DNA aptamers have emerged as novel molecular tools in disease theranostics owing to their high binding affinity and specificity for protein targets, which rely on their ability to fold into distinctive three-dimensional (3D) structures. However, delicate atomic interactions that shape the 3D structures are often ignored when designing and modeling aptamers, leading to inefficient functional optimization. Challenges persist in determining high-resolution aptamer-protein complex structures. Moreover, the experimentally determined 3D structures of DNA molecules with exquisite functions remain scarce. These factors impede our comprehension and optimization of some important DNA aptamers. Here, we performed a streamlined solution NMR-based structural investigation on the 41-nt sgc8c, a prominent DNA aptamer used to target membrane protein tyrosine kinase 7, for cancer theranostics. We show that sgc8c prefolds into an intricate three-way junction (3WJ) structure stabilized by long-range tertiary interactions and extensive base-base stackings. Delineated by NMR chemical shift perturbations, site-directed mutagenesis, and 3D structural information, we identified essential nucleotides constituting the key functional elements of sgc8c that are centralized at the core of 3WJ. Leveraging the well-established structure-function relationship, we efficiently engineered two sgc8c variants by modifying the apical loop and introducing L-DNA base pairs to simultaneously enhance thermostability, biostability, and binding affinity for both protein and cell targets, a feat not previously attained despite extensive efforts. This work showcases a simplified NMR-based approach to comprehend and optimize sgc8c without acquiring the complex structure, and offers principles for the sophisticated structure-function organization of DNA molecules.

Postoperative peritoneal adhesion (PPA) is a prevalent complication of abdominal surgery, posing a significant hindrance to postsurgical recovery. Although several strategies have been developed to alleviate and prevent adhesions, their efficacy remains unsatisfactory. For the first time, we studied the therapeutic effect and mechanism of our recently developed thermally stable oligonucleotide-based mimetics of hepatocyte growth factor (HGF DNA aptamer) to prevent PPA. The HGF DNA aptamer effectively inhibited canonical TGF-β1 signaling transduction, partially suppressing mesothelial mesenchymal transition. Additionally, the aptamer, respectively, upregulated and downregulated the expression of tissue plasminogen activator and plasminogen activator inhibitor 1, thereby enhancing fibrinolytic activity. As a pleiotropic factor, the HGF DNA aptamer also enhanced the migratory and proliferative capacities of mesothelial cells. Finally, the aptamer demonstrated a higher level of effectiveness in preventing PPAs than the commercially available antiperitoneal adhesion barrier, Seprafilm. Due to its therapeutic benefits, excellent stability, biosafety, cost-effectiveness, and versatility, the HGF DNA aptamer demonstrates promise for preventing PPA in future clinical settings.

Overactive or dysregulated cytokine expression is a hallmark of many acute and chronic inflammatory diseases. This is true for acute or chronic infections, neurodegenerative diseases, autoimmune diseases, cardiovascular diseases, cancer, and others. Cytokines such as interleukin-6 (IL-6) are known therapeutic targets and biomarkers for such inflammatory diseases. Platforms for cytokine detection are, therefore, desirable tools for both research and clinical applications. Single-walled carbon nanotubes (SWCNT) are versatile nanomaterials with near-infrared fluorescence that can serve as transducers for optical sensors. When functionalized with an analyte-specific recognition element, SWCNT emission may become sensitive and selective toward the desired target. SWCNT-aptamer sensors are easily assembled, inexpensive, and biocompatible. In this work, we introduced a nanosensor design based on SWCNT and a DNA aptamer specific to IL-6. We first evaluated several SWCNT-aptamer constructs based on this simple direct complexation method, wherein the aptamer both solubilizes the SWCNT and confers sensitivity to IL-6. The sensor limit of detection, 105 ng/mL, lies in the relevant range for pathological IL-6 levels. Upon investigation of sensor kinetics, we found rapid response within seconds of antigen addition which continued over the course of 3 h. We found that this sensor construct is stable and the aptamer is not displaced from the nanotube surface during IL-6 detection. Finally, we investigated the ability of this sensor construct to detect macrophage activation caused by bacterial lipopolysaccharides (LPS) in an in vitro model of disease, finding rapid and sensitive detection of macrophage-expressed IL-6. We are confident that further development of this sensor will have novel implications for diagnosis of acute and chronic inflammatory diseases, in addition to contributing to the understanding of the role of cytokines in these diseases.

Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with KD = 2.1 μM or KD = 1.6 μM toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and anti-hemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10-500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real-time and versatile monitoring of large-scale scFv production.

UNASSIGNED: Oligomeric amyloid beta (oAβ) is a toxic factor that acts in the early stage of Alzheimer\'s disease (AD) and may initiate the pathologic cascade. Therefore, detecting oAβ has a crucial role in the early diagnosis, monitoring, and treatment of AD.
UNASSIGNED: The purpose of this study was to evaluate MRI signal changes in different mouse models and the time-dependent signal changes using our novel gadolinium (Gd)-dodecane tetraacetic acid (DOTA)- ob5 aptamer contrast agent.
UNASSIGNED: We developed an MRI contrast agent by conjugating Gd-DOTA-DNA aptamer called ob5 to evaluate its ability to detect oAβ deposits in the brain using MRI. A total of 10 control mice, 9 3xTg AD mice, and 11 APP/PS/Tau AD mice were included in this study, with the age of each model being 16 or 36 weeks. A T1-weighted image was acquired at the time points before (0 min) and after injection of the contrast agent at 5, 10, 15, 20, and 25 min. The analyses were performed to compare MRI signal differences among the three groups and the time-dependent signal differences in different mouse models.
UNASSIGNED: Both 3xTg AD and APP/PS/Tau AD mouse models had higher signal enhancement than control mice at all scan-time points after injection of our contrast media, especially in bilateral hippocampal areas. In particular, all Tg AD mouse models aged 16 weeks showed a higher contrast enhancement than those aged 36 weeks. For 3xTg AD and APP/PS/Tau AD groups, the signal enhancement was significantly different among the five time points (0 min, 5 min, 10 min, 15 min, 20 min, and 25 min) in multiple ROI areas, typically in the bilateral hippocampus, left thalamus, and left amygdala.
UNASSIGNED: The findings of this study suggest that the expression of the contrast agent in different AD models demonstrates its translational flexibility across different species. The signal enhancement peaked around 15-20 min after injection of the contrast agent. Therefore, our novel contrast agent targeting oAβ has the potential ability to diagnose early AD and monitor the progression of AD.

Type 2 diabetes mellitus (T2DM) is a risk factor for male infertility, but the underlying molecular mechanisms remain unclear. Advanced glycation end products (AGEs) are pathogenic molecules for diabetic vascular complications. Here, we investigated the effects of the DNA aptamer raised against AGEs (AGE-Apt) on testicular and sperm abnormalities in a T2DM mouse model. KK-Ay (DM) and wild-type (non-DM) 4- and 7-week-old male mice were sacrificed to collect the testes and spermatozoa for immunofluorescence, RT-PCR, and histological analyses. DM and non-DM 7-week-old mice were subcutaneously infused with the AGE-Apt or control-aptamer for 6 weeks and were then sacrificed. Plasma glucose, testicular AGEs, and Rage gene expression in 4-week-old DM mice and plasma glucose, testicular AGEs, oxidative stress, and pro-inflammatory gene expressions in 7-week-old DM mice were higher than those in age-matched non-DM mice, the latter of which was associated with seminiferous tubular dilation. AGE-Apt did not affect glycemic parameters, but it inhibited seminiferous tubular dilation, reduced the number of testicular macrophages and apoptotic cells, and restored the decrease in sperm concentration, motility, and viability of 13-week-old DM mice. Our findings suggest that AGEs-Apt may improve sperm abnormality by suppressing AGE-RAGE-induced oxidative stress and inflammation in the testes of DM mice.

