Single-Round DNA Aptamer Selection by Combined Use of Capillary Electrophoresis and Next Generation Sequencing: An Aptaomics Approach for Identifying Unique Functional Protein-Binding DNA Aptamers

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a “DNA Aptaomics” approach to systematically design functional aptamers as well as to obtain high affinity aptamers.     

Electrochemical Immunosensing Chip Using Selective Surface Modification,Capillary‐Driven Microfluidic Control,and Signal Amplification by Redox Cycling

A sensitive electrochemical immunosensing chip is presented by employing (i) selective modification of protein‐resistant surfaces; (ii) fabrication of a stable Ag/AgCl reference electrode; (iii) capillary‐driven microfluidic control; (iv) signal amplification by redox cycling along with enzymatic reaction. Purely capillary‐driven microfluidic control is combined with electrochemical sandwich‐type immunosensing procedure. Selective modification of the surfaces is achieved by chemical reactivity‐controlled patterning and electrochemical deposition. Fluidic control of the immunosensing chip is achieved by spontaneous capillary‐driven flows and passive washing. The detection limit for mouse IgG in the immunosensing chip is 10 pg/mL.     

Near-infrared laser-induced fluorescence detection in capillary electrophoresis

As capillary electrophoresis continues to focus on miniaturization, either through reducing column dimensions or situating entire electrophoresis systems on planar chips, advances in detection become necessary to meet the challenges posed by these electrophoresis platforms. The challenges result from the fact that miniaturization requires smaller load volumes, demanding highly sensitive detection. In addition, many times multiple targets must be analyzed simultaneously (multiplexed applications), further complicating detection. Near-infrared (NIR) fluorescence offers an attractive alternative to visible fluorescence for critical applications in capillary electrophoresis due to the impressive limits of detection that can be generated, in part resulting from the low background levels that are observed in the NIR. Advances in instrumentation and fluorogenic labels appropriate for NIR monitoring have led to a growing number of examples of the use of NIR fluorescence in capillary electrophoresis. In this review, we will cover instrumental components used to construct ultrasensitive NIR fluorescence detectors, including light sources and photon transducers. In addition, we will discuss various types of labeling dyes appropriate for NIR fluorescence and finally, we will present several applications that have used NIR fluorescence in capillary electrophoresis, especially for DNA sequencing and fragment analysis.     

Electrochemical Signal Substance for Multiplexed Immunosensing Interface Construction: A Mini Review

Appropriate labeling method of signal substance is necessary for the construction of multiplexed electrochemical immunosensing interface to enhance the specificity for the diagnosis of cancer. So far, various electrochemical substances, including organic molecules, metal ions, metal nanoparticles, Prussian blue, and other methods for an electrochemical signal generation have been successfully applied in multiplexed biosensor designing. However, few works have been reported on the summary of electrochemical signal substance applied in constructing multiplexed immunosensing interface. Herein, according to the classification of labeled electrochemical signal substance, this review has summarized the recent state-of-art development for the designing of electrochemical immunosensing interface for simultaneous detection of multiple tumor markers. After that, the conclusion and prospects for future applications of electrochemical signal substances in multiplexed immunosensors are also discussed. The current review can provide a comprehensive summary of signal substance selection for workers researched in electrochemical sensors, and further, make contributions for the designing of multiplexed electrochemical immunosensing interface with well signal.     

Non-SELEX selection of aptamers

Aptamers are typically selected from libraries of random DNA (or RNA) sequences by SELEX, which involves multiple rounds of alternating steps of partitioning and PCR amplification. Here we report, for the first time, non-SELEX selection of aptamers-a process that involves repetitive steps of partitioning with no amplification between them. A highly efficient affinity method, non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), was used for partitioning. We found that three steps of NECEEM-based partitioning in the non-SELEX approach were sufficient to improve the affinity of a DNA library to a target protein by more than 4 orders of magnitude. The resulting affinity was higher than that of the enriched library obtained in three rounds of NECEEM-based SELEX. Remarkably, NECEEM-based non-SELEX selection took only 1 h in contrast to several days or several weeks required for a typical SELEX procedure by conventional partitioning methods. In addition, NECEEM-based non-SELEX allowed us to accurately measure the abundance of aptamers in the library. Not only does this work introduce an extremely fast and economical method for aptamer selection, but it also suggests that aptamers may be much more abundant than they are thought to be. Finally, this work opens the opportunity for selection of drug candidates from libraries of small molecules, which cannot be PCR-amplified and thus are not approachable by SELEX.     

