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High Throughput Screening Assays for Bacterial Primases
Author(s) -
Griep Mark A.,
Koepsell Scott A.,
Hinrichs Steven H.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a510-d
Subject(s) - primase , primer (cosmetics) , high throughput screening , dna , computational biology , biology , dna replication , microbiology and biotechnology , biochemistry , chemistry , rna , gene , reverse transcriptase , organic chemistry
Bacterial primase is a DNA‐dependent RNA polymerase that functions at the replication fork to create short oligoribonucleotide polymers. Knockout and conditional mutant experiments have indicated that primase is an essential replication protein in three different bacteria. The essentiality of primase makes it a good target for novel antibiotic development. Since the bacterial primase has many features that differ from the eukaryotic primase, it should be possible to discover inhibitors that are specific to the bacterial primase and that don’t cause side effects with the eukaryotic one. In order to screen libraries of compounds, we needed to develop a high throughput screening assay and a faster method to characterize the primase activity. For many years, the most common assay for measuring primase activity was to incorporate radiolabeled nucleotides into the primer, separate the different lengths of polymers by electrophoresis, and use various imaging methods to characterize them. Recently, one company created a high throughput screening method from this by using scintillation proximity beads instead of electrophoresis. That assay suffers from some correctible design flaws but we sought to develop other methods. One of the assays that we designed uses denaturing high‐pressure liquid chromatography to separate the RNA polymers according to length and sequence. It is much faster than the radioactive method and allows detailed analysis of product lengths and concentrations. The other assay that we have developed is a microplate‐based fluorescence assay that does not use radioactivity and is adaptable to robotic screening methods.