Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion

Abstract

We have systematically investigated the duplex to hairpin conversion of oligoribonucleotides under the aspect of nucleobase methylation. The first part of our study refers to the self-complementary sequence rCGCGAAUUCGCGA, which forms a stable Watson-Crick base paired duplex under various buffer conditions. It is shown that this sequence is forced to adopt a hairpin conformation if one of the central 6 nt is replaced by the corresponding methylated nucleotide, such as 1-methylguanosine N(2),N(2)-dimethylguanosine, N(6),N(6)-dimethyladenosine (m(6)(2)A) or 3-methyluridine. On the other hand, the duplex structure is retained and even stabilized by replacement of a central nucleotide with N(2)-methylguanosine (m(2)G) or N(4)-methylcytidine. A borderline case is represented by N(6)-methyladenosine (m(6)A). Although generally a duplex-preserving modification, our data indicate that m(6)A in specific strand positions and at low strand concentrations is able to effectuate duplex-hairpin conversion. Our studies also include the ssu ribosomal helix 45 sequence motif, rGACCm(2)GGm(6)(2)Am(6)(2)AGGUC. In analogy, it is demonstrated that the tandem m(6)(2)A nucleobases of this oligoribonucleotide prevent duplex formation with complementary strands. Therefore, it can be concluded that nucleobase methylations at the Watson-Crick base pairing site provide the potential not only to modulate but to substantially affect RNA structure by formation of different secondary structure motifs.

Scheme 1. Site-specific methylation of a palindromic duplex. M, N, nucleotides capable of complementary Watson–Crick base pairing; mN, mM, methylated nucleotides. Depending on the kind of methylation the duplex is either converted into a hairpin (A) or the duplex is retained (B). The bold numbers refer to the sequences investigated (compare Table 1).

Scheme 1. Site-specific methylation of a palindromic duplex. M, N, nucleotides capable of complementary Watson–Crick base pairing; mN, mM, methylated nucleotides. Depending on the kind of methylation the duplex is either converted into a hairpin (A) or the duplex is retained (B). The bold numbers refer to the sequences investigated (compare Table 1).

Figure 1

Watson–Crick base pairing pattern of adenine-uracil (A-U) and guanine-cytosine (G-C) (above). Chemical constitution and conformation of methylated nucleotides encountered in cellular RNAs: 1-methylguanosine (m¹G), N²,N²-dimethylguanosine (m²₂G), N⁶,N⁶-dimethyladenosine (m⁶₂A), 3-methyluridine (m³U), 1-methylinosine (m¹I), N²-methylguanosine (m²G), N⁶-methyladenosine (m⁶A) and N⁴-methylcytidine (m⁴C). Preferred conformations of m⁴C, m⁶A and m²G are according to Engel and von Hippel (43).

Figure 2

(A) Melting profiles of rCGCGAAUUCGCGA 3 and rCGCm¹GAAUUCGCGA 4 (3 µM, 1.0 M NaCl, 10 mM Na₂HPO₄, pH 7.0). (B) Dependence of melting transitions on strand concentration c of sequences 3 and 4, (0.15 M or 1.0 M NaCl, 10 mM Na₂HPO₄, pH 7.0): 1/T versus lnc plots. H, hyperchromicity; T, temperature.

Figure 3

Selected ¹H NMR (500 MHz) NH spectra: rCGCGAAUUCGCGA 3 (duplex, assignment according to 42), rCGCm²GAAUUCGCGA 5 (duplex), rCGCm¹GAAUUCGCGA 4 (hairpin) and rCGCm²₂GAAUUCGCGA 6 (hairpin, asterisks indicate signals for competitive duplex formation to a minor extent). Total strand concentrations are as indicated. H₂O/D₂O, 9:1; sodium arsenate buffer, pH 7.4; 25 mM for 3, 5 and 6, 12.5 mM for 4)

Figure 4

(A) Examples of UV melting curves for the sequence rCGCGm⁶AAUUCGCGA 9. Strand concentrations <5 µM result in low hyperchromicites and invariable melting transitions of T_m = 69.5°C, indicating hairpin formation. Higher concentrations yield increased hyperchromicities and a linear 1/T versus lnc correlation (37–313 µM, T_m = 64.2–69.2°C), indicating duplex formation. 150 mM NaCl, 10 mM Na₂HPO₄, pH 7.0. (B) ¹H NMR (500 MHz) NH spectrum of rCGCGm⁶AAUUCGCGA 9 at a concentration of 0.2 mM corroborates formation of a duplex (27°C, 25 mM arsenate buffer, pH 7.4).

Figure 5

UV melting curves of rGACCGGAAGGUC/GACCUUCCGGUC 20 and rGACCm²GGm⁶₂Am⁶₂AGGUC/GACCUUCCGGUC 21. c = 3 µM. 150 mM NaCl, 10 mM Na₂HPO₄, pH 7.0.