Structure Of Aptamer Research Articles

Crystal structures of an HIV-1 integrase aptamer: Formation of a water-mediated A•G•G•G•G pentad in an interlocked G-quadruplex

93del is a 16-nucleotide G-quadruplex-forming aptamer which can inhibit the activity of the HIV-1 integrase enzyme at nanomolar concentration. Previous structural analyses of 93del using NMR spectroscopy have shown that the aptamer forms an interlocked G-quadruplex structure in K+ solution. Due to its exceptional stability and unique topology, 93del has been used in many different studies involving DNA G-quadruplexes, such as DNA aptamer and multimer design, as well as DNA fluorescence research. To gain further insights on the structure of this unique aptamer, we have determined several high-resolution crystal structures of 93del and its variants. While confirming the overall dimeric interlocked G-quadruplex folding topology previously determined by NMR, our results reveal important detailed structural information, particularly the formation of a water-mediated A•G•G•G•G pentad. These insights allow us to better understand the formation of various structural elements in G-quadruplexes and should be useful for designing and manipulating G-quadruplex scaffolds with desired properties.

Advancement of dengue virus NS1 protein detection by 3D-nanoassembly complex gold nanoparticles utilizing competitive sandwich aptamer on disposable electrode

Signal amplification have been centralized in developing the highly reliable biosensor for analyte detection with a narrow detection window. We proposed an aptasensor to provide a highly sensitive early-stage diagnostic platform of dengue virus NS1 protein (DENV-NS1) by dual-approach – colorimetric and electrochemical detection. This work utilized two different aptamers specific to DENV-NS1: One conjugated to gold nanoparticles (AuNPs), forming AuNPs-Apt1 and its complementary sequence aptamer, forming AuNPs-Apt2. The unbound Apt1 of AuNPs-Apt1 by DENV-NS1 were to hybridize to AuNPs-Apt2 and induced a 3D-nanoassembled formation, resulting in DENV-NS1 concentration-dependent plasmonic color change. Occurrence of the hybridization of Apt1 and Apt2, the 3D-assembled hybridized aptamers of AuNPs was incubated with methylene blue (MB) solution, which intercalated a high number of MB molecules within the duplex structure of aptamers, and the complex was captured on the Apt2-conjugated disposable gold electrode (DGE). The developed aptamer-based biosensor showed high sensitivity with colorimetric response down to 1.28 pg/mL and electrochemical approach down to 30 fg/mL of DENV-NS1 with good selectivity. This work showcases an advanced utilization of aptamer and its complementary anti-sense aptamer in signal amplification and nanocarrier for biosensing.

Label-Free Detection of Ochratoxin A Using Aptamer as Recognition Probe at Liquid Crystal-Aqueous Interface

Aqueous interfaces of stimuli-responsive, thermotropic nematic liquid crystals (LC) have been utilized in the design of biosensing platforms for a range of analytes. Owing to the orientational changes in LC, these interfaces can easily report aptamer-target binding events at the LC-aqueous interface. We demonstrate a label-free, simple and robust technique for the detection of Ochratoxin A (OTA) using aptamer as the recognition probe. The self-assembly of CTAB (cetyltrimethylammonium bromide; cationic surfactant) at aqueous-LC interface gives a homeotropic orientation of LC. In presence of negatively charged OTA specific aptamer, aptamer forms a complex with CTAB. Formation of aptamer-CTAB complex results in ordering transition of LCs to planar/tilted. In presence of OTA, OTA forms a strong and stable G-quadruplex structure of aptamer that results in the redistribution of CTAB at LC-aqueous interface and leads to homeotropic orientation of LC. The designed LC aptasensor exhibits a detection limit of 0.1 nM. We observed that the sensitivity of LC aptasensor was affected by the pH and ionic strength. In addition, we demonstrated the applicability of the designed LC aptasensor for the detection of OTA in tap water and apple juice. This approach offers advantages over the conventional detection methods in terms of fabrication, ease of operation, and analysis.

Multivalent Aptamer Approach: Designs, Strategies, and Applications.

