Properties of pseudouridine N1 imino protons located in the major groove of an a-form RNA duplex

The exchangeable N1 imino protons of two pseudouridine (Ψ) bases located at adjacent Internal positions within an undecamer RNA duplex (5 AUA-CψψACCUG/3 UAUGAAUGGUC) can report on the environment of the major groove of an A-form double-stranded nucleic acid. The ψN1 Imlno protons of these residues (which are not involved in interstrand Watson-Crick hydrogen bonding) are protected from chemical exchange with the solvent water and thus are observable in the proton NMR spectrum in H₂O (1). These protons will exchange readily at Increased pH values or upon thermal denaturation of the duplex. The longitudinal (T¹) relaxation times of the ψN1 imino protons In 100 mM NaCI or in 10 mM MgCI₂ and 100 mM NaCI are approximately two-fold faster than those of the ψN3 imino protons which are Involved in Watson-Crick base pairing. With the addition of spermkJine, the ψN1 imino protons become readily exchangeable at a temperature some 20°C below the melting temperature of the duplex.