Capillary Electrophoresis-systematic Evolution Of Ligands Research Articles

Peptide-based CE-SELEX enables convenient isolation of aptamers specifically recognizing CD20-expressing cells

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with Kd estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds.

Highly-efficient selection of interferon gamma-specific aptamers and development of a sensitive fiber-optic evanescent wave aptasensor

Interferon-gamma (IFN-γ) is a critical indicator of the immune response to many infections and diseases. Identifying excellent IFN-γ recognition molecules is of great significance for the early diagnosis and treatment of diseases. Herein, anti-IFN-γ aptamers were selected based on capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) and their affinities were characterized by the biolayer interferometry (BLI) assay. A rational molecular docking strategy was utilized to predict precisely the binding behavior between aptamer and IFN-γ and remove redundant bases of original sequence. Therefore, aptamer IFNG-8 with the highest affinity can be efficiently identified within only three rounds and the truncated aptamer IFNG-8 T with comparable affinity is eventually obtained. With IFNG-8 T as the detection probe, a fiber-optic evanescent wave aptasensor is constructed for the detection of IFN-γ, which shows a wide linear range from 0.1 to 10000 pM and a low limit of detection (LOD) of 0.0718 pM. The developed aptasensor exhibits high selectivity and can be utilized for IFN-γ detection in human serum samples with good recoveries. In summary, the truncated aptamer can serve as a novel optional transducing element and the developed aptasensor provides an innovative methodology for detection of IFN-γ in trace amount.

Selection and Identification of an ssDNA Aptamer for Fibroblast Activation Protein.

As a type II transmembrane serine protease, fibroblast activation protein (FAP) is specifically expressed on the surface of fibroblasts associated with a variety of epithelial-derived malignancies such as pancreatic cancer, breast cancer, and colon cancer. It participates in the processes of tumorigenesis, progression, and immunosuppression. FAP constitutes an important target for tumor treatment; however, the current studies on FAP are mainly related to structural characteristics, enzymatic properties, and biological functions, and aptamers of FAP have not been investigated. In this work, by using recombinant human FAP as the target, five candidate aptamers, which are AptFAP-A1, AptFAP-A2, AptFAP-A3, AptFAP-A4, and AptFAP-A5, were selected by capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX), and their secondary structures were predicted to be mainly stem-loop. Moreover, the CE-laser-induced fluorescence (LIF) method was used to determine the equilibrium dissociation constant KD values between the FAP protein and candidate aptamers, and the KD value was in the low molar range. Finally, Cy5-labeled aptamers were co-incubated with human pancreatic cancer-associated fibroblasts highly expressing FAP protein, and confocal microscopy imaging showed that aptamer AptFAP-A4 had the highest affinities with the cells. The FAP aptamers screened in this study provide a promising direction for the development of rapid tumor diagnosis and targeted therapy.

Biosensors for the detection of organophosphate exposure by a new diethyl thiophosphate-specific aptamer.

An aptamer specifically binding to diethyl thiophosphate (DETP) was constructed and incorporated in an optical sensor and electrochemical techniques to enable the specific measurement of DETP as a metabolite and a biomarker of organophosphate exposure. A DETP-bound aptamer was selected from the library using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). A colorimetric method revealed that the aptamer had the highest affinity for DETP, with a mean Kd value (± SD) of 0.103 ± 0.014µM. The docking results and changes in resistance showed that the selectivity of the aptamer for DETP was higher than that for the similar structures of dithiophosphate (DEDTP) and diethyl phosphate (DEP). The altered amplitude of cyclic voltammetry showed a linear range of DETP detection covering 0.0001-10µg/ml with a limit of detection of 0.007µg/ml. The recovery value of a real sample of pH 7 was 97.2%. The current method showed great promise in using the DETP-specific aptamer to detect the exposure history to organophosphates by measuring their metabolites, although degradation of organophosphate parent compounds might occur.

Selection and characterization of an ssDNA aptamer against thyroglobulin

Thyroglobulin (Tg) is a significant biomarker for the diagnose and postoperative monitoring of differentiated thyroid cancer, and its recognition is urgent due to the rising prevalence. In this study, an ssDNA aptamer against Tg was obtained by capillary electrophoresis-systematic evolution of ligands via exponential enrichment (CE-SELEX). Under the optimized conditions, the sub-library was enriched well through two selection rounds. After high-throughput sequencing, eight candidate sequences were picked out and their affinities towards Tg were observed not in accordance with the order of their frequencies, whereas sequence homology played a significant role in binding affinity. The high-affinity sequence Seq.T-2 with a dissociation constant (Kd) of 3.18 μM was finally selected as the aptamer, and its affinity was confirmed qualitatively by gold nanoparticles colorimetric and quantitatively by thin film interferometry (Kd, 4.51 nM). Besides, molecular docking and dynamics simulation were performed for their binding sites prediction and affinity confirmation. Furthermore, the aptamer was applied for Tg detection, which delivered a detection limit of 5.0 nM as well as with good selectivity, and showed a good linear relationship within a wide range of 10 nM-6.4 μM of Tg spiked into the serum matrix. This study first reported Tg's aptamer which also exhibited the potential in real applications.