Aptamers are short and single-stranded DNA or RNA molecules with highly programmable structures that give them the ability to interact specifically with a large variety of targets, including proteins, cells, and small molecules. Multivalent aptamers refer to molecular constructs that combine two or more identical or different types of aptamers. Multivalency increases the avidity of aptamers, a particularly advantageous feature that allows for significantly increased binding affinities in comparison with aptamer monomers. Another advantage of multivalency is increased aptamer stabilities that confer improved performances under physiological conditions for various applications in clinical settings. The current study aims to review the most recent developments in multivalent aptamer research. The review will first discuss structures of multivalent aptamers. This is followed by detailed discussions on design strategies of multivalent aptamer approaches. Finally, recent developments of the multivalent aptamer approach in biosensing and biomedical applications are highlighted.

Development of electrochemical aptasensors detecting phosphate ions on TMB substrate with epoxy-based mesoporous silica nanoparticles

This study, it is aimed to develop an electrochemical aptasensor that can detect phosphate ions using 3.3′5.5′ tetramethylbenzidine (TMB). It is based on the principle of converting the binding affinity of the target molecule phosphate ion (PO43−) into an electrochemical signal with specific aptamer sequences for the aptasensor to be developed. The aptamer structure served as a gate for the TMB to be released and was used to trap the TMB molecule in mesoporous silica nanoparticles (MSNPs). The samples for this study were characterized by transmission electron spectroscopy (TEM), Brunner-Emmet-Teller, dynamic light scattering&electrophoretic light scattering, and induction coupled plasma atomic emission spectroscopy. According to TEM analysis, MSNPs have a morphologically hexagonal structure and an average size of 208 nm. In this study, palladium-carbon nanoparticles (Pd/C NPs) with catalytic reaction were used as an alternative to the biologically used horseradish peroxidase (HRP) enzyme for the release of TMB in the presence of phosphate ions. The limit of detection (LOD) was calculated as 0.983 μM, the limit of determination (LOQ) was calculated as 3.276 μM, and the dynamic linear phosphate range was found to be 50–1000 μM. The most important advantage of this bio-based aptasensor assembly is that it does not contain molecules such as a protein that cannot be stored for a long time at room temperature, so its shelf life is very long compared to similar systems developed with antibodies. The proposed sensor shows good recovery in phosphate ion detection and is considered to have great potential among electrochemical sensors.

A review on the therapeutic applications of aptamers and aptamer-conjugated nanoparticles in cancer, inflammatory and viral diseases

The advancement in early diagnosis and precise treatments options result in more predictable and powerful health care modalities. Aptamers are known as nucleic acid structures with three-dimensional conformation to selectively bind a target site. Physicochemical properties of aptamers, their conjugation with nanoparticles (NPs) in theranostics applications and their internalization have been found to be of interest in development of aptamer-based drug delivery systems. Therefore, we aimed to present an overview on the structure and generation of aptamers followed by advantages of aptamers-conjugated NPs and their theranostics applications in various diseases such as oncology, inflammatory diseases and viral diseases. Afterward, we discussed several reports on the internalization approaches of aptamers, efficiency of aptamers vs. their analogous, and implications of aptamers in clinical trials. Finally, we discussed the current challenges and future perspectives of actively targeted aptamers for clinical application. In conclusion, this review may hold a great promise for development of aptamer-based therapeutic platforms in clinical trials.

Utilizing the DNA Aptamer to Determine Lethal α-Amanitin in Mushroom Samples and Urine by Magnetic Bead-ELISA (MELISA).

Amanita poisoning is one of the most deadly types of mushroom poisoning. α-Amanitin is the main lethal toxin in amanita, and the human-lethal dose is about 0.1 mg/kg. Most of the commonly used detection techniques for α-amanitin require expensive instruments. In this study, the α-amanitin aptamer was selected as the research object, and the stem-loop structure of the original aptamer was not damaged by truncating the redundant bases, in order to improve the affinity and specificity of the aptamer. The specificity and affinity of the truncated aptamers were determined using isothermal titration calorimetry (ITC) and gold nanoparticles (AuNPs), and the affinity and specificity of the aptamers decreased after truncation. Therefore, the original aptamer was selected to establish a simple and specific magnetic bead-based enzyme linked immunoassay (MELISA) method for α-amanitin. The detection limit was 0.369 μg/mL, while, in mushroom it was 0.372 μg/mL and in urine 0.337 μg/mL. Recovery studies were performed by spiking urine and mushroom samples with α-amanitin, and these confirmed the desirable accuracy and practical applicability of our method. The α-amanitin and aptamer recognition sites and binding pockets were investigated in an in vitro molecular docking environment, and the main binding bases of both were T3, G4, C5, T6, T7, C67, and A68. This study truncated the α-amanitin aptamer and proposes a method of detecting α-amanitin.