Automated acquisition of aptamer sequences

While the in vitro selection of nucleic acid binding species (aptamers) requires numerous liquid-handling steps, these steps are relatively straightforward and the overall process is therefore amenable to automation. Here we demonstrate that automated selection techniques are capable of generating aptamers against a number of diverse protein targets. Automated selection techniques can be integrated with automated analytical methods, including sequencing, determination of binding constants, and structural analysis. The methods that have so far been developed can be further multiplexed, and it should soon be possible to attempt the selection of aptamers against organismal proteomes or metabolomes.     

Selection of smart aptamers by equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM)

We coin a term of "smart aptamers", which describes aptamers with predefined binding parameters of their interaction with the target. Here, we introduce a method for selection of smart aptamers with predefined values of Kd: equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM). Conceptually, a mixture of a target with a DNA (RNA) library is prepared and equilibrated. A plug of the equilibrium mixture is injected into a capillary prefilled with a run buffer containing the target at the concentration identical to the target concentration in the equilibrium mixture. The components of the equilibrium mixture are separated by capillary electrophoresis while equilibrium is maintained between the target and aptamers. The unique feature of ECEEM is that aptamers with different Kd values migrate with different and predictable mobilities. Thus, collecting fractions with different mobilities results in smart aptamers with different and predefined Kd values. In this proof-of-principle work, we used ECEEM to select smart aptamers for MutS protein, for which aptamers have never been previously selected. Three rounds of ECEEM-based selection were sufficient to obtain smart aptamers with Kd values approaching theoretically predicted ones. ECEEM is the first method for aptamer selection whose ability to generate smart aptamers has been experimentally proven.     

Pressure controllable aptamers picking strategy by targets competition

Selection of aptamers with high affinity and good specificity requires multiple rounds of alternating steps of separation and PCR amplification.Herein,we proposed a novel high-efficiency aptamers picking strategy:One-round pressure controllable selection(OPCS).OPCS integrates four types of screening superiority,high-efficiency separation,one-round selection and PCR amplification,synchronous negative selection and targets competition.The controllable screening pressure can be achieved through two approaches,balanced competition by the regulation of protein concentration,and dominant competition by introducing a predatory protein with high concentration.In OPCS process,two proteins were co-incubated with one ssDNA library,and each protein bound its favorable sequences specifically and formed protein-ss D NA complex re spectively.Meanwhile,one protein could supply/sufferthe picking pressure of affinity and specificity to/from another,which eliminated weakly bound or unbound sequences for each other.Two complexes could be separated and collected conveniently,and aptamers for two proteins obtained synchronously with high affinity and good specificity.This strategy not only provides a more effective way for aptamers selection,but shows great potential in other ligands or drugs selection.     

Battery-triggered ultrasensitive electrochemiluminescence detection on microfluidic paper-based immunodevice based on dual-signal amplification strategy

Dual-signal amplification strategy for ultrasensitive electrochemiluminescence (ECL) multiplexed immunoassay on microfluidic paper-based analytical devices (μ-PADs) was demonstrated. This dual-signal amplification technique was achieved by employing graphene oxide-chitosan/gold nanoparticles (GCA) immunosensing platform and [4,4′-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid] (P-acid) functionalized nanoporous silver (P-acid/NPS) signal amplification label. For further low-cost and disposable applications, battery-triggered constant-potential ECL (+1.0 V for P-acid label (vs. Ag/AgCl auxiliary electrode)) was applied on this paper-based immunodevice with the aid of a home-made voltage-tunable power device, allowing the traditional electrochemical workstation to be abandoned. We found that two tumor markers could be sequentially detected in the linear ranges of 0.003–20 and 0.001–10 ng mL⁻¹ with the detection limits down to 1.0 and 0.8 pg mL⁻¹, respectively, by simply reversing the connection mode on two working electrodes. The results exhibited excellent precision and high sensitivity of such immunoassay, and it also demonstrated that this battery-triggered ECL paper-based immunodevice could provide a rapid, simple and simultaneous multiplex immunoassay with high throughput, low-cost and low detection limits for point-of-care testing.     

Ideal‐Filter Capillary Electrophoresis (IFCE) Facilitates the One‐Step Selection of Aptamers

Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides. Herein, we report a highly practical solution for reliable one‐step selection of aptamers. We introduce partitioning by ideal‐filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE‐based partitioning reaches 10⁹, which is ten million times higher than that of typical solid‐phase partitioning methods. One step of IFCE‐based partitioning is sufficient for the selection of a high‐affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.     