Investigation of Library Input on Aptamers Selection Efficiency Using Capillary Electrophoresis

Capillary electrophoresis (CE) is a high-efficiency method for aptamer selection, which exhibits many merits of high resolution, rapid separation, little sample consumption, and no immobilization. However, the influence of injection input on CE systematic evolution of ligands via exponential enrichment (SELEX) is controversial because the low injection volume may result in an insufficient capacity of the oligonucleotides library. In this research, neuron-specific enolase (NSE) was used as a model protein to investigate the effects of library input capacity on aptamer selection for clarifying the validity of CE-SELEX. Four different concentrations of initial libraries (0.1, 1, 10 and 100 μmol/L) were utilized into aptamer selection. The affinity of the initial library and sub-libraries after three rounds of selection were evaluated. The results showed that there was no significant difference in the affinity of four different sub-libraries, and aptamers with micromolar dissociation constant ( K D ) were all obtained after two rounds of selection. In addition, the sequences obtained via high concentration ssDNA library screening were more diverse, but the affinity of them was not enhanced. The NSE aptamer Seq qN-01 obtained by two rounds of selection had a K D of (4.72 ± 0.15) μmol/L, and also presented a good specificity. In conclusion, library input capacity could increase the sequence diversity but had no considerable effects on the efficiency of aptamer selection. CE-SELEX was employed for screening the aptamers of neuron-specific enolase (NSE). Four ssDNA libraries of different concentrations (0.1, 1, 10 and 100 μmol/L) were utilized to investigate the effects of input capacity of library on aptamer selection efficiency by comparing the affinity between rounds, sequence diversity and candidate sequences affinity.

Screening of Aptamer for Breast Cancer Biomarker Calreticulin and Its Application to Detection of Serum and Recognition of Breast Cancer Cell

Calreticulin (CRT) is closely related to cancer, and it is considered to be a novel biomarker for invasion and metastasis of breast cancer, as well as an indicator of staging and prognosis. Here, we screened and identified aptamers of CRT for the first time with capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) technology. The Apt 23 with high affinity and specificity was screened and verified by CE, isothermal titration calorimetry and gold nanoparticles-based colorimetric assay. By using FAM-labeled Apt 23 (FAM-Apt23) as an affinity probe, CRT was detected in the concentration range of 1–1000 nmol/L in serum by laser-induced fluorescence detector with a detection limit of 0.5 nmol/L. FAM-Apt 23 could also be used as an immunofluorescence imaging probe, which could be bound to the surface of breast cancer cells 4T1 with overexpressed CRT. Preliminary studies showed that Apt 23 could be used as aptamer probe for CRT recognition and detection.

Screening Aptamer of Apo-transferrin via Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Environmental Factors Analysis

Aptamer is oligonucleotide with high affinity and specificity toward the target which is obtained via systematic evolution of ligands by exponential enrichment (SELEX). However, due to the complexity of environmental factors and the unknown influence on the selection and application of aptamers, the optimization of multiple environmental factors through conventional experiments requires a huge amount of work and samples, so there are few systematic studies. Capillary electrophoresis (CE)-SELEX has become a highly efficient technique for aptamers screening because of the advantages of CE, e.g. microscale, high efficiency and low cost. In this work, human apo-transferrin (A-TF) was used as a model to study the effects of factors based on CE, including length of ssDNA library, incubation temperature, type and pH value of incubation buffer, metal ions etc. The results showed that shorter ssDNA library had higher affinity with the target protein. Incubation temperature, type and pH value of incubation buffer affected the formation of complex. Low concentrations of K+, Ca2+ and Mg2+ metal ions promoted the formation of complexes. The A-TF aptamer Seq A3 obtained by three rounds of selection had a dissociation constant (KD) of 0.476 μmol/L, and its affinity and specificity were verified via CE-laser induced fluorescence in human serum. The results indicated that Seq A3 could specifically recognize A-TF in serum.

