Aptamer Modification Research Articles

In Silico Approaches for the Identification of Aptamer Binding Interactions to Leptospira spp. Cell Surface Proteins

Aptamers are nucleic acids that can bind with high affinity and specificity to a range of target molecules. However, their functionality relies on their secondary and tertiary structures such that the combination of nucleotides determines their three-dimensional conformation. In this study, the binding mechanisms of candidate aptamers and their interactions with selected target proteins found in the cell surface of Leptospira were predicted to select high-affinity aptamers. Four aptamers were evaluated through molecular modeling and docking using available software and web-based tools, following the workflow previously designed for in silico evaluation of DNA aptamers. The most predominant and highly conserved surface-exposed proteins among pathogenic Leptospira species were used as aptamer targets. The highest number of interactions was seen in aptamers AP5 and AP1. Hydrogen bonds, along with a few hydrophobic interactions, occur in most aptamer-protein complexes. Further analysis revealed serine, threonine, glutamine, and lysine as main protein residues. H-bond interactions occur mostly with polar amino acids, as reflected in the predicted interaction profiles of aptamer-protein complexes. In silico strategies allowed the identification of key residues crucial in aptamer-target interaction during aptamer screening. Such information can be used in aptamer modification for improved binding affinity and accuracy for diagnostics application.

Surface plasmon field enhanced upconversion luminescence for the screening and detection of phenolic environmental estrogens

The endocrine system's interference caused by environmental estrogens (EEs) residue in food is a topic of public concern. Here, we construct an aptasensor for the sensitive detection of EEs based on luminescence resonance energy transfer (LRET). With MoS2 nanosheets acting as the energy acceptor and upconversion luminescence nanoparticles@gold nanoparticles (UCNPs@Au) as the luminescence donor, autofluorescence from food is prevented from interfering. The in-situ deposition of AuNPs not only induces local field enhancement to significantly increase the luminescence intensity of UCNPs, but also conduces to the modification of aptamer through Au-S bond. This aptasensor can respond to multiple estrogens thanks to the choice of a universal aptamer that recognizes phenolic hydroxyl group, and it offers the probability to screen unidentified phenolic estrogens. This method has a high sensitivity and a low limit of detection (LOD), and the satisfactory recovery rates acquired from water and milk samples confirmed its considerable application value.

Development and classification of RNA aptamers for therapeutic purposes: an updated review with emphasis on cancer.

Today, RNA aptamers are being considered promising theranostic tools against a wide variety of disorders. RNA aptamers can fold into complex shapes and bind to diverse nanostructures, macromolecules, cells, and viruses. It is possible to isolate RNA aptamers from a vast pool of nucleic acids via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. As therapeutics, aptamers have great potential because of their ability to bind to proteins and selectively limit their activities with negligible side effects. Several RNA aptamers with potential implications in cancer diagnosis are known to confer a great affinity for single-stranded DNA molecules, long non-coding RNAs, circulating tumor cells, vascular endothelial growth factors, and tissue and sera-derived exosomes in patients with different malignancies. Furthermore, clinical investigations have revealed the efficacy of RNA aptamer-based agents in imaging modalities. This review seeks to deliver new insights into the development, classification, nanomerization, and modification of RNA aptamers, as well as their applications in cancer theranostics. The aptamers' mechanism of action and their interest to clinical trials as theranostic agents are also discussed.

Systematic bio-fabrication of aptamers and their applications in engineering biology.

Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity to bind to specific targets. Compared to antibodies, aptamers are easy to in vitro synthesize with low cost, and exhibit excellent thermal stability and programmability. With these features, aptamers have been widely used in biology and medicine-related fields. In the meantime, a variety of systematic evolution of ligands by exponential enrichment (SELEX) technologies have been developed to screen aptamers for various targets. According to the characteristics of targets, customizing appropriate SELEX technology and post-SELEX optimization helps to obtain ideal aptamers with high affinity and specificity. In this review, we first summarize the latest research on the systematic bio-fabrication of aptamers, including various SELEX technologies, post-SELEX optimization, and aptamer modification technology. These procedures not only help to gain the aptamer sequences but also provide insights into the relationship between structure and function of the aptamers. The latter provides a new perspective for the systems bio-fabrication of aptamers. Furthermore, on this basis, we review the applications of aptamers, particularly in the fields of engineering biology, including industrial biotechnology, medical and health engineering, and environmental and food safety monitoring. And the encountered challenges and prospects are discussed, providing an outlook for the future development of aptamers.