Ovarian cancer and KiSS-1 gene expression: A consideration of the use of Kisspeptin plus Kisspeptin aptamers in diagnostics and therapy

Gynaecological cancers continue to present a significant health burden upon the health of the global female population. This deficit is most prominent with ovarian cancer which possesses the lowest survival rate compared to all other cancers occurring within this anatomical region, with an annual UK-mortality of 7,300. The poor tolerability and selectively of the treatment options that are currently available is likely to have contributed to this high mortality rate thus, demonstrating the need for the development of enhanced therapeutic approaches. Aptamer technology would involve the engineering of specifically sequenced oligonucleotide chains, which bind to macromolecular targets with a high degree of affinity and selectively. Recent in-vitro studies conducted upon the clinical utility of this technique have supported its superiority in targeting individual therapeutic drug targets compared to various other targeting moieties currently within therapeutic use such as, monoclonal antibodies. For this reason, the employment of this technique is likely to be favourable in reducing the incidence of non-specific, chemotherapy-associated adverse effects. Kisspeptin is a naturally expressed polypeptide with an established role in the development of the reproductive system and other proposed roles in influencing the ability of ovarian cancer growths to exhibit the metastasis hallmark. This distinctive feature would indicate the potential for the manipulation of this pathway through the application of aptamer structures in developing a novel prophylactic strategy and improve the long-term outcome for ovarian cancer patients.

Structure-guided development of Pb2+-binding DNA aptamers

Owing to its great threat to human health and environment, Pb2+ pollution has been recognized as a major public problem by the World Health Organization (WHO). Many DNA aptamers have been utilized in the development of Pb2+-detection sensors, but the underlying mechanisms remain elusive. Here, we report three Pb2+-complexed structures of the thrombin binding aptamer (TBA). These high-resolution crystal structures showed that TBA forms intramolecular G-quadruplex and Pb2+ is bound by the two G-tetrads in the center. Compared to K+-stabilized G-quadruplexes, the coordinating distance between Pb2+ and the G-tetrads are much shorter. The T3T4 and T12T13 linkers play important roles in dimerization and crystallization of TBA, but they are changeable for Pb2+-binding. In combination with mutagenesis and CD spectra, the G8C mutant structure unraveled that the T7G8T9 linker of TBA is also variable. In addition to expansion of the Pb2+-binding aptamer sequences, our study also set up one great example for quick and rational development of other aptamers with similar or optimized binding activity.

Structural analysis of thrombin-binding G-aptamers in presence of bivalent ions

The aim of this study was to examine 3D structures of DNA aptamers, thrombin inhibitors. •The main objective was to study 3D structure 15TBA, RE31, NU172 aptamers using the small-angle X-ray scattering method. The size of 15TBA was 4.5 nm, which corresponds to a partially unfolded conformation. The CD spectrum of Nu172 in the presence of 50 mM strontium ions indicates the presence of an antiparallel G-quadruplex, the concentration o f which drops at 50°C. NU172 does not have a rigid structure, apparently due to the presence of a guanine residue in the GT loop. The NU172 aptamer does not form a stable conformation in solution either without ions or with Ba2+ and Sr2+ ions. • It was shown that there is possibility of aptamers transition from one conformation to another dependently on concentration and temperature confirms that the potassium ion is a unique stabilizing ion of natural molecules containing G-quadruplexes

Sensitive detection of patulin based on DNase Ⅰ-assisted fluorescent aptasensor by using AuNCs-modified truncated aptamer

Patulin (PAT) is a mycotoxin that exists in a wide range and is harmful to human health, which has aroused great attention to food safety. In this study, we established a novel assay to detect food samples contaminated by PAT. First of all, we proposed a truncation protocol based on the secondary structure of aptamer. By removing the stem-loop structure one by one, an optimized truncated PAT aptamer (P-30) was obtained. To the best of our knowledge, the mechanism between PAT and P-30 was elaborated in the assisting of molecular docking and circular dichroism for the first time. Furthermore, we developed a sensitive aptasensor platform to detect PAT in food samples. The aptamer modified with gold nanoclusters (AuNCs) was used as fluorescent probe, and BSA-directed synthesized MnO2 nanoflakes (MnO2 NFs) as fluorescence receptor. Additionally, fluorescence signal amplification strategy assisted by DNase Ⅰ successfully improved the sensitivity of aptasensor. The developed assay had a low detection limit of 8.5 ng/L, and can be effectively used in apple and grape juice samples. Thus, the established aptasensor shows a broad application prospect in PAT detection.

