Selection of DNA ligands for protein kinase C-delta.
Mallikaratchy Prabodhika,Stahelin Robert V,Cao Zehui,Cho Wonhwa,Tan Weihong
Chemical communications (Cambridge, England)
Protein kinase Cs are a family of serine and threonine kinases that mediate a wide variety of cellular signalling processes such as cell growth, differentiation, apoptosis and tumor development. We have selected high-affinity DNA aptamers for PKCdelta by capillary electrophoresis based SELEX (systematic evolution of ligands by exponential enrichment, CE-SELEX). We have demonstrated that fluorescently tagged PB9 aptamer can specifically recognize PKCdelta under in vitro conditions. The Kd of the aptamer-protein binding is 122 nM. These aptamers will enable us to apply fluorescently labelled probes to study the spatiotemporal dynamics and activation of individual endogenous PKC isoforms during various cell signalling processes.
Nano aptasensor for protective antigen toxin of anthrax.
Cella Lakshmi N,Sanchez Pablo,Zhong Wenwan,Myung Nosang V,Chen Wilfred,Mulchandani Ashok
We demonstrate a highly sensitive nano aptasensor for anthrax toxin through the detection of its polypeptide entity, protective antigen (PA toxin) using a PA toxin ssDNA aptamer functionalized single-walled carbon nanotubes (SWNTs) device. The aptamer was developed in-house by capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) and had a dissociation constant (K(d)) of 112 nM. The aptasensor displayed a wide dynamic range spanning up to 800 nM with a detection limit of 1 nM. The sensitivity was 0.11 per nM, and it was reusable six times. The aptasensor was also highly selective for PA toxin with no interference from human and bovine serum albumin, demonstrating it as a potential tool for rapid and point-of-care diagnosis for anthrax.
Isolation of DNA aptamers using micro free flow electrophoresis.
Jing Meng,Bowser Michael T
Lab on a chip
A micro free flow electrophoresis (μFFE) device was used to select DNA aptamers for human immunoglobulin E (IgE). The continuous nature of μFFE allowed 1.8 × 10(14) sequences to be introduced over a period of 30 min, a 300-fold improvement in library size over capillary electrophoresis based selections (CE-SELEX). Four rounds of selection were performed within four days. Aptamers with low nM dissociation constants for IgE were identified after a single round of μFFE selection.
Selection of aptamers against Ara h 1 protein for FO-SPR biosensing of peanut allergens in food matrices.
Tran Dinh T,Knez Karel,Janssen Kris P,Pollet Jeroen,Spasic Dragana,Lammertyn Jeroen
Biosensors & bioelectronics
The rising prevalence to food allergies in the past two decades, together with the fact that the only existing therapy is avoidance of allergen-containing food next to the implementation of anti-allergic drugs, urges the need for improved performance of current assays to detect potential allergens in food products. Therein, the focus has been on aptamer-based biosensors in recent years. In this paper we report for the first time the selection of aptamers against one of the most important peanut allergens, Ara h 1. Several Ara h1 DNA aptamers were selected after eight selection rounds using capillary electrophoresis (CE)-SELEX. The selected aptamers specifically recognized Ara h 1 and did not significantly bind with other proteins, including another peanut allergen Ara h 2. The dissociation constant of a best performing aptamer was in the nanomolar range as determined independently by three different approaches, which are surface plasmon resonance, fluorescence anisotropy, and capillary electrophoresis (353 ± 82 nM, 419 ± 63 nM, and 450 ± 60 nM, respectively). Furthermore, the selected aptamer was used for bioassay development on a home-built fiber optic surface plasmon resonance (FO-SPR) biosensor platform for detecting Ara h 1 protein in both buffer and food matrix samples demonstrating its real potential for the development of novel, more accurate aptamer-based biosensors. In conclusion, the reported aptamer holds a great potential for the detection of Ara h 1 in both the medical field and the food sector due to its high affinity and specificity for the target protein.
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.
Kasahara Yuuya,Irisawa Yuuta,Fujita Hiroto,Yahara Aiko,Ozaki Hiroaki,Obika Satoshi,Kuwahara Masayasu
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.
In vitro evolution of functional DNA using capillary electrophoresis.
