Systematic Evolution Research Articles

Evolutionary analysis of MADS-box genes in buckwheat species and functional study of FdMADS28 in flavonoid metabolism

The MADS-box gene family is a transcription factor family that is widely expressed in plants. It controls secondary metabolic processes in plants and encourages the development of tissues like roots and flowers. However, the phylogenetic analysis and evolutionary model of MADS-box genes in Fagopyrum species has not been reported yet. This study identified the MADS-box genes of three buckwheat species at the whole genome level, and conducted systematic evolution and physicochemical analysis. The results showed that these genes can be divided into four subfamilies, with fragment duplication being the main way for the gene family expansion. During the domestication process from golden buckwheat to tartary buckwheat and the common buckwheat, the Ka/Ks ratio indicated that most members of the family experienced strong purification selection pressure, and with individual gene pairs experiencing positive selection. In addition, we combined the expression profile data of the MADS genes, mGWAS data, and WGCNA data to mine genes FdMADS28/48/50 that may be related to flavonoid metabolism. The results also showed that overexpression of FdMADS28 could increase rutin content by decreasing Kaempferol pathway content in hairy roots, and increase the resistance and growth of hairy roots to PEG and NaCl. This study systematically analyzed the evolutionary relationship of MADS-box genes in the buckwheat species, and elaborated on the expression patterns of MADS genes in different tissues under biotic and abiotic stresses, laying an important theoretical foundation for further elucidating their role in flavonoid metabolism.

Selection of ssDNA aptamers and construction of aptameric electrochemical biosensor for the detection of Giardia intestinalis trophozoite protein

Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (Kd), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.

Advancing the Diagnosis of Periprosthetic Joint Infections: Integrated Microfluidic Platform for Alpha-Defensins-Specific Aptamer Selection and Its Analytical Applications.

Periprosthetic joint infections (PJIs) pose a significant challenge in orthopedic surgery, particularly total joint arthroplasty (TJA), due to the potential for implant failure and increased patient morbidity. Early and accurate detection of PJIs is crucial for timely intervention and better patient prognosis. Herein, we successfully screened a high-affinity aptamer targeting alpha-defensin complex human neutrophil protein 1-3 (HNP 1-3; potential PJI biomarkers in synovial fluid [SF]) for the first time using systematic evolution of ligands by exponential enrichment (SELEX) on an integrated microfluidic platform. The compact microfluidic device enabled efficient screening, with each round completed within <2 h, comprising five rounds of positive selection, two rounds of negative selection, and one round of competitive selection. A novel one-aptamer-one-antibody assay was further developed from the optimal aptamer screened, and it could accurately quantify HNP 1-3 in SF within 3 h with only ∼50 μL of SF. The assay demonstrated strong binding affinity and specificity for the target protein in SF. Thirteen PJI SF samples were accurately diagnosed and the assay was accurate over a wide dynamic range (0.32-100 mg/L). This study has showcased a rapid and accurate diagnostic tool for PJI detection, which should see widespread use in the clinic, holding promise for potential analytical applications in orthopedic surgery and improving patient care.

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.

Identification of antibiotic-resistance markers of Edwardsiella tarda using aptamers

Edwardsiella tarda poses significant challenges in the aquaculture sector due to its antibiotic resistance, leading to substantial economic loss in the aquaculture industry. Therefore, it is imperative to identify antibiotic-resistance markers to prevent and control E. tarda. In the present study, we used tetracycline-sensitive (S) and resistant (R) E. tarda strains and six non-target bacterial strains, to develop aptamers through the Systematic Evolution of Ligands by Exponential Enrichment technique. We then conducted comparative analysis of these aptamers based on their relative importance index values and selected six candidate sequences (M1, M13, M15, N57, N69, and N71) for affinity verification. The results showed that M1 had significantly higher affinity towards both the S and R strains compared to the non-target strains. M13 and M15 had significantly higher affinities for the S strain compared to the R strain and non-target strains, and N57, N69, and N71 had significantly higher affinities for R strains relative to the sensitive and non-target strains. Subsequent analysis indicated that N57 and N69 specifically recognized unique sites in the R strain. Therefore, our study presents a novel approach for detecting and controlling antibiotic-resistant strains of E. tarda and identification of drug-resistant markers in bacteria.

