In recent years, aptamers have emerged as versatile molecular tools with promising applications in various fields, including diagnostics and therapeutics. In livestock reproduction, their application holds promise for improving the sorting and identification of X and Y chromosome-bearing sperm cells, which is essential for increasing productivity in the dairy and beef industries.

This study utilized seven rounds of Cell-SELEX using bovine X and Y sperm cells to isolate specific aptamers that target these cells. A comprehensive in-silico analysis was conducted to evaluate the binding interactions between the selected aptamer sequences and the differentially expressed plasma membrane proteins of X and Y sperm cells.

The analysis identified the aptamer sequences APT1X, APT2X, and APT5X as having the most stable interactions with the X sperm surface proteins TLR8 (Toll-like receptor 8), CLRN3, and TLR7 (Toll-like receptor 7), respectively. APT2Y exhibited a relatively high affinity for the protein SCAMP1, a Y-sperm-specific protein. Aptamer‒protein interactions are characterized by hydrogen bonds and hydrophobic contacts. Notably, APT1X formed the greatest number of hydrogen bonds with the polar residues of TLR8, whereas TLR7-APT5X interactions exhibited the greatest number of hydrophobic contacts.

The use of in-silico analysis for evaluating the interaction between candidate aptamer sequences and differentially expressed X and Y bovine sperm proteins provides valuable insights. This approach might facilitate the sorting of bovine X and Y sperm cells, contributing to advancements in livestock reproduction strategies.

Oligonucleotide aptamers are typically identified through a rigorous and time-consuming process known as systematic evolution of ligands by exponential enrichment (SELEX), which requires 20 to 30 iterative rounds to eliminate non/weak binding sequences and enrich tight binding sequences with high affinity. Moreover, inherent experimental biases and non-specific interactions within SELEX could inadvertently exclude high-affinity candidates, leading to a high failure rate. To address these challenges, we proposed DeepAptamer for identifying high-affinity sequences from unenriched early SELEX rounds. As a hybrid neural network model combining convolutional neural networks and bidirectional long short-term memory, DeepAptamer integrated sequence composition and structural features to predict aptamer binding affinities and potential binding motifs. Trained on comprehensive SELEX data, DeepAptamer outperformed existing models in accuracy as substantiated by experimental evidence. More importantly, DeepAptamer effectively identified key nucleotides for target binding. DeepAptamer can efficiently identify high-affinity aptamers against various targets, enhancing its potential to discover promising sequences in initial screening stages and obviating the 20-30 iterative selection rounds required for full enrichment of selection pools. This represented a notable leap forward in aptamer technology, with broad implications for its application across a spectrum of selection targets.

Continuous biosensors have the potential to transform medicine, enabling healthcare to be more preventative and personalized as compared to the current standard of reactive diagnostics. Realizing this transformative potential requires biosensors that can function continuously in vivo without sample preparation and deliver molecular specificity, sensitivity, and high temporal resolution. Molecular switches stand out as a promising solution for creating such sensors for the continuous detection of many different types of molecules. Molecular switches are target-binding receptors designed such that binding causes a conformational change in the switch's structure. This structure switching induces a measurable signal change via reporters incorporated into the molecular switch, enabling highly specific, label-free sensing. However, there remains an outstanding need for generalizable switch designs that can be adapted for the detection of a wide range of molecular targets. In this Account, we chronicle the work our lab has done to develop generalizable molecular switch designs that allow more rapid development of high-performance biosensors across a broad range of biomarkers. Pioneering efforts toward molecular switch-based biosensing have employed aptamers─nucleic acid-based receptors with sequence-specific target affinity. However, most of these early demonstrations relied upon aptamers with intrinsic structure-switching capabilities. To accelerate aptamer switch design for more targets, we have applied rational design and knowledge of an aptamer's structure to engineer switching functionality into pre-existing aptamers. Our designs contained several structural parameters that enabled us to easily tune the sensitivity and binding kinetics of the resulting switches. Using such rationally designed aptamer switches, we demonstrated continuous optical detection of cortisol and dopamine at physiologically relevant concentrations in complex media. In an effort to move beyond aptamers with well-characterized structural properties, we developed a high-throughput screening method that allowed us to simultaneously screen millions of candidates derived from a single aptamer to find sensitive switches without any prior structural knowledge of the parent aptamer. In subsequent work, we reasoned that we could enhance our ability to design a broader range of biosensors by leveraging other classes of receptors besides aptamers. Antibodies offer excellent affinity and specificity for a wide range of targets, but lack the capacity for intrinsic structure switching. We therefore developed a set of strategies to augment antibodies with the capacity to act as molecular switches with a diverse range of target-binding properties. We combined both the high binding affinity of an antibody with the structure-switching capabilities of an aptamer to develop a chimeric switch with 100-fold enhanced sensitivity for a protein target and improved function in interferent-rich samples. In a second design, we developed a competitive immunoassay-inspired scheme to engineer switching behavior into an antibody for minutes-scale temporal resolution with nanomolar sensitivity. We used this competitive antibody-switch to demonstrate the first continuous detection of cortisol directly in whole blood. Together, these advances in molecular switch development have expanded our capability to rapidly engineer new continuous biosensors, thereby increasing opportunities to track health via a wide range of biomarkers to deliver more personalized and preventative medicine.

