Stranded DNA Aptamers Research Articles

334 Identification of novel plasma protein biomarkers for diagnosis and prediction of Alzheimer’s disease in African Americans

OBJECTIVES/GOALS: To identify novel panel of plasma protein biomarkers to improve prediction and diagnosis of Alzheimer’s disease (AD) for African Americans (AA), who are at greater risk of developing AD compared to non-Hispanic White individuals but are underrepresented in AD research. METHODS/STUDY POPULATION: Pre-existing plasma samples from 460 AA individuals with clinical diagnoses of AD, cognitively unimpaired (CU), mild cognitive impairment (MCI), or dementia with Lewy bodies (DLB) will undergo untargeted proteomics using the SomaScan assay, where modified single stranded DNA aptamers bind to protein targets which are quantified by DNA microarray. Protein expression levels will be compared between diagnostic groups to identify differentially expressed proteins. Additional clinical, genetic, and lifestyle factors will be compared with protein expression when available. Proteins of interest, identified by differential protein expression analysis results, will be included in receiver operating characteristic analyses to identify the optimal set of proteins for diagnostic classification. RESULTS/ANTICIPATED RESULTS: A pilot experiment utilizing plasma from 40 individuals identified multiple differentially expressed proteins (DEPs) between AD and non-AD groups. Eight proteins were nominated from the differential protein analysis into a receiver operating characteristic (ROC) analysis based on pvalue and previous implication in AD genome wide association studies. Proteins involved in microglial activation, neuronal adhesion, cell proliferation, and innate immunity were nominated. The ROC model achieved 100% classification accuracy of AD and CU groups using age, sex, and the eight nominated proteins. It is expected that there will be more significant associations when utilizing the full cohort of 460 AA and that DEPs between AD, CU, MCI, and DLB will be identified. DISCUSSION/SIGNIFICANCE: The nomination of a novel panel of plasma biomarkers developed from an AA cohort will directly serve the AA community by improving access to an early and accurate diagnosis of AD. Access to improved prediction and diagnosis will likely improve disease management, thus improving patient outcomes and decreasing burden on families and caregivers.

A novel aptamer-G-quadruplex/hemin self-assembling color system: rapid visual diagnosis of invasive fungal infections

BackgroundThe clinical symptoms of invasive fungal infections (IFI) are nonspecific, and early clinical diagnosis is challenging, resulting in high mortality rates. This study reports the development of a novel aptamer-G-quadruplex/hemin self-assembling color system (AGSCS) based on (1 → 3)-β-D-glucans’ detection for rapid, specific and visual diagnosis of IFI.MethodsWe screened high affinity and specificity ssDNA aptamers binding to (1 → 3)-β-D-glucans, the main components of cell wall from Candida albicans via Systematic Evolution of Ligands by EXponential enrichment. Next, a comparison of diagnostic efficiency of AGSCS and the (1 → 3)-β-D-glucans assay (“G test”) with regard to predicting IFI in 198 clinical serum samples was done.ResultsWater-soluble (1 → 3)-β-D-glucans were successfully isolated from C. albicans ATCC 10,231 strain, and these low degree of polymerization glucans (< 1.7 kD) were targeted for aptamer screening with the complementary sequences of G-quadruplex. Six high affinity single stranded DNA aptamers (A1, A2, A3, A4, A5 and A6) were found. The linear detection range for (1 → 3)-β-D-glucans stretched from 1.6 pg/mL to 400 pg/mL on a microplate reader, and the detection limit was 3.125 pg/mL using naked eye observation. Using a microplate reader, the sensitivity and specificity of AGSCS for the diagnosis of IFI were 92.68% and 89.65%, respectively, which was higher than that of the G test.ConclusionThis newly developed visual diagnostic method for detecting IFI showed promising results and is expected to be developed as a point-of-care testing kit to enable quick and cost effective diagnosis of IFI in the future.

