Speeding up in Vitro Discovery of Structure-Switching Aptamers via Magnetic Cross-Linking Precipitation.

We report here a modified aptamer selection method, magnetic cross-linking precipitation (MCP)-SELEX, for highly efficient library enrichment and aptamer isolation. MCP-SELEX isolates bound aptamers via highly efficient chemical cross-linking between amino groups of target proteins and activated carboxylic acid groups on magnetic beads (>90% coupling efficiency). Importantly, MCP-SELEX avoids surface interferences in conventional target-fixed methods and substantially minimizes nonspecific binding. The enrichment efficiencies of MCP-SELEX for various proteins (PD-L1, ubiquitin, thrombin, and HSA) were all greatly higher than those of the conventional target-bound magnetic bead based-SELEX (MB-SELEX). Antithrombin aptamer with KD of 33 nM was successfully isolated by four rounds of MCP-SELEX. MCP-SELEX also enabled the efficient aptamer isolation by coupling with MB-SELEX or falling-off-SELEX. We identified structure-switching aptamers (SSAs) that specifically bind to HSA with low nanomolar dissociation constant via three rounds of MCP-SELEX and 1 round of falling-off-SELEX. Our HSA SSAs also have ∼3-fold higher specificity against streptavidin relative to thrombin SSAs discovered through falling-off-SELEX only. The enriched library has ∼78-fold higher signal-to-noise ratio (the number of DNAs eluted by 50 nM HSA divided by the number of DNAs self-dissociated in blank buffer) than that obtained by 4 rounds of direct falling-off-SELEX. We finally demonstrated the application of the selected SSA in fluorescent detection of HSA in urine with diagnostic required sensitivity and dynamic range. We expect that MCP-SELEX may be coupled with other selection methods to substantially accelerate aptamer discovery.

An efficient method to evaluate experimental factor influence on in vitro binding of aptamers.

Nucleic acid-based aptamers are promising alternative to antibodies, however, their selection process (SELEX) is challenging. A number of simulations and few experiments have been reported offering insights into experimental factors (EFs) that govern the effectiveness of the selection process. Though useful, these previous studied were either lack of experimental confirmation, or considered limited EFs. A more efficient experimental method is highly desired. In this study, we developed a fast method that is capable to quantitatively probe the influence of multiple EFs. Based on the fact that the aptamer enrichment efficiency is highly affected by background binding, the binding ratio between the numbers of specific aptamer binders and nonspecific (unselected library) binders per bead was used to quantitatively evaluate EF effects. Taking thrombin and streptavidin as models, three previously studied EFs (surface coverage, buffer composition, and DNA concentration) and four never-studied ones (surface chemistry, heat treatment, elution methodology and pool purity) were investigated. The EFs greatly affected binding ratio (ranging from 0.03 ±â€¯0.03 to 14.60 ±â€¯2.30). The results were in good agreement with the literature, suggesting the good feasibility of our method. Our study provides guidance for the choice of EFs not only for aptamer selection, but also for binding evaluation of aptamers.

Selection of Group-Specific Phthalic Acid Esters Binding DNA Aptamers via Rationally Designed Target Immobilization and Applications for Ultrasensitive and Highly Selective Detection of Phthalic Acid Esters.

Phthalic acid esters (PAEs) are ubiquitous in the environment, and some of them are recognized as endocrine disruptors that cause concerns on ecosystem functioning and public health. Due to the diversity of PAEs in the environment, there is a vital need to detect the total concentration of PAEs in a timely and low-cost way. To fulfill this requirement, it is highly desired to obtain group-specific PAE binders that are specific to the basic PAE skeleton. In this study, for the first time we have identified the group-specific PAE-binding aptamers via rationally designed target immobilization. The two target immobilization strategies were adopted to display either the phthalic ester group or the alkyl chain, respectively, at the surface of the immobilization matrix. The former enabled the rapid enrichment of aptamers after four rounds of selection. The top 100 sequences are cytosine-rich (44.7%) and differentiate from each other by only 1-4 nucleotides at limited locations. The top two aptamers all display the nanomolar dissociation constants to both the immobilized target and the free PAEs [dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), bis(2-ethylhexyl) phthalate (DEHP)]. We further demonstrate the applications of the aptamers in the development of high-throughput PAE assays and DEHP electrochemical biosensors with exceptional sensitivity [limit of detection (LOD), 10 pM] and selectivity (>105-fold). PAE aptamers targeting one of the most sought for targets thus offer the promise of convenient, low-cost detection of total PAEs. Our study also provides insights on the aptamer selection and sensor development of highly hydrophobic small molecules.

Light-Induced Reactivity Switch at O2-Activating Bioinspired Copper(I) Complexes.

Using light to unveil unexplored reactivities of earth-abundant metal-oxygen intermediates is a formidable challenge, given the already remarkable oxidation ability of these species in the ground state. However, the light-induced reactivity of Cu-O2 intermediates still remains unexplored, due to the photoejection of O2 under irradiation. Herein, we describe a photoinduced reactivity switch of bioinspired O2-activating CuI complexes, based on the archetypal tris(2-pyridyl-methyl)amine (TPA) ligand. This report represents a key precedent for light-induced reactivity switch in Cu-O2 chemistry, obtained by positioning C-H substrates in close proximity of the active site. Open and caged CuI complexes displaying an internal aryl ether substrate were evaluated. Under light, a Cu-O2 mediated reaction takes place that induces a selective conversion of the internal aryl ether unit to a phenolate-CH2- moiety with excellent yields. This light-induced transformation displays high selectivity and allows easy postfunctionalization of TPA-based ligands for straightforward preparation of challenging heteroleptic structures. In the absence of light, O2 activation results in the standard oxidative cleavage of the covalently attached substrate. A reaction mechanism that supports a monomeric cupric-superoxide-dependent reactivity promoted by light is proposed on the basis of reactivity studies combined with (TD-) DFT calculations.

Bioinspired complexes confined in well-defined capsules: getting closer to metalloenzyme functionalities.

Reproducing the key features offered by metalloprotein binding cavities is an attractive approach to overcome the main bottlenecks of current open artificial models (in terms of stability, efficiency and selectivity). In this context, this featured article brings together selected examples of recent developments in the field of confined bioinspired complexes with an emphasis on the emerging hemicryptophane caged ligands. In particular, we focused on (1) the strategies allowing the insulation and protection of complexes sharing similarities with metalloprotein active sites, (2) the confinement-induced improvement of catalytic efficiencies and selectivities and (3) very recent efforts that have been made toward the development of bioinspired complexes equipped with weakly binding artificial cavities.

A caged tris(2-pyridylmethyl)amine ligand equipped with a Ctriazole-H hydrogen bonding cavity.

A capped bioinspired ligand built from a tris(2-pyridyl-methyl)amine (TPA) unit and surmounted by a triazole-based intramolecular H-bonding secondary sphere was prepared. The resulting cage provides a well-defined cavity combining the hydrophobic nature with H-bonding properties. Its coordinating properties were explored using Zn(II) and Cu(II) metal ions.

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor.

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.