Structure Of Aptamer Research Articles

Aptamer’s Structure Optimization for Better Diagnosis and Treatment of Glial Tumors

Background: Oncological diseases are a major focus in medicine, with millions diagnosed each year, leading researchers to seek new diagnostic and treatment methods. One promising avenue is the development of targeted therapies and rapid diagnostic tests using recognition molecules. The pharmaceutical industry is increasingly exploring nucleic acid-based therapeutics. However, producing long oligonucleotides, especially aptamers, poses significant production challenges. Objectives: This study aims to demonstrate the efficacy of using molecular modeling, supported by experimental procedures, for altering aptamer nucleotide sequences while maintaining their binding capabilities. The focus is on reducing production costs and enhancing binding dynamics by removing nonfunctional regions and minimizing nonspecific binding. Methods: A molecular modeling approach was employed to elucidate the structure of a DNA aptamer, Gli-55, facilitating the truncation of nonessential regions in the Gli-55 aptamer, which selectively binds to glioblastoma (GBM). This process aimed to produce a truncated aptamer, Gli-35, capable of forming similar structural elements to the original sequence with reduced nonspecific binding. The efficiency of the truncation was proved by flow cytometry, fluorescence polarization (FP), and confocal microscopy. Results: The molecular design indicated that the new truncated Gli-35 aptamer retained the structural integrity of Gli-55. In vitro studies showed that Gli-35 had a binding affinity comparable to the initial long aptamer while the selectivity increased. Gli-35 internalized inside the cell faster than Gli-55 and crossed the blood–brain barrier (BBB), as demonstrated in an in vitro model. Conclusions: The success of this truncation approach suggests its potential applicability in scenarios where molecular target information is limited. The study highlights a strategic and resource-efficient methodology for aptamer development. By employing molecular modeling and truncation, researchers can reduce production costs and avoid trial and error in sequence selection. This approach is promising for enhancing the efficiency of therapeutic agent development, particularly in cases lacking detailed molecular target insights.

Palindrome-mediated DNA nanotubes with cell-specific aptamers to improve targeted antitumor effects and reduce toxicity on non-small cell lung cancer.

Chemotherapy is regarded as a widely used and effective treatment strategy for lung cancer, although most conventional chemotherapeutics cause severe toxic side-effects due to their indiscriminate attacks on both cancerous and normal cells. Although nucleic acid nanomaterials are emerging as a promising drug delivery strategy, their clinical applications are limited by rapid degradation by nucleases and difficulties in targeting cancer cells. In this study, we have developed a Rhein-loaded aptamer-based DNA nanotube (DNT-S6@Rhein) for the targeted and efficient therapy of non-small cell lung cancer. Through the palindrome segments, two specified oligonucleotides were hybridized and folded into the well-defined nanotubes (DNT-S6), with the S6 aptamer distributed outside. The obtained nanotubes exhibited excellent serum stability and targeting ability towards A549 cells due to the firm structure and decoration of the S6 aptamer. Rhein, as an antitumor drug and DNA intercalator, can be effectively inserted into the DNT-S6. The drug-loaded nanotubes rapidly disassembled in intracellular environment and then the released Rhein was found to activate cellular apoptotic process and significantly suppress proliferation, migration and invasion of A549 cells. Moreover, DNT-S6@Rhein could efficiently accumulate in tumor regions, offering compelling therapeutic efficacy and biocompatibility under both in vitro and in vivo settings. These findings of this study provide a promising strategy for mitigating the inevitable systemic side-effects of chemotherapy and expand the potential application of DNA nanostructure on targeted drug delivery.

Analyzing aptamer structure and interactions: in silico modelling and instrumental methods

Analyzing aptamer structure and interactions: in silico modelling and instrumental methods

Using Computer Modeling and Experimental Methods to Screen for Aptamers That Bind to the VV-GMCSF-LACT Virus.

