Structure Of Aptamer Research Articles

High-resolution structure of a novel fluorogenic RNA aptamer.

High-resolution structure of a novel fluorogenic RNA aptamer.

Design and demonstration of a temperature-resistant aptamer structure for highly sensitive mercury ion detection with BioFETs

In this study, we developed a temperature-resistant aptamer coupled with extended gate electric double-layer Field-Effect Transistors (FETs) for highly sensitive mercury ion detection. The design of the temperature-resistant aptamer is based on the thermal and structural properties of the hairpin structure. Two aptamer sequences were designed with different melting temperatures (Tm) and compared for their sensitivity. The hairpin structure of the aptamer with a high melting temperature ensures the stable structure prior to the addition of the mercury ions, which allows the formation of thymine–mercury–thymine (T -Hg2+-T) complex in low concentrations of mercury ions. High sensitivity and low detection limit are achieved at elevated temperatures with the aptamer having a high melting temperature. The elevated temperature facilitates the reaction rate, resulting in high sensitivity and a low detection limit. The aptamer with a high melting temperature hairpin structure has shown a remarkable improvement in the limit of detection with a 4 orders of magnitude increase compared to the one with a low melting temperature. The sensor with the high melting temperature aptamer shows an extremely low detection limit of 4.68 × 10−12 M, surpassing previously reported results. The aptamer with a low melting temperature requires mercury ions to stabilize the hairpin structure by forming a T-Hg2+-T complex. The results show that if the melting temperature is lower than the ambient temperature, it is very difficult to detect low concentrations of mercury ions at the ambient temperature. The selectivity of this sequence was also tested against multiple heavy metals, including arsenic (As), lead (Pb), chromium (Cr), and cadmium (Cd) ions. This study has shown how the structural and thermal properties of the aptamer, and the ambient temperature affect the sensitivity. They are strongly correlated to the performance of the sensors and need to be considered in the design of the sequence.

Electrochemical sensor for vascular endothelial growth factor based on self-assembling DNA aptamer structure

Developing vascular endothelial growth factor (VEGF) protein is essential for early cancer diagnosis and cancer treatment monitoring. This study presents the design and characterisation of an electrochemical sensor utilising a self-assembling DNA aptamer structure for the sensitive and selective detection of VEGF. The aptamer structure comprises three different parts of single-stranded DNA that are assembled prior to integration into the sensor. Polypyrrole (Ppy)-based layers were deposited onto screen-printed carbon electrodes (SPCEs) using an electrochemical deposition technique, followed by the entrapment of a self-assembled DNA aptamer structure within electrochemically formed Ppy matrix ((DNA aptamer)/Ppy). The response to the sensor toward VEGF was measured by the pulsed amperometric detection (PAD), highlighting the enhanced performance of DNA aptamer/Ppy configuration compared to bare Ppy. The sensor exhibited high sensitivity, achieving a limit of detection (LOD) of 0.21 nM for VEGF. The interaction behaviour between VEGF in the solution and the immobilise DNA aptamer/Ppy-based structure was analysed using Langmuir isotherm model. The developed electrochemical biosensor in promising for in vitro applications in early cancer diagnostics and treatment monitoring, enabling rapid screening of patient samples.

AptamerRunner: An accessible aptamer structure prediction and clustering algorithm for visualization of selected aptamers

Aptamers are short single-stranded DNA or RNA molecules with high affinity and specificity for targets and are generated using the iterative Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. Next-generation sequencing (NGS) revolutionized aptamer selections by allowing a more comprehensive analysis of SELEX-enriched aptamers as compared to Sanger sequencing. The current challenge with aptamer NGS datasets is identifying a diverse cohort of candidate aptamers with the highest likelihood of successful experimental validation. Herein we present AptamerRunner, an aptamer sequence and/or structure clustering algorithm that synergistically integrates computational analysis with visualization and expertise-directed decision making. The visual integration of networked aptamers with ranking data, such as fold enrichment or scoring algorithm results, represents a significant advancement over existing clustering tools by providing a natural context to depict groups of aptamers from which ranked or scored candidates can be chosen for experimental validation. The inherent flexibility, user-friendly design, and prospects for future enhancements with AptamerRunner has broad-reaching implications for aptamer researchers across a wide range of disciplines.

