Polymer supported synthesis of aminooxyalkylated oligonucleotides, and some applications in the fabrication of microarrays

A new protocol has been described for solid phase preparation of 3′- and 5′-aminooxylalkylated oligonucleotides using commercially available reagents. This involves attachment of linker 4 either with an LCAA-CPG support via succinoylation followed by synthesis (3′-aminooxyalkylated oligomers) or formation of its phosphoramidite 6 followed by coupling with desired oligomer (for generating 5′-aminooxyalkylated oligomers). Both the routes produced modified oligonucleotides in sufficiently high yields and purity (on HPLC) via conventional oligonucleotide synthesis on an automated synthesizer and deprotection step using aqueous ammonia (16 h, 60 °C). Aminooxyalkylated oligonucleotides were used to construct microarrays on glass surface (biochips). The performance of the biochips was evaluated by immobilizing modified oligonucleotides on epoxylated glass microslides under different sets of conditions with respect to pH, temperature and time. Further, the constructed microarrays were successfully used for detection of nucleotide mismatches and bacterial typhoid.

A simple and efficient protocol for solid phase synthesis of 3′- and 5′-aminooxyalkyl-oligonucleotides has been described. The strategy involves the attachment of linker 4 either with an LCAA-CPG followed by synthesis or formation of its phosphoramidite followed by coupling with desired oligomer. Modified oligomers were produced in high yield following the standard deprotection condition. Subsequently, these oligonucleotides were employed for construction of oligonucleotide microarrays on epoxylated glass surface, which resulted in the excellent immobilization and hybridization efficiencies. The constructed microarray was then successfully used for detection of bacterial typhoid.Figure optionsDownload as PowerPoint slide