Screening of Aptamer for Human IgG Fc Fragment by Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment

A DNA aptamer screening method for human immunoglobulin G (IgG) Fc fragments was established based on capillary electrophoresis- systematic evolution of ligands by exponential enrichment (CE-SELEX). The purity, charged properties and non-adsorptivity of the samples of IgG, Fc and Fab were analyzed by capillary zone electrophoresis (CZE), and their affinity with ssDNA libraries was compared. The results showed that IgG formed significant complex with the ssDNA libraries, while Fc and Fab fragments exhibited weak affinity with the ssDNA libraries. Therefore, the use of the Fab fragment as the reverse screening target had no significant effect on the sequence except for the specific binding of Fc. The aptamer screening strategy for Fc fragments was designed. Aptamers (Seq Fc1–3) were obtained under optimized incubation conditions (10 mmol/L Na2HPO4-KH2PO4 (pH 7.17), 0.05 mmol/L Mg2+, incubated at 37 °C for 25 min) by three rounds of CE-SELEX, with KD values of 0.071–0.321 μmol/L, and their affinity and specificity were verified by AuNPs colorimetry assay.

Online reaction based single-step capillary electrophoresis-systematic evolution of ligands by exponential enrichment for ssDNA aptamers selection

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has proven to be an effective technique for aptamers selection. In this study, we present an online reaction based convenient single-step CE-SELEX (ssCE-SELEX) mode with human thrombin (H-Thr) as a model target. The selection progress was monitored through bulk Kd analysis, which showed more than a 1000-fold improvement over the initial library after two rounds of selection. Three selected candidate sequences presented high binding affinities against H-Thr with nanomolar (nM) Kd determined by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM, 56.4–177.1 nM) and CE based non-linear fitting (CE-NLF, 98.2–199.7 nM). They also exhibited high specificities towards H-Thr compared with bovine thrombin, IgG, lysozyme, and lactoferrin. Meanwhile, the Kd results by isothermal titration calorimetry (ITC) confirmed the effective CE in measuring the aptamer affinity. In addition, three candidates were applied as aptasensors in the AuNPs based colorimetric assay, which showed visible color change and good linear relationships (R2 > 0.93) with H-Thr concentration. Furthermore, molecular dynamics (MD) simulation was performed to validate the binding of the three candidates with H-Thr by binding sites and binding free energy. The ssCE-SELEX method avoids off-line incubation, saves time and sample, and may provide a universal and convenient method for aptamers selection.

Low pH capillary electrophoresis application to improve capillary electrophoresis-systematic evolution of ligands by exponential enrichment

In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) as a CE-SELEX variant is proposed. Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein are incubated with a FAM labeled ssDNA library, respectively. Incubation mixture is separated in low pH CE (pH 2.6), where positively charged protein, protein–ssDNA complex and negatively charged ssDNA library migrate oppositely without EOF driven. Analysis of protein–ssDNA complex under positive voltage and unbound ssDNA library under negative voltage by CE–UV are applied for interactive evaluation. By increasing injection time, larger amount protein–ssDNA complex can be collected conveniently at the cathode end whereas ssDNA migrates to anode. Finally, stability of protein–ssDNA complex in low pH CE separation is discussed.

Tracking the emergence of high affinity aptamers for rhVEGF165 during capillary electrophoresis-systematic evolution of ligands by exponential enrichment using high throughput sequencing.

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) is a powerful technique for isolating aptamers for various targets, from large proteins to small peptides with molecular weights of several kilodaltons. One of the unique characteristics of CE-SELEX is the relatively high heterogeneity of the ssDNA pools that remains even after multiple rounds of selection. Enriched sequences or highly abundant oligonucleotide motifs are rarely reported in CE-SELEX studies. In this work, we employed 454 pyrosequencing to profile the evolution of an oligonucleotide pool through multiple rounds of CE-SELEX selection against the target recombinant human vascular endothelial growth factor 165 (rhVEGF165). High throughput sequencing allowed up to 3 × 10(4) sequences to be obtained from each selected pool and compared to the unselected library. Remarkably, the highest abundance contiguous sequence (contig) was only present in 0.8% of sequences even after four rounds of selection. Closer analyses of the most abundant contigs, the top 1000 oligonucleotide fragments, and even the eight original FASTA files showed no evidence of prevailing motifs in the selected pools. The sequencing results also provided insight into why many CE-SELEX selections obtain pools with reduced affinities after many rounds of selection (typically >4). Preferential amplification of a particular short polymerase chain reaction (PCR) product allowed this nonbinding sequence to overtake the pool in later rounds of selection suggesting that further refinement of primer design or amplification optimization is necessary. High affinity aptamers with 10(-8) M dissociation constants for rhVEGF165 were identified. The affinities of the higher abundance contigs were compared with aptamers randomly chosen from the final selection pool using affinity capillary electrophoresis (ACE) and fluorescence polarization (FP). No statistical difference in affinity between the higher abundance contigs and the randomly chosen aptamers was observed, supporting the premise that CE-SELEX selects a uniquely heterogeneous pool of high affinity aptamers.