Aptamer-based Strategies for COVID-19 Detection and Treatment: A Systematic Review Study

Background: Aptamer-based strategies have emerged as promising tools for the detection and treatment of COVID-19, offering advantages such as high specificity, sensitivity, and versatility. This systematic review aims to evaluate the effectiveness and innovation of aptamer-based approaches for COVID-19 detection and treatment. Methods: Following the guidelines of the Cochrane Handbook for Systematic Reviews and the PRISMA 2020 guidelines, a systematic search was conducted across multiple databases up to 2024. The search included studies that utilized aptamers for the diagnosis or therapy of COVID-19. Screening and selection of studies were performed independently by two reviewers, with any disagreements resolved by a third reviewer. Data were extracted regarding study characteristics, aptamer details, and outcomes. Results: In our systematic review, 98 studies from an initial pool of 1541 records met the inclusion criteria for analysis. Aptamers, single-stranded DNA or RNA molecules with unique threedimensional (3D) structures, were extensively explored for COVID-19 detection and treatment. Various aptamer-based assays, including electrochemical sensors, surface plasmon resonance (SPR) biosensors, and lateral flow assays, demonstrated high sensitivity and specificity in detecting SARSCoV- 2 in clinical samples such as saliva, nasal swabs, and wastewater. Several aptamer structures targeting viral proteins like the spike and nucleocapsid proteins were employed. Nucleic Acid Amplification Techniques (NAATs) utilizing aptamers, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based and Loop-mediated Isothermal Amplification (LAMP) assays, showed exceptional sensitivity in detecting viral genetic material. Aptamer-based therapeutic approaches showed potential by blocking viral protein activity or serving as delivery vehicles for therapeutic agents like small interfering RNAs (siRNAs). Despite their advantages, aptamer technologies face limitations such as susceptibility to nuclease degradation and rapid renal clearance, highlighting the need for further optimization. Conclusion: Aptamer-based strategies present promising avenues for COVID-19 detection and treatment. These approaches offer advantages such as high sensitivity, specificity, and rapid detection, making them valuable tools in combating the COVID-19 pandemic. Further research and development are warranted to optimize aptamer-based strategies for widespread application in clinical settings.

Structure-based insights into fluorogenic RNA aptamers.

Fluorogenic RNA aptamers are in vitro-selected RNA molecules capable of binding to specific fluorophores, significantly increasing their intrinsic fluorescence. Over the past decade, the color palette of fluorescent RNA aptamers has greatly expanded. The emergence and development of these fluorogenic RNA aptamers has introduced a powerful approach for visualizing RNA localization and transport with high spatiotemporal resolution in live cells. To date, a variety of tertiary structures of fluorogenic RNA aptamers have been determined using X-ray crystallography or NMR spectroscopy. Many of these fluorogenic RNA aptamers feature base quadruples or base triples in their fluorophore-binding sites. This review summarizes the structure-based investigations of fluorogenic RNA aptamers, with a focus on their overall folds, ligand-binding pockets and fluorescence activation mechanisms. Additionally, the exploration of how structures guide rational optimization to enhance RNA visualization techniques is discussed.

A Branched SELEX Approach Identifies RNA Aptamers That Bind Distinct HIV-1 Capsid Structural Components.

The HIV-1 capsid protein (CA) assumes distinct structural forms during replication, each presenting unique, solvent-accessible surfaces that facilitate multifaceted functions and host factor interactions. However, functional contributions of individual CA structures remain unclear, as evaluation of CA presents several technical challenges. To address this knowledge gap, we identified CA-targeting aptamers with different structural specificities, which emerged through a branched SELEX approach using an aptamer library previously selected to bind the CA hexamer lattice. Subsets were either highly specific for the CA lattice or bound both the CA lattice and CA hexamer. We then evaluated four representatives to reveal aptamer regions required for binding, highlighting interesting structural features and challenges in aptamer structure determination. Further, we demonstrate binding to biologically relevant CA structural forms and aptamer-mediated affinity purification of CA from cell lysates without virus or host modification, supporting the development of structural form-specific aptamers as exciting new tools for the study of CA.

