Aptamer Length Research Articles

Effects of Label-free Fluorescence Aptasensors with Different Aptamer Length on Quenching of Carbon Dots

Label-free fluorescence aptasensors have the advantages of better detection sensitivity, simple manufacturing process and no pollution. In this experiment, the effect of aptamer base number on the performance fluorescence aptasensors has been studied. Thrombin of 15 base number, ATP of 27 base number and DA of 58 base number were selected, and then constructed three kinds of fluorescent probes about Au/Aptamer/CQDs nanostructures with gold nanoparticles and carbon dots. By studying the quenching degree of carbon dots with each probe, the quenching degree of carbon dots fluorescence by ATP probe was the highest, thrombin probe was the worst, and the DA probe was between ATP probe and DA probe. This indicates that with the increase of the aptamer base number in the probe, the more carbon dots are adsorbed on the aptamer and the more fluorescence quenching occurs, but the aptamer will be winding when it has too many bases, leading to the quenching unstable of carbon dots fluorescence. This study provides a useful reference for the construction and detection of fluorescent aptasensors, and has a great potential in the field of small molecule detection.

Fluorescence Polarization-Based Rapid Detection System for Salivary Biomarkers Using Modified DNA Aptamers Containing Base-Appended Bases.

The field of care testing toward the analysis of blood and saliva lacks nowadays simple test techniques for biomarkers. In this study, we have developed a novel nucleobase analog, Ugu, which is a uracil derivative bearing a guanine base at the 5-position. Moreover, we attempted the development of aptamers that can bind to secretory immunoglobulin A (SIgA), which has been examined as a stress marker in human saliva. It was observed that the acquired aptamer binds strongly and selectively to the SIgA dimer (Kd = 13.6 nM) without binding to the IgG and IgA monomers of human serum. Reduction of the aptamer length (41 mer) successfully improved 4-fold the binding affinity (Kd = 3.7 nM), compared to the original, longer aptamer (78 mer). Furthermore, the development of a simple detection system for human saliva samples by fluorescence polarization was investigated, using the reported human salivary α-amylase (sAA) and the SIgA-binding aptamer. Comparison of the present method with conventional enzyme-linked immunosorbent assay techniques highlighted a significant Pearson's correlation of 0.94 and 0.83 when targeting sAA and SIgA, respectively. It is thus strongly suggested that a new simple test of stress markers in human saliva can be quantified quickly without bound/free (B/F) separation.

Universal Design of Structure-Switching Aptamers with Signal Reporting Functionality.

Structure-switching aptamers (SSAs) offer a promising recognition element for sensor development. However, the generation of SSAs via in vitro aptamer selection technologies or postselection engineering is challenging. Inspired by the two-domain structure of antibodies, we have devised a simple, universal strategy for engineering aptamers into SSAs with signal reporting functionality. These constructs consist of a "constant" domain, comprising a split DNAzyme G-quadruplex (G4) region for signal transduction, and a "variable" domain, comprising an aptamer sequence capable of specific target binding. In the absence of target, the G4-SSA construct folds into a parallel G4 structure with high peroxidase catalytic activity. Target binding disrupts the G4 structure, resulting in low enzymatic activity. We demonstrate that this change in DNAzyme activity enables sensitive and specific colorimetric detection of diverse targets including Hg2+, thrombin, sulfadimethoxine, cocaine, and 17β-estradiol. G4-SSAs can also achieve label-free fluorescence detection of various targets using a specific G4-binding dye. We demonstrate that diverse aptamers can be readily engineered into G4-SSA constructs independent of target class, binding affinity, aptamer length, or structure. This design strategy could broadly extend the power, accessibility, and utility of numerous SSA-based biosensors.

In Silico Screening of Aptamers Configuration against Hepatitis B Surface Antigen.

Aptamer has been long studied as a substitute of antibodies for many purposes. However, due to the exceeded length of the aptamers obtained in vitro, difficulties arise in its manipulation during its molecular conjugation on the matrix surfaces. Current study focuses on computational improvement for aptamers screening of hepatitis B surface antigen (HBsAg) through optimization of the length sequences obtained from SELEX. Three original aptamers with affinity against HBsAg were truncated into five short hairpin structured aptamers and their affinity against HBsAg was thoroughly studied by molecular docking, molecular dynamics (MD) simulation, and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method. The result shows that truncated aptamers binding on HBsAg “a” determinant region are stabilized by the dynamic H-bond formation between the active binding residues and nucleotides. Amino acids residues with the highest hydrogen bonds hydrogen bond interactions with all five aptamers were determined as the active binding residues and further characterized. The computational prediction of complexes binding will include validations through experimental assays in future studies. Current study will improve the current in vitro aptamers by minimizing the aptamer length for its easy manipulation.