Abstract LB432: Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme

Abstract Glioblastoma Multiforme (GBM) is one of the most aggressive and lethal brain tumor types with a five-year survival rate of 6.9%. In 2023, more than 14,490 Americans were expected to receive a diagnosis. Although great progress has been achieved in protein target identification and treatment over the last few years, current treatment options are limited, and tumor recurrence occurs at a high rate due to treatment resistance. Aptamers, short single-stranded oligonucleotide ligands with high-affinity binding to molecular targets, are well suited tools for exploring novel targets in highly heterogeneous tumors with complex etiologies like GBM, where clinically relevant biomarkers are limited. In this study, aptamer-based cancer-specific drug target identification was performed by Adaptive Dynamic Artificial Polyligand Targeting (ADAPT) Biotargeting System. In short, a GBM-specific library was enriched through a Systematic Evolution of Ligands by EXponential enrichment (SELEX) process using micro dissected Formalin-Fixed Paraffin-Embedded (FFPE) tissues from GBM patients, along with whole body normal tissue for counterselection. Microdissection of tissue allows the partitioning of different types of cells to focus on specific cells of interest. The enriched libraries have been evaluated on clinical GBM and normal brain control samples through aptamer mediated tissue staining, resulting in preferential binding of enriched libraries compared to controls in the vast majority of GBM patients with little binding to normal brain samples. The final library was subsequently applied in an in-house developed library-based affinity pull-down assay using lysates from FFPE tissue of the same group of GBM patients and normal brain samples, followed by unbiased Mass Spectrometry for protein target identification. Combining the results from this proteomics analysis and data from the internal Whole Transcriptome Sequencing (WTS) database, we were able to identify several targets that are highly expressed in GBM and other cancer lineages. Antibody staining provided further validation of the expression levels and the cellular localization. The targets identified in this study include not only potential novel therapeutic targets, but also well-studied biomarkers and targets with available drugs already in clinical trials, demonstrating the platform has great potential for identifying high affinity binding and selectivity compared to traditional proteomics and other methods in drug target discovery. Citation Format: Xixi Wei, Myunggi An, Radhika Santhanam, Barbara Richardson, Lucas Vu, Justin Saul, Anna Walton, Michael Gee, Daniel Martin, Amanda Hubbard, Alexis Rodin, Matthew Rosenow, Tassilo Hornung. Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB432.

IMPATIENT-qPCR: monitoring SELEX success during in vitro aptamer evolution

SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (Tm) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and Tm alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general.IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher Tm by quantitative PCR with fluorescence dye SYBR Green I.Key points• Fast and easy analysis.• Universal applicability shown for a series of real successful projects.Graphical

Characterization of the complete chloroplast genome sequence of Lycium qingshuiheense (Solanaceae)

Lycium qingshuiheense is a typical drought and salt-alkali-tolerant plant, which has been added to the new species of Lycium in recent years. Here, we first sequenced the complete chloroplast genome of L. qingshuiheense to investigate its evolutionary relationship within the family Solanaceae. Results suggested that the circular complete chloroplast genome of L. qingshuiheense was 154,945 bp in length, including a large single-copy (LSC) of 85,930 bp, a small single-copy (SSC) of 18,203 bp, and two inverted repeats (IRs) of 25,406 bp. The GC content accounts for 37.90% and annotated 131 genes, including 86 protein-coding genes, eight rRNA genes, and 37 tRNA genes. A neighbor-joining phylogenetic tree revealed that L. qingshuiheense was a sister species to L. ruthenicum. Our study provides a new insight into the systematic evolution of Lycium in the Solanaceae family.

Development of an aptamer capable of multidrug resistance reversal for tumor combination chemotherapy

Multidrug resistance (MDR) is a major factor in the failure of many forms of tumor chemotherapy. Development of a specific ligand for MDR-reversal would enhance the intracellular accumulation of therapeutic agents and effectively improve the tumor treatments. Here, an aptamer was screened against a doxorubicin (DOX)-resistant human hepatocellular carcinoma cell line (HepG2/DOX) via cell-based systematic evolution of ligands by exponential enrichment. A 50 nt truncated sequence termed d3 was obtained with high affinity and specificity for HepG2/DOX cells. Multidrug resistance protein 1 (MDR1) is determined to be a possible recognition target of the selected aptamer. Aptamer d3 binding was revealed to block the MDR of the tumor cells and increase the accumulation of intracellular anticancer drugs, including DOX, vincristine, and paclitaxel, which led to a boost to the cell killing of the anticancer drugs and lowering their survival of the tumor cells. The aptamer d3-mediated MDR-reversal for effective chemotherapy was further verified in an in vivo animal model, and combination of aptamer d3 with DOX significantly improved the suppression of tumor growth by treating a xenograft HepG2/DOX tumor in vivo. This work demonstrates the feasibility of a therapeutic DNA aptamer as a tumor MDR-reversal agent, and combination of the selected aptamer with chemotherapeutic drugs shows great potential for liver cancer treatments.

RNA sequences that direct selective ADAR editing from a SELEX library bearing 8-azanebularine

Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs’ ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.

