ALTERATION OF HAIRPIN RIBOZYME SPECIFICITY UTILIZING PCR

We have developed a method by which a researcher can quickly alter the specificity of a trams hairpin ribozyme. Utilizing this PCR method, t wo oligonucleotides, and any target vector, new ribozyme template sequ ences can be generated without the synthesis of longer oligonucleotide s. We have produced templates with altered specificity for both standa rd and modified (larger) ribozymes. After transcription, these ribozym es show specific cleavage activity with the new substrate beta-glucuro nidase (GUS), and no activity against the original substrate (HIV-1, 5 ' leader sequence). Utilizing this technique, it is also possible to p roduce an inactive ribozyme that can be used as an antisense control. Applications of this procedure would provide a rapid and economical sy stem for the assessment of trans ribozyme activity.