Abstract
The chemical synthesis of ribonucleic acids (RNA) with novel chemical modifications is largely driven by the motivation to identify eligible functional probes for the various applications in life sciences. To this end, we have a strong focus on the development of novel fluorinated RNA derivatives that are powerful in NMR spectroscopic analysis of RNA folding and RNA ligand interactions. Here, we report on the synthesis of 2′-SCF3 pyrimidine nucleoside containing oligoribonucleotides and the comprehensive investigation of their structure and base pairing properties. While this modification has a modest impact on thermodynamic stability when it resides in single-stranded regions, it was found to be destabilizing to a surprisingly high extent when located in double helical regions. Our NMR spectroscopic investigations on short single-stranded RNA revealed a strong preference for C2′-endo conformation of the 2′-SCF3 ribose unit. Together with a recent computational study (L. Li, J. W. Szostak, J. Am. Chem. Soc. 2014, 136, 2858–2865) that estimated the extent of destabilization caused by a single C2′-endo nucleotide within a native RNA duplex to amount to 6 kcal mol−1 because of disruption of the planar base pair structure, these findings support the notion that the intrinsic preference for C2′-endo conformation of 2′-SCF3 nucleosides is most likely responsible for the pronounced destabilization of double helices. Importantly, we were able to crystallize 2′-SCF3 modified RNAs and solved their X-ray structures at atomic resolution. Interestingly, the 2′-SCF3 containing nucleosides that were engaged in distinct mismatch arrangements, but also in a standard Watson–Crick base pair, adopted the same C3′-endo ribose conformations as observed in the structure of the unmodified RNA. Likely, strong crystal packing interactions account for this observation. In all structures, the fluorine atoms made surprisingly close contacts to the oxygen atoms of the corresponding pyrimidine nucleobase (O2), and the 2′-SCF3 moieties participated in defined water-bridged hydrogen-bonding networks in the minor groove. All these features allow a rationalization of the structural determinants of the 2′-SCF3 nucleoside modification and correlate them to base pairing properties.
Highlights
Fluorine is hardly encountered in biomolecules and because of this pronounced bioorthogonality, it becomes a highly attractive reporter group
We have explored and rationalized the structural basis of the 2′-SCF3 modification based on various chemical and biophysical methods, including NMR and high-resolution X-ray structure analysis of ribonucleic acids (RNA) that carry the modification at distinct positions and in distinct base pair situations
While the 2′-SCF3 modification has only a minor impact on the thermodynamic stability of an RNA fold when it resides in a single-stranded region, it exerts a surprisingly high degree of destabilization if located in a Watson−Crick base paired helix
Summary
Fluorine is hardly encountered in biomolecules and because of this pronounced bioorthogonality, it becomes a highly attractive reporter group. From structure and dynamics investigations to cellular imaging, have been reported over the past decade.[1−15] Concerning ribonucleic acids (RNA), the potential of fluorine has been explored, mainly relying on labeling patterns with fluorine atoms that were attached at the 5-positions of pyrimidine nucleobases,[8−12] or alternatively, at the ribose 2′-positions along the backbone.[13−15] being powerful, these reporter units rely on a single fluorine atom, and limitations with respect to sensitivity could potentially be encountered. We have originally reported on 2′-trifluoromethylthio-2′deoxy(2′-SCF3) uridine as a potential candidate to achieve RNA trifluoromethylation patterns in a straightforward manner.[16] A first set of NMR spectroscopic applications using this label was significant and diverse.[16] The very preliminary observation that the novel modification, decreased the stability of a double helix to a very significant extent, brings up the questions on the generality of this behavior which is surprising when compared to related derivatives.
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