Effects of O^6-Alkyl Guanosine Residues on RNA Duplex Stability

Abstract

Several short oligoribonucleotide sequences containing modified purine residues of biological significance were synthesized using the phosphotriester method developed in Neilson's laboratory. Variable temperature proton nuclear magnetic resonance (NMR) spectroscopy was used to examine the solution conformations of these oligomers. Studies of the effect of position, type and extent of alkyl modifications on helix structure and stability were undertaken. The triribonucleotide GpCpA was the first trimer shown to form a stable RNA duplex (Tm 33°C) (Alkema, et al, 1981(a)). This duplex contained two G:C base pairs and two 3'-dangling adenosine residues, and had a stability equal to that of the tetramer duplex UpGpCpA, having four Watson Crick base pairs. A series of GpCpN trimers was prepared (N= m⁶A, m⁶₂A, m¹G, m⁶G, e⁶G, m²m⁶G), using GpCpA as a reference, to determine how N-or O-alkylation of the dangling residue affected duplex stability. Both the studies of the N-alkylated (N=m⁶A, m⁶₂A, m¹G) (D'Andrea, et al, 1983) and 0-alkylated (N=m⁶G, e⁶G, m²m⁶G) sequences (present work) led to the conclusion that site and degree of modification were important factors for stability. Comparison of N-versus O-alkylated sequences revealed how the hydrophobic regions surrounding the alkylated nitrogen or alkylated oxygen atoms, and the spatial location of these regions, contributed to duplex stability. Examination of the effect of modified guanosine residues within a short squence, was performed through studies on ApGpNpCpU pentamers (N=m⁶G, e⁶G, m²m⁶G), having N in internal non-base-pairing and. in internal base-pairing positions. No duplex formation was seen, in contrast to studies involving reference compounds : ApGpGpCpU (a qualitative reference), ApGpGpCpU : ApGpUpCpU (Tm 31.4 C), and ApGpGpCpU : ApGpCpCpU (Tm 47.0 C). As no melting temperatures could be calculated for the modified strands, and because their NMR analyses were so similar, no comparisions regarding degree of destabilization, could be made amongst the various modified residues. Never the less, it is clear that O-alkylation of the central G residue significantly disrupts duplex formation, through generation of a centre of great instability. This result sharply contrasts that when the same modified residues are located in terminal, non-bonding positions.