Integration of an Expression Platform in the SELEX Cycle to Select DNA Aptamer Binding to a Disease Biomarker

[Image: see text] Aptamers can be developed for biosensors, diagnostic tools, and therapeutic reagents. These applications usually require a fusion of aptamers and expression platforms. However, the fusion process is usually time-consuming and laborious. In this study, we integrated the deoxyribozyme (I-R3) as an expression platform in the SELEX cycle (called Expression-SELEX) to select aptazymes that can sense diverse molecules. We used the Maple syrup urine disease (MSUD) biomarker L-allo-isoleucine to test the selection model. After five rounds of screening, the cleavage products were sufficiently enriched to be visualized on polyacrylamide gel electrophoresis (PAGE) gel. Through high-throughput sequencing analysis, several candidates were identified. One such candidate, IR3-I-DNA, binds L-allo-isoleucine with a dissociation constant (K(D)) of 0.57 mM. When the ligand was present, the cleavage fraction of IR3-I-DNA increased from 0.3 to 0.5, and its K(obs) value improved from 1.38 min(–1) to 1.97 min(–1). Our selection approach can also be applied to produce aptazymes that can bind to variable ligands and be used more directly as biosensors.