Mendonsa Shaun D,Bowser Michael T
Journal of the American Chemical Society
Electrophoretic selection with capillary electrophoresis (CE) is used, for the first time, to isolate functional nucleic acid sequences using SELEX (systematic evolution of ligands by exponential enrichment). SELEX uses molecular evolution to select functional sequences (aptamers) from random RNA or DNA libraries. Conventional SELEX is usually performed with affinity chromatography, which may introduce significant bias into the selection step. Important biases include the slow kinetics involved in the elution of strongly bound sequences and performing the selection with the target molecule tethered to the stationary support, not in free solution. In this novel CE-SELEX approach, selection occurs in free solution. The nucleic acid sequences that bind the target undergo a mobility shift, migrating at a different rate, allowing them to be separated from the inactive sequences. Thus, there is no need to wash the active sequences off a column as in conventional SELEX, eliminating any kinetic bias. In this work, the viability of CE-SELEX was demonstrated by performing selections against immunoglobulin E (IgE). Anti-IgE aptamers with dissociation constants as low as 40 nM were obtained in only two rounds of selection.
In vitro selection of DNA-based aptamers that exhibit RNA-like conformations using a chimeric oligonucleotide library that contains two different xeno-nucleic acids.
Hagiwara Kenta,Fujita Hiroto,Kasahara Yuuya,Irisawa Yuuta,Obika Satoshi,Kuwahara Masayasu
We successfully generated chimeric DNA aptamers that contained six nucleoside analogs of 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA) in the primer region and multiple guanosine analogs of 2'-deoxy-2'-fluoro-ribonucleic acid (FNA) in the non-primer region using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). Active species enrichment became saturated only after five selection rounds, and we obtained DNA-based xeno-nucleic acid (XNA) aptamers that had high binding affinities for the target human thrombin, with dissociation constant (Kd) values of ≥10 nanomolar. Based on sequence and circular dichroism (CD) analyses, these XNA aptamers exhibited RNA-like conformations, which could cause DNA-based strands to adopt structurally diverse conformations.
Two DNA aptamers against avian influenza H9N2 virus prevent viral infection in cells.
Zhang Yuewei,Yu Ziqiang,Jiang Fei,Fu Ping,Shen Junjun,Wu Wenxue,Li Jinxiang
New antiviral therapy for pandemic influenza mediated by the H9N2 avian influenza virus (AIV) is increasingly in demand not only for the poultry industry but also for public health. Aptamers are confirmed to be promising candidates for treatment and prevention of influenza viral infections. Thus, we studied two DNA aptamers, A9 and B4, selected by capillary electrophoresis-based systemic evolution of ligands by exponential enrichment (CE-SELEX) procedure using H9N2 AIV purified haemagglutinin (HA) as target. Both aptamers had whole-virus binding affinity. Also, an enzyme-linked aptamer assay (ELAA) confirmed binding affinity and specificity against other AIV subtypes. Finally, we studied aptamer-inhibitory effects on H9N2 AIV infection in Madin-Darby canine kidney (MDCK) cells and quantified viral load in supernatant and in cell with quantitative PCR (qPCR). Our data provide a foundation for future development of innovative anti-influenza drugs.
Capillary electrophoresis-SELEX selection of aptamers with affinity for HIV-1 reverse transcriptase.
Mosing Renee K,Mendonsa Shaun D,Bowser Michael T
Capillary electrophoresis-SELEX (CE-SELEX) was used to select ssDNA aptamers with affinity for HIV reverse transcriptase (HIVRT). A library of ssDNA was incubated with HIVRT. Sequences bound to HIVRT were isolated using CE, PCR amplified, and purified, yielding an enriched ssDNA pool suitable for further rounds of selection. Aptamers with dissociation constants as low as 180 pM were isolated after four rounds of selection. This is the first report of aptamers isolated by CE-SELEX with higher affinity than those obtained for the same target using conventional selection techniques. No sequence motifs were identified in the 27 clones sequenced, suggesting that there are many sequences that can bind HIVRT with low picomolar dissociation constants.
The DNA aptamers that specifically recognize ricin toxin are selected by two in vitro selection methods.
Tang Jijun,Xie Jianwei,Shao Ningsheng,Yan Yan
Aptamers which specifically recognize cytotoxin ricin were successfully selected using the two different in vitro selection methods. One selection method was used to isolate aptamers by affinity chromatography. Another selection method, named CE-SELEX, was carried out using CE as a separation approach. The high separation efficiency of CE evidently improved the rate of enrichment and obviously shortened the selection rounds, with near 87.2% binding just after the fourth round of selection. The aptamers A3, C1, and C5, derived from the two selection methods, were found to possess high affinity and specificity for ricin with the Kd values in the low nanomolar range, and did not recognize abrin toxin similar to ricin in the structures and properties, or BSA. Among the aptamers selected, A3 isolated by affinity chromatography shared extensive sequence similarity with C1 and C5 derived from CE-SELEX. They differed by only one base from each other. Their stable secondary structures predicted also had very similar structure motifs, and all folded a long and internal loop-embedded loop stem structure by base pairing. The ELISA and dot-blot analysis also proved that the selected DNA aptamers had the high specificity to ricin toxin.
