Sol-Gel SELEX Circumventing Chemical Conjugation of Low Molecular Weight Metabolites Discovers Aptamers Selective to Xanthine

Abstract

Sensitive detection of the metabolites indicative of a particular disease contributes to improved therapy outcomes. Developing binding reagents for detection of low molecular weight metabolites is hampered by the difficulty with immobilization of targets through appropriate covalent chemical linkage while ensuring that selected reagents retain specificity to unmodified metabolites. To circumvent chemical modification of targets, we employed sol-gel droplets deposited onto a porous silicon chip to entrap a purine metabolite, xanthine, which was found at lower levels in urine samples from patients with non-Hodgkin lymphoma. By sol-gel SELEX (systematic evolution of ligands by exponential enrichment) against xanthine, specific aptamers (KD ∼ 10 μM) with sensitivity of detection at as low as 1 μM were isolated, which bound to other purine metabolites at more than 100-fold lower affinity. In contrast, we failed to isolate xanthine-specific aptamers when SELEX was performed against xanthine covalently linked to polymer resin. This study demonstrates that the sol-gel platform for entrapping low molecular weight metabolites without chemical modifications can be utilized for SELEX to discover aptamers against clinical metabolite markers for diagnosis application.

New Technologies Provide Quantum Changes in the Scale, Speed, and Success of SELEX Methods and Aptamer Characterization

“…These platforms come in two main types; the target molecule is immobilized on micromagnetic beads 33,34,45,46 or encapsulated in sol-gel, a gel-like porous silica material formed as a result of polymerization of multiple components in solution. 47,48,49 Microfluidic SELEX platforms benefit from miniaturization leading to reduced consumption of reagents used for SELEX and creates a more stringent selection in part due to a reduction in available target molecules, and from fluidics to perform extensive washing. 50 Both platforms were shown to be efficient in generating high affinity aptamers to protein targets in 3–6 rounds of selection, 35,51 but successful selection of low nanomolar affinity aptamers to Botulinum Neurotoxin Type A Light Chain (BoNT/A-rLC) in a single round has also been reported.…”

“…45 Specific formulations of sol-gel were developed for encapsulation of small molecules and successfully used for selection of aptamers. 48 A sol-gel-based microfluidic SELEX platform offers a unique advantage, by enabling selection of aptamers to multiple targets in one experiment (multiplex selection), whereas the current magnetic bead-based microfluidic platforms are not multiplexable; therefore, requires a parallel independent selection for each target.…”

“…It would be interesting to compare side-by-side the selection process and the resulting aptamers obtained from Capture-SELEX, or a SELEX done with a target small molecule immobilized on beads or encapsulated in SolGel, which was shown to be feasible for small molecule aptamer selections. 48 …”

Selection of DNA Aptamers Against Botulinum Neurotoxin E for Development of Fluorescent Aptasensor

“…Target BoNTE and negative control BSA were immobilized on sol–gel and spotted on to different porous silicon chips according to the manufacturer's protocol. The sol–gel SELEX was carried out as described previously with a few modifications (Figure ) . Briefly, the sol–gel chip was equilibrated with the binding buffer (25 mM Tris–HCl, pH 8.0, 100 mM NaCl, 25 mM KCl, 10 mM MgCl 2 ) for 5 min and blocked for 1 h with blocking buffer (binding buffer containing 1 mg/mL BSA and 20 µg/mL yeast tRNA).…”

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

“…106 Filtering aptamers: Sol-gel target immobilization A chip-based technology with similar characteristics of affinity chromatography was developed using sol-gel arrays as a solid support for target immobilization ( Figure 6(b)). [119][120][121] Selections are achieved by trapping target molecules within a small drop of highly porous 3D silica matrix. Nucleic acids are gently flowed across a sol-gel spot allowing them to enter and diffuse within the sol-gel and bind to the target in its native state.…”