Oncolytic virotherapy is a promising approach for cancer treatment. However, when introduced into the body, the virus provokes the production of virus-neutralizing antibodies, which can reduce its antitumor effect. To shield viruses from the immune system, aptamers that can cover the membrane of the viral particle are used. Aptamers that specifically bind to the JX-594 strain of the vaccinia virus were developed earlier. However, the parameters for binding to the recombinant virus VV-GMCSF-Lact, developed based on the LIVP strain of the vaccinia virus, may differ due its different repertoire of antigenic determinants on its membrane compared to JX-594. In this work, the spatial atomic structures of aptamers to JX-594 and bifunctional aptamers were determined using molecular modeling. The efficiency of viral particles binding to the aptamers (EC50), as well as the cytotoxicity and stability of the aptamers were studied. The synergistic effect of the VV-GMCSF-Lact combination with the aptamers in the presence of serum was investigated using human glioblastoma cells. This proposed approach allowed us to conduct a preliminary screening of sequences using in silico modeling and experimental methods, and identified potential candidates that are capable of shielding VV-GMCSF-Lact from virus-neutralizing antibodies.

High-resolution structure of a novel fluorogenic RNA aptamer.

High-resolution structure of a novel fluorogenic RNA aptamer.

Electrochemical sensor for vascular endothelial growth factor based on self-assembling DNA aptamer structure

Developing vascular endothelial growth factor (VEGF) protein is essential for early cancer diagnosis and cancer treatment monitoring. This study presents the design and characterisation of an electrochemical sensor utilising a self-assembling DNA aptamer structure for the sensitive and selective detection of VEGF. The aptamer structure comprises three different parts of single-stranded DNA that are assembled prior to integration into the sensor. Polypyrrole (Ppy)-based layers were deposited onto screen-printed carbon electrodes (SPCEs) using an electrochemical deposition technique, followed by the entrapment of a self-assembled DNA aptamer structure within electrochemically formed Ppy matrix ((DNA aptamer)/Ppy). The response to the sensor toward VEGF was measured by the pulsed amperometric detection (PAD), highlighting the enhanced performance of DNA aptamer/Ppy configuration compared to bare Ppy. The sensor exhibited high sensitivity, achieving a limit of detection (LOD) of 0.21 nM for VEGF. The interaction behaviour between VEGF in the solution and the immobilise DNA aptamer/Ppy-based structure was analysed using Langmuir isotherm model. The developed electrochemical biosensor is promising for in vitro applications in early cancer diagnostics and treatment monitoring, enabling rapid screening of patient samples.

Design and demonstration of a temperature-resistant aptamer structure for highly sensitive mercury ion detection with BioFETs

In this study, we developed a temperature-resistant aptamer coupled with extended gate electric double-layer Field-Effect Transistors (FETs) for highly sensitive mercury ion detection. The design of the temperature-resistant aptamer is based on the thermal and structural properties of the hairpin structure. Two aptamer sequences were designed with different melting temperatures (Tm) and compared for their sensitivity. The hairpin structure of the aptamer with a high melting temperature ensures the stable structure prior to the addition of the mercury ions, which allows the formation of thymine–mercury–thymine (T -Hg2+-T) complex in low concentrations of mercury ions. High sensitivity and low detection limit are achieved at elevated temperatures with the aptamer having a high melting temperature. The elevated temperature facilitates the reaction rate, resulting in high sensitivity and a low detection limit. The aptamer with a high melting temperature hairpin structure has shown a remarkable improvement in the limit of detection with a 4 orders of magnitude increase compared to the one with a low melting temperature. The sensor with the high melting temperature aptamer shows an extremely low detection limit of 4.68 × 10−12 M, surpassing previously reported results. The aptamer with a low melting temperature requires mercury ions to stabilize the hairpin structure by forming a T-Hg2+-T complex. The results show that if the melting temperature is lower than the ambient temperature, it is very difficult to detect low concentrations of mercury ions at the ambient temperature. The selectivity of this sequence was also tested against multiple heavy metals, including arsenic (As), lead (Pb), chromium (Cr), and cadmium (Cd) ions. This study has shown how the structural and thermal properties of the aptamer, and the ambient temperature affect the sensitivity. They are strongly correlated to the performance of the sensors and need to be considered in the design of the sequence.