Aptamers are synthetic oligonucleotides that bind with high affinity and specificity to various targets, making them invaluable for diagnostics, therapeutics, and biosensing. Microfluidic platforms can improve the efficiency and scalability of aptamer selection, especially through advancements in systematic evolution of ligands by exponential enrichment (SELEX) methods. Microfluidic SELEX methods are less time-consuming and labor-intensive and include critical steps like library preparation, binding, partitioning, and amplification. This review examines the contributions of microfluidic technology to SELEX-based aptamer identification, with alternative methods like conditional SELEX, in vivo-like SELEX and Non-SELEX for selecting aptamers and also discusses critical SELEX steps over the past decade. This work also examined the integrated microfluidic systems for SELEX, highlighting innovations such as conditional SELEX and in vivo-like SELEX. These advancements provide potential solutions to existing challenges in aptamer selection using conventional SELEX, especially concerning biological samples. A trend toward non-SELEX methods was also reviewed and discussed, wherein nucleic acid amplification was eliminated to improve aptamer selection. Microfluidic platforms have demonstrated versatility not only in aptamer selection but also in various detection applications; they allow for precise control of liquid flow and have been essential in the advancement of therapeutic aptamers, facilitating accurate screening, enhancing drug delivery systems, and enabling targeted therapeutic interventions. Although advances in microfluidic technology are expected to enhance aptamer-based diagnostics, therapeutics, and biosensing, challenges still persist, especially in up-scaling microfluidic systems for various clinical applications. The advantages and limitations of integrating microfluidic platforms with aptamer development are further addressed, emphasizing areas for future research. We also present a perspective on the future of microfluidic systems and aptamer technologies, highlighting their increasing significance in healthcare and diagnostics.

Aptamers, a new category of molecular probes, are overthrowing antibodies in molecular diagnostics. However, there are serious problems with using aptamers for this application including poor or non-specific binding in vivo conditions. Systematic evolution of aptamers is achieved through various approaches including CE-SELEX and Cell-SELEX, each suffering its inevitable weaknesses. The shortcomings of negative selection and the lengthy procedure are Cell-SELEX's main problems, while CE-SELEX is deprived of native targets. Here, we introduced a kind of hybrid CE-Cell-SELEX, named CEC hybrid-SELEX, for addressing these limitations in creating aptamer probes detecting human aspartate β-hydroxylase (ASPH), which is a well-established tumor biomarker, in cancer diagnostic investigations.

In our approach, the selected oligomer pool from the last cycle of CE-SELEX was sequenced and then subjected to 3 additional rounds of Cell-SELEX which provides native ASPH (CEC hybrid-SELEX). High-throughput sequencing was applied to achieve a comprehensive sight of the enriched pools. Further confirmatory investigations on oligomers with higher copy numbers were performed using flow cytometry.

Three selected oligomers, AP-CEC 1, AP-CEC 2, and AP-CEC 3, showing Kd values of 43.09 nM, 34.85 nM, and 35.92 nM, respectively, were achieved based on the affinity assessment of the ASPH-expressing cells.

Our research suggested that CEC hybrid-SELEX could help recognize which oligomers from CE-SELEX are more capable of binding native ASPH in vivo.

Active cancer targeting consists of the selective recognition of overexpressed biomarkers on cancer cell surfaces or within the tumor microenvironment, enabled by ligands conjugated to drug carriers. Nanoparticle (NP)-based systems are highly relevant for such an approach due to their large surface area which is amenable to a variety of chemical modifications. Over the past decades, several studies have debated the efficiency of passive targeting, highlighting active targeting as a more specific and selective approach. The choice of conjugation chemistry for attaching ligands to nanocarriers is critical to ensure a stable and robust system. Among the panel of cancer biomarkers, the epidermal growth factor receptor (EGFR) stands as one of the most frequently overexpressed receptors in different cancer types. The design and development of nanocarriers with surface-bound anti-EGFR ligands are vital for targeted therapy, relying on their facilitated capture by EGFR-overexpressing tumor cells and enabling receptor-mediated endocytosis to improve drug accumulation within the tumor microenvironment. In this review, we examine several examples of the most recent and significant anti-EGFR nanocarriers and explore the various conjugation strategies for NP functionalization with anti-EGFR biomolecules and small molecular ligands. In addition, we also describe some of the most common characterization techniques to confirm and analyze the conjugation patterns.

The detection of pathogens is critical for preventing and controlling health hazards across clinical, environmental, and food safety sectors. Functional nucleic acids (FNAs), such as aptamers and DNAzymes, have emerged as versatile molecular tools for pathogen detection due to their high specificity and affinity. This review focuses on the in vitro selection of FNAs for pathogens, with emphasis on the selection of aptamers for specific biomarkers and intact pathogens, including bacteria and viruses. Additionally, the selection of DNAzymes for bacterial detection is discussed. The integration of these FNAs into colorimetric biosensors has enabled the development of simple, cost-effective diagnostic platforms. Both non-catalytic and catalytic colorimetric biosensors are explored, including those based on gold nanoparticles, polydiacetylenes, protein enzymes, G-quadruplexes, and nanozymes. These biosensors offer visible detection through color changes, making them ideal for point-of-care diagnostics. The review concludes by highlighting current challenges and future perspectives for advancing FNA-based colorimetric biosensing technologies for pathogen detection.

The field of healthcare diagnostics is navigating complex challenges driven by evolving patient demographics and the rapid advancement of new technologies worldwide. In response to these challenges, these biosensors offer distinctive advantages over traditional diagnostic methods, such as cost-effectiveness, enhanced specificity, and adaptability, making their integration with point-of-care (POC) platforms more feasible. In recent years, aptasensors have significantly evolved in diagnostic capabilities through the integration of emerging technologies such as microfluidics, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, wearable devices, and machine learning (ML), driving progress in precision medicine and global healthcare solutions. Moreover, these advancements not only improve diagnostic accuracy but also hold the potential to revolutionize early detection, reduce healthcare costs, and improve patient outcomes, especially in resource-limited settings. This Account examines key advancements, focusing on how scientific breakthroughs, including artificial intelligence (AI), have improved sensitivity and precision. Additionally, the integration of aptasensors with these technologies has enabled real-time monitoring and data analysis, fostering advances in personalized healthcare. Furthermore, the potential commercialization of aptasensor technologies could increase their availability in clinical settings and support their use as widespread solutions for global health challenges. Hence, this review discusses technological improvements, practical uses, and prospects while also focusing on the challenges surrounding standardization, clinical validation, and interdisciplinary collaboration for widespread application. Finally, ongoing efforts to address these challenges are key to ensure that aptasensors can be effectively implemented in diverse healthcare systems.