Tag-free fluorometric aptasensor for detection of chromium(VI) in foods via SYBR Green I signal amplification and aptamer structure transition.

In response to growing concerns regarding heavy metal contamination in food, particularly chromium (Cr)(VI) contamination, this study presented a simple, sensitive and practical method for Cr(VI) detection. A magnetic separation-based capture-exponential enrichment ligand system evolution (SELEX) method was used to identify and characterize DNA aptamers with a high affinity for Cr(VI). An aptamer, Cr-15, with a dissociation constant (Kd) of 4.42 ± 0.44 μmol L-1 was obtained after only eight rounds of selection. Further innovative methods combining molecular docking, dynamic simulation and thermodynamic analysis revealed that CrO4 2- could bind to the 19th and 20th guanine bases of Cr-15 via hydrogen bonds. Crucially, a label-free fluorometric aptasensor based on SYBR Green I was successfully constructed to detect CrO4 2-, achieving a linear detection range of 60-300 nmol L-1 with a lower limit of detection of 44.31 nmol L-1. Additionally, this aptasensor was able to quantitatively detect CrO4 2- in grapes and broccoli within 40 min, with spike recovery rates ranging from 89.22% to 108.05%. The designed fluorometric aptasensor exhibited high selectivity and could detect CrO4 2- in real samples without sample processing or target pre-enrichment. The aptasensor demonstrated its potential as a reliable tool for monitoring Cr(VI) contamination in fruit and vegetable products. © 2024 Society of Chemical Industry.

Aptamer biosensor design for the detection of endocrine-disrupting chemicals small organic molecules using novel bioinformatics methods

Endocrine-disrupting chemicals (EDCs) are substances that can disrupt the normal functioning of hormones.Using aptamers, which are biological recognition elements, biosensors can quickly and accurately detect EDCs in environmental samples. However, the elucidation of aptamer structures by conventional methods is highly challenging due to their complexity.This has led to the development of three-dimensional aptamer structures based on different models and techniques. To do this, we developed a way to predict the 3D structures of the SS DNA needed for this sequence by starting with an aptamer sequence that has biosensor properties specific to bisphenol-A (BPA), one of the chemicals found in water samples that can interfere with hormones. In addition, we will elucidate the intermolecular mechanisms and binding affinity between aptamers and endocrine disruptors using bioinformatics techniques such as molecular docking, molecular dynamics simulation, and binding energies. The outcomes of our study are to compare modeling programs and force fields to see how reliable they are and how well they agree with results found in the existing literature, to understand the intermolecular mechanisms and affinity of aptamer-based biosensors, and to find a new way to make aptamers that takes less time and costs less.

DNA Cryogels with Anisotropic Mechanical and Responsive Properties for Specific Cell Capture and Release.

Due to their programmable stimuli-responsiveness, excellent biocompatibility, and water-rich and soft structures similar to biological tissues, smart DNA hydrogels hold great promise for biosensing and biomedical applications. However, most DNA hydrogels developed to date are composed of randomly oriented and isotropic polymer networks, and the resulting slow response to biotargets and lack of anisotropic properties similar to those of biological tissues have limited their extensive applications. Herein, anisotropic DNA hydrogels consisting of unidirectional void channels internally oriented up to macroscopic length scales were constructed by a directional cryopolymerization method, as exemplified by a DNA-incorporated covalently cross-linked DNA cryogel and a DNA duplex structure noncovalently cross-linked DNA cryogel. Results showed that the formation of unidirectional channels significantly improved the responsiveness of the gel matrix to biomacromolecular substances and further endowed the DNA cryogels with anisotropic properties, including anisotropic mechanical properties, anisotropic swelling/shrinking behaviors, and anisotropic responsiveness to specific biotargets. Moreover, the abundant oriented and long macroporous channels in the gel matrix facilitated the migration of cells, and through the introduction of aptamer structures and thermosensitive polymers, an anisotropic DNA cryogel-based platform was further constructed to achieve the highly efficient capture and release of specific cells. These anisotropic DNA hydrogels may provide new opportunities for the development of anisotropic separation and biosensing systems.