AptamerRunner: An accessible aptamer structure prediction and clustering algorithm for visualization of selected aptamers

Aptamers are short single-stranded DNA or RNA molecules with high affinity and specificity for targets and are generated using the iterative Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. Next-generation sequencing (NGS) revolutionized aptamer selections by allowing a more comprehensive analysis of SELEX-enriched aptamers as compared to Sanger sequencing. The current challenge with aptamer NGS datasets is identifying a diverse cohort of candidate aptamers with the highest likelihood of successful experimental validation. Herein we present AptamerRunner, an aptamer sequence and/or structure clustering algorithm that synergistically integrates computational analysis with visualization and expertise-directed decision making. The visual integration of networked aptamers with ranking data, such as fold enrichment or scoring algorithm results, represents a significant advancement over existing clustering tools by providing a natural context to depict groups of aptamers from which ranked or scored candidates can be chosen for experimental validation. The inherent flexibility, user-friendly design, and prospects for future enhancements with AptamerRunner has broad-reaching implications for aptamer researchers across a wide range of disciplines.

Aptamer-based Strategies for COVID-19 Detection and Treatment: A Systematic Review Study

Background: Aptamer-based strategies have emerged as promising tools for the detection and treatment of COVID-19, offering advantages such as high specificity, sensitivity, and versatility. This systematic review aims to evaluate the effectiveness and innovation of aptamer-based approaches for COVID-19 detection and treatment. Methods: Following the guidelines of the Cochrane Handbook for Systematic Reviews and the PRISMA 2020 guidelines, a systematic search was conducted across multiple databases up to 2024. The search included studies that utilized aptamers for the diagnosis or therapy of COVID-19. Screening and selection of studies were performed independently by two reviewers, with any disagreements resolved by a third reviewer. Data were extracted regarding study characteristics, aptamer details, and outcomes. Results: In our systematic review, 98 studies from an initial pool of 1541 records met the inclusion criteria for analysis. Aptamers, single-stranded DNA or RNA molecules with unique threedimensional (3D) structures, were extensively explored for COVID-19 detection and treatment. Various aptamer-based assays, including electrochemical sensors, surface plasmon resonance (SPR) biosensors, and lateral flow assays, demonstrated high sensitivity and specificity in detecting SARSCoV- 2 in clinical samples such as saliva, nasal swabs, and wastewater. Several aptamer structures targeting viral proteins like the spike and nucleocapsid proteins were employed. Nucleic Acid Amplification Techniques (NAATs) utilizing aptamers, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based and Loop-mediated Isothermal Amplification (LAMP) assays, showed exceptional sensitivity in detecting viral genetic material. Aptamer-based therapeutic approaches showed potential by blocking viral protein activity or serving as delivery vehicles for therapeutic agents like small interfering RNAs (siRNAs). Despite their advantages, aptamer technologies face limitations such as susceptibility to nuclease degradation and rapid renal clearance, highlighting the need for further optimization. Conclusion: Aptamer-based strategies present promising avenues for COVID-19 detection and treatment. These approaches offer advantages such as high sensitivity, specificity, and rapid detection, making them valuable tools in combating the COVID-19 pandemic. Further research and development are warranted to optimize aptamer-based strategies for widespread application in clinical settings.

Structure-based insights into fluorogenic RNA aptamers.

Fluorogenic RNA aptamers are in vitro-selected RNA molecules capable of binding to specific fluorophores, significantly increasing their intrinsic fluorescence. Over the past decade, the color palette of fluorescent RNA aptamers has greatly expanded. The emergence and development of these fluorogenic RNA aptamers has introduced a powerful approach for visualizing RNA localization and transport with high spatiotemporal resolution in live cells. To date, a variety of tertiary structures of fluorogenic RNA aptamers have been determined using X-ray crystallography or NMR spectroscopy. Many of these fluorogenic RNA aptamers feature base quadruples or base triples in their fluorophore-binding sites. This review summarizes the structure-based investigations of fluorogenic RNA aptamers, with a focus on their overall folds, ligand-binding pockets and fluorescence activation mechanisms. Additionally, the exploration of how structures guide rational optimization to enhance RNA visualization techniques is discussed.

A Branched SELEX Approach Identifies RNA Aptamers That Bind Distinct HIV-1 Capsid Structural Components.

