AptaDiff: de novo design and optimization of aptamers based on diffusion models.

Aptamers are single-stranded nucleic acid ligands, featuring high affinity and specificity to target molecules. Traditionally they are identified from large DNA/RNA libraries using $in vitro$ methods, like Systematic Evolution of Ligands by Exponential Enrichment (SELEX). However, these libraries capture only a small fraction of theoretical sequence space, and various aptamer candidates are constrained by actual sequencing capabilities from the experiment. Addressing this, we proposed AptaDiff, the first in silico aptamer design and optimization method based on the diffusion model. Our Aptadiff can generate aptamers beyond the constraints of high-throughput sequencing data, leveraging motif-dependent latent embeddings from variational autoencoder, and can optimize aptamers by affinity-guided aptamer generation according to Bayesian optimization. Comparative evaluations revealed AptaDiff's superiority over existing aptamer generation methods in terms of quality and fidelity across four high-throughput screening data targeting distinct proteins. Moreover, surface plasmon resonance experiments were conducted to validate the binding affinity of aptamers generated through Bayesian optimization for two target proteins. The results unveiled a significant boost of $87.9\%$ and $60.2\%$ in RU values, along with a 3.6-fold and 2.4-fold decrease in KD values for the respective target proteins. Notably, the optimized aptamers demonstrated superior binding affinity compared to top experimental candidates selected through SELEX, underscoring the promising outcomes of our AptaDiff in accelerating the discovery of superior aptamers.

Aptamer-Protein Interactions: From Regulation to Biomolecular Detection.

Serving as the basis of cell life, interactions between nucleic acids and proteins play essential roles in fundamental cellular processes. Aptamers are unique single-stranded oligonucleotides generated by in vitro evolution methods, possessing the ability to interact with proteins specifically. Altering the structure of aptamers will largely modulate their interactions with proteins and further affect related cellular behaviors. Recently, with the in-depth research of aptamer-protein interactions, the analytical assays based on their interactions have been widely developed and become a powerful tool for biomolecular detection. There are some insightful reviews on aptamers applied in protein detection, while few systematic discussions are from the perspective of regulating aptamer-protein interactions. Herein, we comprehensively introduce the methods for regulating aptamer-protein interactions and elaborate on the detection techniques for analyzing aptamer-protein interactions. Additionally, this review provides a broad summary of analytical assays based on the regulation of aptamer-protein interactions for detecting biomolecules. Finally, we present our perspectives regarding the opportunities and challenges of analytical assays for biological analysis, aiming to provide guidance for disease mechanism research and drug discovery.

High-throughput quantitative binding analysis of DNA aptamers using exonucleases.

Aptamers are nucleic acid bioreceptors that have been used in various applications including medical diagnostics and as therapeutic agents. Identifying the most optimal aptamer for a particular application is very challenging. Here, we for the first time have developed a high-throughput method for accurately quantifying aptamer binding affinity, specificity, and cross-reactivity via the kinetics of aptamer digestion by exonucleases. We demonstrate the utility of this approach by isolating a set of new aptamers for fentanyl and its analogs, and then characterizing the binding properties of 655 aptamer-ligand pairs using our exonuclease digestion assay and validating the results with gold-standard methodologies. These data were used to select optimal aptamers for the development of new sensors that detect fentanyl and its analogs in different analytical contexts. Our approach dramatically accelerates the aptamer characterization process and streamlines sensor development, and if coupled with robotics, could enable high-throughput quantitative analysis of thousands of aptamer-ligand pairs.

Selection of allosteric dnazymes that can sense phenylalanine by expression-SELEX.

Aptamers are ligand-binding RNA or DNA molecules and have been widely examined as biosensors, diagnostic tools, and therapeutic agents. The application of aptamers as biosensors commonly requires an expression platform to produce a signal to report the aptamer-ligand binding event. Traditionally, aptamer selection and expression platform integration are two independent steps and the aptamer selection requires the immobilization of either the aptamer or the ligand. These drawbacks can be easily overcome through the selection of allosteric DNAzymes (aptazymes). Herein, we used the technique of Expression-SELEX developed in our laboratory to select for aptazymes that can be specifically activated by low concentrations of l-phenylalanine. We chose a previous DNA-cleaving DNAzyme known as II-R1 as the expression platform for its low cleavage rate and used stringent selection conditions to drive the selection of high-performance aptazyme candidates. Three aptazymes were chosen for detailed characterization and these DNAzymes were found to exhibit a dissociation constant for l-phenylalanine as low as 4.8 µM, a catalytic rate constant improvement as high as 20 000-fold in the presence of l-phenylalanine, and the ability to discriminate against closely related l-phenylalanine analogs including d-phenylalanine. This work has established the Expression-SELEX as an effective SELEX method to enrich high-quality ligand-responsive aptazymes.