AFM and Electrochemical Analysis of Different Biomolecules Using Npy-Specific Aptamer Modified Electrodes

Neurotransmitters and neuropeptides are chemical signals in the brain that produce responses or functions throughout the organism. Measurements of neurotransmitters are important to understand some mechanisms of how they are released off the cell. Different biomolecules can be measured with gold (Au) electrodes because it is a ductile and malleable metal that can detect small changes and can be easily modified. In this work, we use Neuropeptide Y (NPY) specific aptamer to modify the electrodes to take advantage of the binding with NPY molecules that produce conformational changes. We also study Dopamine (DA) and Norepinephrine (NE) because they corelease with NPY and we can monitor the electrochemical difference between these biomolecules. Those modifications and detection can be monitored using Electrochemical Impedance Spectroscopy (EIS) to understand the adsorption properties of molecules that stick to the surface of the electrode and Atomic Force Microscopy (AFM) to measure the topography of the electrodes before and after the aptamer modification. Electrochemical results showed that the capacitance of the Au electrode decreases at higher concentrations of NPY. AFM results showed small changes on the surface of the Au electrodes with the different modifications due to the roughness of the surface. The results obtained give us a better understanding of the use of these techniques for the monitoring of biomolecules.

Synthesis of a Terminal Amino‐Modified Nucleolin Aptamer and Its Paclitaxel Conjugate

AbstractAptamer‐drug conjugate has been developed as a potential class of targeted therapeutics, which using aptamer as tumor recognition elements could targeted deliver the conjugated chemically toxic drug to tumor site. One of the keys to the success of aptamer‐drug conjugate is terminal modification of aptamer to facilitate the conjugation with drugs. Herein, a novel terminal amino‐modified nucleolin aptamer which using solketal as raw material was developed. Moreover, its paclitaxel conjugate wherein the aptamer coupled with paclitaxel derivatives by amidation was also conducted. The easily available raw materials, stable structure, together with its simplicity of operation, gives it the potential for use on an industrial scale.

Covalent and Oriented Immobilization of Antibodies through Systematic Modification of Photoactivatable RNA Hybrid Aptamers

We report a covalent and well-oriented strategy of immobilization of antibodies using photoactivatable Fc-binding RNA aptamers (FcBAs). We prepared several types of FcBAs that were further modified with photoaffinity probes (i.e., benzophenone or diazirine), and evaluated the binding capabilities and the photo-crosslinking efficiency of them via pull-down assays and fluorescence analyses, respectively. Initial moderate photo-crosslinking efficiency (Kd ∼ 110 nM) was substantially improved by multivalent association of FcBAs and structural modification of FcBAs. For a conceptual proof, covalent crosslinking of human IgG on FcBA-tethered solid chips was fabricated and investigated by scanometry, which eventually proved real applicability of the present scheme toward immunoassays.

Selection and identification of a novel ssDNA aptamer targeting human skeletal muscle.

Skeletal muscle disorders have posed great threats to health. Selective delivery of drugs and oligonucleotides to skeletal muscle is challenging. Aptamers can improve targeting efficacy. In this study, for the first time, the human skeletal muscle-specific ssDNA aptamers (HSM01, etc.) were selected and identified with Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The HSM01 ssDNA aptamer preferentially interacted with human skeletal muscle cells in vitro. The in vivo study using tree shrews showed that the HSM01 ssDNA aptamer specifically targeted human skeletal muscle cells. Furthermore, the ability of HSM01 ssDNA aptamer to target skeletal muscle cells was not affected by the formation of a disulfide bond with nanoliposomes in vitro or in vivo, suggesting a potential new approach for targeted drug delivery to skeletal muscles via liposomes. Therefore, this newly identified ssDNA aptamer and nanoliposome modification could be used for the treatment of human skeletal muscle diseases.

