Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors

Abstract

Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and selectivity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose, or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a non-specific organometallic receptor. The method is general and enables broad clinical use of aptamers for detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects.

Fluorescent single-stranded DNA-based assay for detecting unchelated Gadolinium(III) ions in aqueous solution

“…The study was carried out using the strategy [20,22] depicted in Fig. 1a, where the clone is tagged with a fluorescein molecule on the 5’ terminus.…”

“…The in vitro selection process [22] was largely followed with slight modification to the strand sequences. In brief, the oligonucleotides used for SELEX are as follows:

a random library with 22N bases: 5’-GGAGGCTCTCGGGACGAC- N 2 -GGATTTTCC-N 20 -GTGTCCCGATGCTGCAATCGTAAGAAT-3’

biotinylated immobilizing strand: 5’-GTCGTCCCGAGAGCCATA-BioTEG-3’

forward primer: 5’-GGAGGCTCTCGGGACGAC-3’

reverse primer: 5’-ATTCTTACGATTGCAGCATC-3’

biotinylated reverse primer: 5’-biotin-ATTCTTACGATTGCAGCATC-3’

Throughout all rounds of SELEX, the mole ratio of library to immobilizing strand was kept constant at 1:5, respectively.…”

“…The overall K d for the binding between Ln-aptamer and Gd(III) is derived following the equation K d = ([ Ln-aptamer ][Gd(III)])/[ Ln-aptamer -Gd(III)] [22]. This in turn, is calculated by K d, eff1 /K d, eff2 where K d, eff1 = ([ Ln-aptamer ][QS])/[ Ln-aptamer -QS] and K d, eff2 = ([ Ln-aptamer -Gd(III)][QS])/([ Ln-aptamer -QS][Gd(III)]).…”

“…Encouraged by an earlier published work on the development of single stranded DNA molecule (aptamer) that can bind to Zinc(II) [20], we decided to focus our attention on developing an aptamer-based assay for detecting Gd(III) ion. Through a well-established selection and amplification protocol of modified SELEX (Systematic Evolution of Ligands by EXponential enrichment) [20–22], a 44-base long aptamer was isolated. Although the strand is not specific for Gd(III) over other lanthanides, it does not react with many biologically relevant metal ions and its low detection limit allows for its use to detect Gd(III) in GBCA solutions, where no other lanthanides are present.…”

“…The strand ( Ln-aptamer ) was subsequently developed into a fluorescent sensor for the Gd(III) ion using the strategy depicted in Fig. 1a [20,22]. The selectivity of Ln-aptamer was tested against several metal ions, ligands and complexes.…”