Rapid Nuclease-Assisted Selection of High-Affinity Small-Molecule Aptamers.

Aptamers are nucleic acid bioreceptors that have been widely utilized for a variety of biosensing applications, including in vivo detection methods that would not be possible with antibody-based systems. However, it remains challenging to generate high-quality aptamers for small molecule targets, particularly for use under physiological conditions. We present a highly effective aptamer selection technology for small-molecule targets that utilizes the nuclease EcoRI to remove nonspecific or weakly binding sequences in solution phase, rapidly enriching high-affinity target binders within just a few rounds of selection. As proof-of-concept, we used our nuclease-assisted SELEX (NA-SELEX) method to isolate aptamers for a synthetic cannabinoid, AB-FUBINACA. Within five rounds, we identified two highly specific aptamers that exhibit nanomolar affinity at physiological temperature. We also demonstrate the robustness and reproducibility of NA-SELEX by performing the same selection experiment with fresh reagents and libraries, obtaining the same two aptamers as well as two other high-quality aptamer candidates. Finally, we compare NA-SELEX against a conventional library-immobilized SELEX screen for AB-FUBINACA using the same screening conditions, identifying aptamers with 25-100-fold weaker affinity after 11 rounds of selection. NA-SELEX therefore could be an effective selection method for the isolation of high-quality aptamers for small-molecule targets.

Efficient Strategy to Discover DNA Aptamers Against Low Abundance Cell Surface Proteins in Scarce Samples.

Molecular recognition probes targeting cell surface proteins such as aptamers play crucial roles in precise diagnostics and therapy. However, the selection of aptamers against low-abundance proteins in situ on the cell surface, especially in scarce samples, remains an unmet challenge. In this study, we present a single-round, single-cell aptamer selection method by employing a digital DNA sequencing strategy, termed DiDS selection, to address this dilemma. This approach incorporates a molecular identification card for each DNA template, thereby mitigating biases introduced by multiple PCR amplifications and ensuring the accurate identification of aptamer candidates. Through DiDS selection, we successfully obtained a series of high-quality aptamers against cell lines, clinical specimens, and neurons. Subsequent analyses for target identification revealed that aptamers derived from DiDS selection exhibit recognition capabilities for proteins with varying abundance levels. In contrast, multiple rounds of selection resulted in the enrichment of only one aptamer targeting a high-abundance target. Moreover, the comprehensive profiling of cell surfaces at the single-cell level, utilizing an enriched aptamer pool, revealed unique molecular patterns for each cell line. This streamlined approach holds promise for the rapid generation of specific recognition molecules targeting cell surface proteins across a broad range of expression levels and expands its applications in cell profiling, specific probe identification, biomarker discovery, etc.

Biomimetic Bacterial Capsule for Enhanced Aptamer Display and Cell Recognition.

Great effort has been made to encapsulate or coat living mammalian cells for a variety of applications ranging from diabetes treatment to three-dimensional printing. However, no study has reported the synthesis of a biomimetic bacterial capsule to display high-affinity aptamers on the cell surface for enhanced cell recognition. Therefore, we synthesized an ultrathin alginate-polylysine coating to display aptamers on the surface of living cells with natural killer (NK) cells as a model. The results show that this coating-mediated aptamer display is more stable than direct cholesterol insertion into the lipid bilayer. The half-life of the aptamer on the cell surface can be increased from less than 1.5 to over 20 h. NK cells coated with the biomimetic bacterial capsule exhibit a high efficiency in recognizing and killing target cells. Therefore, this work has demonstrated a promising cell coating method for the display of aptamers for enhanced cell recognition.