Online reaction based single-step CE for Protein-ssDNA complex obtainment to assist aptamer selection.
Zhu Chao,Wang Xiaoqian,Li Linsen,Hao Chenxu,Hu Youhao,Rizvi Aysha Sarfraz,Qu Feng
Biochemical and biophysical research communications
CE application in aptamer selection (CE-SELEX) shows more advantages than other selection methods. In this study, an online reaction based single-step CE (ssCE) mode was employed for fast obtaining protein-ssDNA complex. Using human thrombin (H-Thr) and its aptamer Apt29 as models, we accomplished the procedures of mixing, reaction, separation, detection and complex collection in single step online process, which took about 10 min to obtain the H-Thr/Apt29 complex. Important factors, affecting the aptamer and H-Thr interaction (buffer, ratio of aptamer and H-Thr amount), and complex separation and collection (voltage and temperature) were discussed. Later, the online reaction of H-Thr with an 80 nt ssDNA library was realized under optimized conditions, and the H-Thr/ssDNA complex was collected and subjected to PCR. By analyzing the PCR product through capillary gel electrophoresis, the resulting approximative 80 nt DNA length validated the ssDNA sequence in complex. To confirm the availability of ssCE mode, two ssDNA libraries with different lengths (56 nt and 82 nt ssDNA) and three proteins (platelet derived growth factor, PDGF-BB; lactoferrin protein, LF; and single-strand DNA binding protein, SSB) were utilized. Their complex peaks were also observed in electropherograms as expected. Additionally, the online incubation of ssDNA and H-Thr was achieved by stopping the separation voltage for some time when ssDNA passed the H-Thr zone. Our results show the ssCE mode has apparent merits of saving time and sample cost for aptamer selection against protein targets.
Rapidly Neutralizable and Highly Anticoagulant Thrombin-Binding DNA Aptamer Discovered by MACE SELEX.
Wakui Koji,Yoshitomi Toru,Yamaguchi Akane,Tsuchida Maho,Saito Shingo,Shibukawa Masami,Furusho Hitoshi,Yoshimoto Keitaro
Molecular therapy. Nucleic acids
We present a rapidly neutralizable and highly anticoagulant thrombin-binding aptamer with a short toehold sequence, originally discovered by systematic evolution of ligands by exponential enrichment (SELEX) with microbead-assisted capillary electrophoresis (MACE). MACE is a novel CE-partitioning method for SELEX and able to separate aptamers from a library of unbound nucleic acids, where the aptamer and target complexes can be detected reliably and partitioned with high purity even in the first selection cycle. Three selection rounds of MACE-SELEX discovered several TBAs with a nanomolar affinity (K = 4.5-8.2 nM) that surpasses previously reported TBAs such as HD1, HD22, and NU172 (K = 118, 13, and 12 nM, respectively). One of the obtained aptamers, M08, showed a 10- to 20-fold longer prolonged clotting time than other anticoagulant TBAs, such as HD1, NU172, RE31, and RA36. Analyses of the aptamer and thrombin complexes using both bare and coated capillaries suggested that a large number of efficient aptamers are missed in conventional CE-SELEX because of increased interaction between the complex and the capillary. In addition, the toehold-mediated rapid antidote was designed for safe administration. The efficient aptamer and antidote system developed in the present study could serve as a new candidate for anticoagulant therapy.
Selection and characterization of an ssDNA aptamer against thyroglobulin.
Zhu Chao,Li Linsen,Fang Senbiao,Zhao Yi,Zhao Liping,Yang Ge,Qu Feng
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 (K) 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 (K, 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.
[Efficient screening for 8-oxoguanine DNA glycosylase binding aptamers via capillary electrophoresis].
