Binding Aptamer Research Articles

Highly sensitive detection of Salmonella based on dual-functional HCR-mediated multivalent aptamer and amplification-free CRISPR/Cas12a system

BackgroundSalmonella infection severely threatens human health and causes substantial medical and financial concerns. Sensitive and specific detection of Salmonella in food samples is crucial but remains challenging. While some traditional assays for S. typhimurium are reliable, they suffer from various limitations, such as being time-consuming (culture-based methods), involving intricate nucleic molecular extraction (polymerization chain reaction, PCR), and exhibiting inadequate sensitivity (enzyme-linked immunosorbent assay, ELISA). In this case, it is essential to establish a rapid, simple-operation, and sensitive method for monitoring S. typhimurium to preserve food quality and prevent contamination. ResultHerein, an amplification-free detection method for Salmonella was developed by coupling the aptamer magnetic separation with dual-functional HCR (hybridization chain reaction)-scaffold multivalent aptamer and the activity of CRISPR/Cas12a. In the detection system, the dual-functional HCR-scaffold multivalent aptamer with high binding affinity and specificity was fabricated in advance by assembling numerous Salmonella specific aptamers on the long HCR products. In addition to the enhanced affinity, the HCR-multiApt also contains a massive amount of repeated CRISPR-targetable DNA units in its HCR scaffold, which could trigger the trans-cleavage activity of Cas12a. In the presence of target bacteria, the HCR-scaffold multivalent aptamer could attach on the surface of bacteria effectively and amplified the signal of bacteria into CRISPR/Cas12a based fluorescent readout. The proposed detection system allowed for ultrasensitive detection of Salmonella in a linear range from 100 to 107 cfu mL−1 with a LOD (limit of detection) of 2 cfu mL−1. SignificanceThe novel dual-functional HCR-multiApt presents a simple and powerful strategy for improving the aptamer binding affinity toward Salmonella. Simultaneously, integrating this dual-functional HCR-multiApt with the CRISPR/Cas12a system significantly enhances the sensitivity by cascade signal amplification in a nucleic acids amplification-free way. Finally, leveraging the versatility of the aptamer, this highly sensitive method can be further extended for application in the detection of other bacteria, food safety monitoring, or clinical diagnostics.

Identification and characteristics of temperature-sensitive sites of Pseudomonas plecoglossicida based on aptamers

Pseudomonas plecoglossicida can infect Pseudosciaena crocea and cause visceral white spot disease, resulting in substantial economic losses to the P. crocea industry. Pseudomonas plecoglossicida is a temperate-sensitive bacterium and only infects P. crocea below 22 ℃. Aptamers are oligonucleotide molecules selected by the systematic evolution of ligands by exponential enrichment (SELEX) and can identify all kinds of targets. In the present study, five aptamers (M17, M19, M48, M56 and M81) for P. plecoglossicida cultured at 18 ℃ were selected by SELEX and based on comparative analysis of the high frequency sequences. The five aptamers had significantly higher affinities for the pathogenic P. plecoglossicida at 18 ℃ than for the nonpathogenic P. plecoglossicida at 28 ℃ and the other six bacteria (Escherichia coli, Edwardsiella tarda, Aeromonas hydrophila, Vibiro harveryi, Vibiro alginolyticus, Pseudomonas aeruginosa) (P < 0.01). The affinity constants (Kd) and maximum affinity (Am) of these aptamers for P. plecoglossicida were determined, with Kd values of M17 (23.35 ± 6.40), M19 (66.37 ± 14.73), M48 (105.91 ± 21.41), M56 (31.16 ± 5.03) and M81 (52.63 ± 13.82) nmol/L and with Am values of M17 (1041.26 ± 57.08), M19 (2291.76 ± 171.96), M48 (1258.51 ± 21.41), M56 (1307.70 ± 54.01) and M81 (543.27 ± 44.16) nmol/L, respectively. The secondary structures of the aptamers were analyzed, and the aptamer binding proteins of the pathogenic P. plecoglossicida were isolated by magnetic separation and purified by polyacrylamide gel electrophoresis. Mass spectrometry revealed ribosomal proteins to be the main aptamer binding proteins. The secondary and tertiary structures and subcellular localization of these aptamer binding proteins were also studied and analyzed. These findings provide a foundation for the temperature-sensitive pathogenic mechanism of P. plecoglossicida in P. crocea. This is the first report on aptamers that recognize temperature-sensitive sites in P. plecoglossicida.