Selection of G-rich ssDNA aptamers for the detection of enterotoxins of the cholera toxin family

Cholera and milder diarrheal disease are caused by Vibrio cholerae and enterotoxigenic Escherichia coli and are still a prominent public health concern. Evaluation of suspicious isolates is essential for the rapid containment of acute diarrhea outbreaks or prevention of epidemic cholera. Existing detection techniques require expensive equipment, trained personnel and are time-consuming. Antibody-based methods are also available, but cost and stability issues can limit their applications for point-of-care testing. This study focused on the selection of single stranded DNA aptamers as simpler, more stable and more cost-effective alternatives to antibodies for the co-detection of AB5 toxins secreted by enterobacteria causing acute diarrheal infections. Cholera toxin and Escherichia coli heat-labile enterotoxin, the key toxigenicity biomarkers of these bacteria, were immobilized on magnetic beads and were used in a SELEX-based selection strategy. This led to the enrichment of sequences with a high % GC content and a dominant G-rich motif as revealed by Next Generation Sequencing. Enriched sequences were confirmed to fold into G-quadruplex structures and the binding of one of the most abundant candidates to the two enterotoxins was confirmed. Ongoing work is focused on the development of monitoring tools for potential environmental surveillance of epidemic cholera and milder diarrheal disease.

Analysis of breast cancer biomarker HER2 based on single stranded DNA aptamer and enzyme signal amplification

In this study, we described a new electrochemical immunosensor for detecting human epidermal growth factor receptor 2 (HER2) extra-cellular domain, as a breast cancer biomarker, in human serum samples. The immunosensor uses anti-HER2 combined single walled carbon nanotubes (SWNT) to label the glassy carbon electrode (GCE) to enhance the electrochemical signal and present rich antibody domains. The biotin-labeled HER2-specific aptamers were used to recognize target HER2. Then, the streptavidin labeled alkaline phosphatase (AP) as detection probes combined the aptamers and generated electrochemical signals. AP has an excellent catalytic activity to substrate α-NP and significantly amplifies the detection signal for target HER2. Using the optimized experimental conditions, the immunosensor could response down to 0.23 pg mL−1 HER2 with a linear range from 1.0 pg mL−1 to 100 ng mL−1, and showed acceptable accuracy and reproducibility. The designed sensor was used to detect other biomolecules, and found no apparent electrochemical signal, indicating its high specificity. In addition, when the biosensor was used to analyze human serum samples, it also showed sufficient recovery rate and good precision. All the results above indicate that the developed biosensor can provide an excellent potential analysis platform in breast cancer diagnostics.

Virtual screening of truncated single stranded DNA aptamers for Staphylococcal enterotoxin type A

Single stranded DNA (ssDNA)/RNA aptamers, are screened through the labor intensive, iterative Systematic Evolution of Ligand by Exponential Enrichment process (SELEX) method. Complete sequence of screened aptamers never interacts with target or participates in final structure. Hence, in silico tools can be used to redesign a short length aptamer from previously reported aptamers which can have high affinity and specificity to the target. This approach is fast, cost effective, and less laborious than in vitro SELEX towards finding an aptamer sequence with better affinity with the target. Here, Staphylococcal enterotoxin type A (SEA) was used as target. A total of nine aptamers reported for different Staphylococcal food poisoning (SFP) enterotoxins were used as a starting pool. The aptamers were variously truncations and thoroughly analyzed through in silico methods. Three truncated aptamers namely AptSEA1.4, AptSEA2.4 and AptSEA8.4 were found to show higher affinity with target SEA. The computational data was also validated with DOT BLOT assay complemented with image analysis. These results also confirmed that the % specific binding and the dissociation constant (Kd) of truncated aptamers AptSEA1.4, AptSEA2.4 and AptSEA8.4 was better than their original counterparts. The truncated aptamers showed great promise to be used as a capture reagent in developing a sensitive assay for detection of SEA. Communicated by Ramaswamy H. Sarma

A CRISPR/Cas12a-assisted on-fibre immunosensor for ultrasensitive small protein detection in complex biological samples