Aptamers based sensing of pregnancy associated glycoproteins (PAG) of bovine for early pregnancy detection

Tosyl activated magnetic beads were used for aptamer selection against PAG- 7 and 18 proteins of bovine origin. PAG proteins were immobilized on beads with further addition of biotin tagged aptamer library. The recognition of aptamers with PAG was identified by ST-HRP based approach which was colorimetric in nature. The selected aptamers were sequenced and at the same time several new aptamers were identified. Later M-fold structure and G-quadruplex score of aptamers were analyzed for their selection. Those aptamers having high G value and complex structure were chosen. In dot blot assay, aptamers recognized PAG protein in an animal after 42 days of artificial insemination which later given birth to a healthy calf. Further the cross reactivity with serum of 0th day animal (post AI) or with non pregnant animal serum was minimal. Aptamers have also shown interaction with PAG protein of buffalo origin. These selected aptamers have commercial application especially in development of biosensors for early detection of pregnancy in bovine.

Optical and Electrochemical Aptasensors for Sensitive Detection of Aflatoxin B1 and Aflatoxin M1 in Blood Serum, Grape Juice, and Milk Samples.

Aflatoxin food contamination with toxic and carcinogenic impacts on human health is a global concern. We have developed aptasensors for the detection of Aflatoxin M1 (AFM1) and B1 (AFB1) using electrochemical and optical methods. In the first method, an electrochemical aptasensor was designed for the detection of AFM1 based on complementary strand of AFM1 aptamer that was attached onto the gold nanoparticles and a hairpin-shaped AFM1 aptamer. The designed electrochemical aptasensor showed high selectivity toward AFM1 with a limit of detection (LOD) as low as 0.9nM. Moreover, the developed aptasensor was successfully used to detect AFM1 in milk and serum with LODs of 1.8 and 1.2nM, respectively. In the second method, a novel electrochemical aptasensor was developed based on the π-shape structure of AFB1 aptamer. The detection limit was found to be 2pg/mL in buffer. Also, the developed aptasensor was used to analyze AFB1 spiked human serum and grape juice samples, and the recoveries were 95.4-108.1%. In another method, a fluorescent sensing scheme was developed for AFB1 detection based on a hairpin structure of G-quadruplex oligonucleotide-aptamer chimera, streptavidin-coated silica nanoparticles (SNP-streptavidin) and N-methyl mesoporphyrin IX (NMM). The LOD was reported as 8pg/mL with a linear range of 30-900pg/mL. Moreover, the developed sensor could detect AFB1 in serum and grape juice with the LOD of 9.8 and 11.2pg/mL, respectively.

An Electrochemical Aptasensor for the Detection of HER2 as a Breast Cancer Biomarker Based on Gold Nanoparticles-Aptamer Bioconjugates

Inaccurate diagnoses contributes to the high mortality rate of breast cancer. Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer tumors at around 20–30%. This study aims to develop an electrochemical biosensor for HER2 based on a gold nanoparticle-aptamer bioconjugate (AuNP@HER2 aptamer) and investigate the interaction between DNA aptamer and HER2 using computational methods. The bioconjugate was synthesized using maleimide and polyethylene glycol as a linker. The –NH2 group of cysteamine that modified the gold electrode can form a covalent bond with the bioconjugate maleimide. The interaction of the bioconjugated aptamer with HER2 was measured electrochemically based on the [Fe(CN)6]3−/4− redox system. The limit of detection, the linear range of HER2, precision, and accuracy in this study were 1.52 ng mL–1, 0.01 to 15.0 ng mL–1, 0.1298, and 94.06%, respectively. The structure of the DNA aptamer was modeled using mFold, Assemble2, and Chimera, with the interaction between the DNA aptamer and HER2 explored by NPDock. The modeling of the aptamer with HER2 showed that electrostatic interactions dominated the attractive forces. The resulting interaction pattern can be used as a template to improve the binding energy of the aptamer, thus providing insight into the development of aptamer-based biosensors.

