Cell-SELEX is an effective method for generating aptamers that specifically bind molecules in their native state on live cells. It not only uncovers novel potential biomarkers but also provides robust molecular recognition tools for a wide spectrum of applications. In this work, we generate a high affinity aptamer, HL15, through Cell-SELEX. A refined sequence HL15a demonstrated a strong binding affinity to target cells, with a minimal dissociation constant (Kd) of just 1.90 ± 0.49 nM. Subsequent truncation and mutation assays revealed that the core sequence of HL15a forms an antiparallel G-quadruplex structure. Furthermore, the target proteins of aptamer HL15a were identified and confirmed to be ZIP10 and ZIP6, both members of the zinc transporter ZIP family with high homology. Using HL15a as a molecular probe, we detected a range of universal binding affinities to the majority of tumor cells in the 48 cell lines evaluated. Furthermore, HL15a was effectively applied to distinguish high malignancy cancer tissues from both normal and low malignancy tissues in the pathological sections of breast and prostate cancers. Given that ZIP10 and ZIP6 play crucial roles in regulating zinc homeostasis and are implicated in numerous diseases, the aptamer HL15a offers a powerful tool for the study and potential therapeutic intervention of these two proteins.

Aptamers are potent alternatives to antibodies in applications including diagnostics and disease treatment. These synthetic molecules are generated from sequences identified through specific targets within an aptamer pool of random sequences, approximately 10^15 in size, via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. Nevertheless, SELEX encompasses repetitive, time/money-consuming and stochastic methodologies. In this study, we introduce a method for the direct acquisition of target aptamers in a single step, rapidly identifying the aptamers of interest. Single molecules of aptamers are first encapsulated into droplets and amplified therein, and the fluorescence in the droplets will be active upon binding between the aptamers with the target with good affinity. Subsequent identification and sorting of these fluorescing droplets enable the immediate acquisition of desired aptamers without the need for synthesizing them based on selected sequences. This digital selection process bypasses traditional sequencing, thereby reducing stochastic events and costs associated with repeated sequencing, as well as mitigating the uncertainties tied to the synthesis of aptamers. Our proof-of-concept findings suggest that this straightforward yet effective strategy can directly yield aptamers, thereby enhancing the exploration of aptamer biology and promoting the development of aptamer-based applications.

Since cancer biomarkers for lung adenocarcinoma can lead to early intervention and treatment, they have been the focus of much research attention. DNA aptamers, which are functional oligonucleotides, exhibit high specificity and binding affinity to different types of cancer biomarkers. Through DNA aptamer screening, a leucine-rich PPR-motif-containing protein (LRPPRC) was discovered as a potential biomarker for lung adenocarcinoma therapeutics. It is an RNA-binding protein that helps in regulating post-transcriptional gene expression in mitochondria. Interestingly, the first LRPPRC-targeted small-molecule drug showed significant antitumor effects. Apart from biomarker discovery, DNA aptamers have also shown promise in cancer therapeutics, but challenges in the programmable delivery of aptamers have limited applications. Herein, we have addressed these challenges in two steps. First, after obtaining purified protein LRPPRC, we verified aptamer R14 as its high-affinity binding ligand. Second, for programmable delivery, a rectangular DNA tile (RDT) was constructed to improve cellular internalization. In particular, DNA handles on the surface of this DNA nanostructure serve as overhangs for loading multivalent R14, and both A549 and PC9 cells treated with R14-RDT targeted to LRPPRC showed significant inhibition of cancer cell proliferation. We then investigated the molecular mechanism(s) underlying the interaction between multivalent aptamer R14 loaded on an RDT and its cognate target protein such that the result is inhibition of cancer cell proliferation. Based on our findings, we hypothesized that R14-RDT-LRPPRC interaction triggers significant gene transcription and RNA processing events that result in inhibiting mitochondria-related genes and RNA transcriptional processing, while causing an immune inflammatory response that ultimately leads to the inhibition of cancer cell proliferation. Therefore, this research offers an instructive paradigm for programmable loading of a multivalent aptamer onto a two-dimensional DNA nanostructure to improve targeted cancer therapeutics through intervening with the cell's transcriptome.

Combination therapy integrating chemotherapeutic agents with natural bioactive ingredients represents an attractive strategy for esophageal squamous cell carcinoma (ESCC) treatment, yet achieving tumor-specific co-delivery remains a critical challenge. Herein, we report that the combination of luteolin (LUT) and paclitaxel (PTX) exerts a remarkable synergy in ESCC treatment, while concurrently alleviating PTX-induced hepatotoxicity; EA2 aptamer has been identified for its exceptional specificity and strong affinity toward Catenin Alpha 1 protein (CTNNA1) in ESCC cells. Leveraging this specificity, nanosized EA2-modified pH-sensitive liposomes (EA2-PSL-PTX/LUT) are successfully developed with effective co-loading, controlled release, and good biostability. EA2-PSL-PTX/LUT exhibits stimuli-triggered release in the acidic tumor microenvironment and facilitates specific cellular uptake and endosomal escape in ESCC cells. In vivo imaging confirms precise tumor localization, deep tumor penetration, and prolonged retention of the nanocarrier. In vitro and in vivo findings validate that the nanocarrier potentiates synergistic inhibitions of PTX and LUT. Notably, EA2-PSL-PTX/LUT significantly activates the tumor microenvironment by promoting dendritic cell maturation and T cell infiltration. And the immunosuppressive microenvironment has been remodeled by decreasing myeloid-derived suppressor cells and regulatory T cell accumulation. This study provides a strategy for precise delivery of combinational chemotherapeutic drugs for ESCC targeted therapy.