The HIV-1 capsid protein (CA) assumes distinct structural forms during replication, each presenting unique, solvent-accessible surfaces that facilitate multifaceted functions and host factor interactions. However, functional contributions of individual CA structures remain unclear, as evaluation of CA presents several technical challenges. To address this knowledge gap, we identified CA-targeting aptamers with different structural specificities, which emerged through a branched SELEX approach using an aptamer library previously selected to bind the CA hexamer lattice. Subsets were either highly specific for the CA lattice or bound both the CA lattice and CA hexamer. We then evaluated four representatives to reveal aptamer regions required for binding, highlighting interesting structural features and challenges in aptamer structure determination. Further, we demonstrate binding to biologically relevant CA structural forms and aptamer-mediated affinity purification of CA from cell lysates without virus or host modification, supporting the development of structural form-specific aptamers as exciting new tools for the study of CA.

Tag-free fluorometric aptasensor for detection of chromium(VI) in foods via SYBR Green I signal amplification and aptamer structure transition.

In response to growing concerns regarding heavy metal contamination in food, particularly chromium (Cr)(VI) contamination, this study presented a simple, sensitive and practical method for Cr(VI) detection. A magnetic separation-based capture-exponential enrichment ligand system evolution (SELEX) method was used to identify and characterize DNA aptamers with a high affinity for Cr(VI). An aptamer, Cr-15, with a dissociation constant (Kd) of 4.42 ± 0.44 μmol L-1 was obtained after only eight rounds of selection. Further innovative methods combining molecular docking, dynamic simulation and thermodynamic analysis revealed that CrO4 2- could bind to the 19th and 20th guanine bases of Cr-15 via hydrogen bonds. Crucially, a label-free fluorometric aptasensor based on SYBR Green I was successfully constructed to detect CrO4 2-, achieving a linear detection range of 60-300 nmol L-1 with a lower limit of detection of 44.31 nmol L-1. Additionally, this aptasensor was able to quantitatively detect CrO4 2- in grapes and broccoli within 40 min, with spike recovery rates ranging from 89.22% to 108.05%. The designed fluorometric aptasensor exhibited high selectivity and could detect CrO4 2- in real samples without sample processing or target pre-enrichment. The aptasensor demonstrated its potential as a reliable tool for monitoring Cr(VI) contamination in fruit and vegetable products. © 2024 Society of Chemical Industry.

Aptamer biosensor design for the detection of endocrine-disrupting chemicals small organic molecules using novel bioinformatics methods

Endocrine-disrupting chemicals (EDCs) are substances that can disrupt the normal functioning of hormones.Using aptamers, which are biological recognition elements, biosensors can quickly and accurately detect EDCs in environmental samples. However, the elucidation of aptamer structures by conventional methods is highly challenging due to their complexity.This has led to the development of three-dimensional aptamer structures based on different models and techniques. To do this, we developed a way to predict the 3D structures of the SS DNA needed for this sequence by starting with an aptamer sequence that has biosensor properties specific to bisphenol-A (BPA), one of the chemicals found in water samples that can interfere with hormones. In addition, we will elucidate the intermolecular mechanisms and binding affinity between aptamers and endocrine disruptors using bioinformatics techniques such as molecular docking, molecular dynamics simulation, and binding energies. The outcomes of our study are to compare modeling programs and force fields to see how reliable they are and how well they agree with results found in the existing literature, to understand the intermolecular mechanisms and affinity of aptamer-based biosensors, and to find a new way to make aptamers that takes less time and costs less.

DNA Cryogels with Anisotropic Mechanical and Responsive Properties for Specific Cell Capture and Release.

Due to their programmable stimuli-responsiveness, excellent biocompatibility, and water-rich and soft structures similar to biological tissues, smart DNA hydrogels hold great promise for biosensing and biomedical applications. However, most DNA hydrogels developed to date are composed of randomly oriented and isotropic polymer networks, and the resulting slow response to biotargets and lack of anisotropic properties similar to those of biological tissues have limited their extensive applications. Herein, anisotropic DNA hydrogels consisting of unidirectional void channels internally oriented up to macroscopic length scales were constructed by a directional cryopolymerization method, as exemplified by a DNA-incorporated covalently cross-linked DNA cryogel and a DNA duplex structure noncovalently cross-linked DNA cryogel. Results showed that the formation of unidirectional channels significantly improved the responsiveness of the gel matrix to biomacromolecular substances and further endowed the DNA cryogels with anisotropic properties, including anisotropic mechanical properties, anisotropic swelling/shrinking behaviors, and anisotropic responsiveness to specific biotargets. Moreover, the abundant oriented and long macroporous channels in the gel matrix facilitated the migration of cells, and through the introduction of aptamer structures and thermosensitive polymers, an anisotropic DNA cryogel-based platform was further constructed to achieve the highly efficient capture and release of specific cells. These anisotropic DNA hydrogels may provide new opportunities for the development of anisotropic separation and biosensing systems.