Tracking trace amounts of analytes directly from low volumes of complex biological samples remains an ongoing challenge in precision diagnostics, as the commonly used immunosorbent assays have limited sensitivity. Herein, a CRISPR/Cas12a assisted on-fibre immunosensor (CAFI) was developed based on an antibody-analyte-aptamer sandwich structure, in which a single strand DNA aptamer was applied to detect the analyte while triggering the CRISPR/Cas12a fluorescent detection system to amplify the analyte signal. This novel CAFI biosensing system was fabricated on a glass fibre surface with an antifouling PEG polymer brush modified for the detection of a spectrum of small molecules from complex media. In comparison with a conventional ELISA system, CAFI has a 1,000-fold higher sensitivity with the limit of detection for IFN-γ down to 1 fg mL−1 (58.8 aM). It also has a tuneable linear detection range that can be easily adjusted within the range 1 fg mL−1 to 100 pg mL−1 (5 orders of magnitude), meeting the requirements of the demanding diagnostic scenarios. CAFI has successfully been demonstrated by detecting IFN-γ from a diverse complex biological sample type, including human serum, whole blood, perspiration, and saliva. Moreover, CAFI is applicable for the detection of other analytes by simply modifying the capture antibody and detection aptamer, demonstrated here with insulin. All these superior capabilities of CAFI make it a suitable technology to measure proteins in low (100 μL) volume complex biological samples.

A facile method for coal to graphene oxide and its application to a biosensor

The classic methods of synthesis of graphene oxide derived from graphite require harsh oxidation with excessive chemicals (H2SO4, H3PO4, KMnO4, etc.) and multiple processes. In this paper, we present a facile one-pot process using HNO3 to obtain graphene oxide from coal (Coal-GO). Coal has a unique molecular structure comprising of small regions or clusters of graphene-like and aliphatic side chains, for which our process is successfully able to exclude aliphatic compounds while preserving graphene domains during oxidization and subsequent exfoliation. The Coal-GO was converted to reduced graphene oxide (Coal-rGO) by conventional method to produce a few-layered graphene nanosheets in lateral size of 300–700 nm. The surface characteristics of the Coal-GO indicate the persistence of N from the raw coal as well as that introduced in the process. Notably, the Coal-GO shows a considerable amount of COOH compared to the other oxygen groups. Our data indicates that the process is effective in oxidative scissoring of the edges of coal, with minimal effect on swelling of coal towards the c-direction. Lastly, we demonstrate that compared to Graphite-GO, the Coal-GO has superior interaction with single stranded DNA aptamer, which could result in higher sensitivity chemiluminescence resonance energy transfer-based biosensors.

Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy.

Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.

An Aptameric Biolayer Interferometric Assay for Detection of Recombinant Human Erythropoietin-α

The biolayer interferometry (BLI), one promising biosensing technique for real-time, high-sensitive detection of analytes, is a new surface-sensitive spectroscopic technique in which the change of biolayer thickness is reflected by measuring the interferometric phase deflections. In our previous research, an ssDNA aptamer, 39nt, with definite interaction characteristics for recombinant human erythropoietin-α (EPO-α) was screened. In this work, a new aptameric BLI assay of EPO-α based on this aptamer 39nt was established. The biotinylated aptamer 39nt was firstly immobilized onto the optical fiber streptavidin biosensor chip, and the analytes in microliter volume were quickly diffused via high-speed vibration of 2200 r/min. The key parameters such as the orientation of aptamer sequence, the concentration for immobilization, and the overcome of nonspecific adsorption to exhibit the affinity recognition of aptamers were optimized. It was found that the 3'-end biotinylated aptamer 39nt with 50 nmol/L provided the best sensitivity, and the addition of Tween 20 and BSA could efficiently overcome nonspecific adsorption effect. Besides, towards the original designed disposable sensor chip, the affinity between protein EPO-α and ssDNA aptamer 39nt for ca. 10 times was still maintained under optimized regeneration condition, which reflected the operational flexibility of single-channel BLI system. Furthermore, a signal amplification approach of a sandwiched format via wheat germ agglutinin for detection of EPO-α was established, with the linear range of 10–200 nmol/L, and the LOD was 5 nmol/L. This assay was directly applied to the complex matrix such as 50% human plasma with recoveries in the range of 86.7%–104.2% and RSDs less than 15%. The BLI sensing system developed here provided a helpful way for practical applications in label-free protein interaction and detection. A new aptameric biolayer interferometry assay based on single stranded DNA aptamer 39nt was firstly established. Concomitantly with a signal amplification approach of a sandwiched format via wheat germ agglutinin, a sensitive measurement of recombinant human erythropoietin-α in complex matrix such as 50% human plasma were fulfilled.