Small molecules that can bind to specific cells have broad application in cancer diagnosis and treatment. Screening large chemical libraries against live cells is an effective strategy for discovering cell-targeting ligands. The DNA-encoded chemical library (DEL or DECL) technology has emerged as a robust tool in drug discovery and has been successfully utilized in identifying ligands for biological targets. However, nearly all DEL selections have predefined targets, while target-agnostic DEL selections interrogating the entire cell surface remain underexplored. Herein, we systematically optimized a cell-based DEL selection method against cancer cells without predefined targets. A 104.96-million-member DEL was selected against MDA-MB-231 and MCF-7 breast cancer cells, representing high and low metastatic properties, respectively, which led to the identification of cell-specific small molecules. We further demonstrated cell-targeting applications of these ligands in cancer photodynamic therapy and targeted drug delivery. Finally, leveraging the DNA tag of DEL compounds, we identified α-enolase (ENO1) as the cell surface receptor of one of the ligands targeting the more aggressive MDA-MB-231 cells. Overall, this work offers an efficient approach for discovering cell-targeting small molecule ligands by using DELs and demonstrates that DELs can be a useful tool to identify specific surface receptors on cancer cells.

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 represent a distinct category of short nucleotide sequences or peptide molecules characterized by their ability to bind to specific targets with high precision. These molecules are predominantly synthesized through SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology. Recent findings indicate that aptamers may have significant applications in regenerative medicine, particularly in the domain of tissue repair. In comparison to other bioactive agents, aptamers exhibit superior specificity and affinity, are more readily accessible, and can be chemically modified, thereby presenting a promising avenue for the functionalization of tissue engineering materials in tissue repair applications. This review delineates the properties of aptamers and examines the methodologies and advancements related to aptamer-functionalized hydrogels, nanoparticles, and electrospun materials. It categorizes the four primary functions of aptamers in tissue repair, namely regeneration, delivery systems, anti-inflammatory actions, and pro-coagulation effects. Furthermore, the review explores the utilization of aptamer-functionalized tissue engineering materials in the repair of bone, nerve, and vascular tissues, highlighting the mechanisms by which aptamers facilitate tissue growth and repair through regenerative properties and their role in transporting substances that promote repair. Lastly, the review addresses the future prospects and challenges associated with the application of aptamers in tissue repair, offering novel insights and directions for further research and application in this domain.

The disease progression and treatment options of leukemia between different subtypes vary considerably, emphasizing the importance of phenotyping. However, early typing of leukemia remains challenging due to the lack of highly sensitive and specific analytical tools. Herein, we propose a SERS-based platform for the classification of acute lymphoblastic T-cell leukemia (T-ALL) and chronic myeloid leukemia (CML) through the combination of machine learning and microfluidic chips. The ordered arrays in microfluidic channels reshape the microscopic flow field and contacting interfaces, facilitating the uniform and efficient capture of tumor cells. To enable phenotypic analysis, spectrally orthogonal SERS aptamer nanoprobes were applied, providing composite spectral signatures of individual cells in accordance with surface protein expression. Further, machine learning algorithms were employed to analyze the SERS signatures automatically, resulting in an accuracy of 98.6 % for 73 clinical blood samples. The results demonstrate that this platform holds promising potential for clinical leukemia diagnosis and precision medicine.

DNA reaction equilibrium-based calculations have great potential in thermodynamic characterization, but their widespread applications are hindered by significant measurement deviation of equilibrium concentration. Here, we report the advantages of metastable DNA hybridization in reducing quantification deviation of equilibrium concentration and propose a universal and standardized strategy for measuring aptamer binding energy, termed metastable DNA reference calorimetry (MDRC). We built different MDRC-based algorithms tailored to different aptamer binding models, enabling the calculation of thermodynamic parameters for aptamers with one or more binding sites. Our correlative model, considering the cross-effects between different binding sites, showed that for ATP aptamers with two binding sites, binding of the first ATP molecule would decrease its affinity for the second at low temperatures and even completely inhibit this binding at high temperatures. Moreover, the thermodynamic parameters of protein-specific aptamers were calculated to elucidate the universality of the method. The successful analysis of cell-specific aptamers further demonstrated MDRC's applicability in complex biological systems.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated system has displayed promise in visualizing the dynamics of target loci in living cells, which is important for studying genome regulation. However, developing a cell-friendly and rapid transfection method for achieving dynamic and long-term genomic imaging in living cells with high specificity and accuracy is still challenging. Herein, a robust and versatile method is presented that employs a barrel-shaped DNA nanostructure (TUBE) modified with aptamers for loading, protecting, and delivering CRISPR-Cas9 to visualize specific genomic loci in living cells. This approach enables dynamic tracking of target genomic regions (Chr3q29, a repetitive region of chromosome 3) throughout the mitotic process and captures variations in their spatial distribution and quantity accurately. Distinct dynamic behaviors between the Chr3q29 and telomeres are observed, which are linked to their unique chromosomal positions and levels of mobility. High-resolution multicolor labeling of the target genes is achieved, with a high degree of colocalization between the enhanced green fluorescent protein and cyanine-5 channels, facilitating precise imaging of target loci. This method not only supports dynamic genomic imaging but also enables multiplexed tracking, providing a powerful visualization tool for studying cellular processes and genetic interactions in real time within living cells.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection posed a significant threat to public health and the global economy, in vitro diagnosis of the SARS-CoV-2 nucleocapsid protein proved to be an effective way for SARS-CoV-2 infection control in the past years. Tyramide signal amplification (TSA) has been extensively utilized in tissue imaging and pathological diagnosis owing to the powerful signal enhancement. However, the elevated "ALWAYS ON" fluorescence background limited the accuracy and sensitivity of the conventional TSA assay. To achieve an activated "TURN ON" signal, herein, a small molecule, termed dichlorodihydrofluorescein tyramide (T-DCFH), was synthesized for activatable TSA. Under the catalysis of horseradish peroxidase (HRP) with hydrogen peroxide (H2O2), this T-DCFH facilitates the "TURN ON" fluorescence signal. Additionally, as a recognition tool, DNA aptamer has been used for developing in vitro diagnostic approaches. Hence, based on HRP-labeled aptamers binding with SARS-CoV-2 nucleocapsid protein, we achieved aptamers-based activatable TSA detection with a higher signal-to-noise ratio than that of fluorescent dye (FITC)-labeled aptamers, while showing lower background than traditional fluorescein tyramide with "ALWAYS ON". The results demonstrated that the activated T-DCFH significantly enhances the fluorescence signal while diminishing the background noise. By employing multiple aptamers targeting, we offered a timely and accurate in vitro diagnostic approach for future emergent infectious diseases.

