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.
Fujita
K
K
Laboratory of Molecular Microbiology, NIAID, NIH, Bethesda, MD 20892.
Silver
J
J
D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D001709:Biotechnology; D001710:Biotin; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005069:Evaluation Studies as Topic; D015497:HIV-1 / Q000235:genetics; D008280:Magnetics; D008969:Molecular Sequence Data; D012313:RNA / Q000096:biosynthesis; D019809:Streptavidin; D014158:Transcription, Genetic; D014764:Viral Proteins
D001426:Bacterial Proteins; D004279:DNA, Viral; D014764:Viral Proteins; D012313:RNA; D001710:Biotin; D004247:DNA; D019809:Streptavidin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases