Structure Of Aptamer Research Articles

Programming Fluorogenic DNA Probes for Rapid Detection of Steroids

AbstractThe ability of aptamers to recognize a variety of different molecules has fueled their emergence as recognition agents to probe complex media and cells. Many detection strategies require aptamer binding to its target to result in a dramatic change in structure, typically from an unfolded to a folded state. Here, we report a strategy based on forced intercalation (FIT) that increases the scope of aptamer recognition by transducing subtle changes in aptamer structures into fluorescent readouts. By screening a library of green‐fluorescent FIT‐aptamers whose design is guided by computational modeling, we could identify hits that sense steroids like dehydroepiandrosterone sulfate (DHEAS) down to 1.3 μM with no loss in binding affinity compared to the unmodified aptamer. This enabled us to study DHEAS in clinical serum samples with several advantages over gold standard methods, including rapid readout (<30 min), simple instrumentation (plate‐reader), and low sample volumes (10 μL).

Programming Fluorogenic DNA Probes for Rapid Detection of Steroids.

The ability of aptamers to recognize a variety of different molecules has fueled their emergence as recognition agents to probe complex media and cells. Many detection strategies require aptamer binding to its target to result in a dramatic change in structure, typically from an unfolded to a folded state. Here, we report a strategy based on forced intercalation (FIT) that increases the scope of aptamer recognition by transducing subtle changes in aptamer structures into fluorescent readouts. By screening a library of green-fluorescent FIT-aptamers whose design is guided by computational modeling, we could identify hits that sense steroids like dehydroepiandrosterone sulfate (DHEAS) down to 1.3 μM with no loss in binding affinity compared to the unmodified aptamer. This enabled us to study DHEAS in clinical serum samples with several advantages over gold standard methods, including rapid readout (<30 min), simple instrumentation (plate-reader), and low sample volumes (10 μL).

Study of the prostate-specific antigen–aptamer stability in the PSA–aptamer-single wall carbon nanotube assembly by docking and molecular dynamics simulation

ABSTRACT Prostate-specific antigen (PSA) is used to detect prostate cancer. RNA aptamers against this protein have been reported previously. In this work, the stability and interaction mechanism of aptamer with PSA and single-wall carbon nanotube (SWCNT) was studied by computational techniques. Molecular dynamics simulations were used to investigate the interaction of aptamer segments with the surfaces of SWCNT and prostate-specific antigen (PSA) protein in an aqueous solution by two steps. Firstly, the interaction of aptamer and SWCNT was studied by the molecular dynamic simulation method. Our results showed that an aptamer segment can attach to the surface of SWCNT with its helix axis perpendicularly. Our analysis shows that the π–π stacking interactions play an important role in the binding of the aptamer to carbon rings. Secondly, we analysed the interaction of PSA protein with aptamer in the aptamer–SWCNT assemble. We found that the 3D structure of aptamer in the presence of PSA protein and SWCNT was stable. Finally, this study provides evidence about the stability of aptamer as a sensor on the surface of SWCNT (as a fixed surface) for PSA protein as a ligand. This finding can help to improve new biosensors for detecting cancer protein markers.

TISS nanobiosensor for salivary cortisol measurement by aptamer Ag nanocluster SAIE supraparticle structure

The aptamer based signal-on nanobiosensor for measuring salivary cortisol in the nanomolar level is presented for the first time in this study. Cortisol, as a stress biomarker, plays a critical role in physiological mechanisms. Measurement without contamination by needle stress is the main advantage of cortisol measurement in saliva. Salivary cortisol quantity is much lower than serum cortisol, so overcoming the measurement challenge requires new ultrasensitive detection methods. In the presented Target-Induced Structural Switching mode Aptamer (TISS) nanobiosensor, the ssDNA aptamer plays the role of transducer moiety and biorecognition module simultaneously. The aptamer structure has sequence against cortisol in the middle and two cytosine reach compartments in 3' and 5' ends which are designed for Ag-NC supramolecule configuration. The molecule-like properties of the Ag nanocluster create a strong AIE fluorescence signal. Binding aptamer to cortisol causes proximity of Ag-NCs supraparticles and leads to aggregation base fluorescence enhancement. 1 nmol/l as LOD and 1 nmol/L to 900 nmol/L for linearity achieved by presented salivary cortisol nanobiosensor. This assay was performed in one single tube without further stages as a mix and measure method.

