To create new, effective reagents for affinity modification of restriction-modification (R-M) enzymes, a regioselective method for reactive dialdehyde group incorporation into oligonucleotides, based on insertion of a 1-beta-D-galactopyranosylthymine residue, has been developed. We synthesized DNA duplex analogs of the substrates of the Eco RII and Mva I R-M enzymes that contained a galactose or periodate-oxidized galactose residue as single substituents either in the center of the Eco RII (Mva I) recognition site or in the flanking nucleotide sequence. The dependence of binding, cleavage and methylation of these substrate analogs on the modified sugar location in the duplex was determined. Cross-linking of the reagents to the enzymes under different conditions was examined. M. Eco RII covalent attachment to periodate-oxidized substrate analogs proceeded in a specific way and to a large extent depended on the location of the reactive dialdehyde group in the substrate. The yield of covalent attachment to a DNA duplex with a dialdehyde group in the flanking sequence with Eco RII or Mva I methylases was 9-20% and did not exceed 4% for R. Eco RII.