Fluorine-substituted base analogues have proven invaluable as “nonpolar nucleoside isosteres” to probe the physical forces that govern the stabilities of nucleic acids. When paired against natural bases, fluorinated analogues destabilize DNA and RNA helices and exhibit little binding sequence specificity. 5] These observations make Watson–Crick base pairing involving hydrogen bonds to fluorine unlikely. When paired opposite one another, however, a considerable degree of stability is regained, and a selective pairing of fluorinated bases in the context of nucleic acids is observed. Weak C F···H C dipolar interactions have been implicated as acting as stabilizing forces in this case. Apparently, the role of fluorine in molecular recognition strongly depends on the surrounding molecular environment. Similar effects have been observed in the fields of medicinal chemistry and protein design, in which the fluorophilicity/fluorophobicity of the protein environment affects the affinity of fluorine-substituted ligands or the stabilizing influence of fluorine-containing artificial amino acids. With the goal of addressing the influence of the environment on the molecular recognition thermodynamics of organic fluorine, we have undertaken a combined experimental/computational study of fluo ACHTUNGTRENNUNGrobenzene self-pairing in the context of duplex RNA. We report here the first systematic study of the determinants of the surprising stability of fluorobenzene-based self-pairs with increasing fluorine-substitution. Motivated by preliminary modeling results, we synthesized novel ribonucleoside analogues in which the nucleobases are replaced by benzene or fluorine-substituted benzenes, respectively 13] (Scheme 1 and in the Supporting Information). The modified nucleosides were tested in a defined 12-mer RNA duplex (5’-CUU UUC XUU CUU paired with 3’-GAA AAG YAA GAA). The nucleoside analogues were introduced at positions X and Y, respectively, to form a base pair in the duplex. We anticipated that this supramolecular system should be particularly apt for investigation of the molecular recognition properties of organic fluorine. Here we focus on results obtained for homo-self-pairs (that is, positions X and Y were occupied by the same nucleotide) of 1–5. The 2,4,6-trifluorobenzene-substituted nucleoside analogue and the pentafluorinated species were omitted, as steric effects due to bis-ortho substitution result in large destabilization. Likewise, we restrict ourselves to the homologous set of benzene derivatives 1–5 instead of also considering, for example, indoleor benzimidazole-based base analogues. That way we can minimize any influence due to variation in shape or size of the base analogues or stacking interactions (see also below) on the observed stabilities. The CD spectra of the RNA duplexes with the modified bases follow the typical curves for an A-type helix (Figure S2 in the Supporting Information). Thus, the structure of the duplex RNA is not disturbed by incorporation of our modified nucleosides, in agreement with previous findings. The thermodynamic stabilities of the modified RNA duplexes were determined by thermal denaturation as monitored by UV absorbance in a phosphate buffer (20 mm, pH 7.0) containing NaCl (140 mm). The thermodynamic data were extracted from the melting curves by means of a two-state model for the transition from duplex to single strand. Not unexpectedly, our measurements demonstrate that the pairing preference of fluorinated bases is higher in selfpairs (Figure 1; Table 1) than in pairs with natural bases. In both cases, the stability increases incrementally with the number of fluorine substituents in the base analogue, with the largest gain in stability observed in the first two fluorination steps (1!2 : DDG=1.7 kcalmol , 2!3 : 1.4 kcalmol ). Surprisingly, this leads to RNA duplex stabilities with self-paired bases 3, 4, and 5 (11.6, 11.8 and 12.2 kcalmol , respectively) that are similar to or exceed that of the natural AU base pair (11.9 kcalmol ). In stark contrast, in the case of a 12-mer DNA double helix, the presence of two self-pairs of 5 bases resulted in an overall destabilization of the duplex by 4.6 kcalmol 1 compared to the natural AT base pairs, and the stability increase observed on going from two self-pairs of 1 bases to two self-pairs of 5 bases is much less pronounced (DDG=0.6 kcalmol ). What is the molecular origin of the stepwise stability increase and the unexpected overall stability in the RNA case? To address this question, we performed 10 ns molecular dynamics (MD) simulations and free energy calculations together with a structural component analysis for RNA duplexes containing homo-self-pairs of 1–5, including solvent and consider[a] H. Kopitz, Prof. Dr. H. Gohlke Pharmazeutisches Institut Christian-Albrechts-Universit t zu Kiel Gutenbergstr. 76, 24118 Kiel (Germany) Fax: (+49)431-880-1352 E-mail : gohlke@pharmazie.uni-kiel.de [b] Dr. A. Živkovic, Prof. Dr. J. W. Engels Fachbereich Biochemie, Chemie und Pharmazie, Goethe-Universit t Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. Structures of the base analogues that form self pairs. R is always the ribosephosphate moiety. 1=benzene; 2=4-fluorobenzene; 3=2,4-difluorobenzene; 4=2,4,5-trifluorobenzene; 5=2,3,4,5-tetrafluorobenzene.
Search from 250 M+ research papersSearch from 250 M+ research papers
Apr 1, 2012
Matthew William Kellinger + 3
Open Access