Efficient splicing-based RNA regulators for tetracycline-inducible gene expression in human cell culture and C. elegans.

Synthetic riboswitches gain increasing interest for controlling transgene expression in diverse applications ranging from synthetic biology, functional genomics, and pharmaceutical target validation to potential therapeutic approaches. However, existing systems often lack the pharmaceutically suited ligands and dynamic responses needed for advanced applications. Here we present a series of synthetic riboswitches for controlling gene expression through the regulation of alternative splicing. Placing the 5′-splice site into a stem structure of a tetracycline-sensing aptamer allows us to regulate the accessibility of the splice site. In the presence of tetracycline, an exon with a premature termination codon is skipped and gene expression can occur, whereas in its absence the exon is included into the coding sequence, repressing functional protein expression. We were able to identify RNA switches controlling protein expression in human cells with high dynamic ranges and different levels of protein expression. We present minimalistic versions of this system that circumvent the need to insert an additional exon. Further, we demonstrate the robustness of our approach by transferring the devices into the important research model organism Caenorhabditis elegans, where high levels of functional protein with very low background expression could be achieved.

Structural Biology for the Molecular Insight between Aptamers and Target Proteins.

Aptamers are promising therapeutic and diagnostic agents for various diseases due to their high affinity and specificity against target proteins. Structural determination in combination with multiple biochemical and biophysical methods could help to explore the interacting mechanism between aptamers and their targets. Regrettably, structural studies for aptamer–target interactions are still the bottleneck in this field, which are facing various difficulties. In this review, we first reviewed the methods for resolving structures of aptamer–protein complexes and for analyzing the interactions between aptamers and target proteins. We summarized the general features of the interacting nucleotides and residues involved in the interactions between aptamers and proteins. Challenges and perspectives in current methodologies were discussed. Approaches for determining the binding affinity between aptamers and target proteins as well as modification strategies for stabilizing the binding affinity of aptamers to target proteins were also reviewed. The review could help to understand how aptamers interact with their targets and how alterations such as chemical modifications in the structures affect the affinity and function of aptamers, which could facilitate the optimization and translation of aptamers-based theranostics.

Hybrid Aptamer-Molecularly Imprinted Polymer (aptaMIP) Nanoparticles from Protein Recognition-A Trypsin Model.

Aptamers offer excellent potential for replacing antibodies for molecular recognition purposes however their performance can compromise with biological/environmental degradation being a particular problem. Molecularly imprinted Polymers (MIPs) offer an alternative to biological materials and while these offer the robustness and ability to work in extreme environmental conditions, they often lack the same recognition performance. By slightly adapting the chemical structure of a DNA aptamer it is incorporated for use as the recognition part of a MIP, thus creating an aptamer-MIP hybrid or aptaMIP. Here these are developed for the detection of the target protein trypsin. The aptaMIP nanoparticles offer superior binding affinity over conventional MIP nanoparticles (nanoMIPs), with KD values of 6.8 × 10-9 (±0.2 × 10-9 ) m and 12.3 × 10-9 (±0.4 × 10-9 ) m for the aptaMIP and nanoMIP, respectively. The aptaMIP also outperforms the aptamer only (10.3× 10-9 m). Good selectivity against other protein targets is observed. Using surface plasmon resonance, the limit of detection for aptaMIP nanoparticles is twofold lower (2 nm)compared to the nanoMIP (4nm). Introduction of the aptamer as a "macro-monomer" into the MIP scaffold has beneficial effects and offers potential to improve this class of polymers significantly.

