Towards Precision Medicine: Rapid Microfluidic Isolation of Aptamers for Glycan Targets

Abstract

Progress Claims This paper demonstrates a microfluidic approach to isolate aptamers, single-stranded oligonucleotides with affinity and specificity for a target, for glycan targets. The approach is capable of rapidly (<1 day) isolating high affinity (KD <100 nM) aptamers with limited sample consumption (<500 µg of glycan) on a single microfluidic platform without the need for offline processes. The presented approach has potential to generate easily accessible sensors for the emerging glycomics field. Background Glycans, one of the four major classes of molecules making up cells (the others being nucleic acids, proteins and lipids) [1], are involved in all human developmental activities including structural roles, modulatory, intercellular signaling and adhesion [2]. Aberrant expression of glycans, including under- and overexpression, is associated with many diseases, attracting considerable interest in using glycans as disease markers [3]. Thus, the concept of stratifying patients based on the human glycome, similar to the genome or proteome, is developing but methods to rapidly and sensitively analyze glycan samples remains an unmet challenge. Conventional methods for sensor development (e.g. antibodies) require an infeasible amount of purified glycans [4], and glycan specific proteins (lectins) are often of low-affinity and non-specific [5]. Aptamers have been previously used for glycan detection but the process of affinity-selection and PCR amplification needed to isolate aptamers (termed SELEX) is conventionally too time- and resource-intensive for widespread glycan-binding aptamer development [6]. Microfluidics has been applied to SELEX to rapidly isolate aptamers but existing devices still require off-chip processes to complete the process, and moreover have been primarily demonstrated with proteins which, compared to glycans, contain multifunctional surfaces ideal for aptamer isolation [7-8]. Approach We present a microfluidic approach that integrates the entire SELEX process onto a single platform to rapidly isolate glycan targeting aptamers while consuming modest amounts of target glycans (<500 µg) (Fig. 1). The approach is realized in a microfluidic device consisting of three (selection, counter selection, and amplification) functional chambers of identical geometry (~4.5 µL) connected in a microfluidic loop by microchannels. The device was fabricated using standard soft-lithography techniques (Fig. 2). The selection and amplification chambers are each integrated with devoted microheaters and temperature sensors for thermal control. Target-binding oligonucleotides are affinity-selected from a randomized library by incubation with target glycan functionalized microbeads in the selection chamber. Binding oligonucleotides are thermally eluted using the integrated heater located beneath the selection chamber, transferred through the counter selection chamber (containing a non-target glycan functionalized to the beads) to the amplification chamber where the integrated heater is employed for on-chip PCR thermal cycling. The amplified product is then transferred back to the selection chamber as the process repeats. As such, the approach is capable of performing SELEX on a single microchip without relying on offline processes or transfers to additional microchips while using microfluidics to limit glycan consumption. Results Gangliosides GM1, GM3, and IgA hinge region peptide with N-acetylgalactosaminyl (GalNAc) glycosylation were used as targets for three different aptamer isolation processes. Successful affinity-selection was confirmed by incubating randomized library with target-functionalized microbeads and collecting oligomers that were removed by buffer washes and after thermal elution (Fig. 3). Gel electropherograms of a 100 fM DNA sample before on-chip PCR and after on-chip PCR demonstrate successful on-chip amplification (Fig. 4). Four rounds of closed-loop SELEX were performed on-chip (taking ~9 hours) using the approach with gel electropherograms from this process showing successful enrichment of aptamer candidates (Fig. 5). Aptamers were sequenced and characterized by surface plasmon resonance (SPR, Fig. 6). Aptamers of high affinity (GM1 KD: 49.6 nM and GM3 KD: 43.59 nM) were isolated for the ganglioside targets. Aptamers showed high affinity and specificity only for the glycosylation pattern of the peptide, as demonstrated by the 15 nM KD for the glycosylated version of the peptide versus a 3.2 µM KD for the same peptide but without the glycosylation pattern (Fig. 7). All selections consumed <500 µg of glycan sample. We have thus developed an approach enabling the practical generation glycan probes for glycome studies. J. D. Marth, et al., Nat. Cell Biol., 1015, 2008.Varki, et al., Glycobiology, 3-49, 2017.M. J. Kailemia, et al., Anal. Bioanal. Chem., 395-410, 2017.H. J. An, et al., Curr. Opin. Chem. Biol., 601-607, 2009.W.I. Weis, et al., Annual Review of Biochemistry, 441-473, 1996M. Li etal., JACS, 12636-12638, 2008.M. Cho et al., Proc. Nat. Acad. Sci., 15373-15378, 2010.J.Y. Ahn et al., Analytical Chemistry, 2647-2653, 2012. Figure 1