DNA duplexes with reactive dialdehyde groups as novel reagents for cross-linking to restriction-modification enzymes

Abstract

To create new, effective reagents for affinity modification of restriction-modification (R-M) enzymes, a regio-selective method for reactive dialdehyde group incorporation into oligonucleotides, based on insertion of a 1-β-D-galactopyranosylthymine residue, has been developed. We synthesized DNA duplex analogs of the substrates of the EcoRII and Mval R-M enzymes that contained a galactose or periodate-oxidized galactose residue as single substituents either in the center of the EcoRII (Mval) 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.EcoRII 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 EcoRII or Mval methylases was 9-20% and did not exceed 4% for R.EcoRII.