A simple and label-free fluorescent aptasensor for detection of tobramycin: Appropriate for on-site antibiotic monitoring

Herein a simple fluorescent aptasensor is introduced to quantitatively monitor tobramycin (TOB) antibiotic by using the inherent hairpin structure of TOB aptamer and SYBR Green I (SGI) dye, as the biorecognition element and signal reporter, respectively. In the presence of TOB, the preferential binding of the aptamer towards its target induces the conformational change of the aptamer from hairpin-like to a single string structure. Hence, the SGI intercalation is reduced, decreasing the fluorescent intensity. Under the effect of experimental conditions, there was a wide linear relationship between the relative fluorescence emission and TOB concentration in the range of 0.1–6 µM. Besides, the aptasensor could detect TOB with the detection limit as low as 0.063 µM. The developed aptasensor was utilized to determine TOB in human serum and milk samples with the detection limits of 0.043 and 0.034 µM, respectively. The label-free aptasensor with the main features of feasibility, simplicity and high sensitivity holds the superior potential to develop a low-cost sensing method for human diagnostics, food control, and environmental monitoring. The designed aptasensor can be efficiently applied by non-specialist consumers for on-site monitoring of the diverse targets by substituting their specific aptamers.

Tying the knot in the tetrahydrofolate (THF) riboswitch: A molecular basis for gene regulation

Effective gene regulation by the tetrahydrofolate riboswitch depends not only on ligand affinity but also on the kinetics of ligand association, which involves two cooperative binding sites. We have determined a 1.9-Å resolution crystal structure of the ligand-free THF riboswitch aptamer. The pseudoknot binding site ‘unwinds’ in the absence of ligand, whereby the adjacent helical domains (P1, P2, and P3) become disjointed, resulting in rotation and misalignment of the gene-regulatory P1 helix with respect to P3. In contrast, the second binding site at the three-way junction, which is the first to fold, is structurally conserved between apo and holo forms. This suggests a kinetic role for this site, in which binding of the first ligand molecule to the stably folded three-way junction promotes formation of the regulatory pseudoknot site and subsequent binding of the second molecule. As such, these findings provide a molecular basis for both conformational switching and kinetic control.

Selection and identification of an ssDNA aptamer to NB4 cell.

This study was to find the aptamers with high affinity and specificity binding to acute promyelocytic leukemia (APL) NB4 cell line.These aptamers targeted NB4 cells were selected from a random single‐stranded DNA (ssDNA) library of systematic evolution of ligands by exponential enrichment (CELL‐SELEX). The binding rate of FITC‐ssDNA library and NB4 cells was monitored using flow cytometry and fluorescence microscope. After cloned and sequenced, the structure, specificity, and affinity of these candidate aptamers were further analyzed.After a total of 19 rounds of selection, the ssDNA library was enriched and the BR (19.9%) of the 16th round was 12 times of the first round (1.6%). Three enriched aptamers were obtained from 21 positive clones of the 16th round, and the predicted secondary structures of these aptamers were mainly stem‐loop. The aptamer CX9 had the highest affinity, and the equilibrium dissociation constant (Kd) was 16.2 nM. The fluorescence intensity of CX9 binding to NB4 cells was stronger than HL60 and K562 cells under fluorescence microscopy.The study indicates that aptamer CX9 exhibits high affinity and specificity with NB4 cells and lay a foundation for the rapid diagnostic method to detect APL with fluorescence‐labeled aptamer.

The emerging structural complexity of G-quadruplex RNAs.