A fluorescent aptasensor was developed based on target-induced hairpin conformation switch coupled with nicking enzyme-assisted signal amplification (NESA) to detect the oligomeric form of ß-amyolid peptide (AβO) in cerebrospinal fluid. The hairpin DNA probe (HP) was specifically designed to recognize AβO. When AβO is present in the sensing system, it induces an HP conformational switch and triggers the NESA reaction. After evaluating the parameters of the aptasensor system, we selected Hairpin10, which has a 10-nucleotide extended sequence, as the hairpin sequence that interacts with AβO. The quantitative linear range of the proposed aptasensor is from 11.3 to 113 ng mL-1 in artificial cerebrospinal fluid (aCSF), and the detection limit was 7.29 ng mL-1. The present work realized the assay of AβO in aCSF with satisfactory quantitative results.

Leukemia remains a fatal disease for most affected patients, and a simple and effective therapeutic strategy is urgently needed. Targeted delivery chemo-drugs to leukemia cells shows promise, but the diverse subtypes of leukemia make single-ligand nanomedicine often ineffective. Herein, a dual-aptamer decorated, reduction-responsive dimeric prodrug-based nanoparticle (NP), termed SXP-NPs, was developed using the two leukemia-specific aptamers Sgc8c and XQ-2d, a reduction-responsive podophyllotoxin (POD) dimeric prodrug, and DSPE-PEG2000. Because the receptors of XQ-2d (CD71) and Sgc8c (PTK7) are overexpressed in different subtypes of leukemia cells, SXP-NPs can broadly and selectively recognize these leukemia cells after intravenous administration, subsequently releasing POD in response to the intracellular high-reduction environment to kill the leukemia cells. In vitro experiments showed that these simple SXP-NPs can specifically bind to various leukemia cancer cells and kill them. In vivo experiments revealed that SXP-NPs can remarkably reduce spleen weight, decrease white blood cell counts, and extend overall survival in a preclinical leukemia animal model. The in vitro and in vivo validation demonstrated that SXP-NPs offer several advantages, including high drug-loading potential, broad-spectrum recognition of leukemia cells, reduced systemic toxicity, and enhanced therapeutic effects of the drug. Taken together, this study provides a simple and effective strategy for broad-spectrum leukemia therapy and highlights the clinical potential of SXP-NPs.

An aptamer-based electrochemical sensor for the sensitive detection of staphylococcal enterotoxin type A (SEA) is presented. The truncated aptamer AptSEA1.4 used in this work was screened using computational techniques, which reduced the cost of the SELEX screening process. The aptamer-SEA interactions were confirmed by employing circular dichroism (CD) and fluorescence spectroscopy. Afterwards, for developing an electrochemical aptasensor, a fabricated GNR/FTO aptasensor was prepared and characterized using scanning electron microscopy-energy-dispersive X-ray analysis (SEM-EDX), atomic force microscopy (AFM), cyclic voltammetry (CV), and square wave voltammetry (SWV). A detailed investigation of aptamer and SEA interaction in the presence of various experimental conditions was also conducted through SWV and electrochemical impedance spectroscopy (EIS). The aptamer exhibits a strong affinity for SEA, with a dissociation constant (Kd) of 19.93 nM. The aptasensor is sensitive, with a lower limit of detection of 12.44 pg mL-1. It has good stability, repeatability, and specificity and has displayed highly specific and sensitive detection SEA in spiked packaged mixed fruit juice and milk, with a recovery of 95-110%. The aptasensor has high promise for detecting SEA in other food items.

DNAzymes, a class of single-stranded catalytic DNA with good stability, high catalytic activity, and easy synthesis, functionalization and modification properties, have garnered significant interest in the realm of biosensing and bioimaging. Their integration with fluorescent dyes or chemiluminescent moieties has led to remarkable bioimaging outcomes, while DNAzyme-based biosensors have demonstrated robust sensitivity and selectivity in detecting metal ions, nucleic acids, proteins, enzyme activities, exosomes, bacteria and microorganisms. In addition, by delivering DNAzymes into tumor cells, the mRNA therein can be cleaved to regulate the expression of corresponding proteins, which has further propelled the application of DNAzymes in cancer gene therapy and synergistic therapy. This paper reviews the strategies for screening attractive DNAzymes such as SELEX and high-throughput sequencing, and briefly describes the amplification strategies of DNAzymes, which mainly include catalytic hairpin assembly (CHA), DNA walker, hybridization chain reaction (HCR), DNA origami, CRISPR-Cas12a, rolling circle amplification (RCA), and aptamers. In addition, applications of DNAzymes in bioimaging, biosensing, and cancer therapy are also highlighted. Subsequently, the possible challenges of these DNAzymes in practical applications are further pointed out, and future research directions are suggested.

Aptamers are oligonucleotides with antibody-like binding function, selected from large combinatorial libraries. In this study, we modified a DNA aptamer library with N-hydroxysuccinimide esters, enabling covalent conjugation with cognate proteins. We selected for the ability to bind to mouse monoclonal antibodies, resulting in the isolation of two distinct covalent binding motifs. The covalent aptamers are specific for the Fc region of mouse monoclonal IgG1 and are cross-reactive with mouse IgG2a and other IgGs. Investigation into the covalent conjugation of the aptamers revealed a dependence on micromolar concentrations of Cu2+ ions which can be explained by residual catalyst remaining after modification of the aptamer library. The aptamers were successfully used as adapters in the formation of antibody-oligonucleotide conjugates (AOCs) for use in detection of HIV protein p24 and super-resolution imaging of actin. This work introduces a new method for the site-specific modification of native monoclonal antibodies and may be useful in applications requiring AOCs or other antibody conjugates.