毛细管电泳法高效筛选8-氧代鸟嘌呤DNA糖基化酶的核酸适配体

8-氧代鸟嘌呤DNA糖基化酶(OGG1)是人体中重要的功能蛋白,在修复DNA氧化性损伤过程中起关键作用。氧化应激等引起的氧化损伤易导致炎症反应的发生,对OGG1的抑制可以一定程度上起到缓解作用;对癌细胞OGG1的抑制有望作为癌症治疗的新方法。目前的研究多集中于小分子对OGG1功能的影响和调控,而OGG1的适配体筛选尚未见报道。作为功能配体,适配体具有合成简单、高亲和力及高特异性等优点。该文筛选了OGG1的核酸适配体,结合毛细管电泳高效快速的优点建立了两种基于毛细管电泳-指数富集进化(CE-SELEX)技术的筛选方法:同步竞争法和多轮筛选法。同步竞争法利用单链结合蛋白(SSB)与核酸库中单链核酸的强结合能力,与目标蛋白OGG1组成竞争体系,并通过增加SSB浓度来增加竞争筛选压力,以去除与OGG1弱结合的核酸序列,一步筛选即可获得与OGG1强结合的核酸序列。多轮筛选法在相同孵育条件和电泳条件下,经3轮筛选获得OGG1的核酸适配体。比较两种筛选方法的筛选结果,筛选结果中频次最高的3条候选核酸适配体序列一致,其解离常数(K_D)值在1.71~2.64 μmol/L之间。分子对接分析结果表明候选适配体1(Apt 1)可能与OGG1中具有修复氧化性损伤功能的活性口袋结合。通过对两种筛选方法的对比,证明同步竞争法更加快速高效,对其他蛋白核酸适配体筛选方法的选择具有一定的指导意义。得到的适配体有望用于OGG1功能调控,以抑制其修复功能。     

Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue

Aptamers are DNA oligonucleotides capable of binding different classes of targets with high affinity and selectivity. They are particularly attractive as affinity probes in multiplexed quantitative analysis of proteins. Aptamers are typically selected from large libraries of random DNA sequences in a general approach termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX involves repetitive rounds of two processes: (i) partitioning of aptamers from non-aptamers by an affinity method and (ii) amplification of aptamers by the polymerase chain reaction (PCR). New partitioning methods, which are characterized by exceptionally high efficiency of partitioning, have been recently introduced. For the overall SELEX procedure to be efficient, the high efficiency of new partitioning methods has to be matched by high efficiency of PCR. Here we present the first detailed study of PCR amplification of random DNA libraries used in aptamer selection. With capillary electrophoresis as an analytical tool, we found fundamental differences between PCR amplification of homogeneous DNA templates and that of large libraries of random DNA sequences. Product formation for a homogeneous DNA template proceeds until primers are exhausted. For a random DNA library as a template, product accumulation stops when PCR primers are still in excess of the products. The products then rapidly convert to by-products and virtually disappear after only 5 additional cycles of PCR. The yield of the products decreases with the increasing length of DNA molecules in the library. We also proved that the initial number of DNA molecules in PCR mixture has no effect on the by-products formation. While the increase of the Taq DNA polymerase concentration in PCR mixture selectively increases the yield of PCR products. Our findings suggest that standard procedures of PCR amplification of homogeneous DNA samples cannot be transferred to PCR amplification of random DNA libraries: to ensure efficient SELEX, PCR has to be optimized for the amplification of random DNA libraries.     

Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.     

Cover Picture: Electrophoresis 12'2010

Issue no. 12 is a regular issue comprising 19 contributions distributed over five distinct parts. Part I has 7 articles describing novel methodologies pertaining to proteins and proteomics. Part II has 3 research papers on CEC stationary phases and CEC‐MS. Part III is on detection approaches including a review article on the advances and applications of chemiluminescence coupled to CE. Part IV has two papers on enantioseparations, and Part V has four contributions on aptamers, human genetic, preparative FFE and microchannels. Featured articles include: Simplified method for concentration of mitochondrial membrane protein complexes (( 10.1002/elps.201000019 )) Analysis of low‐molecular mass aldehydes in drinking waters through capillary electrophoresis with laser‐induced fluorescence detection (( 10.1002/elps.200900734 )) Selection of aptamers for signal transduction proteins by capillary electrophoresis (( 10.1002/elps.200900543 ))     

