Undecanucleotide dA-dC-dC-dA-Cm-Cm-dG-Cm-dG-Cm-dT (Cm = 2'-O-methylcytidine) was prepared and used as a probe for splitting of 5S rRNA fragment by RNase H (E. coli). It was found that substitution of 2'-deoxycytidine of d(ACCACCGCGCT) by 2'-O-methylcytidine in positions 5, 6, 8 and 10 provided regiospecific splitting of RNA fragment by RNase H in the presence of modified probe between U25 and C26.