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.     

Particle Display: A Quantitative Screening Method for Generating High‐Affinity Aptamers

We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution‐phase aptamers into “aptamer particles”, each displaying many copies of a single sequence on its surface. We then use fluorescence‐activated cell sorting (FACS) to individually measure the relative affinities of >10⁸ aptamer particles and sort them in a high‐throughput manner. Through mathematical analysis, we identified experimental parameters that enable optimal screening, and demonstrate enrichment performance that exceeds the theoretical maximum achievable with conventional selection by many orders of magnitude. We used particle display to obtain high‐affinity DNA aptamers for four different protein targets in three rounds, including proteins for which previous DNA aptamer selection efforts have been unsuccessful. We believe particle display offers an extraordinarily efficient mechanism for generating high‐quality aptamers in a rapid and economic manner, towards accelerated exploration of the human proteome.     

Arginine-modified DNA aptamers that show enantioselective recognition of the dicarboxylic acid moiety of glutamic acid

We have screened glutamic acid-binding aptamers from a modified DNA pool containing arginine residues using the method of systematic evolution of ligands by exponential enrichment (SELEX). Thirty-one modified DNA molecules were obtained from the enriched pool after the 17th round of selection, and their binding affinities for the target were evaluated by binding assays using affinity gels. Three modified DNA molecules having higher affinity were sequenced and we determined their affinity and specificity for the target by surface plasmon resonance (SPR) measurements. The SPR studies indicated that two of these three aptamers distinguished the dicarboxylic acid moiety of the D-isomer from that of the L-isomer; however, the third aptamer did not show enantioselectivity.     

全细胞的核酸适配体筛选的研究进展

核酸适配体(aptamer)是从人工合成的随机单链DNA(ssDNA)或RNA文库中筛选得到的,能够高亲和力、高特异性地与靶标结合的ssDNA或RNA。核酸适配体的靶标范围广,可包括小分子、蛋白质、细胞、微生物等多种靶标。其中以细胞为靶标的适配体在生物感应、分子成像、医学诊断、药物传输和疾病治疗等领域有很大的应用潜能。但全细胞的核酸适配体筛选过程复杂,筛选难度大,筛选的适配体性能不佳是导致目前可用的适配体非常有限的主要原因。由于细胞表面蛋白质在提取纯化过程中分子结构和形态会发生改变,故以膜表面蛋白质为靶标筛选的适配体很难应用于识别整体细胞。以全细胞为靶标的核酸适配体筛选则不需要准确了解细胞表面的分子结构,筛选过程中可保持细胞的天然状态,以全细胞为靶标筛选出的核酸适配体有望直接用于全细胞识别。本文总结了2008~2015年全细胞的核酸适配体筛选的研究进展,介绍了靶细胞的分类、核酸库的设计、筛选条件和方法以及核酸适配体的亲和力表征方法等。并列出全细胞靶标的核酸适配体序列。     

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed K_d values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with K_d values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.     

Selection of aptamers for signal transduction proteins by capillary electrophoresis

High‐affinity aptamers for important signal transduction proteins, i.e. Cdc42‐GTP, p21‐activated kinase1 (PAK1) and MRCK (myotonic dystrophy kinase‐related Cdc42‐binding kinase) α were successfully selected in the low micro‐ to nanomolar range using non‐systematic evolution of ligands by exponential enrichment (SELEX) with at least three orders of magnitude enhancement from their respective bulk affinity of naïve DNA library. In the non‐SELEX procedure, CE was used as a highly efficient affinity method to select aptamers for the desired molecular target through a process that involved repetitive steps of partitioning, known as non‐equilibrium CE of equilibrium mixtures with no PCR amplification between successive steps. Various non‐SELEX conditions including the type, concentration and pH of the run buffer were optimized. Other considerations such as salt composition of selection buffer, protein concentration and sample injection size were also studied for high stringency during selection. After identifying the best enriched aptamer pool, randomly selected clones from the aptamer pool were sequenced to obtain the individual DNA sequences. The dissociation constants (K_d) of these sequences were in the low micromolar to nanomolar range, indicating high affinity to the respective proteins. The best binders were also subjected to sequence alignment to generate a phylogenetic tree. No significant consensus region based on approximately 50 sequences for each protein was observed, suggesting the high efficiency of non‐SELEX for the selection of numerous unique sequences with high selectivity.     

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.     