Design and Validation of a Short Novel Estradiol Aptamer and Exploration of Its Application in Sensor Technology.

The specific and sensitive detection of 17β-estradiol (E2) is critical for diagnosing and treating numerous diseases, and aptamers have emerged as promising recognition probes for developing detection platforms. However, traditional long-sequence E2 aptamers have demonstrated limited clinical performance due to redundant structures that can affect their stability and recognition ability. There is thus an urgent need to further optimize the structure of the aptamer to build an effective detection platform for E2. In this work, we have designed a novel short aptamer that retains the key binding structure of traditional aptamers to E2 while eliminating the redundant structures. The proposed aptamer was evaluated for its binding properties using microscale thermophoresis, a gold nanoparticle-based colorimetric method, and electrochemical assays. Our results demonstrate that the proposed aptamer has excellent specific recognition ability for E2 and a high affinity with a dissociation constant of 92 nM. Moreover, the aptamer shows great potential as a recognition probe for constructing a highly specific and sensitive clinical estradiol detection platform. The aptamer-based electrochemical sensor enabled the detection of E2 with a linear range between 5 pg mL-1 and 10 ng mL-1 (R2 = 0.973), and the detection capability of a definite low concentration level was 5 pg mL-1 (S/N = 3). Overall, this novel aptamer holds great promise as a valuable tool for future studies on the role of E2 in various physiological and pathological processes and for developing sensitive and specific diagnostic assays for E2 detection in clinical applications.

Selection and application of highly specific Salmonella typhimurium aptamers against matrix interference

In practical applications, the structure and performance of aptamers can be influenced by the presence of sample matrices, which interferes with the specific binding between the aptamer and its target. In this work, to obtain aptamer chains resistant to matrix interference, four typical food matrices were introduced as negative selection targets and selection environments in the process of selecting aptamers for Salmonella typhimurium using the systematic evolution of ligands by exponential enrichment (SELEX) technology. As a result, some highly specific candidate aptamers for Salmonella typhimurium (BB-34, BB-37, ROU-8, ROU-9, ROU-14, ROU-24, DAN-3, NAI-12, and NAI-21) were successfully obtained. Based on the characterization results of secondary structure, affinity, and specificity of these candidate aptamers, ROU-24 selected in the pork matrix and BB-34 selected in the binding buffer were chosen to develop label-free fluorescence aptasensors for the sensitive and rapid detection of the Salmonella typhimurium and verify the performance against matrix interference. The ROU-24-based aptasensor demonstrated a larger linear range and better specificity compared to the BB-34-based aptasensor. Meanwhile, the recovery rate of the ROU-24-based aptasensor in real sample detection (ranging from 94.2% to 110.7%) was significantly higher than that of the BB-34-based aptasensor. These results illustrated that the negative selection of food matrices induced in SELEX could enhance specific binding between the aptamer and its target and the performance against matrix interference. Overall, the label-free fluorescence aptasensors were developed and successfully validated in different foodstuffs, demonstrating a theoretical and practical basis for the study of aptamers against matrix interference.

Detailed Protocol for Predicting 3D Structure of DNA Aptamers and Performing In Silico Docking Calculations.

This paper presents a comprehensive protocol for predicting the three-dimensional (3D) structure of DNA aptamers and performing in silico docking calculations. The protocol includes steps for sequence input, structure prediction, sequence modification, structure minimization, and docking. The procedure is executed on a Mac environment utilizing bioinformatics tools such as mfold, RNA Composer, PyMOL, and Hdock. The protocol is intended to provide a guide for researchers in structural biology and drug design.

Conformational preferences of modified nucleobases in RNA aptamers and their effect on Förster resonant energy transfer.

