Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates

TLDR

The authors present a method that uses T7 RNA polymerase and synthetic DNA templates bearing a T7 promoter to produce defined‑length small RNAs. The approach yields milligram quantities of any 12–35‑nt RNA, with partially single‑stranded templates performing as well as plasmid DNA, producing predictable runoff transcripts and abundant 2–6‑nt abortive products, while promoter variants reduce efficiency but broaden the RNA repertoire.

Abstract

A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the −17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3′ terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.