Multiparameter Particle Display (MPPD): A Quantitative Screening Method for the Discovery of Highly Specific Aptamers

Aptamers are a promising class of affinity reagents because they are chemically synthesized, thus making them highly reproducible and distributable as sequence information rather than a physical entity. Although many high‐quality aptamers have been previously reported, it is difficult to routinely generate aptamers that possess both high affinity and specificity. One of the reasons is that conventional aptamer selection can only be performed either for affinity (positive selection) or for specificity (negative selection), but not both simultaneously. In this work, we harness the capacity of fluorescence activated cell sorting (FACS) for multicolor sorting to simultaneously screen for affinity and specificity at a throughput of 10⁷ aptamers per hour. As a proof of principle, we generated DNA aptamers that exhibit picomolar to low nanomolar affinity in human serum for three diverse proteins, and show that these aptamers are capable of outperforming high‐quality monoclonal antibodies in a standard ELISA detection assay.     

In Vitro Selection of a DNA Aptamer Targeting Degraded Protein Fragments for Biosensing

Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxin B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxin B, fresh toxin B, and toxin B spiked into human stool samples. DNA aptamers selected using intact recombinant toxin B failed to recognize degraded toxin B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper‐based analytical device for colorimetric detection of toxin B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer‐selection and paper‐sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.     

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.     

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.     

A simple and rapid approach for measurement of dissociation constants of DNA aptamers against proteins and small molecules via automated microchip electrophoresis

Automated microchip electrophoresis was used as a simple and rapid method to measure effective dissociation constants (K(d,eff)) of aptamers against both large and small molecule targets. Human thrombin, immunoglobulin E (IgE), and adenosine triphosphate (ATP) were selected as model analytes to validate the method, with four ligands including two DNA aptamers for thrombin (two distinct epitopes), an IgE aptamer, and an ATP aptamer. The approach is based on a microchip version of a DNA mobility shift assay. Non-denaturing microchip gel electrophoresis separations of DNA could resolve and quantify unbound from target-bound aptamers when using large molecules as targets. To extend the technique to small molecule targets such as ATP, an aptamer/competitor strategy was used, in which a DNA competitor complementary to the aptamer could be displaced by ATP and electrophoretically resolved. Using an automated microchip electrophoresis platform, parallel separations of 11 titration samples were completed in ~0.5 h. Analytical performance comparisons show that our approach provides significant advantages in minimized reagent consumption (typically tens of pmol of aptamer and target), reduced analysis time, and minimized user interaction when compared to previously reported methods for aptamer K(d) measurement. Moreover, the flexibility and ease of K(d,eff) measurement for aptamers against large and small targets make this a unique and valuable approach that should find widespread use. Finally, the feasibility of using this method during aptamer selection processes (e.g. SELEX) was shown by accurate bulk K(d,eff) measurement of a known thrombin aptamer (THRaptA) spiked into a random-sequence DNA pool at as low as 5.0% (molar %) of the total pool; only ~825 fmol of total binding sequences were needed for an 11-point titration curve.     

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis

The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.     

蛋白质的核酸适配体筛选的研究进展

核酸适配体是通过指数富集系统配体进化(SELEX)筛选获得的,与靶标具有高亲和力和特异性结合的单链DNA或RNA。蛋白质是生命进程中的关键功能分子。近年来,以蛋白质为靶标的适配体筛选在蛋白质相关的基础及应用研究领域受到广泛关注。核酸适配体应用性能的优劣取决于其亲和力、特异性与稳定性。目前,适配体筛选方法的优化主要是提高筛选效率、提升适配体性能及降低筛选成本。适配体主要筛选步骤包括复合物分离、核酸库优化、次级库的富集、适配体序列分析以及亲和力表征等。迄今为止,以蛋白质-核酸复合物的分离为核心步骤的适配体筛选方法有20余种。本文归纳总结了2005年以来以蛋白质为靶标的适配体筛选技术,讨论了各方法的缺陷与局限。介绍了核酸库的设计优化方法、适配体的序列特征,以及常用的亲和力表征方法。     

Acoustic quantification of ATP using a quartz crystal microbalance with dissipation

A quartz crystal microbalance with a dissipation monitoring (QCM-D) sensor was developed for highly sensitive and specific detection of adenosine-5'-triphosphate (ATP) by using an aptamer. The binding of ATP molecules on the aptamer films could be calculated as accurate mass changes using multiple frequency and dissipation measurements. The detection is achieved by calculating the mass changes from conformational rearrangements of the sensor surface upon interaction with the target. The sensor was demonstrated to respond to changes in ATP concentrations in real time suitable for continuous monitoring applications. This sensor showed excellent selectivity toward ATP compared with other chemically similar nucleotide GTP. The feasibility of the sensor was demonstrated by analyzing ATP concentrations in cell culture media with serum. The maximum frequency change was about -2 Hz after injection of 500 μM ATP. The affinity constant of the aptamer was determined to be 49 ± 7.59 μM. The proposed sensor can extend the application of the QCM-D system in medical diagnosis, and could be adopted for the detection of other small molecules with the use of specific aptamers.     

