Efficient<i>in vitro</i>synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

TLDR

The study presents a simple, efficient method for synthesizing pure single‑stranded RNAs of virtually any structure. The method uses an SP6 RNA polymerase system with plasmids containing an SP6 promoter upstream of a polylinker, enabling specific transcription and versatile RNA production for downstream applications. Optimal conditions were established, and the resulting high‑specific‑activity RNA probes markedly improve the sensitivity of nucleic acid blotting and solution hybridization assays.

Abstract

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This invitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for invitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).