Han Shimiao,Zhao Liping,Yang Ge,Qu Feng
Se pu = Chinese journal of chromatography
8-Oxoguanine DNA glycosylase (OGG1) is an important enzyme that plays a key role in oxidative DNA damage repair. OGG1 can specifically recognize and excise 8-oxoG (a product of oxidative damage found in double-stranded DNA) through base excision repair (BER). OGG1 is expressed in normal tissues, and in most tumor tissues. Oxidative cellular damage can produce an inflammatory reaction, alleviating some measure of constitutive OGG1 inhibition. OGG1 inhibition in cancer cells shows some promise as a new method of cancer treatment. Most current OGG1 research focuses on regulating OGG1 with targeted small molecules. To date, no aptamer screen for OGG1 has been reported. Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides that can bind to a target with high affinity and specificity , that can be identified by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers can be used as chemical ligands to regulate intermolecular interactions. In this study, a screen for aptamers with OGG1 affinity was performed for the first time. Capillary electrophoresis (CE) is a microanalytical technique that offers speed and high separation efficiency. In this work, two screening methods based on CE-SELEX technology were established: a one-round pressure controllable selection, and a multi-round selection. The most important criterion for successful one-round pressure controllable selection is to select a competitive target with a different CE migration time than that of the target of interest. We mixed OGG1 with a competitive target and a nucleic acid library for CE analysis. Two proteins competitively bind sequences in the library, forming independent complexes. The concentration of the competitive target is continuously increased until complexes with the target stop decreasing, indicating that the target and the ssDNA library have formed a stable complex. Complexes were collected for PCR amplification, purification, and high-throughput sequencing to obtain high affinity aptamers. This method greatly improves screening efficiency, and reduces non-specific binding to the target, which is helpful for obtaining aptamers with high affinity and specificity. One-round pressure controllable selection for high affinity OGG1 selective aptamers was performed using single strand binding protein (SSB) to competitively and tightly bind nucleic acids in the library. The competitive screening pressure was increased by increasing the SSB concentration to eliminate sequences with low affinity for OGG1 from the random oligonucleotide library. Nucleic acid sequences with high OGG1 affinity were obtainable in one step, and OGG1-ssDNA complexes were collected by creating a timed program on Beckman P/ACE MDQ capillary electrophoresis. Collection occurred from 2.2 to 2.8 min. Under identical incubation and electrophoresis conditions, multiple round selections were conducted by injecting samples of co-incubated nucleic acid library and target into the capillary. After separation under a high-voltage electric field, nucleic acid target complexes were collected, amplified by PCR, purified, and used as an enriched secondary library in the next round of screening. High affinity aptamers were generally obtained within three rounds. Comparing results of the two screening methods, the three candidate aptamer sequences found with the highest frequency were consistent, and displayed values ranging from 1.71 to 2.64 μmol/L. Molecular docking analysis suggests that Apt 1 may bind to the OGG1 active pocket, which functions to repair oxidative damage. Comparison of the two screening methods indicates that one-round pressure controllable selection is more rapid and efficient, providing guidance for the design of other protein aptamer screening methods. The obtained aptamer is expected to be function effectively as an OGG1-mediated DNA repair inhibitor.
Biosensors for the detection of organophosphate exposure by a new diethyl thiophosphate-specific aptamer.
Swainson Napachanok Mongkoldhumrongkul,Aiemderm Pongsakorn,Saikaew Chonnikarn,Theeraraksakul Kanyanat,Rimdusit Pakjira,Kraiya Charoenkwan,Unajak Sasimanas,Choowongkomon Kiattawee
OBJECTIVE: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. RESULTS: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 K value (± SD) of 0.103 ± 0.014 µM. 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 0.0001-10 µg/ml with a limit of detection of 0.007 µg/ml. The recovery value of a real sample of pH 7 was 97.2%. CONCLUSIONS: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.
Investigating the Influences of Random-Region Length on Aptamer Selection Efficiency Based on Capillary Electrophoresis-SELEX and High-Throughput Sequencing.
Zhu Chao,Li Linsen,Yang Ge,Qu Feng
For aptamer selection, the random-region length of an ssDNA library was generally taken in a relatively arbitrary fashion, which may lead to failure for unsuitable target binding. Herein, we coupled high-efficiency capillary electrophoresis (CE)-SELEX and high-throughput sequencing (HTS) to investigate the influences of random-region length. First, one round of selection against programmed cell death-ligand 1 (PD-L1) was performed using ssDNA libraries with random-region lengths of 15, 30, 40, and 60 nt, respectively. A good correlation was observed between candidates' random-region lengths and dissociation constant (), in which the longer sequences presented higher affinity, and the picked Seq 60-1 after one round notably presented a similar affinity toward a reported aptamer through eight rounds. Molecular dynamics (MD) simulation suggested, for PD-L1, the long sequence could supply more noncovalent bonds including hydrogen bonds, electrostatic interactions, and hydrophobic interactions to form a stable protein/aptamer complex. Besides, four other proteins with selective binding performances validated the importance of random-region length. To further investigate how random-region length affects the selection efficiency, a mixed library with random-region lengths ranging from 10 to 50 nt was employed for six rounds of selection against Piezo2. Sequence variations were tracked by HTS, showing the preferential evolution and PCR uncertainty with even higher impact were the main causes. This study suggested random-region length plays a crucial factor, and a mixed library with different random-region sequences can be a worthy choice for increasing the speed of high-affinity aptamer selection. Moreover, the PCR process should be given particular attention in aptamer selection.
Advanced screening and tailoring strategies of pesticide aptamer for constructing biosensor.
