Binding specificity and stability of duplexes formed by modified oligonucleotides with a 4096-hexanucleotide microarray.

Abstract

The binding of oligodeoxynucleotides modified with adenine 2'-O-methyl riboside, 2,6-diaminopurine 2'-O-methyl riboside, cytosine 2'-O-methyl riboside, 2,6-diaminopurine deoxyriboside or 5-bromodeoxyuridine was studied with a microarray containing all possible (4096) polyacrylamide-bound hexadeoxynucleotides (a generic microchip). The generic microchip was manufactured by using reductive immobilization of aminooligonucleotides in the activated copolymer of acrylamide, bis-acrylamide and N-(2,2-dimethoxyethyl) acrylamide. The binding of the fluorescently labeled modified octanucleotides to the array was analyzed with the use of both melting profiles and the fluorescence distribution at selected temperatures. Up to three substitutions of adenosines in the octamer sequence by adenine 2'-O-methyl ribosides (A(m)), 2,6-diaminopurine 2'-O-methyl ribosides (D(m)) or 2,6-diaminopurine deoxyribosides (D) resulted in increased mismatch discrimination measured at the melting temperature of the corresponding perfect duplex. The stability of complexes formed by 2'-O-methyl-adenosine-modified oligodeoxynucleotides was slightly decreased with every additional substitution, yielding approximately 4 degrees C of total loss in melting temperature for three modifications, as followed from microchip thermal denaturation experiments. 2,6-Diaminopurine 2'-O-methyl riboside modifications led to considerable duplex stabilization. The cytosine 2'-O-methyl riboside and 5-bromodeoxyuridine modifications generally did not change either duplex stability or mismatch resolution. Denaturation experiments conducted with selected perfect duplexes on microchips and in solution showed similar results on thermal stabilities. Some hybridization artifacts were observed that might indicate the formation of parallel DNA.