Surprising Base Pairing and Structural Properties of 2′-Trifluoromethylthio-Modified Ribonucleic Acids

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.

Polyfluorinated Groups in Medicinal Chemistry

“…[62]. The synthesis of the 2'-SCF 3 pyrimidine nucleoside containing oligoribonucleotides 76a and 76b ( Figure 5B), has been recently reported [63]. Differently from other 2' modifications such as OCH 3 , OCF 3 or F, which increase or not alter double helix stability, the presence of a SCF 3 substituent in position 2' of oligoribonucleotides seems to have a strong destabilizing impact on the thermodynamic stability of RNA folds when located in a Watson-Crick base paired helix, due to a remarked intrinsic preference for the C2'-endo conformation of 2'-SCF 3 modified nucleosides.…”

19F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR

“…2 0 -Deoxy-2 0 -fluorinated nucleosides are effectively locked in the C3 0 -endo sugar pucker normally associated with A-form RNA geometry (Barchi, Jeong, Siddiqui, & Marquez, 1997;Deleavey & Damha, 2012;Kreutz, Kahlig, Konrat, & Micura, 2005;Reif et al, 1997;Thibaudeau, Plavec, & Chattopadhyaya, 1998). While attractive due to the increased sensitivity associated with three magnetically equivalent 19 F reporters (Fauster, Kreutz, & Micura, 2012), the 2 0 -trifluoromethylthio (2 0 -SCF 3 ) modification proposed by Micura and coworkers decreases the stability of regular A-form helices (Kosutic et al, 2014).…”

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

“…The modification on the ribosyl moiety of nucleotides allows the use of one similar labeling schema for all nucleobases. Using the 2 0 -deoxy-2 0 -trifluoromethylthio (SCF3) unit or other highly fluorinated substitutions can even enhance the sensitivity of 19 F-NMR spectroscopy (Fauster et al 2012;Kiviniemi and Virta 2010;Kosutic et al 2014), in particular for large RNAs. However, uniform labeling of the RNA with 2 0 F-substituted nucleotides can disrupt tertiary interactions that involve the 2 0 OH group and shift the conformational preferences of the ribofuranosyl sugar unit to preferentially adopt the 3 0 -endo conformation and therefore influences the structural behavior (Reif et al 1997).…”