Probing the Limits of Aptamer Affinity with a Microfluidic SELEX Platform

University of Helsinki, Finland
PLoS ONE (Impact Factor: 3.23). 11/2011; 6(11):e27051. DOI: 10.1371/journal.pone.0027051
Source: PubMed


Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K(d)) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K(d) of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K(d).

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    • "However, it is often necessary to use a lot of animals for the production of antibodies, and these antibodies have several limitations in their chemical or physical stability, usefulness for all potential analytes, quality-assured preparations, and effective cost. As the emerging alternatives to antibodies, one can consider antimicrobial peptides (AMPs) [6] [7] [8], aptamers [9] [10], real-time quantitative polymerase chain reaction (PCR) [11], primers with micro-PCR chip [12], and peptide nucleic acid (PNA) probes [13]. "
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    ABSTRACT: Due to their ability of effective binding to multiple target microbes, the antimicrobial peptides (AMPs) have recently received lots of attention as an alternative to antibodies for detecting bacteria. We devel-oped a new biosensing method to detect Escherichia coli (E. coli) by implementing a microfluidic chip designed with a weir inside the channel, in which AMP-labeled microbeads were embedded. We char-acterized the detection rate of the stained E. coli within a certain period of time to examine the detection effectiveness of our device. As the flow rate of the bacterial suspension increases, the detection time to reach the saturation level decreases to less than 30 min, suggesting rapid detection, while the detection efficiency is maintained at a similar level. Except with very low concentrations of E. coli (<10 3 cells/mL), both the detection time and the efficiency do not depend on the E. coli concentration. Our method has the potential to be developed as a novel biosensing platform for rapid and accurate detection of pathogens.
    Sensors and Actuators B Chemical 10/2014; 191:211-218. DOI:10.1016/j.snb.2013.09.105 · 4.10 Impact Factor
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    • "As anticipated from the lack of appreciable binding to IL-6 (the binding of the top-ranked aptamer to IL-6 is shown in Fig. 2), none of the sequences selected against IL-6 reached a multiplicity of 0.1% (Table 1). The failure to isolate aptamers against IL-6 is likely due to the lack of high affinity sequences in our starting library, which contained 1015 sequences out of a possible diversity of 460 ∼ 1036 for the 60-nt variable region, as well as the nature of IL-6 (isoelectric point (pI) 6.2 compared to 9.5 for PDGF-A and 7.6 for VEGF) being less ‘aptogeneic’ to nucleic acid aptamers [28]. "
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    ABSTRACT: SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers. High-throughput sequencing of selected pools revealed that the emergence of highly enriched sequences concurred with the increase in the percentage of reactive aptamers shown by flow cytometry. Particularly, selected DNA aptamers against VEGF were specific and of high affinity (KD = ∼ 1 nM) and demonstrated a potent inhibition of capillary tube formation of endothelial cells, comparable to the effect of a clinically approved anti-VEGF antibody drug, bevacizumab. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cell-SELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens.
    PLoS ONE 03/2014; 9(3):e93052. DOI:10.1371/journal.pone.0093052 · 3.23 Impact Factor
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    • "Aptamers are evolved by an iterative selection method called SELEX (systematic evolution of ligands by exponential enrichment) to specifically recognize and tightly bind their targets by means of well-defined complementary three-dimensional structures [1], [2]. They have been developed against intracellular and extracellular molecules expressed in cancer cells and demonstrated to attenuate the biological functions of their target molecules [3], [4], [5], [6], [7], [8], [9], [10]. Furthermore, DNA aptamers developed against MUC1 have been applied as tools for diagnosis of epithelial tumors as well as imaging of tumor cells in vivo [1], [11], [12], [13]. "
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    ABSTRACT: CD44 adhesion molecules are expressed in many breast cancer cells and have been demonstrated to play a key role in regulating malignant phenotypes such as growth, migration, and invasion. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. The diversity of the biological functions of CD44 is the result of the various splicing variants of these exons. Previous studies suggest that exon v10 of CD44 plays a key role in promoting cancer invasion and metastasis, however, the molecular mechanisms are not clear. Given the fact that exon v10 is in the ectodomain of CD44, we hypothesized that CD44 forms a molecular complex with other cell surface molecules through exon v10 in order to promote migration of breast cancer cells. In order to test this hypothesis, we selected DNA aptamers that specifically bound to CD44 exon v10 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). We selected aptamers that inhibited migration of breast cancer cells. Co-immunoprecipitation studies demonstrated that EphA2 was co-precipitated with CD44. Pull-down studies demonstrated that recombinant CD44 exon v10 bound to EphA2 and more importantly aptamers that inhibited migration also prevented the binding of EphA2 to exon v10. These results suggest that CD44 forms a molecular complex with EphA2 on the breast cancer cell surface and this complex plays a key role in enhancing breast cancer migration. These results provide insight not only for characterizing mechanisms of breast cancer migration but also for developing target-specific therapy for breast cancers and possibly other cancer types expressing CD44 exon v10.
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