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

The binding of oligodeoxynucleotides modified with adenine 2'-O-methyl ribo side, 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 hexadeoxynu cleotides (a generic microchip). The generic microchip was manufactured by using reductive immobilization of aminooligonucleotides in the activated co polymer 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 fluorescen ce distribution at selected temperatures. Up to three substitutions of aden osines 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 deoxyribosi des (D) resulted in increased mismatch discrimination measured at the melti ng temperature of the corresponding perfect duplex. The stability of comple xes formed by 2'-O-methyl-adenosine-modified oligodeoxynucleotides was slig htly decreased with every additional substitution, yielding similar to4 deg reesC of total loss in melting temperature for three modifications, as foll owed from microchip thermal denaturation experiments. 2,6-Diaminopurine 2'- O-methyl riboside modifications led to considerable duplex stabilization. T he cytosine 2'-O-methyl riboside and 5-bromodeoxyuridine modifications gene rally did not change either duplex stability or mismatch resolution. Denatu ration experiments conducted with selected perfect duplexes on microchips a nd in solution showed similar results on thermal stabilities. Some hybridiz ation artifacts were observed that might indicate the formation of parallel DNA.