Two designed adenosine analogs: Synthesis, fluorescence properties and application in studying the U1A protein-U1snRNA complex

The RNA recognition motif (RRM), one of the most common RNA binding domains, contains three highly conserved aromatic amino acids that participate in stacking interactions with RNA bases. In the complex formed between the N-terminal RRM of the U1A protein and stem loop 2 of U1 snRNA, A6 and C7 of stem loop 2 are stacked with one of the highly conserved aromatic amino acids, Phe56. Substitution of Phe56 with Ala resulted in a large destabilization of the complex. Two modified adenosines, A-3CPh and A-4CPh, in which a phenyl group is linked to the adenosine such that it may replace the phenyl group that is eliminated by the Phe56Ala mutation in the complex, were designed. Incorporation of these adenosine analogs into stem loop 2 RNA stabilizes the complex formed with Phe56Ala, but destabilizes the wild type complex. Experiments with other U1A mutant proteins suggest that the 1.8 kcal/mol stabilization of the complex between the Phe56Ala protein and stem loop 2 RNA is due to a specific interaction between the Phe56Ala protein and SL2-A6-4CPh. Because biochemical experiments did not provide direct evidence for whether the proposed structures were attained by the modified complex, molecular dynamics simulations on the modified SL2-A6-4CPh RNA and its complexes with U1A proteins were carried out to investigate the interactions that contribute to the stabilization of the Phe56Ala protein-SL2-A6-4CPh RNA complex. The results from MD simulations suggest that van der Waals interactions, such as the stacking interaction between the tethered phenyl ring and Tyr13 and the hydrophobic contacts between Phe56Ala and the butyl linker might contributed to the observed stabilization. In addition to stabilizing the Phe56Ala-RNA complex, A-3CPh and A-4CPh have interesting fluorescent properties. Although they are not highly fluorescent as monomers, their quantum yields are significantly enhanced in single stranded RNA. This is surprising because usually the quantum yield of fluorophore decreases upon incorporating into RNA. As a result, the quantum yields of A-3CPh and A-4CPh in RNA are higher than those of RNAs containing commonly used fluorescent base analogs. In addition, incorporating these probes into RNA does not change the RNA structure and stability.