Click-PD: A Quantitative Method for Base-Modified Aptamer Discovery

Abstract

Base-modified aptamers that incorporate non-natural chemical moieties can achieve greatly improved affinity and specificity relative to natural DNA or RNA aptamers. However, conventional methods for generating base-modified aptamers require considerable expertise and resources. In this work, we have accelerated and generalized the process of generating basemodified aptamers by combining a click-chemistry strategy with a fluorescence-activated cell sorting (FACS)-based screening methodology that measures the affinity and specificity of individual aptamers at a throughput of ~10^7 per hour. Our "click-PD" strategy offers many advantages. First, almost any chemical modification can be introduced with a commerciallyavailable polymerase. Second, click-PD can screen vast numbers of individual aptamers based on quantitative on-and off-target binding measurements to simultaneously achieve high affinity and specificity. Finally, it requires minimal specialized equipment or reagents besides a FACS instrument which is now widely-available. Using click-PD, we generated a boronic acid-modified aptamer with ~1 μM affinity for epinephrine, a target for which no aptamer has been reported to date. We subsequently generated a mannose-modified aptamer with nanomolar affinity for the lectin concanavalin A (ConA). The strong affinity of both aptamers is fundamentally dependent upon the presence of chemical modifications, and we show that their removal essentially eliminates aptamer binding. Importantly, our ConA aptamer exhibited exceptional specificity, with minimal binding to other structurally-similar lectins. Finally, we show that our aptamer remarkable biological activity. Indeed, to the best of our knowledge, this aptamer is the most potent inhibitor of Con A-mediated hemagglutination reported to date.

Colorimetric Technique for Antimony Detection Based on the Use of Gold Nanoparticles Conjugated with Poly-A Oligonucleotide

“…However, in some cases, the transition from a change in properties occurs abruptly, when saturation with the analyte leads to a loss of system stability. The same characterization of dependence was observed by Gordon et al [37]. Figure 4 clearly demonstrates that sharp changes of optical density can be formally described by four-parametric equation.…”