Surprising lability of biotin-streptavidin bond during transcription of biotinylated DNA bound to paramagnetic streptavidin beads.

We investigated the use of immobilized DNA templates as substrates for bacteriophage RNA polymerases in order to develop a simple method for separating template DNA from synthesized RNA. Double-stranded DNA molecules with a T7 or T3 RNA polymerase promoter at one end and a single biotin moiety at the other end were attached to streptavidin-coated paramagnetic beads and used in transcription reactions. When the biotin was attached by a nucleotide base on the nontemplate strand, the DNA-bead complex was moderately stable and could be used for multiple rounds of RNA synthesis. However, when the biotin was attached through a phosphodiester bond on the template strand, the enzymatic activity of RNA polymerase reversibly dissociated up to 80% of biotinylated DNA from the streptavidin beads. Biotinylated DNA bound to streptavidin beads in this system with a binding constant on the order of 10(12) M-1. These results stress the need for careful evaluation of solid phase adaptations of standard solution reactions in molecular biology.