A novel electrochemical sensor for bisphenol A detection based on nontarget-induced extension of aptamer length and formation of a physical barrier

In this study, a novel electrochemical sensing strategy was developed based on nontarget-induced bridge assembly and aptamer (Apt) extension reaction triggered by terminal deoxynucleotidyl transferase (TdT). Bisphenol A (BPA) was chosen as the target analyte to study the analytical performance of the designed aptasensor. This sensing system takes advantages of electrochemical sensing platforms and detection of an ultra-low level of BPA, due to the formation of bridge on the surface of electrode in the absence of BPA. In the presence of BPA, Apt/BPA complex is formed and inhibits the access of TdT to the 3′-end of Apt, resulting in the lack of bridge assembly and high access of [Fe(CN)6]3-/4- as redox agent to the electrode surface. Thus, a strong redox peak is observed. This aptasensor enabled the selective detection of BPA with the wide linear range of 0.08–15 nM and detection limit as low as 15 pM. Furthermore, the presented sensing platform was successfully applied for the detection of BPA in tap water and grape juice samples.

A colorimetric detection method of pesticide acetamiprid by fine-tuning aptamer length

This work investigates the effect of shortening aptamer sequences on the colorimetric detection of acetamiprid using aptamer-wrapped gold nanoparticles (AuNPs). Truncated 37-mer and 25-mer aptamers were generated by deleting excess flanking nucleotides from parental 49-mer acetamiprid-target aptamer. In comparing the responses of the three sequences, truncated aptamers did not improve the ability to discriminate against other tested pesticides. However, comparison between 49-mer and other shorter aptamers showed that shortening aptamer sequences through removing excess flanking nucleotides outsides of binding region improved colorimetric sensitivity for acetamiprid by 3.3 fold. Due to excess bases, the target-bound aptamer might still adhere to AuNPs, resulting in incomplete dissociation of aptamer from AuNPs and therefore the suppression of aggregation responses. This work provides further insight to the effects of aptamer structure on detection of the target, as well as a method by fine-tuning aptamer length for rapid detection of pesticide residues in environments or food.

Shortening full-length aptamer by crawling base deletion – Assisted by Mfold web server application

Systematic Evolution of Ligands by EXponential enrichment (SELEX) is the method to select the specific aptamer against a wide range of targets. For this process, the initial library usually has a length of random sequences from ∼25 and it reaches over 100 bases. The lengthy sequences have disadvantages such as difficult to prepare, less stable and expensive. It is wise to prefer shorter version of aptamer for a wide range of applications including drug delivery process. It is a common practice to shorten the full-length aptamer by mapping analyses and it is tedious. Here, we used a crawling method to shorten the aptamer by different sequential deletion of bases from both 5′ and 3′ ends, assisted by Mfold web server application. Two different kinds of aptamer with varied lengths (randomized region of 30 and 74 bases) were desired for this study, generated against Influenza A/Panama/2007/1999 (H3N2) and gD protein of Herpes Simplex Virus-1. It was found that shortening the aptamer length by crawling pattern is possible with the assistance of Mfold web server application. The obtained results resemble the shortened aptamer derived by mapping analyses. The proposed strategy is recommended to predict the shorter aptamer without involving any wet experimental section.

Multifaceted analyses of the interactions between human heart type fatty acid binding protein and its specific aptamers

BackgroundAptamer-protein interaction studies have been mainly confined to dissociation constant (Kd) determination. A combinatorial approach involving limited proteolysis mass spectroscopy, molecular docking and CD studies is reported here to elucidate the specific interactions involved. MethodsTo generate aptamers specific for human FABP3, SELEX was performed incorporating counter SELEX cycles against control FABPs and GST tag, followed by their characterization by EMSA, CD and SVD analysis. Based on computationally obtained aptamer-protein complex models, the interacting aptamer, and protein residues were predicted and supported by limited proteolysis experiments. ResultsTwo aptamers N13 and N53 specific for human fatty acid binding protein (FABP3) were isolated with corresponding Kd of 0.0743±0.0142μM and 0.3337±0.1485μM for FABP3 interactions. Both aptamers possess stable B-DNA structures at salt concentration of 100mM and pH range (6–9). The N13 aptamer led interaction involved 3 salt bridges and 2 hydrogen bonds, whereas N53 had 2 salt bridges with 8 hydrogen and 7 hydrophobic interactions. ConclusionsThe aptamers generated are the first to be reported against human FABP3. The higher interaction footprint of N53 incited synergistic conformational changes in both N53 and FABP3 during interaction, leading to a decline in binding affinity in comparison to N13 which corroborated to the calculated Kd values. General significanceThis combinatorial method may be used to retrieve the possible specific binding modes and interaction patterns involved in large aptamer-protein complexes. Thus the method can be exploited to identify the optimum aptamer length for in-depth structure-function studies and its tailored applications.