High-throughput polymerase chain reaction analysis of clinical samples by capillary electrophoresis with UV detection

Routine genetic analysis of large numbers of individuals by polymerase chain reaction (PCR) using capillary electrophoresis is often restricted by the low throughput of standard protocols and the tedious sample preparation process. Here, we demonstrate that capillary electrophoresis with UV detection can be used in PCR-based DNA analysis starting from clinical samples without purification or complicated sample manipulation. After PCR reaction using cheek cells, blood, or HIV-1 gag DNA, the reaction mixtures were injected into a capillary array either on-line or off-line by base stacking. The use of multiplexed absorption detection and the elimination of any purification steps both before and after PCR reaction can potentially provide significant benefits compared to current methods for DNA analysis with regard to time, cost, and labor.     

Selection of cholesterol esterase aptamers using a dual‐partitioning approach

Non‐systematic evolution of ligands by exponential enrichment and other capillary‐based methods have grown in popularity for selection of aptamers since they provide a fast and efficient partitioning method when compared to classical techniques. Despite promising developments in these techniques, a major obstacle needs to be overcome for capillary‐based selections to be widely accepted. Due to the small injection volumes associated with CE, only a small proportion of the nucleic acid library can be partitioned at any one time. In this paper, we propose a new two‐step method for the selection of aptamers which firstly incorporates a nitrocellulose membrane filter followed by CE. This technique allows for nonbinding sequences to be eliminated, reducing the library size before subsequent capillary‐based partitioning, while still reducing the time taken for aptamers to be selected. We demonstrated this technique on the selection of aptamers for cholesterol esterase and the highest binding truncated aptamer CES 4T displayed a K_D of 203 ± 14 nM. In addition, an increase in the number of sequences partitioned was estimated using spectrophotometry and capillary injection volumes. The results suggested that for successful selection a two‐step approach is needed. This hybrid technique could be used to select aptamers that bind to targets both in solution and immobilized onto a stationary phase, allowing the aptamers to be used in different binding environments.     

Patterned Paper Sensors Printed with Long‐Chain DNA Aptamers

There is growing interest in developing printable paper sensors to enable rapid testing of analytes for environmental, food safety, and clinical applications. A major challenge is to find suitable bioinks that are amenable to high‐speed printing and remain functional after printing. We report on a simple and effective approach wherein an aqueous ink composed of megadalton‐sized tandem repeating structure‐switching DNA aptamers (concatemeric aptamers) is used to rapidly create patterned paper sensors on filter paper by inkjet printing. These concatemeric aptamer reporters remain immobilized at the point of printing through strong adsorption but retain sufficient segmental mobility to undergo structure switching and fluorescence signaling to provide both qualitative and quantitative detection of small molecules and protein targets. The convenience of inkjet printing allows for the patterning of internally referenced sensors with multiplexed detection, and provides a generic platform for on‐demand printing of sensors even in remote locations.     

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.     

Effect of buffer,electric field,and separation time on detection of aptamer-ligand complexes for affinity probe capillary electrophoresis

The separation and detection of complexes of aptamers and protein targets by capillary electrophoresis (CE) with laser-induced fluorescence was examined. Aptamer-thrombin and aptamer-immunoglobulin E (IgE) were used as model systems. Phosphate, 3-(N-morpholino)propanesulfonic acid with phosphate, and tris(hydroxyamino)methane-glycine-potassium (TGK) buffer at pH 8.4 were tested as electrophoresis media. Buffer had a large effect with TGK providing the most stable complexes for both protein-aptamer complexes. Conditions that suppressed electroosmotic flow, such as addition of hydroxypropylmethylcellulose to the media or modification of the capillary inner wall with polyacrylamide, were found to prevent detection of complexes. The effect of separation time and electric field were evaluated by monitoring complexes with electric field varied from 150-2850 V/cm and effective column lengths of 3.5 and 7.0 cm. As expected, shorter times on the column greatly increased peak heights for the complexes due to a combination of less dilution by diffusion and less dissociation on the column. High fields were found to have a detrimental effect on detection of complexes. It is concluded that the best conditions for detection of noncovalent complexes involve use of the minimal column length and electric field necessary to achieve separation. The results will be of interest in developing affinity probe CE assays wherein aptamers are used as affinity ligands.