Immune checkpoint blockade (ICB) therapies have demonstrated remarkable clinical success in treating cancer. However, their objective response rate remains suboptimal because current therapies rely on limited immune checkpoints that fail to cover the multiple immune evasion pathways of cancer. To explore potential ICB strategies, we propose a glycoimmune checkpoint elimination (glycoICE) therapy based on targeted editing of sialoglycans on the tumor cell surface using an aptamer-enzyme chimera (ApEC). The ApEC can be readily generated via a one-step bioorthogonal procedure, allowing for large-scale and uniform production. It specifically targets and desialylates cancer cells, disrupting the sialoglycan-Siglec axis to activate immune cells and enhance immunotherapy efficacy, while its high tumor selectivity minimizes side effects from indiscriminate desialylation of normal tissues. Furthermore, the ApEC has the potential to be a versatile platform for specific editing of sialoglycans in different tumor models by adjusting the aptamer sequences to target specific protein markers. This research not only introduces a novel molecular tool for the effective editing of sialoglycans in complex environments, but also provides valuable insights for advancing DNA-based drugs towards in vivo and clinical applications.

Nucleic acids have enabled the fabrication of self-assemblies and dynamic operations. Among different functional nucleic acids, aptamers can specifically bind to a wide range of targets, including proteins, viral antigens, living cells and even tissues, and have thus emerged as molecular recognition tools in molecular medicine. Hence, aptamer-functionalized nucleic acid nanotechnology offers applications of biosensing, bioimaging, and cancer therapy. In this review, after a brief overview of nucleic acid nanotechnology, we focus on the integration of aptamers with nucleic acid nanotechnology, including self-assembly constructions and dynamic molecular manipulations. The emerging applications in molecular medicine are subsequently reviewed with aptamer-based self-assemblies and aptamer-involved dynamic molecular manipulation. For convenience, applications are broadly categorized into biosensing, bioimaging, and cancer therapy. Finally, challenges and potential development of nucleic acid nanotechnology are discussed.

Aptamers have attracted exceptional attention in medical field due to their intrinsic properties equivalent to antibodies such as high target affinity, low immunogenicity and toxicity, cost-effectiveness and ease of synthesis and modification, and good stability under extreme conditions, thereby providing new avenues for basic research and clinical application. Protein tyrosine kinase 7 (PTK7) has been proved to be closely linked with the progression of many types of cancer. The aberrant expression of PTK7 has positioned it as a potential theranostic biomarker for multiple cancers. Aptamer sgc8 was initially identified for its high-affinity binding to PTK7 on the T-cell acute lymphoblastic leukemia cell line (CCRF-CEM) through cell-SELEX (systematic evolution of ligands by exponential enrichment) and subsequently has demonstrated the ability to effectively recognize many types of cancer cells that express PTK7 oncogenic target. The easily modifiable nature of sgc8 facilitates its conjugation with functional agents and drugs. This identification mode and modification approach of aptamers against cancer cells provides a potential strategy for cancer diagnosis and treatment. In this review, we discuss the potential of sgc8 aptamers in early cancer diagnosis and targeted therapy, focusing specifically on their interaction with the oncogenic biomarker PTK7.

Functional nucleic acids (FNAs), possessing specific biological functions beyond their informational roles, have gained widespread attention in disease therapeutics. However, their clinical application is severely limited by their low serum stability in complex physiological environments. In this work, a precise molecular programming strategy is explored to prepare glyconucleic acid aptamers (GNAAs) with high serum stability. Four glyconucleic acid modules compatible with commercial solid-phase synthesis are designed and synthesized. Through precise molecular design, the accurate modification of four different carbohydrate ligands at specific sites of DNA aptamers is achieved. It is demonstrated that glycosylation modification can significantly increase DNA aptamers' serum stability while maintaining their structures and high affinity. The stabilization effect is superior to that of currently commonly used commercial chemical modifications. Moreover, it is confirmed that this approach displays insignificant effects on the DNA aptamers' tumor-targeting ability and metabolism in vivo. This method offers a simple, economical, and efficient strategy for precise glycosylation modification of nucleic acids. This allows to prepare glycosyl functional nucleic acids with high serum stability, which can expand the application scope of functional nucleic acids and promote the practical transformation of functional nucleic acids.