Complexing deoxyribozymes with RNA aptamers for detection of the small molecule theophylline

Biointegrative information processing systems offer a great advantage to autonomous biodevices, as their capacity for biological computation provides the ability to sense the state of more complex environments and better integrate with downstream biological regulation systems. Deoxyribozymes (DNAzymes) and aptamers are of interest to such computational biosensing systems due to the enzymatic properties of DNAzymes and the ligand-inducible conformational structures of aptamers. Herein, we describe a novel method for providing ligand-responsive allosteric control to a DNAzyme using an RNA aptamer. We designed a NOT-logic-compliant E6 DNAzyme to be complementary to an RNA aptamer targeting theophylline, such that the aptamer competitively interacted with either theophylline or the DNAzyme, and disabled the DNAzyme only when theophylline concentration was below a given threshold. Out of our seven designed “complexing aptazymes,” three demonstrated effective theophylline-responsive allosteric regulation (2.84 ± 3.75%, 4.97 ± 2.92%, and 8.91 ± 4.19% activity in the absence of theophylline; 46.29 ± 3.36%, 50.70 ± 10.15%, and 61.26 ± 6.18% activity in the presence of theophylline). Moreover, the same three complexing aptazymes also demonstrated the ability to semi-quantitatively determine the concentration of theophylline present in solution, successfully discriminating between therapeutically ineffective (<20 μM), safe (20–100 μM), and toxic (>100 μM) theophylline concentrations. Our method of using an RNA aptamer for ligand-responsive allosteric control of a DNAzyme expands the way aptamers can be configured for biosensing, and suggests a pathway for embedding DNAzymes to provide enhanced information processing and control of biological systems.

Selection of broad-spectrum aptamer and its application in fabrication of aptasensor for detection of aminoglycoside antibiotics residues in milk

The multi-residues of aminoglycoside antibiotics (AAs) have been reported present in a food product. Taking the broad-spectrum aptamer as recognition element of sensors for simultaneous detection of the class of AAs is of great significance. In this paper, broad-spectrum ssDNA aptamers for AAs with high affinity and specificity were successfully selected by graphene oxide (GO)-SELEX with a group of targets. Further sequence analysis, the truncated aptamer (AAs 03-2) of only 49 nt was observed with higher affinity than original aptamer of 79 nt. The binding mechanism analysis was performed by circular dichroism and molecular docking simulations, which showed the folding structure of aptamer (AAs 03-2) provided binding pockets for AAs and could recognize AAs conserved structure. Subsequently, the affinity of aptamer (AAs 03-2) for other AAs with favorable binding ability was further verified. Finally, the aptamer (AAs 03-2) was used to fabricate electrochemical biosensor based on screen-printed carbon electrode modified by ordered mesoporous carbon for signal amplification. The aptasensor realized simultaneous analysis of AAs with limit of detection of 2.47 nM. Moreover, the proposed aptasensor was successfully applied to evaluate the residual level of AAs in milk, which provides a new method for multi-residues detection of antibiotics.

High-Throughput Selection and Characterisation of Aptamers on Optical Next-Generation Sequencers.

Aptamers feature a number of advantages, compared to antibodies. However, their application has been limited so far, mainly because of the complex selection process. ‘High-throughput sequencing fluorescent ligand interaction profiling’ (HiTS–FLIP) significantly increases the selection efficiency and is consequently a very powerful and versatile technology for the selection of high-performance aptamers. It is the first experiment to allow the direct and quantitative measurement of the affinity and specificity of millions of aptamers simultaneously by harnessing the potential of optical next-generation sequencing platforms to perform fluorescence-based binding assays on the clusters displayed on the flow cells and determining their sequence and position in regular high-throughput sequencing. Many variants of the experiment have been developed that allow automation and in situ conversion of DNA clusters into base-modified DNA, RNA, peptides, and even proteins. In addition, the information from mutational assays, performed with HiTS–FLIP, provides deep insights into the relationship between the sequence, structure, and function of aptamers. This enables a detailed understanding of the sequence-specific rules that determine affinity, and thus, supports the evolution of aptamers. Current variants of the HiTS–FLIP experiment and its application in the field of aptamer selection, characterisation, and optimisation are presented in this review.