FAM Tag Size Separation-Based Capture-Systematic Evolution of Ligands by Exponential Enrichment for Sterigmatocystin-Binding Aptamers with High Specificity.

Sterigmatocystin (ST) is a known toxin whose aptamer has rarely been reported because ST is a water-insoluble small-molecule target with few active sites, leading to difficulty in obtaining its aptamer using traditional target fixation screening methods. To obtain aptamer for ST, we incorporated FAM tag size separation into the capture-systematic evolution of ligands by exponential enrichment and combined it with molecular activation for aptamer screening. The screening process was monitored using a quantitative polymerase chain reaction fluorescence amplification curve and recovery of negative-, counter-, and positive-selected ssDNA. The affinity and specificity of the aptamer were verified by constructing an aptamer-affinity column, and the binding sites were predicted using molecular docking simulations. The results showed that the Kd value of the H Seq02 aptamer was 25.3 nM. The aptamer-affinity column based on 2.3 nmol of H Seq02 exhibited a capacity of about 80 ng, demonstrating better specificity than commercially available antibody affinity columns. Molecular simulation docking predicted the binding sites for H Seq02 and ST, further explaining the improved specificity. In addition, circular dichroism and isothermal titration calorimetry were used to verify the interaction between the aptamer and target ST. This study lays the foundation for the development of a new ST detection method.

Are Aptamers Really Promising as Receptors for Analytical Purposes? Insights into Anti-Lysozyme DNA Aptamers through a Multitechnique Study.

Aptamers are recognition elements increasingly used for the development of biosensing strategies, especially in the detection of proteins or small molecule targets. Lysozyme, which is recognized as an important biomarker for various diseases and a major allergenic protein found in egg whites, is one of the main analytical targets of aptamer-based biosensors. However, since aptamer-based strategies can be prone to artifacts and data misinterpretation, rigorous strategies for multifaceted characterization of the aptamer-target interaction are needed. In this work, a multitechnique approach has been devised to get further insights into the binding performance of the anti-lysozyme DNA aptamers commonly used in the literature. To study molecular interactions between lysozyme and different anti-lysozyme DNA aptamers, measurements based on a magneto-electrochemical apta-assay, circular dichroism spectroscopy, fluorescence spectroscopy, and asymmetrical flow field-flow fractionation were performed. The reliability and versatility of the approach were proved by investigating a SELEX-selected RNA aptamer reported in the literature, that acts as a positive control. The results confirmed that an interaction in the low micromolar range is present in the investigated binding buffers, and the binding is not associated with a conformational change of either the protein or the DNA aptamer. The similar behavior of the anti-lysozyme DNA aptamers compared to that of randomized sequences and polythymine, used as negative controls, showed nonsequence-specific interactions. This study demonstrates that severe testing of aptamers resulting from SELEX selection is the unique way to push these biorecognition elements toward reliable and reproducible results in the analytical field.

Aptamer-Based Multiparameter Analysis for Molecular Profiling of Hematological Malignancies.

The subtypes of hematological malignancies (HM) with minimal molecular profile differences display an extremely heterogeneous clinical course and a discrepant response to certain treatment regimens. Profiling the surface protein markers offers a potent solution for precision diagnosis of HM by differentiating among the subtypes of cancer cells. Herein, we report the use of Cell-SELEX technology to generate a panel of high-affinity aptamer probes that are able to discriminate subtle differences among surface protein profiles between different HM cells. Experimental results show that these aptamers with apparent dissociation constants (Kd) below 10 nM display a unique recognition pattern on different HM subtypes. By combining a machine learning model on the basis of partial least-squares discriminant analysis, 100% accuracy was achieved for the classification of different HM cells. Furthermore, we preliminarily validated the effectiveness of the aptamer-based multiparameter analysis strategy from a clinical perspective by accurately classifying complex clinical samples, thus providing a promising molecular tool for precise HM phenotyping.

Screening of Cross-Reactive Aptamers for the Detection of 24 Quinolones by Using a Liebig's Law-Guided Parallel-Series Strategy.