Critical reviews in food science and nutrition
The rapid development of aptamers has helped address the challenges presented by the wide existed pesticides contaminations. Screening of aptamers with excellent performance is a prerequisite for successfully constructing biosensors, while further tailoring of aptamers with enhanced activity greatly improved the assay performance. Firstly, this paper reviewed the advanced screening strategies for pesticides aptamers, including immobilization screening that preserves the native structures of targets, non-immobilized screening based on nanomaterials, capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX), virtual screening , high-throughput selection, and rational secondary library generation methods, which contributed significantly to improve the success rate of screening, reduce the screening time, and ensure aptamer binding affinity. Secondly, the precise tailoring strategies for pesticides aptamers were modularly elaborated, containing deletion, splitting, elongation, and fusion, which provided various advantages like cost-efficiency, enhanced binding affinity, and new derived functional motifs. Thirdly, the developed aptamer-based biosensors (aptasensors) for pesticide detection were systematically reviewed according to the different signal output modes. Finally, the challenges and future perspectives of pesticide detection are discussed comprehensively.
In vitro selection of aptamers with affinity for neuropeptide Y using capillary electrophoresis.
Mendonsa Shaun D,Bowser Michael T
Journal of the American Chemical Society
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.
Capillary electrophoresis-SELEX selection of catalytic DNA aptamers for a small-molecule porphyrin target.
Yang Jing,Bowser Michael T
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.
Three-dimensional selection of leptin aptamers using capillary electrophoresis and implications for clone validation.
Ashley Jon,Li Sam F Y
Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has been used as a fast and efficient way to select aptamers against protein targets and offers the advantage of separating bound DNA from unbound DNA in a free solution three-dimensional environment. CE-SELEX was used to select aptamers against human leptin protein. Two methods used to validate the aptamers' binding affinity against the target were performed and gave differing results. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) yielded K(D) values in the high nanomolar range, whereas the fluorescence intensity method gave K(D) values in the low micromolar range. These results may suggest that aptamer validation must be carried out in a similar environment to that of the selection partitioning step and the environment in which the aptamer is intended to be used. We also note that affinity binding by fluorescence intensity using microplate readers may be limited to targets that have relatively low k(off) rates, systematic errors may occur when aptamers are validated using two different techniques, and the immobilization of smaller targets onto plate wells can affect the binding of the DNA, giving rise to lower binding affinities.
In vitro selection of BNA (LNA) aptamers.
Kuwahara Masayasu,Obika Satoshi
Artificial DNA, PNA & XNA
Recently, we achieved the first in vitro selection of 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA) aptamers. High-affinity thrombin-binding aptamers (TBAs) were obtained from DNA-based libraries containing 2'-O,4'-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) in the 5'-primer region, using the method of capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX). Furthermore, a similar selection protocol could provide TBAs that contain B/L nucleotides in both primer and random regions. We review technical challenges involved in the generation of various BNA libraries using analogs of B/L nucleoside-5'-triphosphate and polymerase variants and also discuss applications of these libraries to the selection of BNA (LNA) aptamers, as well as future prospects for their therapeutic and diagnostic uses.
Online reaction based single-step capillary electrophoresis-systematic evolution of ligands by exponential enrichment for ssDNA aptamers selection.
Zhu Chao,Li Linsen,Yang Ge,Fang Senbiao,Liu Manjiao,Ghulam Murtaza,Hao Chenxu,Chen Yubao,Qu Feng
Analytica chimica acta
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 K 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) K 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 K 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 (R > 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.
High-efficiency selection of aptamers for bovine lactoferrin by capillary electrophoresis and its aptasensor application in milk powder.
Zhu Chao,Li Linsen,Yang Ge,Irfan Muhammad,Wang Zijian,Fang Senbiao,Qu Feng
Capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX) is a high-efficient technique for aptamers selection, and has been evolved into many modes. In this study, we obtained the aptamer against bovine lactoferrin (BLF) with high affinity (dissociation constant, K = 20.74 ± 6.89 nM) and good specificity (>1000 folds) using single step CE-SELEX (ssCE-SELEX) mode. In the selection process, ssCE demonstrated high-efficiency selection with bulk K reaching at 0.19 ± 0.04 μM by only two rounds, as compared to capillary zone electrophoresis (CZE) mode with K of 0.39 ± 0.03 μM. Next-generation sequencing (NGS) was performed by two methods of high output (Hiseq) and medium output (Miseq) with different sequencing depths, and their same results of high-frequency sequences confirmed the reliability of the obtained sequences. Through affinity analysis, the primer region and single base mutation (SBM) were observed to affect the sequence structure and to result in affinity change. Besides, molecular dynamics (MD) simulation was performed to validate the binding affinity of the candidates with BLF by analyzing binding sites, interaction forces, and binding free energy. Moreover, BLF detection in milk powder matrices was completed successfully with the optimized CE-aptasensor. The signal response was in a good linear relationship (R = 0.9930) with 4-128 nM of BLF and the detection limit was 1 nM. The obtained results of BLF in four milk powder samples were in an acceptable agreement with the labeled concentrations. This study presented a completed CE based process including aptamers selection, affinity characterization, and detection application, which also validated the high-efficiency selection of ssCE-SELEX mode.
Tracking the emergence of high affinity aptamers for rhVEGF165 during capillary electrophoresis-systematic evolution of ligands by exponential enrichment using high throughput sequencing.
Jing Meng,Bowser Michael T
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.
Phosphorothioate-Modified AP613-1 Specifically Targets GPC3 when Used for Hepatocellular Carcinoma Cell Imaging.
Dong Lili,Zhou Hongxin,Zhao Menglong,Gao Xinghui,Liu Yang,Liu Dongli,Guo Wei,Hu Hongwei,Xie Qian,Fan Jia,Lin Jiang,Wu Weizhong
Molecular therapy. Nucleic acids
Glypican-3 (GPC3), the cellular membrane proteoglycan, has been established as a tumor biomarker for early diagnosis of hepatocellular carcinoma (HCC). GPC3 is highly expressed in more than 70% HCC tissues detected by antibody-based histopathological systems. Recently, aptamers, a short single-strand DNA or RNA generated from systematic evolution of ligands by exponential enrichment (SELEX), were reported as potential alternatives in tumor-targeted imaging and diagnosis. In this study, a total of 19 GPC3-bound aptamers were successfully screened by capillary electrophoresis (CE)-SELEX technology. After truncated, AP613-1 was confirmed to specifically target GPC3 with a dissociation constant (K) of 59.85 nM. When modified with a phosphorothioate linkage, APS613-1 targeted GPC3 with a K of 15.48 nM and could be used as a specific probe in living Huh7 and PLC/PRF/5 imaging, GPC3-positive cell lines, but not in L02 or A549, two GPC3-negative cell lines. More importantly, Alexa Fluor 750-conjugated APS613-1 could be used as a fluorescent probe to subcutaneous HCC imaging in xenograft nude mice. Our results indicated that modified AP613-1, especially APS613-1, was a potential agent in GPC3-positive tumor imaging for HCC early diagnosis.
Low pH capillary electrophoresis application to improve capillary electrophoresis-systematic evolution of ligands by exponential enrichment.
Li Qian,Zhao Xinying,Liu Hongyang,Qu Feng
Journal of chromatography. A
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.
Capillary electrophoresis coupled with automated fraction collection.
Huge Bonnie Jaskowski,Flaherty Ryan J,Dada Oluwatosin O,Dovichi Norman J
A fraction collector based on a drop-on-demand ink-jet printer was developed to interface capillary zone electrophoresis with a 96 well microtiter plate. We first evaluated the performance of the collector by using capillary zone electrophoresis to analyze a 1mM solution of tetramethylrhodamine; a fluorescent microtiter plate reader was then used to detect the analyte and characterize fraction carryover between wells. Relative standard deviation in peak height was 20% and the relative standard deviation in migration time was 1%. The mean and standard deviation of the tetramethylrhodamine peak width was 5 ± 1 s and likely limited by the 4-s period between droplet deposition. We next injected a complex mixture of DNA fragments and used real-time PCR to quantify the product in a CE-SELEX experiment. The reconstructed electrophoretic peak was 27 s in duration. Finally, we repeated the experiment in the presence of a 30-µM thrombin solution under CE-SELEX conditions; fractions were collected and next-generation sequencing was used to characterize the DNA binders. Over 25,000 sequences were identified with close matches to known thrombin binding aptamers.
Capillary Electrophoresis for the Selection of DNA Aptamers Recognizing Activated Protein C.
Hamedani Nasim Shahidi,Müller Jens
Methods in molecular biology (Clifton, N.J.)
Capillary electrophoresis-based SELEX (CE-SELEX) is an efficient technique for the isolation of aptamers binding to a wide range of target molecules. CE-SELEX has a number of advantages over conventional SELEX procedures such as the selection of aptamers can be performed on non-immobilized targets, usually within a fewer number of selection cycles. Here we describe a complete procedure of CE-SELEX using activated protein C (APC) as the target protein.
Evolution of multi-functional capillary electrophoresis for high-efficiency selection of aptamers.