A novel dual-mode and label-free aptasensor based methodology for breast cancer tissue marker targeting

Tumor biomarkers are elements that are created by the tumor itself or other host cells in response to cancerous conditions. The detection of HER2, as one of these biomarkers, is important in order to prescribe Herceptin (trastuzumab). So, precise measurement of the HER2 status is vital to ensure that all patients with breast cancer who can use Herceptin are correctly identified. In this method, we used the optical properties of gold nanoclusters (AuNCs) in colorimetric and fluorescence assays in HER2 positive tumor. For this purpose, our method uses a high affinity single strand DNA aptamer against surface exposed HER2 molecules on cells. Here, we have demonstrated that the aptamer with an inserted C12 loop can be used as a convenient scaffold for AuNCs synthesis. So, an aptamer@AuNCs modified cell/tumor-targeting nanostructure was engineered and demonstrated for an efficient HER2 positive cell/tumor imaging under UV illumination. In addition, on the basis of the catalytic activity of AuNCs, by immersing the HER2 positive tumor in a solution containing TMB, the solution color changes to green. Moreover, to compare other fluorescence probes we have developed a label-free aptamer@CdTe quantum dot system with high quantum yield in comparison with nanoclusters for the identification and imaging of HER2-positive cells/tumor. A good linear relationship for HER2 was obtained ranging from 15 to 1.5 × 106 cell/mL with R square value of 0.953 for aptamer@AuNCs and R square value of 0.977 for aptamer@CdTe QDs.

Assembly of Black Phosphorus Nanosheets and MOF to Form Functional Hybrid Thin-Film for Precise Protein Capture, Dual-Signal and Intrinsic Self-Calibration Sensing of Specific Cancer-Derived Exosomes.

As the emerging and noninvasive biomarkers, exosomes play an important role in cancer screening, cancer-related immune response, and the physiological process. The sensitive, specific, and efficient detection of cancer cell-derived exosomes is of significance for early cancer diagnosis of patients. This work developed a novel dual-signal and intrinsic self-calibration aptasensor of exosomes based on a functional hybrid thin-film platform. This platform was constructed via facile assembly of black phosphorus nanosheets (BPNSs) and ferrocene (Fc)-doped metal-organic frameworks (ZIF-67) on indium tin oxide (ITO) slice, followed by combining methylene blue (MB)-labeled single- strand DNA aptamer on ITO slice. The resultant aptamer-BPNSs/Fc/ZIF-67/ITO platform had dual redox-signal responses of MB (labeled on aptamer) and Fc (doped into ZIF-67). In the presence of specific cancer cell-derived exosomes, the redox current of MB regularly reduced and that of Fc (as reference) hardly changed. An intrinsic self-calibration aptasensor was achieved and enabled sensitive detection of exosomes, showing a limit of detection down to 100 particles mL-1. The aptasensor with a capability of precise protein capture can efficiently determine specific cancer cell-derived exosomes in practical human serum and plasma samples from healthy individuals and breast cancer patients. In light of excellent performances, this aptasensor can be expanded to multiple biomarkers from cell line exosomes and is beneficial for exploring advanced techniques for high-performance detection of exosomes derived from different types of cancer cells. This work promotes the development of current techniques for early cancer screening and clinical diagnosis.

In-silico study of single-strand DNA aptamers against cortisol as a potential biomarker receptor in therapeutics and diagnostics

Stress hormone is the number one adversary to the general health in human all over the world. In the presence of stress, cortisol, a glucocorticoid hormone synthesized by the stimulation of adrenal cortex will be secreted. Cortisol has immunosuppressive and anti-inflammatory effects, increases heart rate and blood pressure, and represses growth, and digestive and reproductive activities. Aptamers are synthetically derived nucleic acids has been long studied as a substitute of antibodies for therapeutics and diagnostics purposes. Current study focuses on in-silico approach of molecular docking and molecular dynamics simulation to study the interactions between single-strand DNA aptamers and cortisol in comparisons with the glucocorticoid receptor. In silico study were known to solve a plethora of biological related target study including the latest on COVID19 polymerase which is the inhibitors target protein. In current study, the tertiary conformational structures of ten single-strand DNA aptamers which experimentally derived were designed and docked against cortisol and compared with the binding of glucocorticoid receptor (PDB:4P6X). Binding affinity, number of hydrogen bonds and hydrophobic interactions were analyzed. The aptamer-cortisol complex with the lowest binding energy value was selected for 50 ns (ns) molecular dynamics simulation. Docking result shows that although number of intra-molecular hydrogen bond formed affecting the binding energy value of aptamer-cortisol complexes, hydrophobic interaction surrounding the cortisol is more important in determining the affinity of cortisol towards its receptor. Aptamer-1 shows the lowest binding energy value formed against cortisol (-10.1 kcal/mol) in comparison with other aptamers. Molecular dynamics simulation of Aptamer-1-cortisol complex shows that RMSD of glucocorticoid receptor-cortisol is lower in comparison with Aptamer 1-cortisol, with higher number of intra-molecular hydrogen bonds. Although the stability of receptor-cortisol is higher compared to Aptamer-1 complex, MMPBSA value of the last 10 ns of MD simulation prove that Aptamer-1-cortisol complex has the stability up to 50 ns of simulation. Further study to improve the single strand DNA aptamer binding against cortisol should be considered.

Aptamer-Based Fluorescent Biosensing of Adenosine Triphosphate and Cytochrome c via Aggregation-Induced Emission Enhancement on Novel Label-Free DNA-Capped Silver Nanoclusters/Graphene Oxide Nanohybrids.

Four fluorescent DNA-stabilized fluorescent silver nanoclusters (DNA-AgNCs) were designed and synthesized with differences in lengths of cytosine-rich DNA strand (as the stabilizing agent) and target-specific strand DNA aptamers for adenosine triphosphate (ATP) and cytochrome c (Cyt c). After their nanohybrid formation with graphene oxide (GO), it was unexpectedly found that, depending on the composition of the base and length of the strand DNA aptamer, the fluorescence intensity of three of the nanohybrids significantly enhanced. Our experimental observations and quantum mechanical calculations provided an insight into the mechanisms underlying the behavior of DNA-AgNCs/GO nanohybrids. The enhanced fluorescence was found to be attributed to the aggregation-induced emission enhancement (AIE) characteristic of the DNA-AgNCs adsorbed on the GO surface, as confirmed evidently by both fluorescence and transmission electron microscopies. The AIE is a result of hardness and oxidation properties of GO, which lead to enhanced argenophilic interaction and thus to increased Ag(I)-DNA complex shell aggregation. Consequently, two of the DNA-AgNCs/GO nanohybrids were successfully extended to construct highly selective, sensitive, label-free, and simple aptasensors for biosensing of ATP (LOD = 0.42 nM) and Cyt c (LOD = 2.3 nM) in lysed Escherichia coli DH5 α cells and mouse embryonic stem cells, respectively. These fundamental findings are expected to significantly influence the designing and engineering of new AgNCs/GO-based AIE biosensors.

A DNA aptamer reveals an allosteric site for inhibition in metallo-β-lactamases.

The hydrolysis of β-lactam antibiotics by β-lactamase enzymes is the most prominent antibiotic resistance mechanism for many pathogenic bacteria. Out of this broad class of enzymes, metallo-β-lactamases are of special clinical interest because of their broad substrate specificities. Several in vitro inhibitors for various metallo-β-lactamases have been reported with no clinical efficacy. Previously, we described a 10-nucleotide single stranded DNA aptamer (10-mer) that inhibits Bacillus cereus 5/B/6 metallo-β-lactamase very effectively. Here, we find that the aptamer shows uncompetitive inhibition of Bacillus cereus 5/B/6 metallo-β-lactamase during cefuroxime hydrolysis. To understand the mechanism of inhibition, we report a 2.5 Å resolution X-ray crystal structure and solution-state NMR analysis of the free enzyme. Chemical shift perturbations were observed in the HSQC spectra for several residues upon titrating with increasing concentrations of the 10-mer. In the X-ray crystal structure, these residues are distal to the active site, suggesting an allosteric mechanism for the aptamer inhibition of the enzyme. HADDOCK molecular docking simulations suggest that the 10-mer docks 26 Å from the active site. We then mutated the three lysine residues in the basic binding patch to glutamine and measured the catalytic activity and inhibition by the 10-mer. No significant inhibition of these mutants was observed by the 10-mer as compared to wild type. Interestingly, mutation of Lys50 (Lys78; according to standard MBL numbering system) resulted in reduced enzymatic activity relative to wild type in the absence of inhibitor, further highlighting an allosteric mechanism for inhibition.

Selection and preliminary application of a single stranded DNA aptamer targeting colorectal cancer serum.

Colorectal cancer is one of the common causes of malignant tumors in recent years, thus the discovery of potential compounds that detect the occurrence of colorectal cancer by efficient approaches is necessary. In this study, the method of systematic evolution of ligands by exponential enrichment (SELEX) was used for recognizing serum from colorectal cancer patients by a single-stranded DNA library of aptamers assisted by single-walled carbon nanotubes (SWCNTs) to remove single-stranded DNA with low affinity. Ten rounds of selection were applied using colorectal cancer serum as a target with the serum of healthy individuals as a control. As the result, we have successfully identified four candidate aptamers after high-throughput genome sequencing analysis, comparison analysis and secondary structure prediction. Among them, aptamer Seq-2 exhibited the highest affinity and the strongest selectivity with an equilibrium dissociation constant (Kd) of 11.31 ± 3.25 nM and a Ct difference value of 4.25 ± 0.38 between the colorectal cancer group and the healthy group. Moreover, with fifty negative control serum samples, the positive detection rate of fifty positive serum samples tested by aptamer Seq-2 was over 90%. In particular, aptamer Seq-2 can strongly bind the colorectal cancer serum, less strongly bind the non-colon cancer serum and hardly bind the healthy serum. Therefore, aptamer Seq-2 presents enormous potential for exploring as a tumor diagnostic kit and detecting unknown tumor markers in serum to reflect colorectal cancer.

Development of novel aptamers for low-density lipoprotein particle quantification.

Cardiovascular disease (CVD) remains the leading cause of death worldwide. Low-density lipoprotein cholesterol (LDL-C) is commonly used for CVD risk assessment; however, recent research has shown LDL particle (LDL-P) number to be a more sensitive indicator of CVD risk than both LDL-C and non-high-density lipoprotein cholesterol (HDL-C). Described herein are five single stranded DNA aptamers with dissociation constants in the low picomolar range specific to LDL-P and its subfractions. Furthermore, a set of antisense sequences have been developed and characterized that are capable of binding to the best aptamers and undergoing displacement by LDL-P for use in a simple, affordable diagnostic assay.

In vitro selection and characterization of single stranded DNA aptamers for luteolin: A possible recognition tool

Distinctive bioactivities possessed by luteolin (3′, 4′, 5, 7-tetrahydroxy-flavone) are advantageous for sundry practical applications. This paper reports the in vitro selection and characterization of single stranded-DNA (ssDNA) aptamers, specific for luteolin (LUT). 76-mer library containing 1015 randomized ssDNA were screened via systematic evolution of ligands by exponential enrichment (SELEX). The recovered ssDNA pool from the 8th round was amplified with unlabeled primers and cloned into PSTBlue-1 vector prior to sequencing. 22 of LUT-binding aptamer variants were further classified into one of the seven groups based on their N40 random sequence regions, wherein one representative from each group was characterized. The dissociation constant of aptamers designated as LUT#28, LUT#20 and LUT#3 was discerned to be 107, 214 and 109 nM, respectively with high binding affinity towards LUT. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Furthermore, LUT#3 displayed higher specificity with insignificant binding toward kaempferol and quercetin despite its structural and functional similarity compared to LUT#28 and LUT#20. Further LUT#3 can detect free luteolin within 0.2–1 mM in solution. It was suggested that LUT#3 aptamer were the most suitable for LUT recognition tool at laboratory scale based on the condition tested.

