Selection of Fluorinated Aptamer Targeting RNA Element with Different Chirality

Abstract

The development of RNA aptamers with high specificity and affinity for target molecules is a critical advancement in the field of therapeutic and diagnostic applications. This study presents the selection of a 2’-fluoro modified mirror-image RNA aptamer through the in vitro SELEX process. Using a random RNA library, we performed iterative rounds of selection and amplification to enrich aptamers that bind specifically to the viral frameshift element which contains the opposite chirality. The unnatural chirality of the aptamer improved its enzymatic stability, and the incorporation of 2’-fluoro modifications was crucial in enhancing the binding affinity of the aptamers. After nine rounds of SELEX, the enriched RNA pool was cloned, sequenced, and analyzed, revealing a dominant aptamer sequence. The selected 2’-fluoro modified mirror-image RNA aptamer demonstrated a dissociation constant of approximately 1.6 μM, indicating moderate binding affinity with the target and exceptional stability against nuclease degradation. Our findings highlight the potential of 2’-fluoro modified mirror-image RNA aptamers in enhancing the stability and utility of RNA-based therapeutics and diagnostics, paving the way for future applications in diverse biomedical fields.