Circularization enhances RNA aptamer binding and Stability: Evidence from in-cell NMR

RNA aptamers are emerging as promising molecular probes due to their high specificity and low immunogenicity. However, their clinical potential is often limited by instability under physiological conditions, primarily due to exonucleolytic degradation and structural flexibility. To address these challenges in a model system, we designed a circular RNA aptamer targeting the HIV-1 Tat protein. Enzymatic circularization of the aptamer was performed using T4 RNA ligase, and circularization was confirmed by mobility shift assays and RNase R digestion. Binding affinity was assessed via filter dot blot assay, while structural stability was evaluated using 1D imino proton NMR and CLEANEX-PM experiments. Intracellular stability was monitored using in-cell NMR following transfection into HeLa cells. In our study, the circular aptamer showed approximately tenfold higher binding affinity compared to its linear counterpart, as determined by filter binding assay. NMR analysis indicated improved structural rigidity, preservation of native base pairing, and reduced solvent exchange. Notably, in-cell NMR revealed that the circular aptamer remained detectable up to 18 h post-transfection, whereas the linear aptamer degraded within a few hours. In this system, circularization substantially improved the binding performance and intracellular persistence of the RNA aptamer. These findings demonstrate the feasibility of applying RNA circularization to enhance aptamer functionality in living cells and lay the groundwork for future therapeutic exploration.