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 (psi) bases located at adjacent internal positions within an undecamer RNA duplex (5'AUAC-psi-psi-ACCUG/3'UAUGAAUGGUC) can report on the environment of the major groove of an A-form double-stranded nucleic acid. The psi-N1 imino 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 psi-N1 imino protons in 100 mM NaCl or in 10 mM MgCl2 and 100 mM NaCl are approximately two-fold faster than those of the psi-N3 imino protons which are involved in Watson-Crick base pairing. With the addition of spermidine, the psi-N1 imino protons become readily exchangeable at a temperature some 20-degrees-C below the melting temperature of the duplex.