Advances in RNA Labeling with Trifluoromethyl Groups

Abstract

Fluorine labeling of ribonucleic acids (RNA) in conjunction with ¹⁹ F NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and dynamics, and RNA-ligand interactions. This study presents the first syntheses of 2'-OCF₃ guanosine and uridine phosphoramidites, their incorporation into oligoribonucleotides by solid-phase synthesis and a comprehensive study of their properties. NMR spectroscopic analysis showed that the 2'-OCF₃ modification is associated with preferential C2'-endo conformation of the U and G ribose in single-stranded RNA. When paired to the complementary strand, slight destabilization of the duplex caused by the modification was revealed by UV melting curve analysis. Moreover, the power of the 2'-OCF₃ label for NMR spectroscopy is demonstrated by dissecting RNA pseudoknot folding and its binding to a small molecule. Furthermore, the 2'-OCF₃ modification has potential for applications in therapeutic oligonucleotides. To this end, three 2'-OCF₃ modified siRNAs were tested in silencing of the BASP1 gene which indicated enhanced performance for one of them. Importantly, together with earlier work, the present study completes the set of 2'-OCF₃ nucleoside phosphoramidites to all four standard nucleobases (A, U, C, G) and hence enables applications that utilize the favorable properties of the 2'-OCF₃ group without any restrictions in placing the modification into the RNA target sequence.

Keywords: 19F NMR spectroscopy; RNA solid-phase synthesis; nucleoside modifications; oligonucleotides; trifluoromethyl.