A highly sensitive sandwich-type electrochemical aptasensor using a pair of novel truncated aptamers for the detection of vaspin

Visceral adipose tissue-derived serpin (vaspin) is a biomarker associated with obesity and type 2 diabetes, both of which are prevalent and increasing in numbers worldwide. However, the sensitivity of existing aptasensors for vaspin detection does not yet match the reported serum levels. Aptamer sequences could contain nonessential nucleotides, which can be truncated out to potentially enhance their functionality and applicability. In this study, we developed a novel truncation method, a nonessentials-guided in situ truncation strategy to generate high-affinity aptamers by directly removing the nonessential portions of the aptamer sequences using exonucleases from the aptamer-protein complex kept stably. We applied this novel strategy to the original vaspin-binding aptamers, V1 and V49, resulting in two truncated aptamers, V1(3′-5′)–26 and V49(3′-5′)–30. These truncated aptamers showed enhanced binding affinity and specificity compared to their original aptamers. Confirmed to have different binding sites, this pair of truncated aptamers was successfully incorporated into a sandwich-type electrochemical aptasensor. This aptasensor achieved 16-fold higher sensitivity than the one using untruncated aptamers and enabled specific detection of vaspin in a buffer and even sensitive detection in serum within the physiological range. The reduced size of the aptamers could potentially cover the surface of the sensing platform, blocking nonspecific binding, thereby lowering the blank value, and improving the signal-to-noise ratio and limit of detection. This approach highlights the potential of the nonessentials-guided in situ truncation strategy for developing improved truncated aptamers, examining aptamer binding sites, and enhancing the analytical performance of biosensing platforms.

Probing naphthalene diimide and 3-hydroxypropylphosphate as end-conjugating moieties for improved thrombin binding aptamers: Structural and biological effects

The limitations associated with the in vivo use of the thrombin binding aptamer (TBA or TBA15) have dramatically stimulated the search of suitable chemically modified analogues in order to discover effective and reversible inhibitors of thrombin activity. In this context, we previously proposed cyclic and pseudo-cyclic TBA analogues with improved stability that proved to be more active than the parent aptamer.Herein, we have investigated a novel library of TBA derivatives carrying naphthalene diimide (NDI) moieties at the 3′- or 5′-end. In a subset of the investigated oligonucleotides, additional 3-hydroxypropylphosphate (HPP) groups were introduced at one or both ends of the TBA sequence. Evaluation of the G-quadruplex thermal stability, serum nuclease resistance and in vitro anticoagulant activity of the new TBA analogues allowed rationalizing the effect of these appendages on the activity of the aptamer on the basis of their relative position. Notably, most of the different TBA analogues tested were more potent thrombin inhibitors than unmodified TBA. Particularly, the analogue carrying an NDI group at the 5′-end and an HPP group at the 3′-end, named N-TBA-p, exhibited enhanced G-quadruplex thermal stability (ΔTm + 14° C) and ca. 10-fold improved nuclease resistance in serum compared to the native aptamer. N-TBA-p also induced prolonged and dose-dependent clotting times, showing a ca. 11-fold higher anticoagulant activity compared to unmodified TBA, as determined by spectroscopic methods. Overall, N-TBA-p proved to be in vitro a more efficient thrombin inhibitor than all the best ones previously investigated in our group. Its interesting features, associated with its easy preparation, make it a very promising candidate for future in vivo studies.

Cross-Binding of Adenosine by Aptamers Selected Using Theophylline.

We recently reported that some adenosine binding aptamers can also bind caffeine and theophylline with around 20-fold lower affinities. This discovery led to the current work to examine the cross-binding of adenosine to theophylline aptamers. For the DNA aptamer for theophylline, cross-binding to adenosine was observed, and the affinity was 18 to 38-fold lower for adenosine based on assays using isothermal titration calorimetry and ThT fluorescence spectroscopy. The binding complexes were characterized using NMR spectroscopy, and both adenosine and theophylline showed an overall similar binding structure to the DNA theophylline aptamer, although small differences were also observed. In contrast, the RNA aptamer did not show binding to adenosine, although both aptamers have very similar relative selectivity for various methylxanthines including caffeine. After a negative selection, a few new aptamers with completely different primary sequences for theophylline were obtained and they did not show binding to adenosine. Thus, there are many ways for aptamers to bind theophylline and some can have cross-binding to adenosine. In biology, theophylline, caffeine, and adenosine can bind to the same protein receptors to regulate sleep, and their binding to the same DNA motifs may suggest an early role of nucleic acids in similar regulatory functions.