Quinolones, a widely used class of antibiotics, present significant environmental and health concerns if they excessively remain in the environment and in food. Aptamers specific to quinolones can be applied as bioreceptors for the detection of quinolone residues in the environment and food. The quinolone family contains dozens of different individuals that share the same core structure coupled with various substituents at six different positions. The diversity and complexity of the substitution sites make it a challenge to choose a set of representative molecules that encompass all the desired sites and preserve the core molecular framework for the screening of quinolone-specific aptamers via systematic evolution of ligands by exponential enrichment (SELEX). To address this challenge, we introduce a novel parallel-series strategy guided by Liebig's law for isolating quinolone-specific cross-reactive aptamers by using the library-immobilized SELEX method. Through this approach, we successfully identified 5 aptamers (Apt.AQ01-Apt.AQ05) with high binding affinity and excellent specificity to 24 different quinolone individuals. Among them, Apt.AQ03 showcased optimal performance with affinities ranging from 0.14 to 1.07 µM across the comprehensive set of 24 quinolones, exhibiting excellent specificity against nontarget interferents. The binding performance of Apt.AQ03 was further characterized with microscale thermophoresis, circular dichroism spectra, and an exonuclease digestion assay. By using Apt.AQ03 as a bioreceptor, a fluorescence resonance energy transfer (FRET) aptasensor was developed for the detection of 24 quinolones in milk, achieving a remarkable detection limit of 14.5-21.8 ng/mL. This work not only establishes a robust and effective strategy for selecting cross-reactive aptamers applicable to other small-molecule families but also provides high-quality aptamers for developing various high-throughput and reliable methods for the detection of multiple quinolone residues in food.

Streamlining RNA Aptamer Selection via Unique Molecular Identifiers and High-Throughput Sequencing.

Aptamers are valuable tools for applications such as cell imaging, drug delivery, and therapeutics. RNA aptamers, in particular, exhibit complex structural diversity and flexibility, affording higher affinity and specificity, broader target recognition, and easier chemical modification compared with DNA aptamers. However, traditional selection methods for RNA aptamers are time-consuming and involve numerous rounds of screening, thus limiting their widespread application. To overcome this challenge, we propose an efficient truncated selection approach termed ID-SELEX. This method incorporates a molecular identification marker whereby each template is labeled with a unique molecular identifier, or UMI. Such incorporation helps mitigate biases introduced by multiple polymerase chain reaction (PCR) amplification during high-throughput sequencing, ensuring accurate identification of aptamer candidates. Utilizing ID-SELEX, we successfully identified a panel of high-quality aptamers targeting the human colon cancer cell line HCT-8 in just 2 rounds of selection. Furthermore, we demonstrated the versatility of this strategy by selecting 6 RNA aptamers targeting mouse myoblast cell line C2C12 with only one round of selection. In summary, RNA aptamer selection based on ID-SELEX utilizes high-throughput sequencing and UMI labeling to enable the rapid screening of RNA aptamers across human and murine cell lines. As such, ID-SELEX has the potential to facilitate RNA aptamer discovery, providing a novel molecular tool for biomedical research, clinical applications, and precision medicine.

Biophysical characterization and design of a minimal version of the Hoechst RNA aptamer.

RNA aptamers are oligonucleotides, selected through Systematic Evolution of Ligands by EXponential Enrichment (SELEX), that can bind to specific target molecules with high affinity. One such molecule is the RNA aptamer that binds to a blue-fluorescent Hoechst dye that was modified with bulky t-Bu groups to prevent non-specific binding to DNA. This aptamer has potential for biosensor applications; however, limited information is available regarding its conformation, molecular interactions with the ligand, and binding mechanism. The study presented here aims to biophysically characterize the Hoechst RNA aptamer when complexed with the t-Bu Hoechst dye and to further optimize the RNA sequence by designing and synthesizing new sequence variants. Each variant aptamer-t-Bu Hoechst complex was evaluated through a combination of fluorescence emission, native polyacrylamide gel electrophoresis, fluorescence titration, and isothermal titration calorimetry experiments. The results were used to design a minimal version of the aptamer consisting of only 21 nucleotides. The performed study also describes a more efficient method for synthesizing the t-Bu Hoechst dye derivative. Understanding the biophysical properties of the t-Bu Hoechst dye-RNA complex lays the foundation for nuclear magnetic resonance spectroscopy studies and its potential development as a building block for an aptamer-based biosensor that can be used in medical, environmental or laboratory settings.