Aptamers are single-stranded RNA or DNA molecules that can specifically bind to targets and have found broad applications in cancer early-stage detection, accurate drug delivery, and precise treatment. Although various aptamer screening methods have been developed over the past several decades, the accurate binding site between the target and the aptamer cannot be characterized during a typical aptamer screening process. In this research, we chose a widely used aptamer screened by our group, sgc8c, and its target protein tyrosine kinase 7 (PTK7) as the model aptamer and target and tried to determine the binding site between aptamer sgc8c and PTK7. Through sequential protein truncation, we confirmed that the exact binding site of sgc8c was within the region of Ig 3 to Ig 4 in the extracellular domain of PTK7. Using in vitro expressed Ig (3-4), we successfully acquired the crystal of an sgc8c-Ig (3-4) binding complex. The possible sgc8c-binding amino acid residues on PTK7 and PTK7-binding nucleotide residues on sgc8c were further identified and simulated by mass spectrometry and molecular dynamics simulation and finally verified by aptamer/protein truncation and mutation.

In the rapidly evolving landscape of nanomedicine, aptamers have emerged as powerful molecular tools, demonstrating immense potential in targeted therapeutics, diagnostics, and drug delivery systems. This paper explores the computational features of aptamers in nanomedicine, highlighting their advantages over antibodies, including selectivity, low immunogenicity, and a simple production process. A comprehensive overview of the aptamer development process, specifically the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process, sheds light on the intricate methodologies behind aptamer selection. The historical evolution of aptamers and their diverse applications in nanomedicine are discussed, emphasizing their pivotal role in targeted drug delivery, precision medicine and therapeutics. Furthermore, we explore the integration of artificial intelligence (AI), machine learning (ML), Internet of Things (IoT), Internet of Medical Things (IoMT), and nanotechnology in aptameric development, illustrating how these cutting-edge technologies are revolutionizing the selection and optimization of aptamers for tailored biomedical applications. This paper also discusses challenges in computational methods for advancing aptamers, including reliable prediction models, extensive data analysis, and multiomics data incorporation. It also addresses ethical concerns and restrictions related to AI and IoT use in aptamer research. The paper examines progress in computer simulations for nanomedicine. By elucidating the importance of aptamers, understanding their superiority over antibodies, and exploring the historical context and challenges, this review serves as a valuable resource for researchers and practitioners aiming to harness the full potential of aptamers in the rapidly evolving field of nanomedicine.

Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.

Soluble growth stimulation expressed gene 2 (sST2) is a new generation biomarker in the diagnosis and prognosis of heart failure (HF). Here, the sST2-specific aptamers were selected from a random ssDNA library with the full length of 88 nucleotides (nt) via target-immobilized magnetic beads (MB)-based systematic evolution of ligands by exponential enrichment (SELEX) technology. After eight rounds of selection, six aptamers with the most enrichment were selected. Among, the aptamer L1 showed the high-affinity binding to sST2 with the lowest Kd value (77.3 ± 0.05 nM), which was chosen as the optimal aptamer for further molecular docking. Then, the aptamer L1 was used to construct a graphene oxide (GO) - based fluorescence resonance energy transfer (FRET) biosensor for sST2, which exhibits a linear detection range of 0.1-100 μg/ml and a detection limit of 3.7 ng/ml. The aptasensor was applied to detect sST2 in real samples, with a good correlation and agreement with the traditional enzyme-linked immunosorbent assay (ELISA) when quantitative analyzing the sST2 concentration in serum samples from HF patients. The results show that not only an efficient strategy for screening the practicable aptamer, but also a rapid and sensitive detection platform for sST2 were established.

Scientists have established a connection between environmental exposure to toxins like β-N-methylamino-l-alanine (BMAA) and a heightened risk of neurodegenerative disorders. BMAA is a byproduct from certain strains of cyanobacteria that are present in ecosystems worldwide and is renowned for its bioaccumulation and biomagnification in seafood. The sensitivity, selectivity, and reproducibility of the current analytical techniques are insufficient to support efforts regarding food safety and environment monitoring adequately. This work outlines the in vitro selection of BMAA-specific DNA aptamers via the systematic evolution of ligands through exponential enrichment (SELEX). Screening and characterization of the full-length aptamers was achieved using the SYBR Green (SG) fluorescence displacement assay. Aptamers BMAA_159 and BMAA_165 showed the highest binding affinities, with dissociation constants (Kd) of 2.2 ± 0.1 μM and 0.32 ± 0.02 μM, respectively. After truncation, the binding affinity was confirmed using a BMAA-conjugated fluorescence assay. The Kd values for BMAA_159_min and BMAA_165_min were 6 ± 1 μM and 0.63 ± 0.02 μM, respectively. Alterations in the amino proton region studied using solution nuclear magnetic resonance (NMR) provided further evidence of aptamer-target binding. Additionally, circular dichroism (CD) spectroscopy revealed that BMAA_165_min forms hybrid G-quadruplex (G4) structures. Finally, BMAA_165_min was used in the development of an electrochemical aptamer-based (EAB) sensor that accomplished sensitive and selective detection of BMAA with a limit of detection (LOD) of 1.13 ± 0.02 pM.

Systematic evolution of ligands by exponential enrichment (SELEX) has been used to discover thousands of aptamers since its development in 1990. Aptamers are short single-stranded oligonucleotides capable of binding to targets with high specificity and selectivity through structural recognition. While aptamers offer advantages over other molecular recognition elements such as their ease of production, smaller size, extended shelf-life, and lower immunogenicity, they have yet to show significant success in real-world applications. By analyzing the importance of structured library designs, reviewing different SELEX methodologies, and the effects of chemical modifications, we provide a comprehensive overview on the production of aptamers for applications in drug delivery systems, therapeutics, diagnostics, and molecular imaging.

