The effect of the sulphur atom on the uracil ring was analyzed in different DNA:RNA microhelixes with three nucleotide base‐pairs, including uridine, 2‐thiouridine, 4‐thiouridine, 2,4‐dithiouridine, cytidine, adenosine and guanosine. Distinct backbone and helical parameters were optimized at different density functional (DFT) levels. The Watson‐Crick pair with 2‐thiouridine appears weaker than with uridine, but its interaction with water molecules appears easier. Two types of microhelixes were found, depending on the H‐bond of H2′ hydroxyl atom: A‐type appears with the ribose ring in 3E‐envelope C3′‐endo, and B‐type in 2E‐envelope C2′‐endo. B‐type is less common but it is more stable and with higher dipole‐moment. The sulphur atoms significantly increase the dipole‐moment of the microhelix, as well as the rise and propeller twist parameters. Simulations with four Na atoms H‐bonded to the phosphate groups, and further hydration with explicit water molecules were carried out. A re‐definition of the numerical value calculation of several base‐pair and base‐stacking parameters is suggested.