Cell-specific internalization study of an aptamer from whole cell selection

Nucleic acid aptamers have been shown many unique applications as excellent probes in molecular recognition. However, few examples are reported which show that aptamers can be internalized inside living cells for aptamer functional studies and for targeted intracellular delivery. This is mainly due to the limited number of aptamers available for cell-specific recognition, and the lack of research on their extra- and intracellular functions. One of the major difficulties in aptamers' in vivo application is that most of aptamers, unlike small molecules, cannot be directly taken up by cells without external assistance. In this work, we have studied a newly developed and cell-specific DNA aptamer, sgc8. This aptamer has been selected through a novel cell selection process (cell-SELEX), in which whole intact cells are used as targets while another related cell line is used as a negative control. The cell-SELEX enables generation of multiple aptamers for molecular recognition of the target cells and has significant advantages in discovering cell surface binding molecules for the selected aptamers. We have studied the cellular internalization of one of the selected aptamers. Our results show that sgc8 is internalized efficiently and specifically to the lymphoblastic leukemia cells. The internalized sgc8 aptamers are located inside the endosome. Comparison studies are done with the antibody for the binding protein of sgc8, PTK7 (Human protein tyrosine kinase-7) on cell surface. We also studied the internalization kinetics of both the aptamer and the antibody for the same protein on the living cell surface. We have further evaluated the effects of sgc8 on cell viability, and no cytotoxicity is observed. This study indicates that sgc8 is a promising agent for cell-type specific intracellular delivery.     

Immobilization-free screening of aptamers assisted by graphene oxide

Graphene oxide (GO) has the ability to separate free short ssDNA in heterogeneous solution. This feature is applied as a label free platform for screening of aptamers that bind to their target with high affinity and specificity. Herein, we report an aptamer selection strategy for Nampt protein based on GO.     

RNA aptamers that bind flavin and nicotinamide redox cofactors

RNA molecules that specifically bind riboflavin (Rb) and beta-nicotinamide mononucleotide (NMN) have been isolated by in vitro selection. A simple structural motif containing intramolecular G-quartets was found to bind tightly to oxidized riboflavin (Kd = 1-5 micromolar). DNA versions of the consensus sequence also bind, but with weaker affinity. DMS protection experiments show that the quartet structure of these aptamers is stabilized by interaction with the flavin. As a measure of their redox specificity, the aptamers do not show significant differential binding between oxidized and reduced forms of a 5-deazariboflavin derivative that is a close structural analog of riboflavin. In contrast to the lack of redox specificity of the riboflavin aptamers, RNAs selected for binding to the nicotinamide portion of NAD discriminate between NAD and NADH in solution by over an order of magnitude. A mutagenized pool based on one of the NMN aptamer sequences was used to reselect for NMN binding. Comparison of the reselected sequences led to the identification of the binding region of the aptamer. A complex secondary structure containing two interacting stem-loops is proposed for the minimal NMN-binding RNA. The same mutagenized pool was used to select for increased discrimination between NMN and NMNH. From these reselected sequences, a mutation within the binding region was identified that increases specificity for NMN. These experiments show that RNA can bind these cofactors with low micromolar affinity and, in the case of nicotinamide cofactors, can discriminate between the two redox states. These cofactor binding motifs may provide a framework for generating new ribozymes that catalyze redox reactions similar to those found in basic metabolic pathways.     

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.     

Two-Dimensional Electrophoresis-Based Selection of Aptamers Against an Unidentified Protein in a Tissue Sample

A fully automated two-dimensional electrophoresis (2DE) system was employed for DNA aptamer selection against an unidentified protein in a mouse liver tissue extract as a model target. A 2DE-based systematic evolution of ligands by exponential enrichment (2DE-SELEX) was demonstrated for aptamer selection against a single protein spot that was separated on a nitrocellulose membrane. After four iterative 2DE-SELEX cycles, the oligonucleotide pool was sequenced and aptamer sequences were identified. A blotting assay showed that an identified aptamer with a stable stem–loop structure had specific binding activity against the target protein. The 2DE-SELEX was shown to be promising for the development of aptamers against unidentified proteins in complex samples for proteomic analysis and biomarker discovery.

Supplemental materials are available for this article. Go to the publisher's online edition of Analytical Letters to view the supplemental file.