Polymerase activities and RNA structures in the atomic force microscope.

Abstract

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 estimates of the activity of DNA polymerases such as Sequenase, Taq polymerase, and AMV reverse transcriptase and RNA polymerases (RNAP) such as Escherichia coli RNAP were obtained from AFM images of the nucleic acids after polymerase reactions. DNA polymerases were assayed via replication of the single-stranded φX-174 virion. RNAP was assayed via transcription, using a rolling circle DNA template that produces long strands of RNA. In some cases, AFM was better than agarose gel electrophoresis for assaying DNA polymerase 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 concatamer and an RNA with 90% purines.