Design and Validation of a Short Novel Estradiol Aptamer and Exploration of Its Application in Sensor Technology.

The specific and sensitive detection of 17β-estradiol (E2) is critical for diagnosing and treating numerous diseases, and aptamers have emerged as promising recognition probes for developing detection platforms. However, traditional long-sequence E2 aptamers have demonstrated limited clinical performance due to redundant structures that can affect their stability and recognition ability. There is thus an urgent need to further optimize the structure of the aptamer to build an effective detection platform for E2. In this work, we have designed a novel short aptamer that retains the key binding structure of traditional aptamers to E2 while eliminating the redundant structures. The proposed aptamer was evaluated for its binding properties using microscale thermophoresis, a gold nanoparticle-based colorimetric method, and electrochemical assays. Our results demonstrate that the proposed aptamer has excellent specific recognition ability for E2 and a high affinity with a dissociation constant of 92 nM. Moreover, the aptamer shows great potential as a recognition probe for constructing a highly specific and sensitive clinical estradiol detection platform. The aptamer-based electrochemical sensor enabled the detection of E2 with a linear range between 5 pg mL-1 and 10 ng mL-1 (R2 = 0.973), and the detection capability of a definite low concentration level was 5 pg mL-1 (S/N = 3). Overall, this novel aptamer holds great promise as a valuable tool for future studies on the role of E2 in various physiological and pathological processes and for developing sensitive and specific diagnostic assays for E2 detection in clinical applications.

Selection and application of highly specific Salmonella typhimurium aptamers against matrix interference

In practical applications, the structure and performance of aptamers can be influenced by the presence of sample matrices, which interferes with the specific binding between the aptamer and its target. In this work, to obtain aptamer chains resistant to matrix interference, four typical food matrices were introduced as negative selection targets and selection environments in the process of selecting aptamers for Salmonella typhimurium using the systematic evolution of ligands by exponential enrichment (SELEX) technology. As a result, some highly specific candidate aptamers for Salmonella typhimurium (BB-34, BB-37, ROU-8, ROU-9, ROU-14, ROU-24, DAN-3, NAI-12, and NAI-21) were successfully obtained. Based on the characterization results of secondary structure, affinity, and specificity of these candidate aptamers, ROU-24 selected in the pork matrix and BB-34 selected in the binding buffer were chosen to develop label-free fluorescence aptasensors for the sensitive and rapid detection of the Salmonella typhimurium and verify the performance against matrix interference. The ROU-24-based aptasensor demonstrated a larger linear range and better specificity compared to the BB-34-based aptasensor. Meanwhile, the recovery rate of the ROU-24-based aptasensor in real sample detection (ranging from 94.2% to 110.7%) was significantly higher than that of the BB-34-based aptasensor. These results illustrated that the negative selection of food matrices induced in SELEX could enhance specific binding between the aptamer and its target and the performance against matrix interference. Overall, the label-free fluorescence aptasensors were developed and successfully validated in different foodstuffs, demonstrating a theoretical and practical basis for the study of aptamers against matrix interference.

Detailed Protocol for Predicting 3D Structure of DNA Aptamers and Performing In Silico Docking Calculations.

This paper presents a comprehensive protocol for predicting the three-dimensional (3D) structure of DNA aptamers and performing in silico docking calculations. The protocol includes steps for sequence input, structure prediction, sequence modification, structure minimization, and docking. The procedure is executed on a Mac environment utilizing bioinformatics tools such as mfold, RNA Composer, PyMOL, and Hdock. The protocol is intended to provide a guide for researchers in structural biology and drug design.

Conformational preferences of modified nucleobases in RNA aptamers and their effect on Förster resonant energy transfer.

