Journal of Molecular Biology
Regular articleThe RNA i-motif1
Introduction
Protonation of C-rich oligodeoxy and oligoribonucleotides results in the formation of hemiprotonated structures Akinrimisi et al 1963, Hartman and Rich 1965, Michelson et al 1967. The C-rich oligodeoxynucleotides associate into four-stranded structures composed of two parallel-stranded duplexes in a head-to-tail orientation, in which hemiprotonated C·C+ pairs are intercalated in a so-called i-motif Gehring et al 1993, Leroy et al 1993. Only deoxy i-motif structures have been reported. So far, the structure formed by C-rich oligoribonucleotides has not been characterized.
We have investigated by NMR the solution structure of four C-rich RNA sequences: r(UC5), r(C5U), r(C5) and r(C3U). They all associate around pH 4.3 into multimers exhibiting the NMR features commonly observed in DNA i-motif structures. In line with previous investigations Michelson et al 1967, Lacroix et al 1996, Collin and Gehring 1998, we found that they are much less stable than their DNA equivalents. The poor stability of the multimer of r(C3U) and the presence of multiple intercalated forms of r(C5) and r(C5U) prevented further investigations. We thus focused on r(UC5), the NMR spectrum of which allows structural investigations. r(UC5) associates into two different i-motif tetramers. The intercalation topology of the major species, U1 C2 C3 C4 C5 C6 , leaves empty the potential intercalation site between U1 and C2, in contrast to the topology of [d(TC5)]4, which provides maximum intercalation (Gehring et al., 1993).
The i-motif core of [r(UC5)]4 is closely related to that of the DNA structures, but its NMR spectrum exhibits several specific features. In order to characterize the structural changes induced by the 2′-OH groups, we compared the RNA i-motif structure with that of [d(TC2)]4 (Leroy & Guéron, 1995), the monomeric i-motif of a sequence related to the human telomeric sequence, d(CCCTAA5mCCCTAACCCUAACCCT) (Phan et al.,2000) and the crystal structure of [d(C4)]4 (Chen et al.,1994).
The nucleic acid bases have two distinct faces. According to Lavery’s definition (Lavery et al., 1992), the faces of an anti nucleoside oriented towards the 5′ and the 3′-directions are designated as the black and white faces, respectively. In the i-motif, the intercalation in a head-to-tail orientation of two hemiprotonated duplexes brings into mutual contact the black faces of each other and the white faces of each other’s bases, and results in alternating black and white steps. Two topologies maximize the intercalation of the C·C+ pairs. In the 5′ E-topology, the outermost C·C+ pairs are formed by the cytidine residues at the 5′-extremity of each cytidine stretch. In the 3′ E-topology, they are formed by the cytidine residues at the 3′-end.
Section snippets
Results
The 1D proton spectrum of r(UC5) shows three clusters of exchangeable protons at the frequencies of the imino (16–15 ppm), and of the cis (around 9 ppm) and trans amino protons (8.8 to 8 ppm) of hemiprotonated pairs, demonstrating the formation of structures containing hemiprotonated cytidine residues (Figure 1 and Table 1). The presence of two uridine imino proton peaks (11.17 and 10.8 ppm) of unequal intensities discloses the formation of two species in proportions of 75/25. The minor and the
Discussion
The existence of hemiprotonated structures of ribo- and deoxyribocytidine homopolymers was deduced in pioneering investigations based on melting experiments and pH-metric titrations followed by optical methods. For 30 years, it has been assumed that poly(rC) and poly(dC) associate into parallel duplexes at acidic pH. The ultraviolet (UV), optical rotatory dispersion (ORD) and infra red (IR) spectra indicate the presence of hemiprotonated pairs in both oligomers, but cannot distinguish between a
Materials and methods
The oligoribonucleotides were synthesized on the 2 μmol scale with a Pharmacia gene assembler using polystyrene beads for primer support and phenoxyacetyl β-RNA phosphoramidites. The synthesis was performed with 5-ethylthio-1H-tetrazole as activator with the same protocol as for DNA synthesis, except that the coupling time was increased to 20 minutes. The terminal 5′-O-dimethoxytrityl group was removed after synthesis. The oligoribonucleotide was cleaved from the support and the bases were
Acknowledgements
We thank Dr Maurice Guéron for stimulating discussions during this study and help in the preparation of the manuscript. This work was supported by grant 9272 (19 Decembre 1997) from the Association pour la Recherche contre le Cancer.
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