Systematic evaluation of biophysical and functional characteristics of selenomethylene-locked nucleic acid-mediated inhibition of miR-21

Abstract

miRNAs constitute an important layer of gene regulation mediated by sequence-specific targeting of mRNAs. Aberrant expression of miRNAs contributes to a host of pathological states. Promoting cancer, miR-21 is upregulated in variety of cancers and promotes tumor progresion by suppressing a network of tumor suppressor genes. Here we describe a novel class of bicyclic RNA analogues, selenomethylene-locked nucleic acid (SeLNA), that display high affinity, improved metabolic stability, and increased potency for miR-21 inhibition. The thermal stability (T_m) for duplexes was increased significantly with incorporation of SeLNA monomers as compared to that of the unmodified DNA–RNA hybrid. A comprehensive thermodynamic profile obtained by isothermal titration calorimetry revealed a favorable increase in the enthalpy of hybridization for SeLNA containing DNA and target RNA heteroduplexes. SeLNA modifications displayed remarkable binding affinity for miR-21 target RNA with a K_a of ≤1.05 × 10⁸ M^–1. We also observed enhanced serum stability for SeLNA–RNA duplexes with a half-life of ≤36 h. These in vitro results were well correlated with the antisense activity in cancer cells imparting up to ~91% inhibition of miR-21. The functional impact of SeLNA modifications on miR-21 inhibition was further gauged by investigating the migration and invasion characterisitics of cancer cells, which were drastically reduced to ~49 and ~55%, respectively, with SeLNA having four such modifications. Our findings demonstrate SeLNA as a promising candidate for therapeutics for disease-associated miRNAs.