Förster resonant energy transfer (FRET) can be utilized in the study of tertiary structures of RNA aptamers, which bind specific fluorophoric ligands to form a fluorogenic aptamer complex. By introducing the emissive nucleobase analog 4-cyanoindole into the fluorogenic Chili RNA aptamer a FRET pair was established. The interpretation of studies aiming to investigate those tertiary structures using FRET, however, relies on prior knowledge about conformational properties of the nucleobase, which govern exciton transfer capabilities. Herein we employed classical molecular dynamics combined with Förster exciton theory to elucidate the preferred orientation relative to proximate bases and the influence on exciton transfer efficiency in multiple substitution sites. We did this by comparing the chromophoric distances emergent from MD simulations with experimental FRET data based on structural data of the native aptamer. We present the outlined methodology as a means to reliably evaluate future nucleobase analogue candidates in terms of their structural behavior and emergent exciton transfer properties as exemplified in the study of the preferred orientation of 4-cyanoindole in the Chili RNA aptamer.

Exploration of SAM-I riboswitch inhibitors: In-Silico discovery of ligands to a new target employing multistage CADD approaches

Targeting riboswitches, regulatory elements responsible for the expression of essential genes, is taking central stage in the new era of antibacterial medications discovery due to the emergence of antibiotic resistance. The S-Adenosyl methionine-I (SAM-I) riboswitch works through transcription termination in a negative feedback manner modulated by the natural ligand SAM. SAM-I riboswitch is specific to bacteria and found mainly in gram-positive bacteria such as Bacillus anthracis. Analyzing the interactions of the co-crystallized structure of SAM-I riboswitch aptamer with its native ligand SAM clarified the needed chemical structural features to achieve binding. Acknowledging those features, structure-based and ligand-based pharmacophore models were built for filtration use in screening the OTAVA Chemical library and the Pubchem database. For further filtration enhancement, the physicochemical properties of SAM were used as a second filtration criterion. Compounds obtained as output from previous steps were energy minimized, and the lowest energy conformer structures were docked to SAM-I using MOE, v.2019.01. S-score and ligand interactions were used to assess the best hits. This yielded eight promising compounds to which molecular dynamics (MD) simulations with SAM-I aptamer were applied using GROMACS 2020.3 package affirming stable binding interactions and binding energetics similar to SAM. Moreover, pharmacokinetic and drug-like properties of those eight hits were assessed using SWISS-ADME. According to the combined computational methods and PK/Tox assessment, compound 20 was the most promising and thus can be considered a lead for future evaluation and optimization as a candidate new antibacterial agent targeting a new biomolecule eliciting a new mechanism of action.

Magnetic lanthanide sensor with self-ratiometric time-resolved luminescence for accurate detection of epithelial cancerous exosomes.

Fluorescence-based LB (liquid biopsy) offers a rapid means of detecting cancer non-invasively. However, the widespread issue of sample loss during purification steps will diminish the accuracy of detection results. Therefore, in this study, we introduce a magnetic lanthanide sensor (MLS) designed for sensitive detection of the characteristic protein, epithelial cell adhesion molecule (EpCAM), on epithelial tumor exosomes. By leveraging the inherent multi-peak emission and time-resolved properties of the sole-component lanthanide element, combined with the self-ratiometric strategy, MLS can overcome limitations imposed by manual operation and/or sample complexity, thereby providing more stable and reliable output results. Specifically, terbium-doped NaYF4 nanoparticles (NaYF4:Tb) and deformable aptamers terminated with BHQ1 were sequentially introduced onto superparamagnetic silica-decorated Fe3O4 nanoparticles. Prior to target binding, emission from NaYF4:Tb at 543 nm was partially quenched due to the fluorescence resonance energy transfer (FRET) from NaYF4:Tb to BHQ1. Upon target binding, changes in the secondary structure of aptamers led to the fluorescence intensity increasing since the deconfinement of distance-dependent FRET effect. The characteristic emission of NaYF4:Tb at 543 nm was then utilized as the detection signal (I1), while the less changed emission at 583 nm served as the reference signal (I2), further reporting the self-ratiometric values of I1 and I2 (I1/I2) to illustrate the epithelial cancerous features of exosomes while ignoring possible sample loss. Consequently, over a wide range of exosome concentrations (2.28 × 102-2.28 × 108 particles per mL), the I1/I2 ratio exhibited a linear increase with exosome concentration [Y(I1/I2) = 0.166 lg (Nexosomes) + 3.0269, R2 = 0.9915], achieving a theoretical detection limit as low as 24 particles per mL. Additionally, MLS effectively distinguished epithelial cancer samples from healthy samples, showcasing significant potential for clinical diagnosis.