MXene-incorporated C60NPs and Au@Pt with dual-electric signal outputs for accurate detection of Mycobacterium tuberculosis ESAT-6 antigen

Rapid and effective detection of Mycobacterium tuberculosis (MTB) is the crux of minimizing tuberculosis (TB) spread. Consequently, a new electrochemical aptasensor based on dual-signal output for ultrasensitive detection of MTB early secreted antigenic target 6 (ESAT-6) antigen was developed. Especially, a new nanocomposite MXene/C60NPs/Au@Pt was synthesized for signal generation and amplification. In this biosensing architecture, dual independent signal outputs were achieved by coupling the electrochemical redox activity of fullerene nanoparticles (C60NPs) with the effective electrocatalytic activity of Au@Pt nanoparticles. MXene possesses a large specific surface area, allowing densely loaded of these two electroactive materials, further improved sensing capability. In addition, specific ESAT-6 antigen binding aptamers were attached to Au@Pt to create the tracer label. With a typical sandwich format along with the introduction of the gold nanoparticle-loaded molybdenum disulfide (MoS2–Au) as the sensing interface, the limit of detection (LOD) of the proposed aptasensor was 2.88 fg mL−1 (DPV measurement) and 13.50 fg mL−1 (IT measurement), respectively, with a broad linear range of 100 fg mL−1 to 50 ng mL−1. Significantly, it exhibited better specificity and accuracy with a sensitivity of 97.5% and a specificity of 96.7% to distinguish healthy donors, other lung diseases and TB patients compared to commercial ELISA assay, holding a promising prospect in clinical diagnosis.

Development and testing of nanoparticles delivery for P7C3 small molecule using injury models.

The use of nanoparticles (NPs) has emerged as a potential tool for safe and effective drug delivery. In the present study, we developed small molecule P7C3-based NPs and tested its efficacy and toxicity along with the tissue specific aptamer-modified P7C3 NPs. The P7C3 NPs were prepared using poly (D, L-lactic-co-glycolic acid) carboxylic acid (PLGA-COOH) polymer, were conjugated with skeletal muscle-specific RNA aptamer (A01B P7C3 NPs) and characterized for its cytotoxicity, cellular uptake, and wound healing in vitro. The A01B P7C3 NPs demonstrated an encapsulation efficiency of 30.2 ± 2.6%, with the particle size 255.9 ± 4.3nm, polydispersity index of 0.335 ± 0.05 and zeta potential of + 10.4 ± 1.8mV. The FTIR spectrum of P7C3 NPs displayed complete encapsulation of the drug in the NPs. The P7C3 NPs and A01B P7C3 NPs displayed sustained drug release in vitro for up to 6 days and qPCR analysis confirmed A01B aptamer binding to P7C3 NPs. The C2C12 cells viability assay displayed no cytotoxic effects of all 3 formulations at 48 and 72h. In addition, the cellular uptake of A01B P7C3 NPs in C2C12 myoblasts demonstrated higher uptake. In vitro assay mimicking wound healing showed improved wound closure with P7C3 NPs. In addition, P7C3 NPs significantly decreased TNF-α induced NF-κB activity in the C2C12/NF-κB reporter cells after 24-hour treatment. The P7C3 NPs showed 3-4-fold higher efficacy compared to P7C3 solutions in both wound-closure and inflammation assays in C2C12 cells. Furthermore, the P7C3 NPs showed 3-4-fold higher efficacy in reducing the infarct size and protected mouse hearts from ex vivo ischemia-reperfusion injury. Overall, this study demonstrates the safe and effective delivery of P7C3 NPs.

Electrostatic-Mediated Binding of DNA to Lysozymes: Evaluation of Aptamer-Based Assays for Highly Positively Charged Targets.

