High-Throughput Discovery of Aptamers for Sandwich Assays

Abstract

Sandwich assays are among the most powerful tools in molecular detection. These assays use "pairs" of affinity reagents so that the detection signal is generated only when both reagents bind simultaneously to different sites on the target molecule, enabling highly sensitive and specific measurements in complex samples. Thus, the capability to efficiently screen affinity reagent pairs at a high throughput is critical. In this work, we describe an experimental strategy for screening "aptamer pairs" at a throughput of 10 aptamer pairs per hour-which is many orders of magnitude higher than the current state of the art. The key step in our process is the conversion of solution-phase aptamers into "aptamer particles" such that we can directly measure the simultaneous binding of multiple aptamers to a target protein based on fluorescence signals and sort individual particles harboring aptamer pairs via the fluorescence-activated cell-sorter instrument. As proof of principle, we successfully isolated a high-quality DNA aptamer pair for plasminogen activator inhibitor 1 (PAI-1). Within only two rounds of screening, we discovered DNA aptamer pairs with low-nanomolar sensitivity in dilute serum and excellent specificity with minimal off-target binding even to closely related proteins such as PAI-2.

Microfluidic methods for aptamer selection and characterization

“…MMS was first demonstrated using the protein streptavidin as the target, and later applied to platelet-derived growth factor-BB to obtain three aptamers with K d ’s below 3 nM. 75 The process has also been incorporated into special selections, such as obtaining orthogonal aptamer pairs for sandwich assays, 76 aptamers with a long binding lifetime, 77 and self-reporting aptamer biosensors. 78…”

Applications of flow cytometry sorting in the pharmaceutical industry: A review

“…Aptamers are an appealing alternative to antibodies because they are chemically synthesized and renewable, 86 but they have limited applications due to major limitations such as degradation, limited duration of action, and cross‐reactivity 87 . Csdordas et al 88 developed a particle display method to screen for aptamers pairs for sandwich assays. This procedure allowed for the simultaneous measurement via fluorescence for both aptamer pairs and was conducted to successfully screen an aptamer pair for plasminogen activator inhibitor‐1.…”

Pseudo aptamer expands aptamer’s applications

“…The major interaction forces in the binding process are defined to be hydrogen bond and charge-charge interaction that are provided from native phosphodiester polymeric linker and the bases on loops. [27][28][29][30] The concept of aptamer ligands binding to proteins was reported early 1980s for the study of human immunodeficiency virus (HIV) and adenovirus. 31,32 In this investigation it had shown that the viruses have a high force of affinity and specificity for the encoding of small numbers of RNAs that bind to viruses or cellular proteins.…”