Signaling redox probe/DNA aptamer complexes on a new POP/2D WSe2 composite-based immunosensor towards the simultaneous detection of three-protein overexpression as an alternative severe SARS-COV-2 infection diagnosis

The coronavirus disease 2019 (COVID-19) has become a global pandemic in which patients with symptoms are infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical investigation of human immune response cytokines or/and overexpressed proteins at trace cut-off levels can be valuable to monitor body function failures and, eventually, improve the prognosis for patients. Nevertheless, the currently available technologies are still limited. Herein, we provide the first sandwich-like immunosensor that has the potential to detect multiple protein biomarkers in a single measurement. The sandwich-like structure was created by coating a three-screen printed carbon electrode (3SPCE) array with a porous organic polymer (POP)/2D WSe2 nanocomposite. This system enables the simultaneous immunoassay of C-reactive protein (CRP), native cardiac troponin I (cTnI), and procalcitonin (PCT), which together provide a panel of symptom-diagnostic biomarkers for the presence and severity of COVID-19 infection. The biomarker detection principle relies on three antibodies as bioreceptor probes bound on the modified 3SPCE and three aptamers to generate three redox probe complexes as signal amplifiers. After incorporating three redox species (Cd2+, methylene blue (MB), and Ag+) into the aptamer structure, well-differentiated oxidation peaks can be achieved, indicating the concentrations of these three proteins. Under the optimized conditions of the stepwise fabrications, the linear dynamic range of three-protein detection by our proposed multiplex biosensor ranges from 100 to 50,000 pg mL–1. The limits of detection (LODs) are as low as 62, 81, and 44 pg mL–1 for the detection of CRP, cTnI, and PCT, respectively. As proof of the concept, this multiplexed assay demonstrates outstanding analytical performance with excellent reproducibility, high sensitivity and selectivity, and acceptable recoveries of the detection in human serum. Therefore, the multiplexed sensor could be an alternative tool in the field of clinical analysis for the diagnosis of COVID-19 severity or other emerging infectious diseases.

Selection of Aptamers for Use as Molecular Probes in AFM Detection of Proteins.

Currently, there is great interest in the development of highly sensitive bioanalytical systems for diagnosing diseases at an early stage, when pathological biomarkers are present in biological fluids at low concentrations and there are no clinical manifestations. A promising direction is the use of molecular detectors-highly sensitive devices that detect signals from single biomacromolecules. A typical detector in this class is the atomic force microscope (AFM). The high sensitivity of an AFM-based bioanalysis system is determined by the size of the sensing element of an atomic force microscope-the cantilever-the radius of the curvature of which is comparable to that of a biomolecule. Biospecific molecular probe-target interactions are used to ensure detection system specificity. Antibodies, aptamers, synthetic antibodies, and peptides can be used as molecular probes. This study has demonstrated the possibility of using aptamers as molecular probes for AFM-based detection of the ovarian cancer biomarker CA125. Antigen detection in a nanomolar solution was carried out using AFM chips with immobilized aptamers, commercially available or synthesized based on sequences from open sources. Both aptamer types can be used for antigen detection, but the availability of sequence information enables additional modeling of the aptamer structure with allowance for modifications necessary for immobilization of the aptamer on an AFM chip surface. Information on the structure and oligomeric composition of aptamers in the solution was acquired by combining small-angle X-ray scattering and molecular modeling. Modeling enabled pre-selection, before the experimental stage, of aptamers for use as surface-immobilized molecular probes.

