In Vitro Selection of New DNA Aptamers for Human Vascular Endothelial Growth Factor 165.

Two DNA aptamers that bind the heparin-binding domain (HBD) of the human vascular endothelial growth factor 165 (VEGF-165) have been previously reported. Although VEGF-165 is a homodimeric protein and the two aptamers have different sequences and secondary structures, the aptamers appear to occupy the same binding site and cannot form a 2 : 1 aptamer/protein complex, thus making them unsuitable for creating a higher-affinity dimeric DNA aptamer. This has motivated us to conduct a new in vitro selection experiment to search for new VEGF-165-binding DNA aptamers with different properties. We undertook a multistream selection strategy in which the concentration of VEGF-165 was varied significantly. We carried out 11 rounds of selection, and next-generation sequencing was conducted for every round in each stream. From comprehensive sequence analysis, we identified four classes of DNA aptamers, of which two were reported before, but two are new DNA aptamers. One of the new aptamers exhibits a unique property that has never been observed before: it is capable of forming the 2 : 1 aptamer/protein complex with VEGF-165. This work has expanded the repertoire of VEGF-165-binding DNA aptamers and creates a possibility to engineer a higher affinity homodimeric aptamer for VEGF-165.

Unraveling Determinants of Affinity Enhancement in Dimeric Aptamers for a Dimeric Protein.

High-affinity aptamers can be derived de novo by using stringent conditions in SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments or can be engineered post SELEX via dimerization of selected aptamers. Using electrophoretic mobility shift assays, we studied a series of heterodimeric and homodimeric aptamers, constructed from two DNA aptamers with distinct primary sequences and secondary structures, previously isolated for VEGF-165, a homodimeric protein. We investigated four factors envisaged to impact the affinity of a dimeric aptamer to a dimeric protein: (1) length of the linker between two aptamer domains, (2) linking orientation, (3) binding-site compatibility of two component aptamers in a heterodimeric aptamer, and (4) steric acceptability of the two identical aptamers in a homodimeric aptamer. All heterodimeric aptamers for VEGF-165 were found to exhibit monomeric aptamer-like affinity and the lack of affinity enhancement was attributed to binding-site overlap by the constituent aptamers. The best homodimeric aptamer showed 2.8-fold better affinity than its monomeric unit (Kd = 13.6 ± 2.7 nM compared to 37.9 ± 14 nM), however the barrier to further affinity enhancement was ascribed to steric interference of the constituent aptamers. Our findings point to the need to consider the issues of binding-site compatibility and spatial requirement of aptamers for the development of dimeric aptamers capable of bivalent recognition. Thus, determinants highlighted herein should be assessed in future multimerization efforts.

Colorimetric Detection of Uranyl Using a Litmus Test.

Ingestion of water containing toxic contaminants above levels deemed safe for human consumption can occur unknowingly since numerous common contaminants in drinking water are colorless and odorless. Uranyl is particularly problematic as it has been found at dangerous levels in sources of drinking water. Detection of this heavy metal-ion species in drinking water currently requires sending a sample to a laboratory where trained personnel use equipment to perform the analysis and turn-around times can be long. A pH-responsive colorimetric biosensor was developed to enable detection of uranyl in water which coupled the uranyl-specific 39E DNAzyme as a recognition element, and an enzyme capable of producing a pH change as the reporter element. The rapid colorimetric assay presented herein can detect uranyl in lake and well water at concentrations relevant for environmental monitoring, as demonstrated by the detection of uranyl at levels below the limits set for drinking water by major regulatory agencies including the World Health Organization (30 µg/L). This simple and inexpensive DNAzyme-based assay enabled equipment-free visual detection of 15 µg/L uranyl, using both solution-based and paper-based pH-dependent visualization strategies.

Colorimetric Detection of Bacteria Using Litmus Test.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.