Liposome-based drug delivery technologies have showed potential in enhancing medication safety and efficacy. Innovative drug loading and release mechanisms highlighted in this review of next-generation liposomal formulations. Due to poor drug release kinetics and loading capacity, conventional liposomes have limited clinical use. Scientists have developed new liposomal carrier medication release control and encapsulation methods to address these limits. Drug encapsulation can be optimized by creating lipid compositions that match a drug's charge and hydrophobicity. By selecting lipids and adding co-solvents or surfactants, scientists have increased drug loading in liposomal formulations while maintaining stability. Nanotechnology has also created multifunctional liposomes with triggered release and personalized drug delivery. Surface modification methods like PEGylation and ligand conjugation can direct liposomes to disease regions, improving therapeutic efficacy and reducing off-target effects. In addition to drug loading, researchers have focused on spatiotemporal modulation of liposomal carrier medication release. Stimuli-responsive liposomes release drugs in response to bodily signals. Liposomes can be pH- or temperature-sensitive. To improve therapeutic efficacy and reduce systemic toxicity, researchers added stimuli-responsive components to liposomal membranes to precisely control drug release kinetics. Advanced drug delivery technologies like magnetic targeting and ultrasound. Pro Drug, RNA Liposomes approach may improve liposomal medication administration. Magnetic targeting helps liposomes aggregate at illness sites and improves drug delivery, whereas ultrasound-mediated drug release facilitates on-demand release of encapsulated medicines. This review also covers recent preclinical and clinical research showing the therapeutic promise of next-generation liposomal formulations for cancer, infectious diseases, neurological disorders and inflammatory disorders. The transfer of these innovative liposomal formulations from lab to clinical practice involves key difficulties such scalability, manufacturing difficulty, and regulatory limits.

We present a novel DNA molecular machine (RCA-D-Walker) that integrates a DNAzyme-based molecular beacon with RCA-based vectors for miRNA imaging in tumor cells. It can accurately target tumor cells through the sgc8 aptamer. The target miRNA can restore the DNAzyme's ability to cleave the substrate, which in turn produces an amplified fluorescent signal. The RCA-D-Walker exhibits enhanced tumor cell targeting, improved cell permeability, and greater resistance to nuclease degradation. Utilizing this strategy, we achieved accurate and efficient imaging of miRNA-21 in tumor cells.

Nucleic acids, because of their precise pairing and simple composition, have emerged as excellent materials for the formation of gels. The application of DNA hydrogels in the diagnosis and therapy of cancer has expanded significantly through research on the properties and functions of nucleic acids. Functional nucleic acids (FNAs) such as aptamers, Small interfering RNA (siRNA), and DNAzymes have been incorporated into DNA hydrogels to enhance their diagnostic and therapeutic capabilities. This review discusses various methods for forming DNA hydrogels, with a focus on pure DNA hydrogels. We then explore the innovative applications of DNA hydrogels in cancer diagnosis and therapy. DNA hydrogels have become essential biomedical materials, and this review provides an overview of current research findings and the status of DNA hydrogels in the diagnosis and therapy of cancer, while also exploring future research directions.

As medical advancements turn most cancers into manageable chronic diseases, new challenges arise in cancer recurrence monitoring. Detecting circulating tumor cells (CTCs) is crucial for monitoring cancer recurrence, but the current methods are cumbersome and costly. This study developed a new CTC detection system combining DNA aptamer recognition, hybridization chain reaction (HCR) technology, and DNA logic devices, enabling the one-step recognition of CTCs by identifying multiple membrane proteins. After catalytically active Au nanoparticles were attached through reduction synthesis in situ onto the DNA hybridization strands of the CTCs surface, a 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric reaction was used to detect CTCs concentration via peroxidase-like catalysis. With this CTCs detection reporting system, we achieved an LOD of 4 cells/mL using an ultraviolet-visible (UV-vis) spectrophotometer. At certain concentrations, CTCs could even be detected visually without the need for an instrument. The development of this CTCs detection reporting system provided a convenient, reliable, and cost-effective detection strategy for widespread CTCs-based cancer recurrence monitoring.

Accurate identification and isolation of target cells are crucial for precision diagnosis and treatment. DNA aptamer-based logic devices provide a distinct advantage in this context, as they can logically analyze multiple cell surface markers with high efficiency. However, the susceptibility of natural DNA (D-DNA) to degradation can compromise the sensitivity and specificity of these devices, potentially leading to false-positive and false-negative results, particularly in complex biological environments. To address this issue, dual- and triple-aptamer-based cell-surface logic devices are designed and developed using mirror-image L-DNA, a chiral molecule of D-DNA with high biostability. These devices allow for simultaneous analysis of multiple cell surface proteins, achieving greater specificity in cell identification and isolation than D-DNA-based logic devices. The L-DNA probes realized 98.7% and 70.5% sensitivities in FBS buffer with dual- and triple-aptamer-based logic devices for target cell identification, while D-DNA probes only showed 27.9% and 0.1%. It is believed that the high stability of L-DNA and the high efficiency of the devices for labeling cell subpopulations will have broad applications in the life sciences, biomedical engineering, and personalized medicine.

Cells are fundamental units of life. The coordination of cellular functions and behaviors relies on a cascade of molecular networks. Technologies that enable exploration and manipulation of specific molecular events in living cells with high spatiotemporal precision would be critical for pathological study, disease diagnosis, and treatment. Framework nucleic acids (FNAs) represent a novel class of nucleic acid materials characterized by their monodisperse and rigid nanostructure. Leveraging their exceptional programmability, convenient modification property, and predictable atomic-level architecture, FNAs have attracted significant attention in diverse cellular applications such as cell recognition, imaging, manipulation, and therapeutic interventions. In this perspective, we will discuss the utilization of FNAs in living cell systems while critically assessing the opportunities and challenges presented in this burgeoning field.

