Homogeneous detection of nucleic acids by transient-state polarized fluorescence

We describe a transient-state polarized fluorescence-based method for detecting nucleic acids. An active ester of the phthalocyanine dye La Jolla Blue was coupled to an oligonucleotide containing an amino group at its 5' end, and the conjugate was purified by HPLC chromatography. We monitored the hybridization characteristics of the conjugate with complementary oligonucleotides and RNA as targets by transient-state polarized fluorescence measurements. The method was comparable in sensitivity to isotopic and nonisotopic heterogeneous detection systems and was capable of detecting 1 fmol of a 382-base-long RNA transcript from human immunodeficiency virus type (HIV-1) generated in a self-sustained sequence replication (3SR) reaction.