Click-Particle Display 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 base-modified 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 ∼107 per hour. Our “click-particle display (PD)” strategy offers many advantages. First, almost any chemical modification can be introduced with a commercially available polymerase. Second, click-PD can screen vast numbers of individual aptamers on the basis of quantitative on- and off-target binding measurements to simultaneously achieve high affinity and specificity. Finally, the increasing availability of FACS instrumentation in academia and industry allows for easy adoption of click-PD in a broader scientific community. 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 (Con A). 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 Con A aptamer exhibited exceptional specificity, with minimal binding to other structurally similar lectins. Finally, we show that our aptamer has remarkable biological activity. Indeed, 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.…”

Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides

“…Expanding on these alkyne handles, a useful scheme introduced by Mayers’ group [ 89 ] realized modified nucleic acids nicknamed “clickmers” as scaffolds to which almost any azide-bearing functional group can be conjugated to an alkyne-modified (at C5 site) dU via facile copper-catalyzed azide-alkyne cycloaddition (CuAAC) or “click chemistry” [ 90 ]. These modular nucleic acid templates can be easily adapted with large chemical moieties such as long carbohydrate chains and polycyclic compounds that are normally incompatible with polymerase-mediated evolution methods [ 90 , 91 ]. Though a glycan chemical modification, for example, is identical at every dU nucleotide, Krauss’s group postulates that composition diversity in the random glycosylated sequence library itself yields structural diversity in how glycan groups are clustered within self-folded glycosylated oligonucleotides to enable a tighter fit to their HIV antibody target [ 92 , 93 ].…”

Dynamic changes in DNA populations revealed by split–combine selection

“…7,8 Recently, we and others established a so-called click-SELEX procedure, in which Cu(I)-catalyzed azide alkynecoupling (CuAAC) is used to introduce chemical modications into DNA libraries at the C5-position of uridine or the 2 0 -position of the deoxyribose position. [4][5][6]9,10 Aptamers obtained by click-SELEX are termed clickmers. Here, we extended this approach towards a split-combine procedure enabling the simultaneous screening of ve or even more different modications, permitting the rapid identication of chemical modi-cations that best mediate binding to a target molecule.…”

“…Subsequently, the ve libraries were mixed at equimolar concentrations and subjected to a split-combine click-SELEX procedure (Scheme 1b). 9,15 Aer incubation, separation and the recovery of bound species, the single-stranded DNA of the amplied library was produced and divided into ve samples (Scheme 1b, split). Each of them was modied with one of the ve azides, separately, and aer pooling (Scheme 1b, combine), this library was subjected to the next selection cycle.…”