Cell surface components, specifically glycans, play a significant role in several biological functions like cell structure, crosstalk between cells, and eventual target recognition of the cells for therapeutics. The dense layer of glycans, i. e., glycocalyx, could differ in taxon, species, and cell type. Glycans are coupled with lipids and proteins to form glycolipids, glycoproteins, proteoglycans, and glycosylphosphatidylinositol-anchored proteins, making their study challenging. However, understanding glycosylation at the cellular level is vital for fundamental research and advancing glycan-targeted therapy. Among different pathways, metabolic glycan labelling uses the natural metabolic processes of the cell to introduce abiotic functionality into glycan residues. The Bertozzi group pioneered metabolic oligosaccharide engineering using glycan salvage pathways to convert monosaccharides with unnatural modifications. This eventually results in the probe becoming part of the complex cellular glycan structures via click chemistry using copper. On the other hand, the boronic acid-based probe can recognise carbohydrates in a single step without any chemical modification of the surface. This review discusses the significance of glycans as biomarkers for different diseases and the necessity to evaluate them in situ within the physiological environment. The review also discusses the prospect of this field and its potential applications.

The era of molecular medicine arose as we began to diagnose and treat diseases based on understanding how genes, proteins, and cells work, providing optimal therapeutic care through molecular profiling. Central to molecular medicine is molecular recognition, which is underpinned by techniques involving omics analysis, gene editing, and targeted agents. Recent advancements in these tools not only expand our understanding of biological processes but also aid in the development of diagnostic and treatment modalities at the molecular level, thus bridging the gap between medical research and clinical applications. This perspective traces the development of molecular tools, highlighting, along the way, their pivotal role in advancing molecular medicine for the global health of people.

Trophoblast cell surface antigen 2 expressed in several malignant cancers promotes tumor growth and metastasis via several signal transduction pathways. Trop2 is reputed as a prospective biomarker and therapeutic target. Trophoblast cell surface antigen 2-targeted agents, including antibodies, antibody conjugates and therapeutic combinations, could be utilized to fight cancers. To develop an effective drug targeting strategy, we resorted to a new trophoblast cell surface antigen 2-targeted anticancer treatment through aptamer conjugated with chemotherapeutic drug. This study identified trophoblast cell surface antigen 2-specific ssDNA aptamers using engineered trophoblast cell surface antigen 2 overexpression cells for cell-SELEX. The obtained ssDNA aptamer bound to trophoblast cell surface antigen 2 overexpressed cells with nanomolar affinity and was specific for several tumor cell types which express trophoblast cell surface antigen 2 abundantly. Significant cytotoxicity against HT29 cell by the conjugate of trophoblast cell surface antigen 2 aptamer and Emtansine was observed while resulting negligible therapeutic effect on human normal intestinal epithelial cell line HIEC in vitro, indicating that the conjugate shows potential as a promising therapeutic agent. Furthermore, the isolated aptamer demonstrated the ability for the targeted delivery, resulting excellent therapeutic effectiveness of aptamer-drug conjugate for xenograft tumor model of mice with human colorectal cancer.

Nucleic acid aptamers have been used in the past for the development of diagnostic methods against a number of targets such as bacteria, pesticides, cancer cells etc. In the present study, six rounds of Cell-SELEX were performed on a ssDNA aptamer library against X-enriched sperm cells from Sahiwal breed cattle. Sequencing was used to examine the aptamer sequences that shown affinity for sperm carrying the X chromosome in order to find any possible X-sperm-specific sequences. Out of 35 identified sequences, 14 were selected based on bioinformatics analysis like G-Score and Mfold structures. Further validation of their specificity was done via fluorescence microscopy. The interaction of biotinylated-aptamer with sperm was also determined by visualizing the binding of streptavidin coated magnetic beads on the head region of the sperm under bright field microscopy. Finally, a real-time experiment was designed for the validation of X-sperm enrichment by synthesized aptamer sequences. Among the studied sequences, aptamer 29a exhibited a higher affinity for X sperm compared to Y sperm in a mixed population of sperm cells. By using aptamer sequence 29a, we obtained an enrichment of 70% for X chromosome bearing sperm cells.

The protein tyrosine kinase-7 (PTK7) is an evolutionarily conserved transmembrane receptor that has emerged as a potential therapeutic target for human tumors. PTK7 is a pseudokinase that is involved in the modulation of the Wnt signaling pathway through interactions with other receptors. These interactions result in targeted gene activation that regulates cell polarity, migration, and proliferation during embryogenesis. Aside of this role during development, PTK7 has been shown as overexpressed in numerous cancers including colon carcinoma, leukemia, neuroblastoma, hepatoma, and ovarian cancer. The activity of PTK7 and the direct correlation with poor prognosis have fostered preclinical investigations and phase I clinical trials, aiming at inhibiting PTK7 and inducing antitumoral effects. In this review, we provide an exhaustive overview of the diverse approaches that use PTK7 as a new molecular target for cancer therapy in different tumor types. We discuss current therapies and future strategies including chimeric antigen receptor-T cells, antibody-drug conjugates, aptamers, based on up-to-date literature and ongoing research progress.

Tumor-associated antigens (TAAs) are not exclusively expressed in cancer cells, inevitably causing the "on target, off tumor" effect of molecular recognition tools. To achieve precise recognition of cancer cells, by using protein tyrosine kinase 7 (PTK7) as a model TAA, a DNA molecular logic circuit Aisgc8 was rationally developed by arranging H+-binding i-motif, ATP-binding aptamer, and PTK7-targeting aptamer Sgc8c in a DNA sequence. Aisgc8 output the conformation of Sgc8c to recognize PTK7 on cells in a simulated tumor microenvironment characterized by weak acidity and abundant ATP, but not in a simulated physiological environment. Through in vitro and in vivo results, Aisgc8 demonstrated its ability to precisely recognize cancer cells and, as a result, displayed excellent performance in tumor imaging. Thus, our studies produced a simple and efficient strategy to construct DNA logic circuits, opening new possibilities to develop convenient and intelligent precision diagnostics by using DNA logic circuits.