G-quadruplexes (G4s) are four-stranded nucleic acid structures that arise from the stacking of G-quartets, cyclic arrangements of four guanines engaged in Hoogsteen base-pairing. Until recently, most RNA G4 structures were thought to conform to a sequence pattern in which guanines stacking within the G4 would also be contiguous in sequence (e.g., four successive guanine trinucleotide tracts separated by loop nucleotides). Such a sequence restriction, and the stereochemical constraints inherent to RNA (arising, in particular, from the presence of the 2′-OH), dictate relatively simple RNA G4 structures. Recent crystallographic and solution NMR structure determinations of a number of in vitro selected RNA aptamers have revealed RNA G4 structures of unprecedented complexity. Structures of the Sc1 aptamer that binds an RGG peptide from the Fragile-X mental retardation protein, various fluorescence turn-on aptamers (Corn, Mango, and Spinach), and the spiegelmer that binds the complement protein C5a, in particular, reveal complexity hitherto unsuspected in RNA G4s, including nucleotides in syn conformation, locally inverted strand polarity, and nucleotide quartets that are not all-G. Common to these new structures, the sequences folding into G4s do not conform to the requirement that guanine stacks arise from consecutive (contiguous in sequence) nucleotides. This review highlights how emancipation from this constraint drastically expands the structural possibilities of RNA G-quadruplexes.

Cobalt-doped tungsten trioxide nanorods decorated with Au nanoparticles for ultrasensitive photoelectrochemical detection of aflatoxin B1 based on aptamer structure switch

Herein, we report cobalt-doped tungsten trioxide (Co-WO3) nanorods/Au nanoparticles (AuNPs) for sensitive photoelectrochemical (PEC) sensing of aflatoxin B1 (AFB1) based on aptamer structure switch and horseradish peroxidase (HRP)-induced biocatalytic precipitation. Because of the decreased band gap and the increased visible light absorption of Co-WO3 nanorods, Co-WO3 nanorods show higher PEC activity than WO3 nanorods under the visible light irradiation. Co-WO3 nanorods decorated with AuNPs offer superior PEC activity to Co-WO3 nanorods due to the surface plasmon resonance effect. To construct a PEC aptasensor, short DNA strands (DNA1) are immobilized on the Co-WO3-AuNPs modified indium-tin oxide electrode. HRP-labeled aptamer hybridizes with the DNA1 on the photoelectrode in the absence of AFB1, and precipitations generated by HRP catalysis can quench the photocurrent of the photoelectrode. In the presence of AFB1, HRP-labeled aptamer does not hybridize with the DNA1 due to the formation of aptamer-AFB1 complex, and thus high photocurrent signal is achieved. The PEC aptasensor for AFB1 detection shows a broad linear range from 5 fg mL−1 to 10 ng mL−1, with a detection limit of 1 fg mL−1 (S/N = 3). We believe the high-performance Co-WO3/AuNPs active material coupled with the simple PEC sensing strategy will provide a promising sensing platform for detecting small biomolecules.

Designing of peptide aptamer targeting the receptor-binding domain of spike protein of SARS-CoV-2: an in silico study.

Short synthetic peptide molecules which bind to a specific target protein with a high affinity to exert its function are known as peptide aptamers. The high specificity of aptamers with small-molecule targets (metal ions, dyes and theophylline; ATP) is within 1 pM and 1 μM range, whereas with the proteins (thrombin, CD4 and antibodies) it is in the nanomolar range (which is equivalent to monoclonal antibodies). The recently identified coronavirus (SARS-CoV-2) genome encodes for various proteins, such as envelope, membrane, nucleocapsid, and spike protein. Among these, the protein necessary for the virus to enter inside the host cell is spike protein. The work focuses on designing peptide aptamer targeting the spike receptor-binding domain of SARS-CoV-2. The peptide aptamer has been designed by using bacterial Thioredoxin A as the scaffold protein and an 18-residue-long peptide. The tertiary structure of the peptide aptamer is modeled and docked to spike receptor-binding domain of SARS CoV2. Molecular dynamic simulation has been done to check the stability of the aptamer and receptor-binding domain complex. It was observed that the aptamer binds to spike receptor-binding domain of SARS-CoV-2 in a similar pattern as that of ACE2. The aptamer–receptor-binding domain complex was found to be stable in a 100 ns molecular dynamic simulation. The aptamer is also predicted to be non-antigenic, non-allergenic, non-hemolytic, non-inflammatory, water-soluble with high affinity toward ACE2 than serum albumin. Thus, peptide aptamer can be a novel approach for the therapeutic treatment for SARS-CoV-2.Graphical abstract