Lysozymes are a highly popular protein target for the development of aptamer-based biosensors. Because a lysozyme is a polycation and DNA is a polyanion, it is essential to separate the contribution of nonspecific electrostatic interactions from specific aptamer binding. In this study, various factors affecting the binding of DNA and lysozymes, including the DNA sequence, DNA length, pH, and salt concentration, were explored using fluorescence polarization. We concluded that direct fluorescence polarization and fluorescence intensity changes are unlikely to be directly applicable for aptamer-based biosensors to detect lysozymes because all of the tested DNA sequences showed binding. These fundamental studies confirm the dominant role of electrostatic binding. We further evaluated three other methods, including label-free fluorescent detection using a DNA staining dye, label-free colorimetric detection using gold nanoparticles, and a fluorescent sensor based on the strand displacement reaction. In each case, we focused on a random DNA sequence that is not expected to bind to the lysozyme as an aptamer. Of all the methods, only the strand displacement strategy can be potentially used to evaluate aptamer binding, as the other methods all responded to nonaptamer sequences. This study provides valuable insights for assaying aptamer binding to cationic proteins that can exhibit a nonspecific attraction to DNA.

A deep learning-enabled smartphone platform for rapid and sensitive colorimetric detection of dimethoate pesticide.

A novel deep learning-enabled smartphone platform is developed to assist a colorimetric aptamer biosensor for fast and highly sensitive detection of dimethoate. The colorimetric determination of dimethoate is based on the specific binding of dimethoate and aptamer, which leads to the aggregation of AuNPs in high-concentration NaCl solution, resulting in an obvious color change from red to blue. This color change provides sufficient data for self-learning enabled by a convolutional neural network (CNN) model, which is established to predict dimethoate concentration based on images acquired from a smartphone. To enhance user-friendliness for non-experts, the CNN model is then embedded into a smartphone app, enabling offline detection of dimethoate pesticide in real environments within just 15min using a pre-configured colorimetric probe. The developed platform exhibits superior performance, achieving a regression coefficient of 0.9992 in the concentration range of 0-10μM. Moreover, the app's performance is found to be consistent with the ELISA kit. These remarkable findings demonstrate the potential of combining colorimetric biosensors with smartphone-based deep learning methods for the development of portable and affordable tools for pesticide detection.

Surface Functionalisation of Self-Assembled Quantum Dot Microlasers with a DNA Aptamer.

The surface functionalisation of self-assembled colloidal quantum dot supraparticle lasers with a thrombin binding aptamer (TBA-15) has been demonstrated. The self-assembly of CdSSe/ZnS alloyed core/shell microsphere-shape CQD supraparticles emitting at 630 nm was carried out using an oil-in-water emulsion technique, yielding microspheres with an oleic acid surface and an average diameter of 7.3 ± 5.3 µm. Surface modification of the microspheres was achieved through a ligand exchange with mercaptopropionic acid and the subsequent attachment of TBA-15 using EDC/NHS coupling, confirmed by zeta potential and Fourier transform IR spectroscopy. Lasing functionality between 627 nm and 635 nm was retained post-functionalisation, with oleic acid- and TBA-coated microspheres exhibiting laser oscillation with thresholds as low as 4.10 ± 0.37 mJ·cm-2 and 7.23 ± 0.78 mJ·cm-2, respectively.

Aptamer-based technology for detecting Bacillus subtilis in soil.

The uncertainty associated with the impact of a bioinoculant on soil microbial community and, as a consequence, on soil quality, as well as the need to define its persistence, has prompted the demand for an accurate detection and tracking of the presence and the quantification of a target microbial inoculant in soil. Although DNA or RNA-based molecular detection are well established and commonly applied in this regard, alternative ligands such as DNA-aptamers have several advantages over them, such as low cost, ease of modification, ease of immobilisation on lab-on-chip or nanosensors, high stability and not thermolability. In this study, we used a toggle-cell SELEX method to isolate, select and characterise ssDNA (single-strand DNA) aptamers to detect a Bacillus subtilis strain which is being tested as a plant growth promoting rhizobacteria (PGPR) formulation. Two ssDNA aptamers (patenting application n.102022000022590) showed strong affinity and specificity for B. subtilis strains, with values of the kinetic parameters Kd (dissociation constant) in the nanomolar range and Bmax (maximum intensity of binding) around 1. Validation of the suitability of the aptamers was validated on three inoculated soils characterised by different chemical-physical features and in soil from a field trial with the formulated B. subtilis PCM/B 00105 strain. These are considered significant features to monitor B. subtilis strains in soil, practical to optimise bioinoculant application methods, support regulatory processes and foster the shift of agricultural production toward more sustainable cropping systems. KEY POINTS: • First DNA aptamers binding a B. subtilis strain included in a bioinoculum formulation. • First DNA aptamer binding B. subtilis in soil. • Aptamer may be a method for microbial inoculant detection in soil.