Targeted protein degradation (TPD) has been established as a viable alternative to attenuate the function of a specific protein of interest in both biological and clinical contexts. The unique TPD mode-of-action has allowed previously undruggable proteins to become feasible targets, expanding the landscape of "druggable" properties and "privileged" target proteins. As TPD continues to evolve, a range of innovative strategies, which do not depend on recruiting E3 ubiquitin ligases as in proteolysis-targeting chimeras (PROTACs), have emerged. Here, we present an overview of direct lysosome- and proteasome-engaging modalities and discuss their perspectives, advantages, and limitations. We outline the chemical composition, biochemical activity, and pharmaceutical characteristics of each degrader. These alternative TPD approaches not only complement the first generation of PROTACs for intracellular protein degradation but also offer unique strategies for targeting pathologic proteins located on the cell membrane and in the extracellular space.

For decades, antibodies have remained the archetypal binding proteins that can be rapidly produced with high affinity and specificity against virtually any target. A conventional antibody is still considered the prototype of a binding molecule. It is therefore not surprising that antibodies are routinely used in basic scientific and biomedical research, analytical workflows, molecular diagnostics etc. and represent the fastest growing sector in the field of biotechnology. However, several limitations associated with conventional antibodies, including stringent requirement of animal immunizations, mammalian cells for expression, issues on stability and aggregation, bulkier size and the overall time and cost of production has propelled evolution of concepts along alternative antigen binders. Rapidly evolving protein engineering approaches and high throughput screening platforms have further complemented the development of myriads of classes of non-conventional protein binders including antibody derived as well as non-antibody based molecular scaffolds. These non-canonical binders are finding use across disciplines of which diagnostics and therapeutics are the most noteworthy.

More than 30 years ago, in 1990, three independent research groups published several papers demonstrating that genetics could be performed in vitro in the absence of living organisms or cells [...].

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.

Aphanizomenon is a genus of cyanobacteria that is filamentous and nitrogen-fixing and inhabits aquatic environments. This genus is known as one of the major producers of cyanotoxins that can affect water quality after the bloom period. In this study, an electrochemical aptasensor is demonstrated using a specific aptamer to detect Aphanizomenon sp. ULC602 for the rapid and sensitive detection of this bacterium. The principal operation of the generated aptasensor is based on the conformational change in the aptamer attached to the electrode surface in the presence of the target bacterium, resulting in a decrease in the current peak, which is measured by square-wave voltammetry (SWV). This aptasensor has a limit of detection (LOD) of OD750~0.3, with an extension to OD750~1.2 and a sensitivity of 456.8 μA·OD750-1·cm-2 without interference from other cyanobacteria. This is the first aptasensor studied that provides rapid detection to monitor the spread of this bacterium quickly in a targeted manner.

Among all the paralytic shellfish toxins (PSTs)-producing algae, Alexandrium tamarense is one of the most widespread harmful species posing a serious threat to marine resources and human health. Therefore, it is extremely important to establish a rapid and accurate monitoring method for A. tamarense that can provide early warnings of harmful algal blooms (HABs) caused by this alga and limit the contamination due to PSTs. In this study, an ssDNA library was first obtained by whole cell systematic evolution of ligands by exponential enrichment after 18 consecutive rounds of iterative screening. After sequencing in combination with subsequent multiple alignment of sequences and secondary structure simulation, the library could be classified into 2 families, namely, Family1 and Family2, according to sequence similarity. Flow cytometry was used to test the affinity and cross-reactivity of Ata19, Ata6, Ata25 and Ata29 belonging to Family2. Ata19 was selected to be modified by truncation, through which a new resultant aptamer named as Ata19-1-1 was obtained. Ata19-1-1 with a KD of 75.16 ± 11.10 nM displayed a much higher affinity than Ata19. The specificity test showed that Ata19-1-1 has the same discrimination ability as Ata19 and can at least distinguish the target microalga from other microalgae. The observation under a fluorescence microscopy showed that the A. tamarense cells labeled with Ata19-1-1 are exhibiting bright green fluorescence and could be easily identified, factually confirming the binding of the aptamer with target cells. In summary, the aptamer Ata19-1-1 produced in this study may serve as an ideal molecular recognition element for A. tamarense, which has the potential to be developed into a novel detection method for this harmful alga in the future.

Control interventions steer the evolution of molecules, viruses, microorganisms or other cells towards a desired outcome. Applications range from engineering biomolecules and synthetic organisms to drug, therapy and vaccine design against pathogens and cancer. In all these instances, a control system alters the eco-evolutionary trajectory of a target system, inducing new functions or suppressing escape evolution. Here, we synthesize the objectives, mechanisms and dynamics of eco-evolutionary control in different biological systems. We discuss how the control system learns and processes information about the target system by sensing or measuring, through adaptive evolution or computational prediction of future trajectories. This information flow distinguishes pre-emptive control strategies by humans from feedback control in biotic systems. We establish a cost-benefit calculus to gauge and optimize control protocols, highlighting the fundamental link between predictability of evolution and efficacy of pre-emptive control.

Nowadays there are numerous discovered natural RNA variations participating in different cellular processes and artificial RNA, e. g., aptamers, riboswitches. One of the required tasks in the investigation of their functions and mechanism of influence on cells and interaction with targets is the prediction of RNA secondary structures. The classic thermodynamic-based prediction algorithms do not consider the specificity of biological folding and deep learning methods that were designed to resolve this issue suffer from homology-based methods problems. Herein, we present a method for RNA secondary structure prediction based on deep learning - AliNA (ALIgned Nucleic Acids). Our method successfully predicts secondary structures for non-homologous to train-data RNA families thanks to usage of the data augmentation techniques. Augmentation extends existing datasets with easily-accessible simulated data. The proposed method shows a high quality of prediction across different benchmarks including pseudoknots. The method is available on GitHub for free (https://github.com/Arty40m/AliNA).

Aptamers are single-stranded oligonucleotides that bind to their targets via specific structural interactions. To improve the properties and performance of aptamers, modified nucleotides are incorporated during or after a selection process such as systematic evolution of ligands by exponential enrichment (SELEX). We summarize the latest modified nucleotides and strategies used in modified (mod)-SELEX and post-SELEX to develop modified aptamers, highlight the methods used to characterize aptamer-target interactions, and present recent progress in modified aptamers that recognize different targets. We discuss the challenges and perspectives in further advancing the methodologies and toolsets to accelerate the discovery of modified aptamers, improve the throughput of aptamer-target characterization, and expand the functional diversity and complexity of modified aptamers.

Organosilica nanoparticles that contain responsive organic building blocks as constitutive components of the silica network offer promising opportunities for the development of innovative drug formulations, biomolecule delivery, and diagnostic tools. However, the synthetic challenges required to introduce dynamic and multifunctional building blocks have hindered the realization of biomimicking nanoparticles. In this study, capitalizing on our previous research on responsive nucleic acid-based organosilica nanoparticles, we combine the supramolecular programmability of nucleic acid (NA) interactions with sol-gel chemistry. This approach allows us to create dynamic supramolecular bridging units of nucleic acids in a silica-based scaffold. Two peptide nucleic acid-based monoalkoxysilane derivatives, which self-assemble into a supramolecular bis-alkoxysilane through direct base pairing, were chosen as the noncovalent units inserted into the silica network. In addition, a bridging functional NA aptamer leads to the specific recognition of ATP molecules. In a one-step bottom-up approach, the resulting supramolecular building blocks can be used to prepare responsive organosilica nanoparticles. The supramolecular Watson-Crick-Franklin interactions of the organosilica nanoparticles result in a programmable response to external physical (i.e., temperature) and biological (i.e., DNA and ATP) inputs and thus pave the way for the rational design of multifunctional silica materials with application from drug delivery to theranostics.

Maintaining a stable level of potassium is crucial for proper bodily function because even a slight imbalance can result in serious disorders like hyperkalemia and hypokalemia. Therefore, detecting and monitoring potassium ion (K+) levels are of utmost importance. Various biosensors have been developed for rapid K+ detection, with aptamer-based biosensors garnering significant attention due to their high sensitivity and specificity. This review focuses on aptamer-based biosensors for K+ detection, providing an overview of their signal generation strategies, including electrochemical, field-effect transistor, nanopore, colorimetric, and fluorescent systems. The analytical performance of these biosensors is evaluated comprehensively. In addition, factors that affect their efficiency, such as their physicochemical properties, regeneration for reusability, and linkers/spacers, are listed. Lastly, this review examines the major challenges faced by aptamer-based biosensors in K+ detection and discusses potential future developments.

Immunosensors are a special class of biosensors that employ specific antibodies for biorecognition of the target analyte. Immunosensors that target disease biomarkers may be exploited as tools for disease diagnosis and/or follow-up, offering several advantages over conventional analytical techniques, such as rapid and easy analysis of patients' samples at the point-of-care. Autoimmune diseases have been increasingly prevalent worldwide in recent years, while the COVID-19 pandemic has also been associated with autoimmunity. Consequently, demand for tools enabling the early and reliable diagnosis of autoimmune diseases is expected to increase in the near future. To this end, interest in immunosensors targeting autoimmune disease biomarkers, mainly, various autoantibodies and specific pro-inflammatory proteins (e.g., specific cytokines), has been rekindled. This review article presents most of the immunosensors proposed to date as potential tools for the diagnosis of various autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis. The signal transduction and the immunoassay principles of each immunosensor have been suitably classified and are briefly presented along with certain sensor elements, e.g., special nano-sized materials used in the construction of the immunosensing surface. The main concluding remarks are presented and future perspectives of the field are also briefly discussed.

Severe acute pancreatitis (AP) is associated with a high mortality rate. Cold-inducible RNA binding protein (CIRP) can be released from cells in inflammatory conditions and extracellular CIRP acts as a damage-associated molecular pattern. This study aims to explore the role of CIRP in the pathogenesis of AP and evaluate the therapeutic potential of targeting extracellular CIRP with X-aptamers. Our results showed that serum CIRP concentrations were significantly increased in AP mice. Recombinant CIRP triggered mitochondrial injury and ER stress in pancreatic acinar cells. CIRP-/- mice suffered less severe pancreatic injury and inflammatory responses. Using a bead-based X-aptamer library, we identified an X-aptamer that specifically binds to CIRP (XA-CIRP). Structurally, XA-CIRP blocked the interaction between CIRP and TLR4. Functionally, it reduced CIRP-induced pancreatic acinar cell injury in vitro and L-arginine-induced pancreatic injury and inflammation in vivo. Thus, targeting extracellular CIRP with X-aptamers may be a promising strategy to treat AP.

The non-specificity of standard anticancer therapies has profound detrimental consequences in clinical treatment. Therapeutic specificity can be precisely achieved using cutting-edge ligands. Small synthetic oligonucleotide-ligands chosen through Systematic evolution of ligands by exponential enrichment (SELEX) would be an unceasing innovation in using nucleic acids as aptamers, frequently referred to as "chemical antibodies." Aptamers act as externally controlled switching materials that can attach to various substrates, for example, membrane proteins or nucleic acid structures. Aptamers pose excellent specificity and affinity for target molecules and can be used as medicines to suppress tumor cell growth directly. The creation of aptamer-conjugated nanoconstructs has recently opened up innovative options in cancer therapy that are more effective and target tumor cells with minor toxicity to healthy tissues. This review focuses on a comprehensive description of the most capable classes of aptamer-tethered nanocarriers for precise recognition of cancer cells with significant development in proficiency, selectivity, and targetability for cancer therapy. Existing theranostic applications with the problems and future directions are also highlighted.

