoparticles, then the aptamer was hybridized with the complementary oligonucleotide to form the duplex structure as a probe, this probe could be used for the specific recognition for thrombin. In the presence of thrombin, the aptamer prefer to form the G-quarter structure with thrombin, resulting in the dissociation of the duplex of the probe and the release of the gold labeled oligonucleotide. Upon this, we were able to detect thrombin through the detection of the electrochemical signal of gold nanoparticles. The strategy combines with the high specificity of aptamer and the excellent characteristics of nanoparticles. This assay is simple, rapid, sensitive and highly specific, it does not require labeling of thrombin, and it could be applied to detect thrombin in complex real sample. The method shows great potential in other protein analysis and in disease diagnosis.

A novel low-cost and easy to develop functionalization platform. Case study: aptamer-based detection of thrombin by surface plasmon resonance.

A novel low-cost platform to assess biomolecular interactions was investigated using surface plasmon resonance and an aptamer-based assay for thrombin detection. Gold SPR surface functionalized with a carboxylated cross-linked BSA film (cBSA) and commercially available carboxymethylated dextran chip (CM5) were used as immobilization platforms for the thrombin binding aptamer. The high end commercial instrument Biacore 3000 and a custom made FIA set-up involving TI Spreeta sensor (TSPR2K23) were used to assess different concentrations of thrombin within the range 0.1-150 nM both in buffer and in a complex matrix (plasma) using the obtained aptasensors. Based on data derived from both CM5 and cBSA platforms, the cBSA aptasensor exhibited good selectivity, stability and regeneration ability, both in buffer and in complex matrices (plasma), comparable with CM5.

Rapid and ultra-sensitive detection of AMP using a fluorescent and magnetic nano-silica sandwich complex.

We report here a novel AMP biosensor based on the aptamer-induced disassembly of fluorescent and magnetic nano-silica sandwich complexes with a direct detection limit of 0.1 microM.