Strain-induced modulation of electronic structure in correlated Dirac semimetal Pv-CaIrO3 epitaxial thin films

Perovskite CaIrO3 is theoretically predicted to be a Dirac node semimetal near the Mott transition, which possesses a considerable interplay between electron correlations and spin–orbit coupling. Electron correlations can significantly tune the behavior of relativistic Dirac fermions. Here, we have grown high-quality perovskite CaIrO3 thin films on different substrates using oxide molecular beam epitaxy to modulate both electron correlations and Dirac electron states. Through in situ angle-resolved photoemission spectroscopy, we demonstrate a systematic evolution of the bandwidth and effective mass of Jeff=1/2 band in perovskite CaIrO3 induced by strain. The bandwidth of the Jeff=1/2 band undergoes an evident increase under in-plane compressive strain, which could be attributed to the weakening of electron correlations. The compressive strain can potentially shift the position of the Dirac node relative to the Fermi level and play a vital role in the transition from hole-type to electron-type transport characteristics. Our work provides a feasible approach for manipulating the topological Dirac electron states by engineering the strength of electron correlations.

Review—Aptamers and Biomimetic Receptors in Biosensing: Innovations and Applications

The fields of biosensing have been transformed by the discovery of extraordinary molecular recognition components, such as aptamers and biomimetic receptors. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a method used to select aptamers, or short sequences of single-stranded DNA (ssDNA) or RNA (ssRNA), based on their unique binding affinity to target molecules. Molecularly imprinted polymers (MIPs) are a type of biomimetic receptor that mimics the selectivity of natural receptors inside a synthetic matrix. They make it possible to identify pathogens, and illness biomarkers with accuracy. Aptamers and biomimetic receptors play crucial roles in various fields including diagnostics, therapeutics, and biosensing. Their high specificity, versatility, and adaptability enable targeted detection, drug delivery, and biomolecule manipulation, thereby contributing to advancements in personalized medicine, biotechnology, and disease diagnosis. Aptamers and biomimetic receptors have been combined with cutting-edge technologies, like nanotechnology and lab-on-a-chip systems, to create biosensors that are quick, portable, and extremely sensitive. These recognition features are anticipated to become more important as technology develops, helping to address global issues, advance biosensing capabilities, and raise people’s standard of living everywhere. Recent advancements and innovation on Aptamers and Biomimetic Receptors in Biosensing have been discussed in this review article.

Aptamer Technologies in Neuroscience, Neuro-Diagnostics and Neuro-Medicine Development.

Aptamers developed using in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology are single-stranded nucleic acids 10-100 nucleotides in length. Their targets, often with specificity and high affinity, range from ions and small molecules to proteins and other biological molecules as well as larger systems, including cells, tissues, and animals. Aptamers often rival conventional antibodies with improved performance, due to aptamers' unique biophysical and biochemical properties, including small size, synthetic accessibility, facile modification, low production cost, and low immunogenicity. Therefore, there is sustained interest in engineering and adapting aptamers for many applications, including diagnostics and therapeutics. Recently, aptamers have shown promise as early diagnostic biomarkers and in precision medicine for neurodegenerative and neurological diseases. Here, we critically review neuro-targeting aptamers and their potential applications in neuroscience research, neuro-diagnostics, and neuro-medicine. We also discuss challenges that must be overcome, including delivery across the blood-brain barrier, increased affinity, and improved in vivo stability and in vivo pharmacokinetic properties.

Optimizing energy efficiency in heterogeneous networks: An integrated stochastic geometry approach with novel sleep mode strategies and QoS framework.

The quest for energy efficiency (EE) in multi-tier Heterogeneous Networks (HetNets) is observed within the context of surging high-speed data demands and the rapid proliferation of wireless devices. The analysis of existing literature underscores the need for more comprehensive strategies to realize genuinely energy-efficient HetNets. This research work contributes significantly by employing a systematic methodology, utilizing This model facilitates the assessment of network performance by considering the spatial distribution of network elements. The stochastic nature of the PPP allows for a realistic representation of the random spatial deployment of base stations and users in multi-tier HetNets. Additionally, an analytical framework for Quality of Service (QoS) provision based on D-DOSS simplifies the understanding of user-base station relationships and offers essential performance metrics. Moreover, an optimization problem formulation, considering coverage, energy maximization, and delay minimization constraints, aims to strike a balance between key network attributes. This research not only addresses crucial challenges in creating EE HetNets but also lays a foundation for future advancements in wireless network design, operation, and management, ultimately benefiting network operators and end-users alike amidst the growing demand for high-speed data and the increasing prevalence of wireless devices. The proposed D-DOSS approach not only offers insights for the systematic design and analysis of EE HetNets but also systematically outperforms other state-of-the-art techniques presented. The improvement in energy efficiency systematically ranges from 67% (min side) to 98% (max side), systematically demonstrating the effectiveness of the proposed strategy in achieving higher energy efficiency compared to existing strategies. This systematic research work establishes a strong foundation for the systematic evolution of energy-efficient HetNets. The systematic methodology employed ensures a comprehensive understanding of the complex interplay of network dynamics and user requirements in a multi-tiered environment.

