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

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.

Bridged cyclic oligoribonucleotides--towards models for codon-anticodon pairing.

Only three base pairs make up for stable double helices of regular A-type if both helix ends are bridged by flexible non-nucleotide linkers. These cyclic oligoribonucleotides are used as model systems for codon-anticodon pairing in order to reveal base stacking effects arising from structurally relevant bases in the direct neighbourhood of the core triplet duplex.

Flexible non-nucleotide linkers as loop replacements in short double helical RNAs.

Ethylene glycol oligomers have been studied systematically as non-nucleotide loop replacements in short hairpin oligoribonucleotides. Structural optimization concerns the length of the linkers and is based on the thermodynamic stabilities of the corresponding duplexes. The optimum linker is derived from heptakis (ethylene glycol) provided that the duplex end to be bridged comprises solely the terminal base pair; the optimum linker is derived from hexakis(ethylene glycol) if a dangling unpaired nucleotide is incorporated into the loop. Moreover, these linkers have been compared to other commonly used linker types which consist of repeating units of tris- or tetrakis(ethylene glycol) phosphate, or of 3-hydroxypropane-1-phosphate. In all cases, the correlation between linker length and duplex stability is independent of the kind of counter ions used (Na(+), Na(+)/Mg(2+), K(+)or Li(+)). Furthermore, all duplexes with non-nucleotide loop replacements are less stable than those with the corresponding standard nucleotide loop. The results corroborate that the linkers are solvent-exposed and do not specifically interfere with the terminal nucleotides at the bridged duplex end.

Pyranosyl-RNA: chiroselective self-assembly of base sequences by ligative oligomerization of tetranucleotide-2',3'-cyclophosphates (with a commentary concerning the origin of biomolecular homochirality).

BACKGROUND: Why did Nature choose furanosyl-RNA and not pyranosyl-RNA as her molecular genetic system? An experimental approach to this problem is the systematic comparison of the two isomeric oligonucleotide systems with respect to the chemical properties that are fundamental to the biological role of RNA, such as base pairing and nonenzymic replication. Pyranosyl-RNA has been found to be not only a stronger, but also a more selective pairing system than natural RNA; both form hairpin structures with comparable ease. Base sequences of pyranosyl-RNA can be copied by template-controlled replicative ligation of short activated oligomers (e.g. tetramer-2',3'-cyclophosphates) under mild and potentially natural conditions. The copying proceeds with high regioselectivity as well as chiroselectivity: homochiral template sequences mediate the formation of the correct (4'-->2')-phosphodiester junction between homochiral tetramer units provided they have the same sense of chirality as the template. How could homochiral template sequences assemble themselves in the first place? RESULTS: Higher oligomers of pyranosyl-RNA can self-assemble in dilute solutions under mild conditions by ligative oligomerization of tetramer-2',3'-cyclophosphates containing hemi self-complementary base sequences. The only side reaction that effectively competes with ligation is hydrolytic deactivation of 2',3'-cyclophosphate end groups. The ligation reaction is highly chiroselective; it is slower by at least two orders of magnitude when one of the (D)-ribopyranosyl units of a homochiral (D)-tetramer-2',3'-cyclophosphate is replaced by a corresponding (L)-unit, except when the (L)-unit is at the 4' end of the tetramer and carries a purine, when the oligomerization rate can be approximately 10% of that shown for a homochiral isomer. The oligomerization of homochiral tetramers is not, or only weakly, inhibited by the presence of the non-oligomerizing diastereomers. CONCLUSIONS: Available data on the chiroselective self-directed oligomerization of tetramer-2',3'-cyclophosphates allow us to extrapolate that sets of tetramers with different but mutually fitting base sequences can be expected to co-oligomerize stochastically and generate sequence libraries consisting of predominantly homochiral (D)- and (L)-oligomers, starting from the racemic mixture of tetramers containing all possible diastereomers. Such a capability of an oligonucleotide system deserves special attention in the context of the problem of the origin of biomolecular homochirality: breaking molecular mirror symmetry by de-racemization is an intrinsic property of such a system whenever the constitutional complexity of the products of co-oligomerization exceeds a critical level.