Polydiacetylene-coated polyvinylidene fluoride strip aptasensor for colorimetric detection of zinc(II)

We report a new polydiacetylene (PDA) sensor strip for simple visual detection of zinc ions in aqueous solution. The specificity of this sensor comes from Zn2+ DNA aptamer probes conjugated onto PDA. Effects of aptamer length and structure on the sensitivity of PDA’s color transition were first investigated. PDA conjugated with the optimal aptamer sequence was then coated onto a strip of polyvinylidene fluoride membrane and photopolymerized by UV exposure. The newly developed sensor successfully exhibited a blue-to-red chromatic change in a semi-quantitative manner in response to zinc ions. No discernable change was observed in solutions containing other common ions. Advantages of this sensor include its ease of fabrication, high specificity, and equipment-free detection, all of which are desirable for in-field applications and use in resource-limited settings.

Designing Anti-Influenza Aptamers: Novel Quantitative Structure Activity Relationship Approach Gives Insights into Aptamer – Virus Interaction

This study describes the development of aptamers as a therapy against influenza virus infection. Aptamers are oligonucleotides (like ssDNA or RNA) that are capable of binding to a variety of molecular targets with high affinity and specificity. We have studied the ssDNA aptamer BV02, which was designed to inhibit influenza infection by targeting the hemagglutinin viral protein, a protein that facilitates the first stage of the virus’ infection. While testing other aptamers and during lead optimization, we realized that the dominant characteristics that determine the aptamer’s binding to the influenza virus may not necessarily be sequence-specific, as with other known aptamers, but rather depend on general 2D structural motifs. We adopted QSAR (quantitative structure activity relationship) tool and developed computational algorithm that correlate six calculated structural and physicochemical properties to the aptamers’ binding affinity to the virus. The QSAR study provided us with a predictive tool of the binding potential of an aptamer to the influenza virus. The correlation between the calculated and actual binding was R2 = 0.702 for the training set, and R2 = 0.66 for the independent test set. Moreover, in the test set the model’s sensitivity was 89%, and the specificity was 87%, in selecting aptamers with enhanced viral binding. The most important properties that positively correlated with the aptamer’s binding were the aptamer length, 2D-loops and repeating sequences of C nucleotides. Based on the structure-activity study, we have managed to produce aptamers having viral affinity that was more than 20 times higher than that of the original BV02 aptamer. Further testing of influenza infection in cell culture and animal models yielded aptamers with 10 to 15 times greater anti-viral activity than the BV02 aptamer. Our insights concerning the mechanism of action and the structural and physicochemical properties that govern the interaction with the influenza virus are discussed.

Colorimetric detection with aptamer–gold nanoparticle conjugates: effect of aptamer length on response

A riboflavin binding aptamer (RBA) was used in combination with gold nanoparticles (AuNPs) to detect riboflavin in vitro. The RBA–AuNP conjugates (RBA–AuNPs) responded colorimetrically to the presence of riboflavin and this response could be followed by the naked eye. This system was used as a model to study how modifications on the aptamer sequence affect the RBA–AuNPs’ stability and their response to their target. To mimic primers and other sequence modifications typically used in aptamer work, the RBA was extended by adding extra bases to its 5′ end. These extra bases were designed to avoid interactions with the RBA binding site. The response of these RBA–AuNPs was evaluated and compared. Dynamic light scattering and UV-aggregation kinetics studies showed that the length of the aptamer significantly affected the RBA–AuNPs’ stability and, as a consequence, the magnitude of the detection response to riboflavin. The addition of thymine nucleotides instead of random tails to the RBA showed that the effects observed were not specific to the sequence used. This study shows that modifications of the aptamer sequence provide a means to improve the stability of aptamer–AuNPs conjugates and their sensing response.