Computational generation and characterization of IsdA-binding aptamers with single-molecule FRET analysis.

Staphylococcus aureus is a major foodborne bacterial pathogen. Early detection of S. aureus is crucial to prevent infections and ensure food quality. The iron-regulated surface determinant protein A (IsdA) of S. aureus is a unique surface protein necessary for sourcing vital iron from host cells for the survival and colonization of the bacteria. The function, structure, and location of the IsdA protein make it an important protein for biosensing applications relating to the pathogen. Here, we report an in-silico approach to develop and validate high-affinity binding aptamers for the IsdA protein detection using custom-designed in-silico tools and small-molecule Fluorescence Resonance Energy Transfer (smFRET) measurements. We utilized in-silico oligonucleotide screening methods and metadynamics-based methods to generate 10 aptamer candidates and characterized them based on the Dissociation Free Energy (DFE) of the IsdA-aptamer complexes. Three of the aptamer candidates were shortlisted for smFRET experimental analysis of binding properties. Limits of detection in the low picomolar range were observed for the aptamers, and the results correlated well with the DFE calculations, indicating the potential of the in-silico approach to support aptamer discovery. This study showcasesa computational SELEXmethod in combination with single-molecule binding studies decipheringeffectiveaptamersagainst S. aureusIsdA, protein. Theestablished approach demonstrates the abilityto expediteaptamerdiscovery that has the potential to cutcosts and predict binding efficacy.The application can be extendedto designingaptamersfor various protein targets, enhancing molecular recognition, and facilitating the development of high-affinityaptamersfor multiple uses. This article is protected by copyright. All rights reserved.

SELEX based aptamers with diagnostic and entry inhibitor therapeutic potential for SARS-CoV-2

Frequent mutation and variable immunological protection against vaccination is a common feature for COVID-19 pandemic. Early detection and confinement remain key to controlling further spread of infection. In response, we have developed an aptamer-based system that possesses both diagnostic and therapeutic potential towards the virus. A random aptamer library (~ 1017 molecules) was screened using systematic evolution of ligands by exponential enrichment (SELEX) and aptamer R was identified as a potent binder for the SARS-CoV-2 spike receptor binding domain (RBD) using in vitro binding assay. Using a pseudotyped viral entry assay we have shown that aptamer R specifically inhibited the entry of a SARS-CoV-2 pseudotyped virus in HEK293T-ACE2 cells but did not inhibit the entry of a Vesicular Stomatitis Virus (VSV) glycoprotein (G) pseudotyped virus, hence establishing its specificity towards SARS-CoV-2 spike protein. The antiviral potential of aptamers R and J (same central sequence as R but lacking flanked primer regions) was tested and showed 95.4% and 82.5% inhibition, respectively, against the SARS-CoV-2 virus. Finally, intermolecular interactions between the aptamers and the RBD domain were analyzed using in silico docking and molecular dynamics simulations that provided additional insight into the binding and inhibitory action of aptamers R and J.

Magnesium Ion-Driven Folding and Conformational Switching Kinetics of Tetracycline Binding Aptamer: Implications for in vivo Riboswitch Engineering

Engineering in vitro selected RNA aptamers into in vivo functional riboswitches represents a long-standing challenge in molecular biology. The highly specific aptamer domain of the riboswitch undergoes a conformational adjustment in response to ligand sensing, which in turn exerts the regulatory function. Besides essential factors like structural complexity and ligand binding kinetics, the active role of magnesium ions in stabilizing RNA tertiary structures and assisting in ligand binding can be a vital criterion. We present spectroscopic studies on the magnesium ion-driven folding of the Tetracycline binding aptamer. Using fluorescent labels, the aptamer pre-folding and subsequent ligand binding is monitored by magnesium titration experiments and time-resolved stopped-flow measurements. A minimum concentration of 0.5 mM magnesium is required to fold into a magnesium ion-stabilized binding-competent state with a preformed binding pocket. Tetracycline binding causes a pronounced conformational change that results in the establishment of the triple helix core motif, and that further propagates towards the closing stem. By a dynamic acquisition of magnesium ions, a kink motif is formed at the intersection of the triple helix and closing stem regions. This ultimately entails a stabilization of the closing stem which is discussed as a key element in the regulatory function of the Tetracycline aptamer.