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.

Amplifying DNA conjugated affinity ligands can improve the sensitivity and multiplicity of cell imaging and play a crucial role in comprehensively deciphering cellular heterogeneity and dynamic changes during development and disease. However, the development of one-step, controllable, and quantitative DNA amplification methods for multiplexed imaging of live-cell membrane proteins is challenging. Here, we introduce the template adhesion reaction (TAR) method for assembling amplifiable DNA sequences with different affinity ligands, such as aptamers or antibodies, for amplified and multiplexed imaging of live-cell membrane proteins with high quantitative fidelity. The precisely controllable TAR enables proportional amplification of membrane protein targets with variable abundances by modulating the concentration ratios of hairpin templates and primers, thus allowing sensitive visualization of multiple membrane proteins with enhanced signal-to-noise ratios (SNRs) without disturbing their original ratios. Using TAR, we achieved signal-enhanced imaging of six proteins on the same live-cell within 1-2 h. TAR represents an innovative and programmable molecular toolkit for multiplexed profiling of membrane proteins in live-cells.

Functional nucleic acids (FNAs) have attracted increasing attention in recent years due to their diverse physiological functions. The understanding of their conformational recognition mechanisms has advanced through nucleic acid tailoring strategies and sequence optimization. With the development of the FNA tailoring techniques, they have become a methodological guide for nucleic acid repurposing. Therefore, it is necessary to systematize the relationship between FNA tailoring strategies and the development of nucleic acid multifunctionality. This review systematically categorizes eight types of FNA multifunctionality, and introduces the traditional FNA tailoring strategy from five aspects, including deletion, substitution, splitting, fusion and elongation. Based on the current state of FNA modification, a new generation of FNA tailoring strategy, called the high-content tailoring strategy, was unprecedentedly proposed to improve FNA multifunctionality. In addition, the multiple applications of rational tailoring-driven FNA performance enhancement in various fields were comprehensively summarized. The limitations and potential of FNA tailoring and repurposing in the future are also explored in this review. In summary, this review introduces a novel tailoring theory, systematically summarizes eight FNA performance enhancements, and provides a systematic overview of tailoring applications across all categories of FNAs. The high-content tailoring strategy is expected to expand the application scenarios of FNAs in biosensing, biomedicine and materials science, thus promoting the synergistic development of various fields.

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.

The rapid and accurate detection of SARS-CoV-2, particularly its spike receptor-binding domain (S-RBD), was crucial for managing the COVID-19 pandemic. This study presents the development and optimization of two types of aptasensors: quartz crystal microbalance (QCM) and electrochemical sensors, both employing thiol-modified DNA aptamers for S-RBD detection. The QCM aptasensor demonstrated exceptional sensitivity, achieved by optimizing aptamer concentration, buffer composition, and pre-treatment conditions, with a limit of detection (LOD) of 0.07 pg/mL and a linear range from 1 pg/mL to 0.1 µg/mL, and a significant frequency change was observed upon target binding. The electrochemical aptasensor, designed for rapid and efficient preparation, utilized a one-step modification process that reduced the preparation time to 2 h while maintaining high sensitivity and specificity. Electrochemical impedance spectroscopy (EIS) enabled the detection of S-RBD concentrations as low as 132 ng/mL. Both sensors exhibited high specificity, with negligible non-specific interactions observed in the presence of competing proteins. Additionally, the QCM aptasensor's functionality and stability were verified in biological fluids, indicating its potential for real-world applications. This study highlights the comparative advantages of QCM and electrochemical aptasensors in terms of preparation time, sensitivity, and specificity, offering valuable insights for the development of rapid, sensitive, and specific diagnostic tools for the detection of SARS-CoV-2 and other viruses.

Rheumatoid arthritis (RA) is an autoimmune disease characterised by aggressive fibroblast-like synoviocytes (FLSs). Very few RA patients-derived FLSs (RA-FLSs)-specific surface signatures have been identified, and there is currently no approved targeted therapy for RA-FLSs. This study aimed to screen therapeutic aptamers with cell-targeting and cytotoxic properties against RA-FLSs and to uncover the molecular targets and mechanism of action of the screened aptamers.

We developed a cell-specific and cytotoxic systematic evolution of ligands by exponential enrichment (CSCT-SELEX) method to screen the therapeutic aptamers without prior knowledge of the surface signatures of RA-FLSs. The molecular targets and mechanisms of action of the screened aptamers were determined by pull-down assays and RNA sequencing. The therapeutic efficacy of the screened aptamers was examined in arthritic mouse models.

We obtained an aptamer SAPT8 that selectively recognised and killed RA-FLSs. The molecular target of SAPT8 was nucleolin (NCL), a shuttling protein overexpressed on the surface and involved in the tumor-like transformation of RA-FLSs. Mechanistically, SAPT8 interacted with the surface NCL and was internalised to achieve lysosomal degradation of NCL, leading to the upregulation of proapoptotic p53 and downregulation of antiapoptotic B-cell lymphoma 2 (Bcl-2) in RA-FLSs. When administrated systemically to arthritic mice, SAPT8 accumulated in the inflamed FLSs of joints. SAPT8 monotherapy or its combination with tumour necrosis factor (TNF)-targeted biologics was shown to relieve arthritis in mouse models.

