NSF Award
9460234
Novel phosphoramidite reagents are proposed which will be used to incorporate gold cluster complexes and combined fluorescent/gold labels into oligonucleotides during automated solid state synthesis. The labels will be synthetically modified to withstand the synthesis and deprotection conditions. Gold and fluorescent/gold conjugated phosphoramidites will be prepared in forms specific for labeling at the 5' end of the oligonucleotide, at the 3' end via derivatization of a controlled pore glass support, and at any intermediate position; the synthesis of an adenosine phosphoramidite labeled in the 2' position is also proposed. The labels will be incorporated into oligonucleotide probes which will be tested in hybridization studies on blots, and in situ electron microscopy hybridization studies. The sensitivity, penetration and resolution of the labeled nucleotides will be evaluated relative to analogous biotinylated probes detected by a secondary probe bearing the same label. It is anticipated that synthetic probes which incorporate the new labels will achieve unprecedented resolution in the electron microscope, and blot sensitivities equal to or better than current radioisotopic or chemiluminescent techniques.
