A LOW-MOLECULAR-WEIGHT ARTIFICIAL RNA OF UNIQUE SIZE WITH MULTIPLE PROBE TARGET REGIONS

Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously bee n shown to be expressed from a plasmid borne growth rate regulated pro moter in E. coli. These aRNAs accumulate to high levels and their dete ction is a promising tool for studies in molecular microbial ecology a nd in environmental monitoring. Herein a new construct is described wh ich illustrates the versatility of detection that is possible with aRN As. This 3xPen aRNA construct carries a 72 nucleotide insert with thre e copies of a unique 17 base probe target sequence. This aRNA is 160 n ucleotides in length and again accumulates to high levels in the E. co li cytoplasm without incorporating into ribosomes. The 3xPen aRNA illu strates two improvements in detection. First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence , easily quantifiable peak, on a high resolution gel profile of low mo lecular weight RNAs. Second, the existence of multiple probe targets r esults in a nearly commensurate increase in signal when detection is b y hybridization. These aRNAs are naturally amplified and carry sequenc e segments that are not found in known rRNA sequences. It thus may be possible to detect them directly. An experimental step involving RT-PC R or PCR amplification of the gene could therefore be avoided.