Enhanced Functional Properties of Three DNA Origami Nanostructures as Doxorubicin Carriers to Breast Cancer Cells

Previous studies have shown that chemotherapeutic efficacy could be enhanced with targeted drug delivery. Various DNA origami nanostructures have been investigated as drug carriers. Here, we compared drug delivery functionalities of three similar DNA origami nanostructures, Disc, Donut, and Sphere, that differ in structural dimension. Our results demonstrated that Donut was the most stable and exhibited the highest Dox-loading capacity. MUC1 aptamer modification in our nanostructures increased cellular uptake in MUC1-high MCF-7. Among the three nanostructures, unmodified Donut exerted the highest Dox cytotoxicity in MCF-7, and MUC1 aptamer modification did not further improve its effect, implicating that Dox delivery by Donut was efficient. However, all Dox-loaded nanostructures showed comparable cytotoxicity in MDA-MB-231 due to the innate sensitivity of this cell line to Dox. Our results successfully demonstrated that functional properties of DNA origami nanocarriers could be tuned by structural design, and three-dimensional Donut appeared to be the most efficient nanocarrier.

Multifunctional biosensor constructed by Ag-coating magnetic-assisted unique urchin core porous shell structure for dual SERS enhancement, enrichment, and quantitative detection of multi-components inflammatory markers

The simultaneous, precise, and quantitative detection of multi-components inflammatory markers (IMs) in sepsis serum by surface-enhanced Raman scattering (SERS) remains a challenging problem. A novel, multifunctional biosensor with dual enrichment and enhancement was designed for the ultrasensitive and quantitative analysis of multi-components IMs. The biosensor contains SERS tags-unique urchin core/porous shell (CPS) structure modified with Raman reporters (RaRs), magnetic assist-Ag coated Fe3O4 magnetic nanoparticles (Ag MNPs) modified with internal standard (IS), and then aptamer (Apt) modification to form the sandwich structure (Ag MNPs/IMs/CPS). This multifunctional sensor used for IMS detection has the following innovations: The intensity ratio IRaRs/IIS with Lg CIMs present a good and wide linear relationship to achieve the simultaneous, precise, and quantitative detection of IMS in serum; The detection results display ultrasensitivity, and the limit of detection (LOD) for CRP, IL-6, and PCT is 100 fg/mL, 0.1 fg/mL, and 1.0 fg/mL, which is lower than other detection techniques; The calculated data of clinical blood samples of sepsis by this SERS method is consistent with the hospital results, and can provide more compositional data of IMs. Thus, this combined approach developed a sensing platform for rapid screening, accurate evaluation, early warning, and diagnosis of sepsis.

Intracellular Self‐Assembly Driven Nucleus‐Targeted Photo‐Immune Stimulator with Chromatin Decompaction Function for Robust Innate and Adaptive Antitumor Immunity

AbstractEfficient nuclear DNA damage and release is highly recommended to improve the photodynamic immunotherapy by eliciting innate immune response yet remains challenging. Herein, an intracellular self‐assembly driven nucleus‐targeted photo‐immune stimulator (PIS) with chromatin decompaction function is reported for innate and adaptive antitumor immunity co‐activation. The PIS consists of vorinostat (SAHA)‐loaded manganese‐porphyrin metal‐organic framework (Mn (III)‐TCPP MOF) with further modification of AS1411 aptamer. The PIS can be efficiently internalized by tumor cells and disassembled under the intracellular overexpressed glutathione (GSH). Notably, the released AS1411 is able to be self‐assembled with photosensitizer TCPP in situ within cells, driving nucleus‐targeted delivery of TCPP; meanwhile, the loaded SAHA can induce chromatin decompaction, cooperatively promoting TCPP‐mediated photodynamic nuclear DNA damage and cytosolic release under laser irradiation. In addition, the released manganese ions (Mn2+) further enhance the cytosolic DNA/cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon gene (STING) pathway mediated innate immunity, which synergizes with PDT‐induced immunogenic cell death to achieve co‐activation of innate and adaptive immunity. Compared to traditional PDT, the self‐assembly driven nucleus‐targeted PDT system with chromatin decompaction show significantly enhanced efficacy for inhibiting primary tumor growth and distant metastasis in several xenograft tumor models, mechanistically by promoting the maturation of dendritic cells and tumor infiltration of natural killer cell, cytotoxic T lymphocytes.

