Quantitative selection and parallel characterization of aptamers

Abstract

Aptamers are promising affinity reagents that are potentially well suited for high-throughput discovery, as they are chemically synthesized and discovered via completely in vitro selection processes. Recent advancements in selection, sequencing, and the use of modified bases have improved aptamer quality, but the overall process of aptamer generation remains laborious and low-throughput. This is because binding characterization remains a critical bottleneck, wherein the affinity and specificity of each candidate aptamer are measured individually in a serial manner. To accelerate aptamer discovery, we devised the Quantitative Parallel Aptamer Selection System (QPASS), which integrates microfluidic selection and nextgeneration sequencing with in situ-synthesized aptamer arrays, enabling simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel. After using QPASS to select aptamers for the human cancer biomarker angiopoietin-2 (Ang2), we in situ synthesized arrays of the selected sequences and obtained equilibrium dissociation constants (K d ) for every aptamer in parallel. We thereby identified over a dozen high-affinity Ang2 aptamers, with K d as low as 20.5 ± 7.3 nM. The same arrays enabled us to quantify binding specificity for these aptamers in parallel by comparing relative binding of differentially labeled target and nontarget proteins, and by measuring their binding affinity directly in complex samples such as undiluted serum. Finally, we show that QPASS offers a compelling avenue for exploring structure−function relationships for large numbers of aptamers in parallel by coupling array-based affinity measurements with next-generation sequencing data to identify nucleotides and motifs within the aptamer that critically affect Ang2 binding.

A Label-Free Aptamer-Based Fluorescent Assay for Cadmium Detection

“…The lake water was first analyzed by inductively coupled plasma mass spectrometry, and no Cd 2+ was detected. After spiking the lake water with Cd 2+ at various concentrations (10,50, and 100 ng/mL), the samples were analyzed using our fluorescence sensor. The recovery rates were between 89.93% and 98.1%, demonstrating the excellent performance of this sensor in practical application.…”

“…Aptamers are single-stranded nucleic acid sequences that are selected via a process known as systematic evolution of ligands by exponential enrichment. They are utilized as molecular recognition elements that can bind various targets with high affinity and specificity [10]. PG is an asymmetric cyanine dye that does not fluoresce when free.…”

Devices and approaches for generating specific high-affinity nucleic acid aptamers

“…For this reason, microarrays have also been used post-selection to screen enriched pools which contain significantly fewer unique sequences and even fewer aptamer candidates, which can be identified through sequencing and chosen based on population metrics such as multiplicity or enrichment. [105][106][107] Aptamers isolated from selections can also be studied and optimized through mutations and assayed on microarrays to identify key features which are critical to the highest affinity aptamer such as the minimal aptamer structure, structure-function relationships, as well as conserved sequence motifs. [107][108][109] The majority of these selection microarrays rely on the imaging and detection of fluorescently labeled molecules (target or library).…”

Exploring the mutational robustness of nucleic acids by searching genotype neighbourhoods in sequence space

“…12 Figure S1). Limited by a lack of high-throughput and parallel experimental technologies, [14][15][16][17][18] an exhaustive search is extremely difficult. Thus, although the problem of finding a functional aptamer in a sequence space has been successfully addressed by SELEX, the greater challenge is to optimize the found aptamers towards better ligand-binding affinity or selectivity, i.e., the inference of local fitness maximum to global fitness maximum in a sequence space, seems unsolvable.…”

“…http://dx.doi.org/10.1101/091389 doi: bioRxiv preprint first posted online Dec. 3, 2016; (Supplementary Text S1). The L-Arm-binding aptamer consists of a stem region (bases 1-7 and 18-24) and a non-canonical region (bases [8][9][10][11][12][13][14][15][16][17], which form the binding pocket 19,[29][30][31] ( Figure 1B). Base C9, which is stacked by a reversed Hoogsteen mismatch pair, A8-C17, and a WatsonCrick pair, G10-C16, forms two hydrogen bonds with L-Arm on its Watson-Crick edge.…”