With the merits of easy synthesis, strong modifiability, and high affinity, aptamers have been broadly applied for protein targeting in bioanalysis, diagnosis, and therapeutics. The selection of protein-targeted aptamers is currently largely dependent on solid-liquid separation by using different types of nano- or micro-beads. However, the use of beads inescapably introduces unwanted nonspecific binding and thus affects selection efficiency. In order to sidestep this obstacle, we herein report an integrated technique to facilitate the discovery and development of protein-targeting aptamers by incorporating formaldehyde cross-linking with phase separation (FCPS). The feasibility and universality of FCPS were confirmed by the successful selection of two aptamers that could target various antibodies. Unlike traditional approaches, the proposed technique avoids the use of beads and enables the rapid generation of aptamers after only one to three rounds of selection. The as-selected aptamers were further used to regulate and control antibody activity, showing potential applications in biomedicine.

Cronobacter spp. are opportunistic foodborne pathogens typically detected in contaminated powdered infant formula (PIF). Thus, the rapid detection and control of Cronobacter spp. are required to prevent outbreaks, necessitating the development of specific aptamers. In this study, we isolated aptamers specific to all seven species of Cronobacter (C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C. condimenti, and C. universalis) using a newly proposed sequential partitioning method. This method avoids the repeated enrichment steps, reducing the total aptamer selection time compared with the conventional systematic evolution of ligands by the exponential enrichment (SELEX) process. We isolated four aptamers showing high affinity and specificity for all seven species of Cronobacter, with dissociation constants of 3.7-86.6 nM. This represents the first successful isolation of aptamers for multiple targets using the sequential partitioning method. Further, the selected aptamers could effectively detect Cronobacter spp. in contaminated PIF.

Leptospirosis is a potentially life-threatening zoonosis caused by pathogenic Leptospira. The major hurdle of the diagnosis of Leptospirosis lies in the issues associated with current methods of detection, which are time-consuming, tedious and the need for sophisticated, special equipments. Restrategizing the diagnostics of Leptospirosis may involve considerations of the direct detection of the outer membrane protein, which can be faster, cost-saving and require fewer equipments. One such promising marker is LipL32, which is an antigen with high amino acid sequence conservation among all the pathogenic strains. In this study, we endeavored to isolate an aptamer against LipL32 protein via a modified SELEX strategy known as tripartite-hybrid SELEX, based on 3 different partitioning strategies. In this study, we also demonstrated the deconvolution of the candidate aptamers by using in-house Python-aided unbiased data sorting in examining multiple parameters to isolate potent aptamers. We have successfully generated an RNA aptamer against LipL32 of Leptospira, LepRapt-11, which is applicable in a simple direct ELASA for the detection of LipL32. LepRapt-11 can be a promising molecular recognition element for the diagnosis of leptospirosis by targeting LipL32.

Riboswitches are structured noncoding RNA domains used by many bacteria to monitor the concentrations of target ligands and regulate gene expression accordingly. In the past 20 years over 55 distinct classes of natural riboswitches have been discovered that selectively sense small molecules or elemental ions, and thousands more are predicted to exist. Evidence suggests that some riboswitches might be direct descendants of the RNA-based sensors and switches that were likely present in ancient organisms before the evolutionary emergence of proteins. We provide an overview of the current state of riboswitch research, focusing primarily on the discovery of riboswitches, and speculate on the major challenges facing researchers in the field.

Early cancer identification is crucial for providing patients with safe and timely therapy. Highly dependable and adaptive technologies will be required to detect the presence of biological markers for cancer at very low levels in the early stages of tumor formation. These techniques have been shown to be beneficial in encouraging patients to develop early intervention plans, which could lead to an increase in the overall survival rate of cancer patients. Targeted drug delivery (TDD) using aptamer is promising due to its favorable properties. Aptamer is suitable for superior TDD system candidates due to its desirable properties including a high binding affinity and specificity, a low immunogenicity, and a chemical composition that can be simply changed.Due to these properties, aptamer-based TDD application has limited drug side effect along with organ damages. The development of aptasensor has been promising in TDD for cancer cell treatment. There are biomarkers and expressed molecules during cancer cell development; however, only few are addressed in aptamer detection study of those molecules. Its great potential of attachment of binding to specific target molecule made aptamer a reliable recognition element. Because of their unique physical, chemical, and biological features, aptamers have a lot of potential in cancer precision medicine.In this review, we summarized aptamer technology and its application in cancer. This includes advantages properties of aptamer technology over other molecules were thoroughly discussed. In addition, we have also elaborated the application of aptamer as a direct therapeutic function and as a targeted drug delivery molecule (aptasensor) in cancer cells with several examples in preclinical and clinical trials.

Aptamers are single-stranded oligonucleotides (RNA or DNA) with a typical length between 25 and 100 nucleotides which fold into three-dimensional structures capable of binding to target molecules. Specific aptamers can be isolated against a large variety of targets through efficient and relatively cheap methods, and they demonstrate target-binding affinities that sometimes surpass those of antibodies. Consequently, interest in aptamers has surged over the past three decades, and their application has shown promise in advancing knowledge in target analysis, designing therapeutic interventions, and bioengineering. With emphasis on their therapeutic applications, aptamers are emerging as a new innovative class of therapeutic agents with promising biochemical and biological properties. Aptamers have the potential of providing a feasible alternative to antibody- and small-molecule-based therapeutics given their binding specificity, stability, low toxicity, and apparent non-immunogenicity. This Review examines the general properties of aptamers and aptamer-protein interactions that help to understand their binding characteristics and make them important therapeutic candidates.