Label-free biosensing of mercury(II) in milk using an aptamer-gated graphene field-effect transistor

• A specific Hg(II) biosensor based on aptamer-modified SGGT is developed. • The sensitivity and selectivity were improved by aptamer and BSA modification. • The biosensor had a high recovery rate for Hg(II) evaluation in real milk samples. Due to the environmental pollution by the growing urban, agricultural and industrial disposals, dairy products are susceptible to heavy metal pollution in the production process. As one of the most toxic heavy metals, mercury cause serious damage to the environment and even public health. Effective monitoring of Hg(II) in large number of diary samples requires the development of rapid assaying methods. In this research work, we report the preparation of a label-free biosensor for Hg(II) using an aptamer-gated graphene field-effect transistor (Apt-SGGT) with the gate electrode modified by a structure-switching aptamer against Hg(II) and blocked with bovine serum albumin. The aptamer immobilized on the gate electrode acts as both a receptor and a reporter for Hg(II) by specifically recognizing Hg(II) through stable T-Hg 2+ -T coordination complex, triggering the folding of the single-stranded aptamer into a duplex structure. The conformational change results in the redistribution of the surface charge on the gate electrode, and produces a large channel current response by the inherent field-effect amplification. This sensing mechanism can effectively enhance the selectivity of the SGGT-based biosensor to mercury ions by significantly reducing the unspecific responses of other interfering cations. The detection limit of Hg(II) by Apt-SGGT sensor was as low as 5 nM, and the linear detection range was from 5 to 200 nM. The sensor can realize the detection of mercury ion residues in milk with a high recovery rate, and has a good application prospect in the detection and analysis of heavy metal residues in complex systems.

Molecular Engineering of Aptamer Self-Assemblies Increases in Vivo Stability and Targeted Recognition.

Functionally modified aptamer conjugates are promising tools for targeted imaging or treatment of various diseases. However, broad applications of aptamer molecules are limited by their in vivo instability. To overcome this challenge, current strategies mostly rely on covalent chemical modification of aptamers, a complicated process that requires case-by-case sequence design, multiple-step synthesis, and purification. Herein, we report a covalent modification-free strategy to enhance the in vivo stability of aptamers. This strategy simply utilizes one-step molecular engineering of aptamers with gold nanoclusters (GNCs) to form GNCs@aptamer self-assemblies. Using Sgc8 as a representative aptamer, the resulting GNCs@Sgc8 assemblies enhance cancer-cell-specific binding and sequential internalization by a receptor-mediated endocytosis pathway. Importantly, the GNCs@aptamer self-assemblies resist nuclease degradation for as long as 48 h, compared to the degradation of aptamer alone at 3 h. In parallel, the tumor-targeted recognition and retention of GNCs@aptamer self-assemblies are dramatically enhanced, indicated by a 9-fold signal increase inside the tumor compared to the aptamer alone. This strategy is to avoid complicated chemical modification of aptamers and can be extended to all aptamers. Our work provides a simple, effective, and universal strategy for enhancing the in vivo stability of any aptamer or its conjugates, thus expanding their imaging and therapeutic applications.

High-fidelity biosensing of dNTPs and nucleic acids by controllable subnanometer channel PaMscS

Current tools for dNTP analysis mainly rely on expensive fluorescent labeling, mass spectrometry or electrochemistry. Single-molecule assay by protein nanopores with an internal diameter of ca. 1–3.6 nm provides a useful tool for dNTP sensing. However, the most commonly used protein nanopores require additional modifications to enable dNTP detection. In this study, the PaMscS channel (mechanosensitive channel of small conductance from Pseudomonas aeruginosa) embedded in the bilayer lipid membrane (BLM) of E. coli polar lipid extract was applied as a nanopore for single molecular sensing. Two mutants of PaMscS nanopores on the side portal region (PaMscS W130A and PaMscS K180R) were selected for direct dNTP or pyrophosphoric acid (PPi) detection without aptamer or protein modification. Notably, the PaMscS mutant pore can be adjusted by regulation of osmolarity differences, which is crucial for the optimal detection of specific molecules. In addition, we established a PaMscS-based diagnosis method for the rapid sensing of disease-associated nucleic acids by monitoring the consumption of dNTPs, with 86% specificity and 100% sensitivity among 22 clinical samples. This protein nanopore, without aptamer or modification, paves a new way for dNTPs, PPi direct sensing and nucleic acid detection with low cost but high versatility.

