2-Thiouracil deprived of thiocarbonyl function preferentially base pairs with guanine rather than adenine in RNA and DNA duplexes.

2-Thiouracil-containing nucleosides are essential modified units of natural and synthetic nucleic acids. In particular, the 5-substituted-2-thiouridines (S2Us) present in tRNA play an important role in tuning the translation process through codon-anticodon interactions. The enhanced thermodynamic stability of S2U-containing RNA duplexes and the preferred S2U-A versus S2U-G base pairing are appreciated characteristics of S2U-modified molecular probes. Recently, we have demonstrated that 2-thiouridine (alone or within an RNA chain) is predominantly transformed under oxidative stress conditions to 4-pyrimidinone riboside (H2U) and not to uridine. Due to the important biological functions and various biotechnological applications for sulfur-containing nucleic acids, we compared the thermodynamic stabilities of duplexes containing desulfured products with those of 2-thiouracil-modified RNA and DNA duplexes. Differential scanning calorimetry experiments and theoretical calculations demonstrate that upon 2-thiouracil desulfuration to 4-pyrimidinone, the preferred base pairing of S2U with adenosine is lost, with preferred base pairing with guanosine observed instead. Therefore, biological processes and in vitro assays in which oxidative desulfuration of 2-thiouracil-containing components occurs may be altered. Moreover, we propose that the H2U-G base pair is a suitable model for investigation of the preferred recognition of 3'-G-ending versus A-ending codons by tRNA wobble nucleosides, which may adopt a 4-pyrimidinone-type structural motif.

Desulfuration of 2-thiouridine with hydrogen peroxide in the physiological pH range 6.6-7.6 is pH-dependent and results in two distinct products.

The 2-thiomodified nucleosides, located at first position of tRNAs anticodon, may constitute a primary target for oxidative attack under conditions of oxidative stress. Desulfuration of 2-thiouridine (S2U) was investigated in the (1)H NMR scale in the presence of 100mM H2O2 and phosphate buffer in the physiological pH range, from pH 6.6 to 7.6. The obtained data demonstrate an intriguing result that within one unit of the pH range uridine is the major product of the S2U desulfuration in the pH 7.6, while the 4-pyrimidinone nucleoside (H2U) is dominant in pH 6.6. The possible desulfuration pathway and the biological importance of the transformation of S2U either to U or H2U are discussed in the context of the tRNA oxidative damage.

Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides.

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.

The 2-thiouridine unit in the RNA strand is desulfured predominantly to 4-pyrimidinone nucleoside under in vitro oxidative stress conditions.

The 2-thiouridine (S2U) unit in the RNA strand is predominantly desulfured with H(2)O(2) to 4-pyrimidinone nucleoside (H2U). The resulting H2U-RNA exhibits significantly lower binding affinity to its complementary strand and in certain conditions undergoes strand scission. These results may explain the tRNA loss of biological function in oxidative stress conditions.