Zhu Chao,Yang Ge,Ghulam Murtaza,Li Linsen,Qu Feng
Aptamers have drawn considerable attention as newly emerging molecular recognition elements in clinical diagnostics, drug delivery, therapeutics, environmental monitoring, and food safety analyses. As the in vitro screening antibody analogs, aptamers are enabled to recognize various types of targets with high affinity and specificity like or even superior to antibodies. However, the restrictions and inefficiency of selection have been hampering their wider application. Among various modified systematic evolution of ligands by exponential enrichment (SELEX) methods, capillary electrophoresis (CE)-SELEX holds multiple functions and advantages with the powerful qualitative and quantitative analysis capabilities, less consumption of sample and analytical reagent, natural binding environment, higher screening efficiency, and availability in multiple modes. This review summarizes the key developments in the area of CE-SELEX by leading research groups, including our teams' ten years of research and experience to help researchers fully understand and utilize CE-SELEX. Aptamers' history, applications, as well as the SELEX developments, have been briefly described; the advantages of CE-SELEX are highlighted compared with the conventional SELEX methods. Further, we describe some essential CE-SELEX models and provide an overview of the CE-SELEX, including the targets and ssDNA library, every technical point in the selection process, and post-SELEX protocol. We expect this review will inspire more researchers to have insight into the screening problems from CE-SELEX viewpoint and will help to improve the selection efficiency and probability of success to meet the growing needs of aptamers' discovery in bioanalytical and medical fields.
Isolating aptamers using capillary electrophoresis-SELEX (CE-SELEX).
Mosing Renee K,Bowser Michael T
Methods in molecular biology (Clifton, N.J.)
SELEX (systematic evolution of ligands by exponential enrichment) is a process for isolating DNA or RNA sequences with high affinity and selectivity for molecular targets from random sequence libraries. These sequences are commonly referred to as aptamers. The process typically requires 10-15 cycles of enrichment, PCR amplification and nucleic acid purification to obtain high-affinity aptamers. We have demonstrated that using capillary electrophoresis (CE) as an enrichment step greatly improves the efficiency of the process. CE-SELEX is capable of isolating high-affinity aptamers in as little as 2-4 rounds of selection, shortening the process time from several weeks to as little as a few days.
Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing.
Eaton Rachel M,Shallcross Jamie A,Mael Liora E,Mears Kepler S,Minkoff Lisa,Scoville Delia J,Whelan Rebecca J
Analytical and bioanalytical chemistry
The development of novel affinity probes for cancer biomarkers may enable powerful improvements in analytical methods for detecting and treating cancer. In this report, we describe our use of capillary electrophoresis (CE) as the separation mechanism in the process of selecting DNA aptamers with affinity for the ovarian cancer biomarker HE4. Rather than the conventional use of cloning and sequencing as the last step in the aptamer selection process, we used high-throughput sequencing on an Illumina platform. This data-rich approach, combined with a bioinformatics pipeline based on freely available computational tools, enabled the entirety of the selection process-and not only its endpoint-to be characterized. Affinity probe CE and fluorescence anisotropy assays demonstrate the binding affinity of a set of aptamer candidates identified through this bioinformatics approach. Graphical Abstract A population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.
Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection.
Ruff Patrick,Pai Rekha B,Storici Francesca
ISRN molecular biology
Aptamers are short nucleic acid or peptide sequences capable of binding to a target molecule with high specificity and affinity. Also known as "artificial antibodies," aptamers provide many advantages over antibodies. One of the major hurdles to aptamer isolation is the initial time and effort needed for selection. The systematic evolution of ligands by exponential enrichment (SELEX) is the traditional procedure for generating aptamers, but this process is lengthy and requires a large quantity of target and starting aptamer library. A relatively new procedure for generating aptamers using capillary electrophoresis (CE), known as CE-SELEX, is faster and more efficient than SELEX but requires laser-induced fluorescence (LIF) to detect the aptamer-target complexes. Here, we implemented an alternative system without LIF using real-time- (RT-) PCR to indirectly measure aptamer-target complexes. In three rounds of selection, as opposed to ten or more rounds common in SELEX protocols, a specific aptamer for bovine serum albumin (BSA) was obtained. The specificity of the aptamer to BSA was confirmed by electrophoretic mobility shift assays (EMSAs), an unlabeled competitor assay, and by a supershift assay. The system used here provides a cost effective and a highly efficient means of generating aptamers.
2',4'-BNA/LNA aptamers: CE-SELEX using a DNA-based library of full-length 2'-O,4'-C-methylene-bridged/linked bicyclic ribonucleotides.
Kasahara Yuuya,Irisawa Yuuta,Ozaki Hiroaki,Obika Satoshi,Kuwahara Masayasu
Bioorganic & medicinal chemistry letters
DNA-based aptamers that contain 2'-O,4'-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) over the entire length were successfully obtained using a capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) method. A modified DNA library was prepared with an enzyme mix of KOD Dash and KOD mutant DNA polymerases. Forty 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA) aptamers were isolated from an enriched pool and classified into six groups according to their sequence. 2',4'-BNA/LNA aptamers of groups V and VI bound human thrombin with K(d) values in the range of several 10 nanomolar levels.
