Cyano-modification on Uridine Decrease the Base Pairing Stability and Specificity through Neighbouring Disruption in RNA Duplex.

5-Cyanomethyluridine (cnm(5)U) and 5-cyanouridine (cn(5)U), the two uridine analogues, were synthesized and incorporated into RNA oligonucleotides. Base-pairing stability and specificity studies in RNA duplexes indicated that cnm(5)U slightly decreased the stability of the duplex but retained the base-pairing preference. In contrast, cn(5)U dramatically decreased both base-pairing stability and specificity between U:A and other noncanonical U:G, U:U, and U:C pairs. In addition, the cn(5)U:G pair was found to be stronger than the cn(5)U:A pair and the other mismatched pairs in the context of a RNA duplex; this implied that cn(5)U might slightly prefer to recognize G over A. Our mechanistic studies by molecular simulations showed that the cn(5)U modification did not directly affect the base pairing of the parent nucleotide; instead, it weakened the neighboring base pair in the 5 ' side of the modification in the RNA duplexes. Consistent with the simulation data, replacing the Watson-Crick A:U pair to a mismatched C:U pair in the 5 '-neighboring site did not affect the overall stability of the duplex. Our work reveals the significance of the electron-withdrawing cyano group in natural tRNA systems and provides two novel building blocks for constructing RNA-based therapeutics.