Interrogating Aptamer Chemical Space Through Modified Nucleotide Substitution Facilitated by Enzymatic DNA Synthesis.

Chemical modification of aptamers is an important step to improve their performance and stability in biological media. This can be performed either during their identification (mod-SELEX) or after the in vitro selection process (post-SELEX). In order to reduce the complexity and workload of the post-SELEX modification of aptamers, we have evaluated the possibility of improving a previously reported, chemically modified aptamer by combining enzymatic synthesis and nucleotides bearing bioisosteres of the parent cubane side-chains or substituted cubane moieties. This method lowers the synthetic burden often associated with post-SELEX approaches and allowed to identify one additional sequence that maintains binding to the PvLDH target protein, albeit with reduced specificity. In addition, while bioisosteres often improve the potency of small molecule drugs, this does not extend to chemically modified aptamers. Overall, this versatile method can be applied for the post-SELEX modification of other aptamers and functional nucleic acids.

Selection and Characterization of DNA Aptamers for Cytidine and Uridine.

Cytidine and uridine are two essential pyrimidine ribonucleotides, and accurate detection of these nucleosides holds significant biological importance. While many aptamers were reported to bind purines, little success was achieved for pyrimidine binding. This study employs the library-immobilization capture-SELEX technique to isolate aptamers capable of selectively binding to cytidine and uridine. First, a selection was performed using a mixture of cytidine and uridine as the target. This selection led to the isolation of a highly selective aptamer for cytidine with a dissociation constant (Kd ) of 0.9 µM as determined by isothermal titration calorimetry (ITC). In addition, a dual-recognition aptamer was also discovered, which exhibited selective binding to both cytidine and uridine. Subsequently, a separate selection was carried out using uridine as the sole target, and the resulting uridine aptamer displayed a Kd of 4 µM based on a thioflavin T fluorescence assay and a Kd of 102 µM based on ITC. These aptamers do not have a strict requirement of metal ions for binding, and they showed excellent selectivity since no binding was observed with their nucleobases or nucleotides. This study has resulted three aptamers for pyrimidines, which can be employed in biosensors and DNA switches.

Using Exonucleases for Aptamer Characterization, Engineering, and Sensing.

Aptamers are short, single-stranded nucleic acids that have been selected from random libraries to bind specific molecules with high affinity via an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX). They have been generated for diverse targets ranging from metal ions to small molecules to proteins and have demonstrated considerable promise as biorecognition elements in sensors for applications including medical diagnostics, environmental monitoring, food safety, and forensic analysis. While aptamer sensors have made great strides in terms of sensitivity, specificity, turnaround time, and ease of use, several challenges have hindered their broader adoption. These include inadequate sensitivity, bottlenecks in aptamer binding characterization, and the cost and labor associated with aptamer engineering. In this Account, we describe our successes in using nuclease enzymes to address these problems. While working with nucleases to enhance the sensitivity of split aptamer sensors via enzyme-assisted target recycling, we serendipitously discovered that the digestion of DNA aptamers by exonucleases is inhibited when an aptamer is bound to a ligand. This finding served as the foundation for the development of three novel aptamer-related methodologies in our laboratory. First, we used exonucleases to truncate nonessential nucleotides from aptamers to generate structure-switching aptamers in a single step, greatly simplifying the aptamer engineering process. Second, we used exonucleases to develop a label-free aptamer-based detection platform that can utilize aptamers directly obtained from in vitro selection to detect analytes with ultralow background and high sensitivity. Through this approach, we were able to detect analytes at nanomolar levels in biological samples, with the capacity for achieving multiplexed detection by using molecular beacons. Finally, we used exonucleases to develop a high throughput means of characterizing aptamer affinity and specificity for a variety of ligands. This approach has enabled more comprehensive analysis of aptamers by greatly increasing the number of aptamer candidates and aptamer-ligand pairs that can be tested in a single experiment. We have also demonstrated the success of this method as a means for identifying new mutant aptamers with augmented binding properties and for quantifying aptamer-target affinity. Our enzymatic technologies can greatly streamline the aptamer characterization and sensor development process, and with the adoption of robotics or liquid handling systems in the future, it should be possible to rapidly identify the most suitable aptamers for a particular application from hundreds to thousands of candidates.