Applying joint theoretical experimental research to aptamer modeling

The aim of the research. In this work we studied the structure of LC-18 DNA aptamer, which exhibits specifi c binding to lung adenocarcinoma cells. Obtaining the 3D structure of the aptamer is necessary for understanding the mechanism of binding of the aptamer to the target. Th erefore, the aim of the research was modeling of the LC-18 aptamer spatial structure using combination of theoretical methods: DNA folding tools, quantum-chemical calculations and molecular dynamic simulations. Material and methods. Th e secondary structure of the LC-18 aptamer was predicted by using OligoAnalyzer and MFold online soft ware under the conditions typical small-angle X-ray scattering (SAXS) experiment. Th e molecular modeling of the aptamer was carried out using the Avogadro program. For prediction of the structure two computational methods were used: quantum-mechanical method with third-order density-functional tight-binding (DFTB3) and molecular dynamics (MD) with force fi elds. Results. In this paper it was shown that molecular simulations can predict structures from the SAXS experiments. OligoAnalyzer and MFold web servers have been used to generate a set of several likely models. However, more accurate calculations have showed that these models do not predict the relative importance of isomers. Meanwhile, application of quantum-chemical and molecular dynamics calculations have showed reliable molecular structures which have a small deviations from the experimental SAXS curves. Conclusion. Th is study demonstrates the approach for modeling 3D structures of DNA-aptamers in solution using both experimental and theoretical methods. It could be very helpful in designing more effi cient aptamers based on results obtained from molecular simulations.

Computational approach to design of aptamers to the receptor binding domain of SARS-CoV-2

The aim of the research. In this work, in silico selection of DNA-aptamers to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was performed using molecular modeling methods. Material and methods. A new computational approach to aptamer in silico selection is based on a cycle of simulations, including the stages of molecular modeling, molecular docking, molecular dynamic simulations, and quantum chemical calculations. To verify the obtained calculated results flow cytometry, fluorescence polarization, and small-angle X-ray scattering methods were applied. Results. An initial library consisted of 256 16-mer oligonucleotides was modeled. Based on molecular docking results, the only one aptamer (Apt16) was selected from the library as a starting aptamer to the RBD protein. For Apt16/RBD complex, molecular dynamic and quantum chemical calculations revealed the pairs of nucleotides and amino acids whose contribution to the binding between aptamer and RBD is the largest. Taking into account these data, Apt16 was subjected to the structure modifi cations in order to increase the binding with the RBD. Th us, a new aptamer Apt25 was designed. Th e procedure of 1) aptamer structure modeling/modifi cation, 2) molecular docking, 3) molecular dynamic simulations, 4) quantum chemical calculations was performed several times. As a result, four aptamers (Apt16, Apt25, Apt27, Apt31) to the RBD were designed in silico without any preliminary experimental data. Binding of the each modeled aptamer to the RBD was studied in terms of interactions between residues in protein and nucleotides in the aptamers. Based on the simulation results, the strongest binding with the RBD was predicted for two Apt27 and Apt31aptamers. The calculated results are in good agreement with experimental data obtained by flow cytometry, fluorescence polarization, and small-angle X-ray scattering methods. Conclusion. Th e proposed computational approach to selection and refi nement of aptamers is universal and can be used for wide range of molecular ligands and targets

Predicting the aptamer SYL3C-EpCAM complex's structure with the Martini-based simulation protocol.