Analysis of Genetic Diversity and Structure of Eight Populations of Nerita yoldii along the Coast of China Based on Mitochondrial COI Gene.

Nerita yoldii is a euryhaline species commonly found in the intertidal zone. To investigate the genetic diversity of 233 N. yoldii individuals from eight locations along the coast of China, we utilized the mitochondrial COI gene as a molecular marker. A total of 34 haplotypes were detected, exhibiting a mean haplotype diversity (Hd) of 0.5915 and a mean nucleotide diversity (Pi) of 0.0025, indicating high levels of genetic diversity among all populations. An analysis of molecular variance (AMOVA) indicated that the primary source of genetic variation occurs within populations. In addition, neutral tests and mismatch analyses suggested that N. yoldii populations may have experienced bottleneck events. Moderate genetic differentiation was observed between Xiapu and other populations, excluding the Taizhou population, and may be attributed to the ocean currents. Intensively studying the genetic variation and population structure of N. yoldii populations contributes to understanding the current population genetics of N. yoldii in the coastal regions of China. This not only provides a reference for the study of other organisms in the same region but also lays the foundation for the systematic evolution of the Neritidae family.

In vitro SELEX and application of an African swine fever virus (ASFV) p30 protein specific aptamer

The African swine fever virus (ASFV) has caused severe economic losses in the pig industry. To monitor ASFV spread, the p30 protein has been identified as an ideal infection marker due to its early and long-term expression during the ASFV infection period. Timely monitoring of ASFV p30 enables the detection of ASFV infection and assessment of disease progression. Aptamers are an outstanding substitute for antibodies to develop an efficient tool for ASFV p30 protein detection. In this study, a series of aptamer candidates were screened by in vitro magnetic bead-based systematic evolution of ligands by exponential enrichment (MB-SELEX). An aptamer (Atc-20) finally showed high specificity and affinity (Kd = 140 ± 10 pM) against ASFV p30 protein after truncation and affinity assessment. Furthermore, an aptamer/antibody heterogeneous sandwich detection assay was designed based on Atc20, achieving a linear detection of ASFV p30 ranging from 8 to 125 ng/ml and a detection limit (LOD) of 0.61 ng/ml. This assay showed good analytical performances and effectively detected p30 protein in diluted serum samples, presenting promising potential for the development of ASFV biosensors.

Development of Better Aptamers: Structured Library Approaches, Selection Methods, and Chemical Modifications

AbstractSystematic 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.

Development of Better Aptamers: Structured Library Approaches, Selection Methods, and Chemical Modifications.

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.

Cosmological evolution of metallicity correlation functions from the Auriga simulations

ABSTRACT We study the cosmological evolution of the two-point correlation functions of galactic gas-phase metal distributions using the 28 simulated galaxies from the Auriga Project. Using mock observations of the z = 0 snapshots to mimic our past work, we show that the correlation functions of the simulated mock observations are well matched to the correlation functions measured from local galaxy surveys. This comparison suggests that the simulations capture the processes important for determining metal correlation lengths, the key parameter in metallicity correlation functions. We investigate the evolution of metallicity correlations over cosmic time using the true simulation data, showing that individual galaxies undergo no significant systematic evolution in their metal correlation functions from z ∼ 3 to today. In addition, the fluctuations in metal correlation length are correlated with but lag ahead fluctuations in star formation rate. This suggests that re-arrangement of metals within galaxies occurs at a higher cadence than star formation activity, and is more sensitive to the changes of environment, such as galaxy mergers, gas inflows/outflows, and fly-bys.

Aptamers targeting SARS-CoV-2 nucleocapsid protein exhibit potential anti pan-coronavirus activity

Emerging and recurrent infectious diseases caused by human coronaviruses (HCoVs) continue to pose a significant threat to global public health security. In light of this ongoing threat, the development of a broad-spectrum drug to combat HCoVs is an urgently priority. Herein, we report a series of anti-pan-coronavirus ssDNA aptamers screened using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). These aptamers have nanomolar affinity with the nucleocapsid protein (NP) of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and also show excellent binding efficiency to the N proteins of both SARS, MERS, HCoV-OC43 and -NL63 with affinity KD values of 1.31 to 135.36 nM. Such aptamer-based therapeutics exhibited potent antiviral activity against both the authentic SARS-CoV-2 prototype strain and the Omicron variant (BA.5) with EC50 values at 2.00 nM and 41.08 nM, respectively. The protein docking analysis also evidenced that these aptamers exhibit strong affinities for N proteins of pan-coronavirus and other HCoVs (−229E and -HKU1). In conclusion, we have identified six aptamers with a high pan-coronavirus antiviral activity, which could potentially serve as an effective strategy for preventing infections by unknown coronaviruses and addressing the ongoing global health threat.