Advances in nucleic acid aptamer-based detection of respiratory virus and bacteria: a mini review.

Respiratory pathogens infecting the human respiratory system are characterized by their diversity, high infectivity, rapid transmission, and acute onset. Traditional detection methods are time-consuming, have low sensitivity, and lack specificity, failing to meet the needs of rapid clinical diagnosis. Nucleic acid aptamers, as an emerging and innovative detection technology, offer novel solutions with high specificity, affinity, and broad target applicability, making them particularly promising for respiratory pathogen detection. This review highlights the progress in the research and application of nucleic acid aptamers for detecting respiratory pathogens, discussing their selection, application, potential in clinical diagnosis, and future development. Notably, these aptamers can significantly enhance the sensitivity and specificity of detection when combined with detection techniques such as fluorescence, colorimetry and electrochemistry. This review offers new insights into how aptamers can address the limitations of traditional diagnostic methods and advance clinical diagnostics. It also highlights key challenges and future research directions for the clinical application of nucleic acid aptamers.

DAPTEV: Deep aptamer evolutionary modelling for COVID-19 drug design.

Typical drug discovery and development processes are costly, time consuming and often biased by expert opinion. Aptamers are short, single-stranded oligonucleotides (RNA/DNA) that bind to target proteins and other types of biomolecules. Compared with small-molecule drugs, aptamers can bind to their targets with high affinity (binding strength) and specificity (uniquely interacting with the target only). The conventional development process for aptamers utilizes a manual process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX), which is costly, slow, dependent on library choice and often produces aptamers that are not optimized. To address these challenges, in this research, we create an intelligent approach, named DAPTEV, for generating and evolving aptamer sequences to support aptamer-based drug discovery and development. Using the COVID-19 spike protein as a target, our computational results suggest that DAPTEV is able to produce structurally complex aptamers with strong binding affinities.

In silico selection of aptamers against SARS-CoV-2.

Aptamers are molecular recognition elements that have been extensively deployed in a wide array of applications ranging from diagnostics to therapeutics. Due to their unique properties as compared to antibodies, aptamers were also largely isolated during the COVID-19 pandemic for multiple purposes. Typically generated by conventional SELEX, the inherent drawbacks of the process including the time-consuming, cumbersome and resource-intensive nature catalysed the move to adopt in silico approaches to isolate aptamers. Impressive performances of these in silico-derived aptamers in their respective assays have been documented thus far, bearing testimony to the huge potential of the in silico approaches, akin to the traditional SELEX in isolating aptamers. In this study, we provide an overview of the in silico selection of aptamers against SARS-CoV-2 by providing insights into the basic steps involved, which comprise the selection of the initial single-stranded nucleic acids, determination of the secondary and tertiary structures and in silico approaches that include both rigid docking and molecular dynamics simulations. The different approaches involving aptamers against SARS-CoV-2 were illuminated and the need to verify these aptamers by experimental validation was also emphasized. Cognizant of the need to continuously improve aptamers, the strategies embraced thus far for post-in silico selection modifications were enumerated. Shedding light on the steps involved in the in silico selection can set the stage for further improvisation to augment the functionalities of the aptamers in the future.

APIPred: An XGBoost-Based Method for Predicting Aptamer-Protein Interactions.

Aptamers are single-stranded DNA or RNA oligos that can bind to a variety of targets with high specificity and selectivity and thus are widely used in the field of biosensing and disease therapies. Aptamers are generated by SELEX, which is a time-consuming procedure. In this study, using in silico and computational tools, we attempt to predict whether an aptamer can interact with a specific protein target. We present multiple data representations of protein and aptamer pairs and multiple machine-learning-based models to predict aptamer-protein interactions with a fair degree of selectivity. One of our models showed 96.5% accuracy and 97% precision, which are significantly better than those of the previously reported models. Additionally, we used molecular docking and SPR binding assays for two aptamers and the predicted targets as examples to exhibit the robustness of the APIPred algorithm. This reported model can be used for the high throughput screening of aptamer-protein pairs for targeting cancer and rapidly evolving viral epidemics.