Aptamers (small single strand DNA/RNAs) such as SYL3C are considered as ideal alternatives to antibodies in cancer related research studies. However, 3D structure predictions for aptamers and aptamer-protein complexes are scarce due to the high cost of experimental measurements and unreliable computer-based methods. Thus aptamers' diagnostic and therapeutic applications are severely restricted. To meet the challenge, we proposed a Martini-based aptamer-protein complex prediction protocol. By combining the base-base contact map from simulation and secondary structure prediction from various tools, improved secondary structure predictions can be obtained. This method reduced the risk of providing incorrect or incomplete base pairs in secondary structure prediction. Thus 3D structure modeling based on the secondary structure can be more reliable. We introduced the soft elastic network to the hairpin folded regions of the Martini ssDNAs to preserve their canonical structure. Using our protocol, we predicted the first 3D structure of the aptamer SYL3C and the SYL3C-EpCAM complex. We believe that our work could contribute to the future aptamer-related research studies and medical implications.

Computational approach to design of aptamers to the receptor binding domain of sars-cov-2

The aim of the research. In this work, in silico selection of DNA-aptamers to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was performed using molecular modeling methods. Material and methods. A new computational approach to aptamer in silico selection is based on a cycle of simulations, including the stages of molecular modeling, molecular docking, molecular dynamic simulations, and quantum chemical calculations. To verify the obtained calculated results flow cytometry, fluorescence polarization, and small-angle X-ray scattering methods were applied. Results. An initial library consisted of 256 16-mer oligonucleotides was modeled. Based on molecular docking results, the only one aptamer (Apt16) was selected from the library as a starting aptamer to the RBD protein. For Apt16/RBD complex, molecular dynamic and quantum chemical calculations revealed the pairs of nucleotides and amino acids whose contribution to the binding between aptamer and RBD is the largest. Taking into account these data, Apt16 was subjected to the structure modifications in order to increase the binding with the RBD. Thus, a new aptamer Apt25 was designed. The procedure of 1) aptamer structure modeling/modification, 2) molecular docking, 3) molecular dynamic simulations, 4) quantum chemical calculations was performed sev-eral times. As a result, four aptamers (Apt16, Apt25, Apt27, Apt31) to the RBD were designed in silico without any preliminary experimental data. Binding of the each modeled aptamer to the RBD was studied in terms of interactions between residues in protein and nucleotides in the aptamers. Based on the simulation results, the strongest binding with the RBD was predicted for two Apt27 and Apt31aptamers. The calculated results are in good agreement with experimental data obtained by flow cytometry, fluorescence polarization, and small-angle X-ray scattering methods. Conclusion. The proposed computational approach to selection and refinement of aptamers is universal and can be used for wide range of molecular ligands and targets. Key words. © ARTYUSHENKO P. V., MIRONOV V. A., MOROZOV D. I., SHCHUGOREVA I. A., BORBONE N., TOMILIN F. N., KICHKAILO A. S.

Structure approaches to study of DNA aptamers in solution

The high potential of aptamers – specifi c molecular agents based on short single-stranded nucleic acids – makes high demands on the molecules under development for the effi ciency of interaction with target biomolecules. In this work, approaches are considered for studying the spatial structure of DNA aptamers in solution using various complementary methods, which make it possible to obtain a more complete picture of the formation of the structure and conformational changes, to track the interaction with the target protein, the tendency to oligomerization, and to characterize the spatial structure of both individual molecules and complexes.

Bio-inspired dynamic biomolecule assembling for fine regulation of protein activity.

A versatile approach for the fine control of DNA-based hierarchical assembly via dual stimuli and two assembly strategies is developed. Moreover, with a reasonable design of functional thrombin aptamer structures on the formed DNA nanoassembly, it can achieve precise regulation of thrombin activity.

Comparative evaluation of clinical manifestations and risk of death in patients admitted to hospital with covid-19 and seasonal influenza: cohort study

<h3>ABSTRACT</h3> RNA nanotechnology uses motifs from nature as well as aptamers from in vitro selection to construct nanostructures and devices for applications in RNA medicine and synthetic biology. The RNA...