Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA

Abstract Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson–Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.


Towards a comprehensive understanding of RNA deamination -Synthesis and properties of xanthosine-modified RNA
. Overview of synthesized RNAs and mass spectrometric analysis. 33 Supporting Table 2. Complete set of thermodynamic data of xanthosine modified RNA. 34 Supporting Table 3. X-ray data collection and crystallographic refinement statistics. 35

1-(2-Iodoethyl)-4-nitrobenzene
Triphenylphosphane (2.09 g, 7.96 mmol, 1.33 eq) and imidazole (0.542 g, 7.96 mmol, 1.33 eq) were dissolved in anhydrous toluene. A solution of iodine (2.02 g, 7.96 mmol, 1.33) eq in toluene (20 mL) was added dropwise and the solution was stirred for 30 minutes. 2-(4-Nitrophenyl)-ethanol (1.00 g, 5.98 mmol, 1 eq) in toluene (20 mL) was added and the reaction mixture was stirred for 3 hours at room temperature. Sat. aqu. sodium bicarbonate solution (20 mL) was added and stirring was continued for 10 minutes. The organic phase was separated and stirred with LiI (1.06 g, 7.96 mmol, 1.33 eq) for another 10 minutes. The solution was extracted once with sat. aqu. sodium thiosulfate and twice with water. The triphenylphosphinoxide can then be directly precipitated from the solution with petroleum ether and filtrated. The solvent is evaporated to dryness and the crude product is purifies via column chromatography (SiO2, 10-15% ethyl acetate in cyclohexane). Yield: 1.40 g of compound 10 as a brown oil (85%). TLC  [c] Errors for DH and DS were determined from at least three independent measurements; in general, errors arising from noninfinite cooperativity of two-state transitions and from the assumption of a temperatureindependent enthalpy, are typically 10−15%. Additional error is introduced when free energies are extrapolated far from melting transitions; errors for DG are typically 3−5%.
Supporting Table 3. X-ray data collection and crystallographic refinement statistics.  Supporting Figure 8. UV-melting profile analysis of RNA duplex IIc at pH 7 and pH 6.
Supporting Figure 9. UV-melting profile analysis of RNA duplexes IIc at pH 5 and IId at pH 7.
Supporting Figure 10. UV-melting profile analysis of RNA duplex IId at pH 6 and pH 5.
Supporting Figure 11. UV-melting profile analysis of RNA duplex IIe at pH 7 and pH 6.
Supporting Figure 12. UV-melting profile analysis of RNA duplex IIe at pH 5.
Supporting Figure 13. UV-melting profile analysis of RNA hairpin IIIa at pH 7.
Supporting Figure 14. UV-melting profile analysis of RNA hairpin IIIb at pH 7.
Supporting Figure 15. UV-melting profile analysis of RNA hairpin IIIc at pH 7.