Capillary Electrophoresis–Systematic Evolution of Ligands by Exponential Enrichment Selection of Base- and Sugar-Modified DNA Aptamers: Target Binding Dominated by 2′-O,4′-C-Methylene-Bridged/Locked Nucleic Acid Primer

Chemically modified DNA aptamers specific to human α-thrombin were obtained from oligodeoxyribonucleotide (ODN) libraries by using a capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) method. These libraries contained 2'-O,4'-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) in the primer region and/or C5-modified thymidine bearing N(6)-ethyladenine (t) in the nonprimer region. Modified DNA aptamers showed high binding affinities to the target, with dissociation constants (Kd) values in the range of subnanomolar to several ten nanomolar levels. The introduction of base modification significantly suppressed the frequency of G-quadruplex motifs, which are often seen in thrombin-binding DNA aptamers. The resulting alternatives contained the 10-mer consensus sequence t5Gt2G2, which is frequently found in modified DNA aptamers with subnanomolar protein binding affinities. Furthermore, some base- and sugar-modified DNA aptamers with the 12-mer consensus sequence t2G2tC(A/G)A2G2t displayed binding activities that were dependent on the presence of B/L nucleotides in the primer region. Such aptamers were interestingly not recovered from a natural DNA library or from DNA libraries modified with either B/L nucleotides or t's. This emerging characteristic binding property will enable the creation of a direct selection methodology for DNA-based molecular switches that are triggered by chemical conversion of B/L nucleotides introduced to constant sequence regions in ODN libraries.

Modified capillary electrophoresis based measurement of the binding between DNA aptamers and an unknown concentration target

The recognition of targets such as biomacromolecules, viruses and cells by their aptamers is crucial in aptamer-based biosensor platforms and research into protein function. However, it is difficult to evaluate the binding constant of aptamers and their targets that are hard to purify and quantify, especially when the targets are undefined. Therefore, we aimed to develop a modified capillary electrophoresis based method to determine the dissociation constant of aptamers whose targets are hard to quantify. A protein target, human thrombin, and one of its aptamers were used to validate our modified method. We demonstrated that the result calculated by our method, only depending on the aptamer's concentrations, was consistent with the classical method, which depended on the concentrations of both the aptamers and the targets. Furthermore, a series of DNA aptamers binding with avian influenza virus H9N2 were confirmed by a four-round selection of capillary electrophoresis-systematic evolution of ligands by exponential enrichment, and we identified the binding constant of these aptamers by directly using the whole virus as the target with the modified method. In conclusion, our modified method was validated to study the interaction between the aptamer and its target, and it may also advance the evaluation of other receptor-ligand interactions.

Capillary Electrophoresis–SELEX Selection of Catalytic DNA Aptamers for a Small-Molecule Porphyrin Target

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has previously been used to select aptamers for large-molecule targets such as proteins, lipopolysaccharides, and peptides. For the first time, we have performed CE-SELEX selection for a small-molecule target, N-methyl mesoporphyrin (NMM), with a molecular weight of only 580 g/mol. DNA aptamers with high-nanomolar to low-micromolar dissociation constants were achieved after only three rounds of selection. This corresponds to an >50-fold improvement in affinity over the random library. Two out of eight randomly chosen aptamers were found to catalyze the metal insertion reaction of mesoporphyrin with 1.7- and 2.0-fold rate enhancements, respectively.

Selection and Characterization of DNA Aptamers for Egg White Lysozyme

We have selected aptamers binding to lysozyme from a DNA library using capillary electrophoresis-systematic evolution of ligands by exponential enrichment. During the selection process the dissociation constant of the ssDNA pool decreased from the micromolar to the low nanomolar range within five rounds of selection. The final aptamer had a dissociation constant of 2.8 ± 0.3 nM, 6.1 ± 0.5 nM, and 52.9 ± 9.1 nM as determined by fluorescence anisotropy, surface plasmon resonance and affinity capillary electrophoresis respectively. The aptamers were successfully challenged for specificity against other egg white proteins. The high affinity aptamers open up possibilities for the development of aptamer based food and medical diagnostics.

In Vitro Selection of Aptamers with Affinity for Neuropeptide Y Using Capillary Electrophoresis

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) was used to select aptamers for neuropeptide Y (NPY). This is the first example of a CE-SELEX selection for aptamers that bind a target molecule smaller than itself. One of the limitations of CE-SELEX is that the aptamer must exhibit a significant mobility shift when it binds the target to facilitate fraction collection. Before this study, it was not clear if smaller targets would be capable of inducing a large enough shift in mobility for CE-SELEX to be successful. NPY is a 36-amino acid peptide (MW = 4272 g/mol), much smaller than the 80-base ssDNA used in the selection ( approximately 25 kDa). NPY binding aptamers with 300-1000 nM dissociation constants were obtained after only four rounds of selection. The specificity of the aptamers was tested using human pancreatic polypeptide (hPP). hPP is a 36-amino acid peptide with approximately 50% homology with NPY. Aptamers with up to 42-fold selectivity for NPY over hPP were observed.