Phosphoramidite building blocks of ribostamycin (3 and 4), that may be incorporated at any position of the oligonucleotide sequence, were synthesized. The building blocks, together with a previously described neomycin-modified solid support, were applied for the preparation of aminoglycoside-2′-O-methyl oligoribonucleotide fusions. The fusions were used to clamp a single strand DNA sequence (a purine-rich strand of c-Myc promoter 1) to form triple helical 2′-O-methyl RNA/DNA-hybrid constructs. The potential of the aminoglycoside moieties to stabilize the triple helical constructs were studied by UV-melting profile analysis.