Synthesis and Hybridization Properties of RNA Oligomers Containing Biologically Relevant 5-Aminomethyl-2-thiouridines...

2-Thiouridines with C5-aminomethyl substituents (nm⁵S²U, mnm⁵S²U, cmnm⁵S²U, τm⁵S²U and inm⁵S²U, here denoted as xnm⁵S²U) are widespread at the wobble position (position 34 or first anticodon letter) of several bacterial and mitochondrial tRNAs. Wobble xnm⁵S²Us are known to recognize purines at the third position of weak and intermediate codons (5'NNA3 and 5'NNG3). Their absence is associated with bacterial virulence defects, mitochondrial dysfunction and human diseases. Previous data suggested that mnm5 in bacterial mnm⁵S²U34-tRNAs facilitates base pairing with G3 and decreases base pairing with A3. The geometry of mnm⁵S²U 34-G3 was solved in the crystal mRNA-tRNA-ribosome complex indicating the unique mnm5-mediated zwitterionic nature of wobble mnm⁵S²U.

In this work we investigate the hybridization and structural properties of a representative group of naturally existing xnm5-2-thiouridines by thermodenaturation and circular dichroism studies of xnm⁵S²U-containing RNA duplexes.

Our results demonstrate the general tendency of xnm5-substituents to decrease the stability of xnm⁵S²U-A duplexes. A correlation between an increase abundance of ionized xnm⁵S²U and decreased base pairing specificity with A was found, confirming that the zwitterionic form of xnm⁵S²U facilitates the formation of xnm⁵S²U-G pair at physiological conditions. In addition, we report the first post-synthetic strategy for xnm⁵S²U-RNA synthesis, applicable also to Cy3-labeled oligomers.

Our findings suggest that the biological importance of xnm⁵S²Us relates to the tunning of mRNA codons reading through enhanced specificity of interactions with G3.