CSCT-SELEX could be a promising strategy for developing cell-targeting and cytotoxic aptamers. SAPT8 aptamer selectively ablates RA-FLSs via modulating NCL-p53/Bcl-2 signalling, representing a potential alternative or complementary therapy for RA.

Oncological diseases represent a significant global health challenge, with high mortality rates. Early detection is crucial for effective treatment, and aptamers, which demonstrate superior specificity and stability compared to antibodies, offer a promising avenue for diagnostic advancement. This study presents the design, development and evaluation of a quartz crystal microbalance (QCM) sensor functionalized with the T2-KK1B10 aptamer for the sensitive and specific detection of Chronic Myeloid Leukemia (CML) K562 cells. The research focuses on optimizing the biorecognition layer by adjusting the aptamer conditions, demonstrating the sensor's ability to detect these CML cells with high specificity and sensitivity. The aptamer-modified QCM sensor operates on the principle of mass change detection upon binding of target cells. By employing the Langmuir isotherm model, the performance of the sensor was optimized for the capture of CML cells from biological samples with LOD of 263 K562 cells. The sensor was also successfully regenerated multiple times without sensitivity loss. Validation of the sensor's performance was conducted under controlled laboratory settings, followed by extensive testing utilizing human lyophilized plasma and clinical samples from patients. The sensor exhibited high sensitivity and specificity in the detection of CML cells within clinical specimens, thereby illustrating its potential for practical clinical deployment. This research presents a novel approach to the early diagnosis of CML, facilitating timely intervention and enhanced patient outcomes. The developed aptasensor demonstrates potential for broader application in cancer diagnostics and personalized medicine.

The efficacy of neurotherapeutic drugs hinges on their ability to traverse the blood-brain barrier and access the brain, which is crucial for treating or alleviating neurodegenerative diseases (NDs). Given the absence of definitive cures for NDs, early diagnosis and intervention become paramount in impeding disease progression. However, conventional therapeutic drugs and existing diagnostic approaches must meet clinical demands. Consequently, there is a pressing need to advance drug delivery systems and early diagnostic methods tailored for NDs. Certain aptamers endowed with specific functionalities find widespread utility in the targeted therapy and diagnosis of NDs. DNA nanoflowers (DNFs), distinctive flower-shaped DNA nanomaterials, are intricately self-assembled through rolling ring amplification (RCA) of circular DNA templates. Notably, imbuing DNFs with diverse functionalities becomes seamlessly achievable by integrating aptamer sequences with specific functions into RCA templates, resulting in a novel nanomaterial, aptamer-bound DNFs (ADNFs) that amalgamates the advantageous features of both components. This article delves into the characteristics and applications of aptamers and DNFs, exploring the potential or application of ADNFs in drug-targeted delivery, direct treatment, early diagnosis, etc. The objective is to offer prospective ideas for the clinical treatment or diagnosis of NDs, thereby contributing to the ongoing efforts in this critical field.

Rationale: Neovascular ocular diseases (NODs) represent the leading cause of visual impairment globally. Despite significant advances in anti-angiogenic therapies targeting vascular endothelial growth factor (VEGF), persistent challenges remain prevalent. As a proof-of-concept study, we herein demonstrate the effectiveness of targeted degradation of VEGF with bispecific aptamer-based lysosome-targeting chimeras (referred to as VED-LYTACs). Methods: VED-LYTACs were constructed with three distinct modules: a mannose-6-phosphate receptor (M6PR)-binding motif containing an M6PR aptamer, a VEGF-binding module with an aptamer targeting VEGF, and a linker essential for bridging and stabilizing the two-aptamer structure. The degradation efficiency of VED-LYTACs via the autophagy-lysosome system was examined using an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. Subsequently, the anti-angiogenic effects of VED-LYTACs were evaluated using in vitro wound healing assay, tube formation assay, three-dimensional sprouting assay, and ex vivo aortic ring sprouting assay. Finally, the potential therapeutic effects of VED-LYTACs on pathological retinal neovascularization and vascular leakage were tested by employing mouse models of NODs. Results: The engineered VED-LYTACs promote the interaction between M6PR and VEGF, consequently facilitating the translocation and degradation of VEGF through the lysosome. Our data show that treatment with VED-LYTACs significantly suppresses VEGF-induced angiogenic activities both in vitro and ex vivo. In addition, intravitreal injection of VED-LYTACs remarkably ameliorates abnormal vascular proliferation and leakage in mouse models of NODs. Conclusion: Our findings present a novel strategy for targeting VEGF degradation with an aptamer-based LYTAC system, effectively ameliorating pathological retinal angiogenesis. These results suggest that VED-LYTACs have potential as therapeutic agents for managing NODs.

This review delves into the advancements in molecular recognition through enhanced SELEX (Systematic Evolution of Ligands by Exponential Enrichment) platforms and post-aptamer modifications. Aptamers, with their superior specificity and affinity compared to antibodies, are central to this discussion. Despite the advantages of the SELEX process-encompassing stages like ssDNA library preparation, incubation, separation, and PCR amplification-it faces challenges, such as nuclease susceptibility. To address these issues and propel aptamer technology forward, we examine next-generation SELEX platforms, including microfluidic-based SELEX, capillary electrophoresis SELEX, cell-based aptamer selection, counter-SELEX, in vivo SELEX, and high-throughput sequencing SELEX, highlighting their respective merits and innovations. Furthermore, this article underscores the significance of post-aptamer modifications, particularly chemical strategies that enhance aptamer stability, reduce renal filtration, and expand their target range, thereby broadening their utility in diagnostics, therapeutics, and nanotechnology. By synthesizing these advanced SELEX platforms and modifications, this review illuminates the dynamic progress in aptamer research and outlines the ongoing efforts to surmount existing challenges and enhance their clinical applicability, charting a path for future breakthroughs in this evolving field.

