Properties of a U1/mRNA 5′ Splice Site Duplex Containing Pseudouridine As Measured by Thermodynamic and NMR Methods

Three RNA undecamers, ⁵AUACΨψACCUG (ψ = pseudouridine), ⁵AUACUUACCUG, and their complementary 11-mer ^5′CAGGUAAGUAU, have been chemically synthesized by phosphite triester chemistry on a controlled-pore glass (CPG) support. The two duplexes formed with these molecules, ⁵AUACΨψACCUG/⁵CAGGUAAGUAU and ⁵AUACUUACCUG/⁵CAGGUAAGUAU, represent the 5′ end of human U1 snRNA paired to the mRNA consensus 5′ splice site. In one undecamer, pseudouridines are incorporated at those positions corresponding to the native in vivo U1 snRNA, while the other (control) undecamer contains only uridine. Surprisingly, the NMR data show that the extra imino proton of the pseudouridines, which is found in the major groove and is presumably not hydrogen bonded, is clearly visible in the imino proton NMR spectrum at pH 6. This result suggests that the structure of the RNA restricts access of solvent to the major groove, slowing the exchange of the pseudouridine NH1 imino proton. A comparison of the thermodynamic properties of the two duplexes show that the free energy of duplex formation is unchanged by the substitution of pseudouridine for uridine.