AIDSWEEKLY Plus; Monday, December 8, 2003
Staff Medical Writers

In a recent study from the United States, "A fluorescence resonance energy transfer assay readily applicable to 96-well and 384-well microplate formats with robotic operation was developed to enable high-throughput screening for inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT)-associated RNase H activity, an underexplored target for antiretroviral development."

"The assay substrate is an 18-nucleotide 3'-fluorescein-labeled RNA annealed to a complementary 18-nucleotide 5'-Dabcyl- modified DNA," explained M.A. Parniak and coauthors at the University of Pittsburgh.

"The intact duplex has an extremely low background fluorescent signal and provides up to 50-fold fluorescent signal enhancement following hydrolysis," they said. "The size and sequence of the duplex are such that HIV-1 RT-RNase H cuts the RNA strand close to the 3' end. The fluorescein-labeled ribonucleotide fragment readily dissociates from the complementary DNA at room temperature with immediate generation of a fluorescent signal."

"This assay is rapid, inexpensive, and robust, providing Z' factors of 0.8 and coefficients of variation of about 5%," according to the report. "The assay can be carried out both in real-time (continuous) and in 'quench' modes; the latter requires only two addition steps with no washing and is thus suitable for robotic operation."

"Several chemical libraries totaling more than 106,000 compounds were screened with this assay in approximately one month," the researchers concluded.

Parniak and colleagues published their study in Analytical Biochemistry (A fluorescence-based high-throughput screening assay for inhibitors of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H activity. Anal Biochem, 2003;322(1):33-39).

For additional information, contact M.A. Parniak, University of Pittsburgh, Department of Medicine, 930 Scaife Hall, Pittsburgh, PA 15261, USA.

Publisher contact information for the journal Analytical Biochemistry is: Academic Press Inc., Elsevier Science, 525 B St., Ste. 1900, San Diego, CA 92101-4495, USA.

The information in this article comes under the major subject areas of AIDS & HIV, Biotechnology and Pharmaceutical & Drug Development.

This article was prepared by AIDS Weekly editors from staff and other reports.

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