Alteration of hairpin ribozyme specificity utilizing PCR.
We have developed a method by which a researcher can quickly alter the specificity of a trans hairpin ribozyme. Utilizing this PCR method, two oligonucleotides, and any target vector, new ribozyme template sequences can be generated without the synthesis of longer oligonucleotides. We have produced templates with altered specificity for both standard and modified (larger) ribozymes. After transcription, these ribozymes show specific cleavage activity with the new substrate beta-glucuronidase (GUS), and no activity against the original substrate (HIV-1, 5' leader sequence). Utilizing this technique, it is also possible to produce an inactive ribozyme that can be used as an antisense control. Applications of this procedure would provide a rapid and economical system for the assessment of trans ribozyme activity.
DeGrandis
P
P
Department of Biological Science, Northern Illinois University, DeKalb 60115, USA.
Hampel
A
A
Galasinski
S
S
Borneman
J
J
Siwkowski
A
A
Altschuler
M
M
D001483:Base Sequence; D017931:DNA Primers; D005966:Glucuronidase / Q000096:biosynthesis; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000378:metabolism; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D021382:Protein Sorting Signals; D016337:RNA, Catalytic; D005966:Glucuronidase