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

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.