Förster resonant energy transfer (FRET) can be utilized in the study of tertiary structures of RNA aptamers, which bind specific fluorophoric ligands to form a fluorogenic aptamer complex. By introducing the emissive nucleobase analog 4-cyanoindole into the fluorogenic Chili RNA aptamer a FRET pair was established. The interpretation of studies aiming to investigate those tertiary structures using FRET, however, relies on prior knowledge about conformational properties of the nucleobase, which govern exciton transfer capabilities. Herein we employed classical molecular dynamics combined with Förster exciton theory to elucidate the preferred orientation relative to proximate bases and the influence on exciton transfer efficiency in multiple substitution sites. We did this by comparing the chromophoric distances emergent from MD simulations with experimental FRET data based on structural data of the native aptamer. We present the outlined methodology as a means to reliably evaluate future nucleobase analogue candidates in terms of their structural behavior and emergent exciton transfer properties as exemplified in the study of the preferred orientation of 4-cyanoindole in the Chili RNA aptamer.

Real-time monitoring of vancomycin using a split-aptamer surface plasmon resonance biosensor.

Real-time monitoring of therapeutic drugs is crucial for treatment management and pharmacokinetic studies. We present the optimization and affinity tuning of split-aptamer sandwich assay for real-time monitoring of the narrow therapeutic window drug vancomycin, using surface plasmon resonance (SPR). To achieve reversible, label-free sensing of small molecules by SPR, we adapted a vancomycin binding aptamer in a sandwich assay format through the split-aptamer approach. By evaluating multiple split sites within the secondary structure of the original aptamer, we identified position 27 (P27) as optimal for preserving target affinity, ensuring reversibility, and maximizing sensitivity. The assay demonstrated robust performance under physiologically relevant ranges of pH and divalent cations, and the specific ternary complex formation of the aptamer split segments with the analyte was confirmed by circular dichroism spectroscopy. Subsequently, we engineered a series of split-aptamer pairs with increasing complementarity in the stem regions, improving both the affinity and limit of detection up to 10-fold, as compared to the primary P27 pair. The kinetics of the engineered split-aptamer pairs were evaluated, revealing fast association and dissociation rates, confirming improved affinity and detection limits across variants. Most importantly, the reversibility of the assay, essential for real-time monitoring, was maintained in all pairs. Finally, the assay was further validated in complex biological matrices, including the cerebrospinal fluid from dogs and diluted plasma from rats, demonstrating functionality in biological environments and stability exceeding 9 hours. Our study paves the way for applications of split-aptamers in real-time monitoring of small molecules, with potential implications for in vivo therapeutic drug monitoring and pharmacokinetic studies.

Exploration of SAM-I riboswitch inhibitors: In-Silico discovery of ligands to a new target employing multistage CADD approaches

Targeting riboswitches, regulatory elements responsible for the expression of essential genes, is taking central stage in the new era of antibacterial medications discovery due to the emergence of antibiotic resistance. The S-Adenosyl methionine-I (SAM-I) riboswitch works through transcription termination in a negative feedback manner modulated by the natural ligand SAM. SAM-I riboswitch is specific to bacteria and found mainly in gram-positive bacteria such as Bacillus anthracis. Analyzing the interactions of the co-crystallized structure of SAM-I riboswitch aptamer with its native ligand SAM clarified the needed chemical structural features to achieve binding. Acknowledging those features, structure-based and ligand-based pharmacophore models were built for filtration use in screening the OTAVA Chemical library and the Pubchem database. For further filtration enhancement, the physicochemical properties of SAM were used as a second filtration criterion. Compounds obtained as output from previous steps were energy minimized, and the lowest energy conformer structures were docked to SAM-I using MOE, v.2019.01. S-score and ligand interactions were used to assess the best hits. This yielded eight promising compounds to which molecular dynamics (MD) simulations with SAM-I aptamer were applied using GROMACS 2020.3 package affirming stable binding interactions and binding energetics similar to SAM. Moreover, pharmacokinetic and drug-like properties of those eight hits were assessed using SWISS-ADME. According to the combined computational methods and PK/Tox assessment, compound 20 was the most promising and thus can be considered a lead for future evaluation and optimization as a candidate new antibacterial agent targeting a new biomolecule eliciting a new mechanism of action.