Polymerase activities and RNA structures in the atomic force microscope

The structures of the reaction products are the basis for novel polymerase assays using the atomic force microscope (AFM). Polymerases are the enzymes involved in transcription and replication of DNA Rapid semiquantitative es timates of the activity of DNA polymerases such as Sequenase, Tag polymeras e, and AMV reverse transcriptase and RNA polymerases (RNAP) such as Escheri chia coli RNAP were obtained from AFM images of the nucleic acids after pol ymerase reactions. DNA polymerases were assayed via replication of the sing le-stranded phi X-174 virion. RNAP was assayed via transcription, using a r olling circle DNA template that produces long strands of RNA. In some cases , AFM was better than agarose gel electrophoresis for assaying DNA polymera se activity, since aggregation prevented the DNA from entering the agarose gel. Extended molecules of single-stranded RNA synthesized with the rolling circle DNA template showed varied conformations and degrees of stretching. Some structural differences were observed between two RNAs-a ribozyme conc atamer and an RNA with 90% purines. (C) 1999 Academic Press.