A smartphone-based inverse opal hydrogel film aptasensor for mycotoxin detection by the naked eye

Ochratoxin A (OTA), a carcinogenic mycotoxins, poses a significant threat to human health. The current detection methods exhibit advantages such as high sensitivity and good selectivity. However, The methods applicability for on-site detection remains hindered by complex operational procedures and the requirement for bulky testing instruments. Here, to address this limitation, we present a novel portable platform for naked-eye detection of OTA using photonic crystal (PC) inverse opal hydrogel films based on the smartphone. The inverse opal hydrogel film was fabricated by integrating hydrogel within PC templates, while a binding aptamer was employed to selectively recognize OTA. In the absence of OTA, the film displayed a vibrant red. With increasing OTA concentration, the diffraction peak progressively shifted toward the blue region of the spectrum. Leveraging a portable microscope, and captured the color change using a smartphone and developed a self-written algorithm to extract the H value of the image, enabling quantitative detection of OTA concentration. This portable platform has demonstrated exceptional reliability and convenience, boasting a remarkable detection limit of 1 μg/mL. Furthermore, its versatility extends to the detection of other mycotoxins, making it a promising tool for broader mycotoxin detection applications.

Mechanisms of ssDNA aptamer binding to Cd2+ in aqueous solution: A molecular dynamics study

ssDNA aptamers have been increasingly used to detect heavy metal ions as recognition elements due to their high affinity and specificity. However, the specific recognition and binding mechanisms between aptamers and most heavy metals were still unclear, which limits the development of aptamer-based detection methods. In this work, the interaction mechanisms of CD-2-1 aptamers with Cd2+ in aqueous solutions were investigated using molecular dynamic simulations. The most stable complex was found where Cd2+ binding at aptamer's stem-loop junction and preferred at the phosphate backbone or bases. Noteworthily, two binding modes of Cd2+ combining aptamer in aqueous solution were discovered: direct and indirect. In the former mode, Cd2+ directly coordinated O atoms of bases. For the latter, Cd2+ connected to bases with coordinated water molecules as bridges. Electrostatic interaction was found to be the main driving force, and differences of residues role between two binding modes were elucidated. Buffer molecules in aqueous solutions can stabilize aptamer-Cd2+ complex by hydrogen bonds. This study revealed the specific interaction mechanisms of aptamer with Cd2+ at an atomic level, which provided theoretical references for aptamer-based Cd2+ detection methods establishment as well as an efficient technical route of screening potential aptamers for heavy metal ions.

Targeting triple-negative breast cancer cells with a β1-integrin binding aptamer

Targeted therapies have increased the treatment options for triple-negative breast cancer patients. However, the paucity of targetable biomarkers and tumor heterogeneity have limited the ability of precision-guided interventions to live up to their full potential. As affinity-targeting ligands, aptamers show high selectivity toward target molecules. Compared with antibodies, aptamers have lower molecular weight, increased stability during transportation, reduced immunogenicity, and increased tissue uptake. Recently, we reported discovery of the GreenB1 aptamer, which is internalized in cultured triple-negative MDA-MB-231 human breast cancer cells. We show that the GreenB1 aptamer specifically targets β1-integrin, a protein linked previously to breast cancer cell invasiveness and migration. Aptamer binds to β1-integrin with low nanomolar affinity. Our findings suggest potential applications for GreenB1-guided precision agents for diagnosis and therapy of cancers overexpressing β1-integrin.