Suppression of FOXM1 Transcriptional Activities via a Single-Stranded DNA Aptamer Generated by SELEX

The transcription factor FOXM1 binds to its consensus sequence at promoters through its DNA binding domain (DBD) and activates proliferation-associated genes. The aberrant overexpression of FOXM1 correlates with tumorigenesis and progression of many cancers. Inhibiting FOXM1 transcriptional activities is proposed as a potential therapeutic strategy for cancer treatment. In this study, we obtained a FOXM1-specific single stranded DNA aptamer (FOXM1 Apt) by SELEX with a recombinant FOXM1 DBD protein as the target of selection. The binding of FOXM1 Apt to FOXM1 proteins were confirmed with electrophoretic mobility shift assays (EMSAs) and fluorescence polarization (FP) assays. Phosphorthioate-modified FOXM1 Apt (M-FOXM1 Apt) bound to FOXM1 as wild type FOXM1 Apt, and co-localized with FOXM1 in nucleus. M-FOXM1-Apt abolished the binding of FOXM1 on its consensus binding sites and suppressed FOXM1 transcriptional activities. Compared with the RNA interference of FOXM1 in cancer cells, M-FOXM1 Apt repressed cell proliferation and the expression of FOXM1 target genes without changing FOXM1 levels. Our results suggest that the obtained FOXM1 Apt could be used as a probe for FOXM1 detection and an inhibitor of FOXM1 transcriptional functions in cancer cells at the same time, providing a potential reagent for cancer diagnosis and treatment in the future.

Chronopotentiometric sensing of specific interactions between lysozyme and the DNA aptamer

Specific DNA-protein interactions are vital for cellular life maintenance processes, such as transcriptional regulation, chromosome maintenance, replication and DNA repair, and their monitoring gives valuable information on molecular-level organization of those processes. Here, we propose a new method of label-free electrochemical sensing of sequence specific binding between the lysozyme protein and a single stranded DNA aptamer specific for lysozyme (DNAapta) that exploits the constant current chronopotentiometric stripping (CPS) analysis at modified mercury electrodes. Specific lysozyme-DNAapta binding was distinguished from nonspecific lysozyme-DNA interactions at thioglycolic acid-modified mercury electrodes, but not at the dithiothreitol-modified or bare mercury electrodes. Stability of the surface-attached lysozyme-DNAapta layer depended on the stripping current (Istr) intensity, suggesting that the integrity of the layer critically depends on the time of its exposure to negative potentials. Stabilities of different lysozyme-DNA complexes at the negatively polarized electrode surface were tested, and it was shown that structural transitions of the specific lysozyme-DNAapta complexes occur in the Istr ranges different from those observed for assemblies of lysozyme with DNA sequences capable of only nonspecific lysozyme-DNA interactions. Thus, the CPS allows distinct discrimination between specific and non-specific protein-DNA binding and provides valuable information on stability of the nucleic acid-protein interactions at the polarized interfaces.

Immuno Affinity SELEX for Simple, Rapid, and Cost-Effective Aptamer Enrichment and Identification against Aflatoxin B1.

Aflatoxins are naturally occurring mycotoxins that contaminate food and agro commodities, leading to acute and chronic health conditions in human and animals. In the present work, an attempt was made to generate high-affinity single stranded DNA aptamers that specifically bind to Aflatoxin B1 (AFB1) by a modified Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure with the aid of Immunoaffinity columns. Ten rounds of SELEX and alternating three counter SELEX rounds with a cocktail of related and other mycotoxins were performed to enhance the specificity. Resultant 105 aptamers were clustered into 12 groups according to their primary sequence homology. Candidates with lowest Gibbs free energy (dG value) and unique stem loop structures were selected for further characterization. Aptamers, AFLA5, AFLA53, and AFLA71 exhibiting lower Kd values (50.45 ± 11.06, 48.29 ± 9.45, and 85.02 ± 25.74 nM) were chosen for development of ELONA and determination of purification ability of toxin. The detection limit (LOD) of AFLA5 and AFLA71 was 20 and 40 ng/ml, respectively. HPLC analysis implied that selected aptamers were able to recover and quantify 82.2 to 96.21% (LOQ – 53.74 ng) and 78.3 to 94.22% (LOQ – 66.75 ng) of AFB1 from spiked corn samples, respectively. These findings indicate, immunoaffinity based SELEX can pave an alternative approach to screen aptamers against mycotoxin detection and purification.