The thermodynamic stability of RNA duplexes and hairpins containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines.

The N6-alkyladenosines and 2-methylthio-N6-alkyladenosines make up over half of the population of all naturally modified adenosines and they are present in the transfer ribonucleic acids (tRNA) at position 37. We measured effects of N6-alkyladenosines and 2-methylthio-N6-alkyladenosines on the thermodynamic stability of RNA duplexes containing a U-A(Mod) base pair at internal and terminal duplex positions, as well as containing modified adenosines as a 3'-terminal unpaired nucleotide. Beside naturally modified adenosines such as N6-isopentenyladenosine (i6A), N6-methyladenosine (m6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A) and 2-methylthio-N6-methyladenosine (ms2m6A), we studied several artificial modifications to evaluate the steric and electronic effects of N6-alkyl substituents. Moreover, some N6-alkyladenosines and 2-methylthio-N6-alkyladenosines were placed in hairpins at positions corresponding to nucleotide 37 of the tRNA anticodon arm, and the thermodynamic stability of those hairpins was studied. The stability of the modified RNA hairpins was measured in standard melting buffer containing 1 M sodium chloride as well as in physiological buffer containing 10 mM magnesium chloride and 150 mM potassium chloride. The results obtained indicate that the nature of the adenosine modification and the position of U-A(Mod) base pairs within the duplex influence the thermodynamic stability of RNA duplexes. For most of the modification, the destabilization of duplexes was observed. Moreover, we found that the buffer composition and the structure of the modified adenosine very significantly affect the thermodynamic stability of RNA.