Molecularly Engineered Aptamers Targeting Tumor Tissue and Cancer Cells for Efficient in Vivo Recognition and Enrichment

Molecularly Engineered Aptamers Targeting Tumor Tissue and Cancer Cells for Efficient in Vivo Recognition and Enrichment

Aptamer functionalized and reduced graphene oxide hybridized porous polymers SPE coupled with LC-MS for adsorption and detection of human α-thrombin.

In this study, reduced graphene oxide (rGO) hybridized high internal phase emulsions were developed and polymerized as porous carriers for aptamer (5'/5AmMC6/-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3') modification to enrich human α-thrombin from serum. The structure and properties of the materials were confirmed by scanning electron microscope (SEM), Fourier transform infrared spectroscope (FT-IR), and X-ray photoelectron spectra (XPS). The adsorption ability and selectivity were studied and the thrombin was detected with liquid chromatography-mass spectrometry (LC-MS). The adsorption of thrombin onto the sorbent was achieved within 30min and the desorption was realized using 5.0mL of acetonitrile/water (80/20, v/v). The thrombin was quantified by LC-MS according to its characteristic peptide sequence of ELLESYIDGR.

Lab-on-a-ZnO-Submicron-Particle Sensor Array for Monitoring AD upon Cd2+ Exposure with CSF Tau441% as an Effective Hallmark.

In this study, based on the posttreatment strategy, blue-color-emissive ZnO submicron particles (B-ZnO SMPs) and red-color-emissive ZnO submicron particles (R-ZnO SMPs) were obtained from rationally designed Zn-infinite coordination polymer (ICP) precursors. After modification of thiol-containing aptamers, diverse spectral changes in the ultraviolet and visible regions of B- and R-ZnO SMPs toward different tau species were explored to construct a lab-on-a-ZnO-submicron-particle sensor array. Assisted by principal component analysis (PCA), the unique fingerprints of the sensor array enabled the simultaneous differentiation and quantitative detection of different tau species (tau381, tau410, and tau441) for the first time. Furthermore, the dynamic changes of tau441% (the ratio of the two most reported representative 4R isoform (full-length tau441) and 3R isoform (tau381)) in cerebrospinal fluid (CSF) during the Alzheimer's disease (AD) onset of Cd2+-exposed rats could also be monitored by the lab-on-a-ZnO-submicron-particle sensor array, which was supposed to be an effective hallmark and highly correlated with the formation of neurofibrillary tangles (NFTs). This study not only provides a further insight into the involvement of subchronic Cd2+ exposure in the tau etiology of AD but also offers more comprehensive and effective information about the asymptomatic stage of AD upon environmental risk, which has potential applications in the early diagnosis and therapy.

Magnetic pollen-like silica nanoparticles for efficient capture of circulating tumor cells

<p indent=0mm>Aptamer-modified magnetic pollen-like silica nanoparticles were synthesized and used as a substrate for the efficient capture of circulating tumor cells. Silica spikes on the surface of nanoparticles can not only provide more binding sites for the modification of aptamer, but also interact with the microvillus and pseudopods on the cell surface, thus improving the capture efficiency of circulating tumor cells (CTCs). The capture efficiency was up to 85%, and the cell viability was nearly 100%. The mimic clinical sample array showed that this strategy achieved a good capture performance even in the complex system and showed a good potential in the clinical applications.

AFM and Electrochemical Analysis of Npy Using Npy-Specific Aptamer-Modified Gold Electrodes

Measurements of neurotransmitters are important to understand some mechanisms of how they are released off the cell. Neurotransmitters and neuropeptides are chemical signals in the brain that produce responses or functions throughout the organism. These biomolecules can be measured with gold (Au) electrodes because it is a ductile and malleable metal that can detect small changes and can be easily modified. In this work, we use NPY-specific aptamer to modify the electrodes to take the advantage of the binding with NPY molecules that produces conformational changes. Those modifications and detection can be monitored using Electrochemical Impedance Spectroscopy (EIS), to understand the adsorption properties of molecules that stick to the surface of the electrode and Atomic Force Microscopy (AFM) to measure the topography of the electrodes before and after the aptamer modification. AFM results showed small changes on the surface of the Au electrodes with the different modifications due to the roughness of the surface. Electrochemical results showed that the capacitance of the gold electrode decreases at higher concentrations of NPY. The results obtained give us a better understanding of the use of these techniques for the monitoring of biomolecules.

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.