Evaluation the effect of nanoparticles on the structure of aptamers by analyzing the recognition dynamics of aptamer functionalized nanoparticles

Aptamer-functionalized nanoparticles have been widely studied as targeted probes in biomedical applications for targeted therapy and imaging. The rigidity of the nanoparticle could stabilized the spatial structure of the aptamer, ensuring the selectivity and affinity for target recognition in the complex environment. The main aim of this article study was to explore the effect of the spatial structure of aptamer in the interaction between aptamer nanoprobes and receptors. We designed and synthesized aptamer functionalized nanoparticle systems with different derivation lengths, and developed a unique kinetic analysis to quantify affinity interactions. The system used silver decahedral nanoparticles (Ag10NPs), which was then chemically functionalized with thrombin (or IgE) aptamers of different tail lengths to produced different nanoprobes, and employed thrombin (or IgE) as target on a surface plasmon resonance (SPR) biosensor to evaluate the binding of these nanoprobes. Kinetic analysis of the SPR binding curve was performed to evaluated the affinity between nanoprobes and targets. Under the premise of eliminating multivalent interactions, we found that the distance between aptamer and nanoparticle could affect the affinity between nanoprobe and target. Furthermore, we found that keeping a certain distance between aptamer and nanoparticle could effectively improved the recognition efficiency of the aptamer nanoprobe and target. It shows that the rigidity of nanomaterials could maintain the spatial structure of the aptamer.

Target Affinity and Structural Analysis for a Selection of Norovirus Aptamers.

Aptamers, single-stranded oligonucleotides that specifically bind a molecule with high affinity, are used as ligands in analytical and therapeutic applications. For the foodborne pathogen norovirus, multiple aptamers exist but have not been thoroughly characterized. Consequently, there is little research on aptamer-mediated assay development. This study characterized seven previously described norovirus aptamers for target affinity, structure, and potential use in extraction and detection assays. Norovirus-aptamer affinities were determined by filter retention assays using norovirus genotype (G) I.1, GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney virus-like particles. Of the seven aptamers characterized, equilibrium dissociation constants for GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney ranged from 71 ± 38 to 1777 ± 1021 nM. Four aptamers exhibited affinity to norovirus GII.4 strains; three aptamers additionally exhibited affinity toward GII.3 and GI.7. Aptamer affinity towards GI.1 was not observed. Aptamer structure analysis by circular dichroism (CD) spectroscopy showed that six aptamers exhibit B-DNA structure, and one aptamer displays parallel/antiparallel G-quadruplex hybrid structure. CD studies also showed that biotinylated aptamer structures were unchanged from non-biotinylated aptamers. Finally, norovirus aptamer assay feasibility was demonstrated in dot-blot and pull-down assays. This characterization of existing aptamers provides a knowledge base for future aptamer-based norovirus detection and extraction assay development and aptamer modification.

The role of SAXS and molecular simulations in 3D structure elucidation of a DNA aptamer against lung cancer

Aptamers are short, single-stranded DNA or RNA oligonucleotide molecules that function as synthetic analogs of antibodies and bind to a target molecule with high specificity. Aptamer affinity entirely depends on its tertiary structure and charge distribution. Therefore, length and structure optimization are essential for increasing aptamer specificity and affinity. Here, we present a general optimization procedure for finding the most populated atomistic structures of DNA aptamers. Based on the existed aptamer LC-18 for lung adenocarcinoma, a new truncated LC-18 (LC-18t) aptamer LC-18t was developed. A three-dimensional (3D) shape of LC-18t was reported based on small-angle X-ray scattering (SAXS) experiments and molecular modeling by fragment molecular orbital or molecular dynamic methods. Molecular simulations revealed an ensemble of possible aptamer conformations in solution that were in close agreement with measured SAXS data. The aptamer LC-18t had stronger binding to cancerous cells in lung tumor tissues and shared the binding site with the original larger aptamer. The suggested approach reveals 3D shapes of aptamers and helps in designing better affinity probes.