We developed an aptamer-based fluorescence resonance energy transfer (FRET) assay capable of recognizing therapeutic monoclonal antibody bevacizumab and rapidly quantifying its concentration with just one mixing step. In this assay, two fluorescent dyes (fluorescein and tetramethylrhodamine) labeled aptamers bind to two Fab regions on bevacizumab, and FRET fluorescence is observed when both dyes come into close proximity. We optimized this assay in three different formats, catering to a wide range of analytical needs. When applied to hybridoma culture samples in practical settings, this assay exhibited a signal response that was concentration-dependent, falling within the range of 50-2000 μg/mL. The coefficients of determination (r2) ranged from 0.998 to 0.999, and bias and precision results were within ±24.0 % and 20.3 %, respectively. Additionally, during thermal and UV stress testing, this assay demonstrated the ability to detect denatured samples in a manner comparable to conventional Size Exclusion Chromatography. Notably, it offers the added advantage of detecting decreases in binding activity without changes in molecular weight. In contrast to many existing process analytical technology tools, this assay not only identifies bevacizumab but also directly measures the quality attributes related to mAb efficacy, such as the binding activity. As a result, this assay holds great potential as a valuable platform for providing highly reliable quality attribute information in real-time. We consider this will make a significant contribution to the worldwide distribution of high-quality therapeutic mAbs in various aspects of antibody manufacturing, including production monitoring, quality control, commercial lot release, and stability testing.

BACKGROUND: Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein.
RESULTS: In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E - 7, 2.004E - 8, and 1.68E - 8 M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033 μg/ml for Yer 21, Yer 24, and Yer 25, respectively.
CONCLUSIONS: This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

Interleukin (IL)-23 inhibitor monoclonal antibodies shown significant efficacy in treating autoimmune diseases. DNA or RNA aptamers exhibit comparable specificity to antibodies, are cost-effective, non-immunogenic, and do not have batch to batch variation. This study aimed to characterize a single-stranded DNA (ssDNA) aptamer targeting human IL-23. The alpha subunit of IL-23 (P19) and intact IL-23 were cloned, expressed, and the proteins finally were purified through Ni2+-iminodiacetic acid affinity chromatography. The selection and characterization of ssDNA aptamer against P19 were conducted using the protein-systematic evolution of ligands by exponential enrichment (SELEX). Dot blot assay was carried out to monitor binding of the aptamer output of SELEX rounds, to P19 protein. The dissociation constant (Kd) of aptamers with positive results in dot blot assay, determined based on their binding to IL-23 using an ELISA method. Recombinant P19 and IL-23 proteins were 26 and 72 kDa, respectively, observed on SDS-PAGE .12 %. The aptamers output from 7, 8, 9, 10, 11, and 12 rounds of the SELEX was monitored by dot blot assay, revealing that the aptamer from the round 8 has stronger luminescent signal and was selected for TA-cloning. After analyzing the biotinylated aptamers from clones, positive clones in dot blot assay and ELISA were sequenced. Finally, the Kd calculation revealed three aptamers with high affinity, named A23P3, A23P6, and A23P15 with Kd values of 1.37, 2.139, and 2.88 nM, respectively. Results of this study introduced three specific anti-IL-23 ssDNA aptamers with high affinity, which could be utilized for therapeutic and diagnostic purposes.

The ongoing SARS-CoV-2 pandemic has underscored the urgent need for versatile and rapidly deployable antiviral strategies. While vaccines have been pivotal in controlling the spread of the virus, the emergence of new variants continues to pose significant challenges to global health. Here, our study focuses on a novel approach to antiviral therapy using DNA aptamers, short oligonucleotides with high specificity and affinity for their targets, as potential inhibitors against the spike protein of SARS-CoV-2 variants Omicron and JN.1. Our research utilizes steered molecular dynamics (SMD) simulations to elucidate the binding mechanisms of a specifically designed DNA aptamer, AM032-4, to the receptor-binding domain (RBD) of the aforementioned variants. The simulations reveal detailed molecular insights into the aptamer-RBD interaction, demonstrating the aptamer\'s potential to maintain effective binding in the face of rapid viral evolution. Our work not only demonstrates the dynamic interaction between aptamer-RBD for possible antiviral therapy but also introduces a computational method to study aptamer-protein interactions.