Anti HER-2 aptamer functionalized gold nanoparticles of dasatinib for targeted chemo-radiotherapy in breast cancer cells

In the present study, gold nanoparticles functionalized with anti HER-2 aptamer were designed for effective targeted delivery of dasatinib (DSB) to breast cancer cells. Anti HER-2 aptamer attached to porous or plain gold nanoparticles were compared for dasatinib delivery. Activated drug with succinic anhydride and L-cysteine linker was used for conjugation of DSB to gold nanoparticles. The loading efficiency of the activated drug on plain and porous gold nanoparticles was 52 and 68 %, respectively, which was significantly more than the loading of free DSB in gold nanoparticles (1–2.5 %). The anti HER-2 aptamer was conjugated to porous gold nanoparticles loaded with the activated drug. Various characterization techniques such as FESEM, TEM, AFM, zeta potential and ICP-MS were used to confirm the binding of the drug to gold nanoparticles. 1HNMR and FTIR spectroscopic analyses were employed to examine the structural characteristics of the conjugated drug. These analytical techniques confirmed the successful incorporation of succinyl and thiol groups onto the drug molecule. The amount of aptamer binding to different types of gold nanoparticles was obtained from the intensity of the light emitted from the bands observed in electrophoresis gel and due to the presence of porosity in porous gold nanoparticles, the amount of aptamer conjugation on porous gold nanoparticles increased compared to plain ones. Cell cytotoxicity and cellular uptake were evaluated by MTT assay and TEM in BT-474 and MCF-7 cells. Aptamer-functionalized porous gold nanoparticles containing activated dasatinib showed higher cytotoxicity and cellular uptake than modified DSB-loaded nanoparticles and un-activated DSB. The combination of radiation therapy with the modified dasatinib attached to porous gold nanoparticles and aptamer demonstrated a notable reduction in the IC50 values for both the BT-474 and MCF-7 cell lines. Specifically, the IC50 value for the BT-474 cells decreased from 6.95 μM (for unmodified dasatinib) to 2.57 μM, while for the MCF-7 cells, it decreased from 13.97 μM to 8.57 μM. These findings indicate a significant improvement in the efficacy of the modified dasatinib compared to its unmodified counterpart when used in conjunction with radiation therapy.

In silico molecular docking validation of procalcitonin-binding aptamer and sepsis diagnosis

BackgroundProcalcitonin (PCT) is a protein that can be used as a biomarker for sepsis detection. Sepsis is a disease where early diagnosis is crucial. Using PCT for sepsis diagnosis can be a new alternative that overcomes limitations of traditional sepsis diagnostic methods.ObjectiveTo develop a sepsis diagnostic platform for PCT detection using a novel material called aptamer.ResultsAptamers that could specifically bind to PCT were selected and various molecular biology analysis methods were utilized to confirm the binding affinity between selected aptamers and PCT. Additionally, in silico structural analysis was conducted to gain a more detailed understanding of the binding structure between the aptamer and PCT using the results of molecular experiments as supporting evidence. Ultimately, an aptamer-based PCT detection platform was developed and its ability to detect PCT in general and serum samples with high sensitivity and specificity was confirmed.ConclusionThrough this study, we were able to develop a technique for early disease diagnosis using aptamer-based protein detection. We also performed aptamer binding validation studies using a combination of molecular validation and in silico validation methods.

Terminal Alkyne-Modified DNA Aptamers with Enhanced Protein Binding Affinities.

Nucleic acid-based receptors, known as aptamers, are relatively fast to discover and manufacture but lack the diverse functional groups of protein receptors (e.g., antibodies). The binding properties of DNA aptamers can be enhanced by attaching abiotic functional groups; for example, aromatic groups such as naphthalene slow dissociation from proteins. Although the terminal alkyne is a π-electron-rich functional group that has been used in small molecule drugs to enhance binding to proteins through noncovalent interactions, it remains unexplored for enhancing DNA aptamer binding affinity. Here, we demonstrate the utility of the terminal alkyne for improving the binding of DNA to proteins. We prepared a library of 256 terminal-alkyne-bearing variants of HD22, a DNA aptamer that binds the protein thrombin with nanomolar affinity. After a one-step thrombin-binding selection, a high-affinity aptamer containing two alkynes was discovered, exhibiting 3.2-fold tighter thrombin binding than the corresponding unmodified sequence. The tighter binding was attributable to a slower rate of dissociation from thrombin (5.2-fold slower than HD22). Molecular dynamics simulations with enhanced sampling by Replica Exchange with Solute Tempering (REST2) suggest that the π-electron-rich alkyne interacts with an asparagine side chain N-H group on thrombin, forming a noncovalent interaction that stabilizes the aptamer-protein interface. Overall, this work represents the first case of terminal alkynes enhancing the binding properties of an aptamer and underscores the utility of the terminal alkyne as an atom economical π-electron-rich functional group to enhance binding affinity with minimal steric perturbation.