Automated Microfluidic Selection of Aptamers against Carbohydrates

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 DESCRIPTION (provided by applicant): Oligonucleotide-based receptors or aptamers are isolated from large libraries of oligonucleotides through an evolutionary process known as an in vitro selection and amplification (SELEX). Aptamers are readily adapted even in non-specialist laboratories for different functions, such as in biosensor and imaging applications, for affinity purifications, or as antagonists and in vivo targeting moieties. Once isolated and characterized, aptamers become true off-the-shelf synthetic reagents, that is, they are readily custom-made and custom- modified, being available within days from the conception of the experiment. There is by now a substantial number of idiosyncratic examples of aptamers binding to individual oligosaccharides. Also, we recently made important breakthroughs in optimization of coupled selection and counterselection (elimination of binding to closely related targets) protocols in order to isolate specific aptamers against challenging targets, including individual monosaccharides. These advances together argue that it is possible with the right selection conditions to isolate specific and high-affinity aptamers binding to any saccharide motif with an arbitrary number (three-, four-, five-, or more) of monomeric units. Furthermore, based on our progress in the development of microfluidic devices for rapid (within one day) SELEX we posit that identification of very specific binders to structural motifs (epitopes) in the context of largr glycans can be now performed in a fully automated, inject-and-collect manner, with ease of isolation limited only by the availability of synthetic and natural materials for selections. This proposal is focused on the implementation, optimization, and validation of microfluidic SELEX procedures for isolation of oligonucleotide-based receptors targeting arbitrary glycan motifs. Specifically, in our first aim we will take commercially available sets of related gangliosides and demonstrate that optimized selection protocols, consisting of individual affinity-capture and affinity-elimination steps ("modules"), can lead to specific binding to a particular epitope, eithe as in a whole glycan or as in a part of a larger motif. In our second aim we will then implement the optimized procedures in microfluidic devices, first on the same example glycans that were used to optimize procedures (gangliosides), and then validating our devices on new examples (e.g., on high-mannose glycans). The optimized device will be robust and reliable as well as allow for shortened procedure times. Individual selection and counterselection procedures, focusing on particular glycan motifs that should or should not bind to aptamers, will be readily implemented and programmed in these devices. As a result of this work, we will be ready to rapidly (within one day) isolate glycan-binding aptamers via integrated and automated microfluidic SELEX, as well as scale up the devices for parallelized isolation of aptamers against a large number of glycans, ultimately allowing aptamers with reproducible properties to become routinely and broadly available synthetic reagents in glycomics research.