Aberrant phosphorylation of receptor tyrosine kinases (RTKs) is usually involved in tumor initiation, progression, and metastasis. However, developing specific and efficient molecular tools to regulate RTK phosphorylation remains a considerable challenge. In this study, we reported novel aptamer-based chimeras to inhibit the phosphorylation of RTKs, such as c-Met and EGFR, by enforced recruitment of a protein tyrosine phosphatase receptor type F (PTPRF). Our studies revealed that aptamer-based chimeras displayed a generic and potent inhibitory effect on RTK phosphorylation induced by growth factor or auto-dimerization in different cell lines and modulated cell biological behaviors by recruiting PTPRF. Furthermore, based on angstrom accuracy of the DNA duplex, the maximum catalytic radius of PTPRF was determined as ∼25.84 nm, providing a basis for the development of phosphatase-recruiting strategies. Taken together, our study provides a generic methodology not only for selectively mediating RTK phosphorylation and cellular biological processes but also for developing novel therapeutic drugs.

Among the various targeting ligands for drug delivery, aptamers have attracted much interest in recent years because of their smaller size compared to antibodies, ease of modification, and better batch-to-batch consistency. In addition, aptamers can be selected to target both known and even unknown cell surface biomarkers. For drug loading, liposomes are the most successful vehicle and many FDA-approved formulations are based on liposomes. In this paper, aptamer-functionalized liposomes for targeted drug delivery are reviewed. We begin with the description of related aptamers selection, followed by methods to conjugate aptamers to liposomes and the fate of such conjugates in vivo. Then a few examples of applications are reviewed. In addition to intravenous injection for systemic delivery and hoping to achieve accumulation at target sites, for certain applications, it is also possible to have aptamer/liposome conjugates applied directly at the target tissue such as intratumor injection and dropping on the surface of the eye by adhering to the cornea. While previous reviews have focused on cancer therapy, the current review mainly covers other applications in the last four years. Finally, this article discusses potential issues of aptamer targeting and some future research opportunities.

Malignant tumors are one of the leading causes of death worldwide, imposing a substantial economic and social burden. Early detection is the key to improving cure rates and reducing mortality rates, which requires the development of sensitive early detection technologies. Signal amplification techniques play a crucial role in aptamer-based early detection of tumors and are increasingly garnering attention from researchers.

To investigate the current research status, developmental trajectories, and hotspots in signal amplification for aptamer-based tumor detection through bibliometric analysis.

English publications pertaining to signal amplification in aptamer-based tumor detection were retrieved from the Web of Science Core Collection database. VOSviewer and CiteSpace software were employed to analyze various information within this field, including countries, institutions, authors, co-cited authors, journals, co-cited journals, cited references, and keywords.

A total of 757 publications were included in this study. China accounted for 85.47% of all publications, with Nanjing University (China) emerging as the institution with the highest publication output. The most influential authors and journals were Hasanzadeh M. from Iran and "Biosensors and Bioelectronics", respectively. Exosomes and carcinoembryonic antigen (CEA) stood out as the most researched tumor-related molecules. Currently, the predominant signal amplification technique, nanomaterial, and signal transduction method were identified as hybridization chain reactions, gold nanoparticles, and electrochemical methods, respectively. Over the past 3 years, exosomes, CEA, electrochemical biosensors, and nanosheets have emerged as research hotspots, exhibiting a robust burst of intensity.

This study is the first bibliometric analysis of literature on signal amplification in aptamer-based tumor detection and elucidates the current status, hotspots, and prospective research directions within this realm. Additionally, it provides an important reference for researchers.

Esophageal cancer ranks the seventh in cancer incidence and the sixth in cancer death. Esophageal squamous cell carcinoma (ESCC) accounts for approximately 90% of the total cases of esophageal cancer. Chemotherapy is the most effective drug-based method for treatment of esophageal cancer. However, severe side effects of traditional chemotherapy limit its treatment efficacy. Targeted chemotherapy can deliver chemotherapeutic drugs to cancer cells and specifically kill these cells with reduced side effects. In the work, the bivalent aptamer-DNA carrier (BAD) was designed by using an ESCC cell-specific aptamer as the recognition molecule and a GC base-rich DNA sequence as the drug carrier. With doxorubicin (Dox) as chemotherapeutic drugs, the bivalent aptamer-DNA-Dox conjugate (BADD) was constructed for targeted killing of ESCC cells. Firstly, the truncated A2(35) aptamer with a retained binding ability was obtained through optimization of an intact A2(80) aptamer and was used to fuse with DNA carrier sequences for constructing the BAD through simple DNA hybridization. The results of gel electrophoresis and flow cytometry analysis showed that the BAD was successfully constructed and had a stronger binding affinity than monovalent A2(35). Then, the BAD was loaded with Dox drugs to construct the BADD through noncovalent intercalation. The results of fluorescence spectra and flow cytometry assays showed that the BADD was successfully constructed and can bind to target cells strongly. Confocal imaging further displayed that the BADD can be specifically internalized into target cells and release Dox. The results of CCK-8 assays, Calcein AM/PI staining, and wound healing assays demonstrated that the BADD can specifically kill target cells, but not control cells. Our results demonstrate that the developed BADD can specifically deliver doxorubicin to target ESCC cells and selectively kill these cells, offering a potentially effective strategy for targeted chemotherapy of ESCC.