Screening and Identifying a Novel ssDNA Aptamer against Alpha-fetoprotein Using CE-SELEX.
Dong Lili,Tan Qiwen,Ye Wei,Liu Dongli,Chen Haifeng,Hu Hongwei,Wen Duo,Liu Yang,Cao Ya,Kang Jingwu,Fan Jia,Guo Wei,Wu Weizhong
Alpha-fetoprotein (AFP) is a liver cancer associated protein and has long been utilized as a serum tumor biomarker of disease progression. AFP is usually detected in HCC patients by an antibody based system. Recently, however, aptamers generated from systematic evolution of ligands by exponential enrichment (SELEX) were reported to have an alternative potential in targeted imaging, diagnosis and therapy. In this study, AFP-bound ssDNA aptamers were screened and identified using capillary electrophoresis (CE) SELEX technology. After cloning, sequencing and motif analysis, we successfully confirmed an aptamer, named AP273, specifically targeting AFP. The aptamer could be used as a probe in AFP immunofluorescence imaging in HepG2, one AFP positive cancer cell line, but not in A549, an AFP negative cancer cell line. More interesting, the aptamer efficiently inhibited the migration and invasion of HCC cells after in vivo transfection. Motif analysis revealed that AP273 had several stable secondary motifs in its structure. Our results indicate that CE-SELEX technology is an efficient method to screen specific protein-bound ssDNA, and AP273 could be used as an agent in AFP-based staining, diagnosis and therapy, although more works are still needed.
Single-Strand DNA-Like Oligonucleotide Aptamer Against Proprotein Convertase Subtilisin/Kexin 9 Using CE-SELEX: PCSK9 Targeting Selection.
Sattari Roohollah,Palizban Abbasali,Khanahmad Hossein
Cardiovascular drugs and therapy
BACKGROUND:Proprotein convertase subtilisin/kexin 9 (PCSK9) serves a key regulatory function in the metabolism of low-density lipoprotein (LDL)-cholesterol (LDL-C) through interaction with the LDL receptor (LDLR) followed by its destruction that results in the elevation of the plasma levels of LDL-C. The aims of the present study were to separate and select a number of single-stranded DNA (ssDNA) aptamers against PCSK9 from a library pool (n > 10) followed by their characterization. METHODS:The aptamers obtained from the DNA-PCSK9 complexes which presented the highest affinity against PCSK9 were separated and selected using capillary electrophoresis evolution of ligands by exponential enrichment (CE-SELEX). The selected aptamers were amplified and cloned into a T/A vector. The plasmids from the positive clones were extracted and sequenced. The Mfold web server was used to predict the secondary structure of the aptamers. RESULTS:Following three rounds of CE-SELEX, the identified anti-PCSK9 ssDNA aptamers, namely aptamer 1 (AP-1) and aptamer 2 (AP-2), presented half maximal inhibitory concentrations of 325 and 327 nM, lowest dissociation constants of 294 and 323 nM, and most negative Gibbs free energy values of - 9.17 and - 8.28 kcal/mol, respectively. CONCLUSION:The results indicated that the selected aptamers (AP-1 and AP-2) induced potent inhibitory effects against PCSK9. Further in vivo studies demand to find out AP-1 and AP-2 aptamers as suitable candidates, instead of antibodies, for using in therapeutic purposes in patients with hypercholesterolemia and cardiovascular disease.
Azobenzene-modified DNA aptamers evolved by capillary electrophoresis (CE)-SELEX method.
Morihiro Kunihiko,Hasegawa Osamu,Kasahara Yuuya,Mori Shohei,Kasai Tatsuro,Kuwahara Masayasu,Obika Satoshi
Bioorganic & medicinal chemistry letters
Chemically modified aptamers have recently emerged as important materials for nucleic acid based therapeutics and diagnostic tools. Here, we report in vitro evolution of azobenzene-modified DNA aptamers by capillary electrophoresis (CE)-SELEX method. Azobenzene has been considered to be a fascinating functional group due to its trans-cis photo-isomerization property. We harnessed C5-azobenzene-modified 2'-deoxyuridine (dU) as a azobenzene-tethered unit and subjected it to CE-SELEX with human thrombin. The obtained dU-modified aptamer showed strong binding affinity toward human thrombin and could be reversibly photo-isomerized by different wavelengths of light. This work demonstrates that CE-SELEX is a powerful method to obtain chemically modified aptamers and dU is an excellent photo-responsive nucleoside for nucleic acid photo-switches.