The principal presumption of phage display biopanning is that the naïve library contains an unbiased repertoire of peptides, and thus, the enriched variants derive from the affinity selection of an entirely random peptide pool. In the current study, we utilized deep sequencing to characterize the widely used Ph.DTM-12 phage display peptide library (New England Biolabs). The next-generation sequencing (NGS) data indicated the presence of stop codons and a high abundance of wild-type clones in the naïve library, which collectively result in a reduced effective size of the library. The analysis of the DNA sequence logo and global and position-specific frequency of amino acids demonstrated significant bias in the nucleotide and amino acid composition of the library inserts. Principal component analysis (PCA) uncovered the existence of four distinct clusters in the naïve library and the investigation of peptide frequency distribution revealed a broad range of unequal abundances for peptides. Taken together, our data provide strong evidence for the notion that the naïve library represents substantial departures from randomness at the nucleotide, amino acid, and peptide levels, though not undergoing any selective pressure for target binding. This non-uniform sequence representation arises from both the M13 phage biology and technical errors of the library construction. Our findings highlight the paramount importance of the qualitative assessment of the naïve phage display libraries prior to biopanning.

Enteric colibacillosis is a common disease in nursing and weanling pigs. It is caused by the colonization of the small intestine by enterotoxigenic strains of Escherichia coli (ETEC) that make use of specific fimbria or pili to adhere to the absorptive epithelial cells of the jejunum and ileum. Once attached, and when both the immunological systems and the gut microbiota are poorly developed, ETEC produce one or more enterotoxins that can have local and, further on, systemic effects. These enterotoxins cause fluid and electrolytes to be secreted into the intestinal lumen of animals, which results in diarrhea, dehydration, and acidosis. From the diversity of control strategies, antibiotics and zinc oxide are the ones that have contributed more significantly to mitigating post-weaning diarrhea (PWD) economic losses. However, concerns about antibiotic resistance determined the restriction on the use of critically important antimicrobials in food-producing animals and the prohibition of their use as growth promoters. As such, it is important now to begin the transition from these preventive/control measures to other, more sustainable, approaches. This review provides a quick synopsis of the currently approved and available therapies for PWD treatment while presenting an overview of novel antimicrobial strategies that are being explored for the control and treatment of this infection, including, prebiotics, probiotics, synbiotics, organic acids, bacteriophages, spray-dried plasma, antibodies, phytogenic substances, antisense oligonucleotides, and aptamers.

Aptamers are a group of synthetic single-stranded nucleic acids. They are generated from a random library of single-stranded DNA or RNA by a technology named systematic evolution of ligands by exponential enrichment (SELEX). SELEX is a repetitive process to select and identify suitable aptamers that show high affinity and specificity towards target cells. Great strides have been achieved in the design, construction, and use of aptamers up to this point. However, only a small number of aptamer-based applications have achieved widespread commercial and clinical acceptance. Additionally, finding more effective ways to acquire aptamers with high affinity remains a challenge. Therefore, it is crucial to thoroughly examine the existing dearth and advancement in aptamer-related technologies. This review focuses on aptamers that are generated by SELEX to detect pathogenic microorganisms and mammalian cells, as well as in cell-internalizing SELEX for diagnostic and therapeutic purposes. The development of novel aptamer-based biosensors using optical and electrical methods for microbial detection is reported. The applications and limitations of aptamers are also discussed.

Clinical diagnostics rely heavily on the detection and quantification of cancer biomarkers. The rapid detection of cancer-specific biomarkers is of great importance in the early diagnosis of cancers and plays a crucial role in the subsequent treatments. There are several different detection techniques available today for detecting cancer biomarkers. Because of target-related conformational alterations, high stability, and target variety, aptamers have received considerable interest as a biosensing system component. To date, several sensitivity-enhancement strategies have been used with a broad spectrum of nanomaterials and nanoparticles (NPs) to improve the limit and sensitivity of analyte detection in the construction of innovative aptasensors. The present article aims to outline the research developments on the potential of DNAzymes-based aptasensors for cancer biomarker detection.

Simple, highly sensitive detection technologies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for the effective implementation of public health policies. We used the systematic evolution of ligands by exponential enrichment with a modified DNA library, including a base-appended base (uracil with a guanine base at its fifth position), to create an aptamer with a high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. The aptamer had a dissociation constant of 1.2 and < 1 nM for the RBD and spike trimer, respectively. Furthermore, enzyme-linked aptamer assays confirmed that the aptamer binds to isolated authentic SARS-CoV-2 wild-type and B.1.617.2 (delta variant). The binding signal was larger that of commercially available anti-SARS-CoV-2 RBD antibody. Thus, this aptamer as a sensing element will enable the highly sensitive detection of SARS-CoV-2.

Aptamers are short single-stranded DNA or RNA oligonucleotides capable of binding with high affinity and specificity to target molecules. Because of their durability and ease of synthesis, aptamers are used in a wide range of biomedical fields, including the diagnosis of diseases and targeted delivery of therapeutic agents. The aptamers were selected using a process called systematic evolution of ligands by exponential enrichment (SELEX), which has been improved for various research purposes since its development in 1990. In this protocol, we describe a modified SELEX method that rapidly produces high aptamer screening yields using two types of magnetic beads. Using this method, we isolated an aptamer that specifically binds to an antimicrobial peptide. We suggest that by conjugating a small therapeutic-specific aptamer to a gold nanoparticle-based delivery system, which enhances the stability and intracellular delivery of peptides, aptamers selected by our method can be used for the development of therapeutic agents utilizing small therapeutic peptides.