Interfacing Aptamer-Modified Nanopipettes with Neuronal Media and Ex Vivo Brain Tissue.

Aptamer-functionalized biosensors exhibit high selectivity for monitoring neurotransmitters in complex environments. We translated nanoscale aptamer-modified nanopipette sensors to detect endogenous dopamine release in vitro and ex vivo. These sensors employ quartz nanopipettes with nanoscale pores (ca. 10 nm diameter) that are functionalized with aptamers that enable the selective capture of dopamine through target-specific conformational changes. The dynamic behavior of aptamer structures upon dopamine binding leads to the rearrangement of surface charge within the nanopore, resulting in measurable changes in ionic current. To assess sensor performance in real time, we designed a fluidic platform to characterize the temporal dynamics of nanopipette sensors. We then conducted differential biosensing by deploying control sensors modified with nonspecific DNA alongside dopamine-specific sensors in biological milieu. Our results confirm the functionality of aptamer-modified nanopipettes for direct measurements in undiluted complex fluids, specifically in the culture media of human-induced pluripotent stem cell-derived dopaminergic neurons. Moreover, sensor implantation and repeated measurements in acute brain slices was possible, likely owing to the protected sensing area inside nanoscale DNA-filled orifices, minimizing exposure to nonspecific interferents and preventing clogging. Further, differential recordings of endogenous dopamine released through electrical stimulation in the dorsolateral striatum demonstrate the potential of aptamer-modified nanopipettes for ex vivo recordings with unprecedented spatial resolution and reduced tissue damage.

Advancements in Rapid and Affordable Diagnostic Testing for Respiratory Infectious Diseases: Evaluation of Aptamer Beacon Technology for Rapid and Sensitive Detection of SAR-CoV-2 in Breath Condensate.

The demand for rapid and efficient diagnostic point-of-care tests for respiratory infectious diseases has become increasingly critical in the current landscape. The emphasis on accessibility has been underscored over the past year, making it crucial to have biological components that exhibit fast and accurate kinetics. The foundation for precise, swift, and effective testing relies on the availability of highly responsive biological agents. Two published aptamer DNA sequences designated Song and MSA52 and their truncated internal stem-loop structures were studied for their potential to serve as aptamer beacons for rapid COVID detection. The candidate beacons were covalently labeled with Atto 633 dye attached to their 5' ends and Iowa Black quencher attached to their 3' ends. The whole aptamer structures exhibited the greatest fluorescence signal intensities and higher fluorescence background than their truncated internal stem-loop beacon structures suggesting that the distance between fluorophores and quenchers was greater for the whole aptamer beacon candidates versus the isolated stem-loop structures. Beacon candidates were tested against two heat- or gamma radiation-killed SARS-CoV-2 Washington 1/2020 virus samples and three different COVID spike (S) proteins to test their effectiveness. Despite the higher background fluorescence, the whole aptamer beacons showed better signal-to-noise ratios and were selected for further investigation. Limit of detection (LOD) studies revealed that both the whole Song and whole MSA52 aptamer beacon candidates had a LOD of 9.61 × 103 genome equivalents in phosphate-buffered saline using the red channel of a Promega Quantus™ fluorometer which correlated well with confirmatory spectrofluorometry. Cross-reactivity studies using numerous COVID variants, related coronaviruses, and other common respiratory pathogens suggested greater COVID selectivity for the whole MSA52 versus the whole Song aptamer beacon candidate, indicating promise for specific COVID detection. Importantly, both whole aptamer beacon candidates exhibited very rapid "bind and detect" fluorescence increases within the first 1-2min of mixing the beacons with killed SARS-CoV-2 viruses in 100µl samples. Overall, this work illustrates the strong potential for aptamer beacons for rapid, on-site detection and presumptive diagnosis of COVID in breath condensates or other small liquid samples. This research highlights the strong potential of aptamer beacons for addressing the need for fast and convenient diagnostic tools in global health contexts, especially in resource-limited settings.