FUNCTIONAL-CHARACTERIZATION OF RIBOZYMES EXPRESSED USING U1 AND T7 VECTORS FOR THE INTRACELLULAR CLEAVAGE OF ANF MESSENGER-RNA

Hammerhead ribozymes targeted to various GUC or GUA sites on rat atria l natriuretic factor (ANF) mRNA were developed. The catalytic activity of ribozymes to four of these sites, synthesized by transcription off synthetic oligodeoxynucleotide duplexes, was studied in detail. Invit ro, ribozyme-mediated cleavage was highly Mg2+-dependent, anti at conc entrations approaching those found intracellularly, the rate but not t he extent of cleavage was markedly reduced. To test for cellular activ ity, synthetic genes encoding the ribozymes were cloned between the in itiation and termination sequences of the U1snRNA gene or between the T7RNA polymerase promoter and terminator sequences in pSP64. Both cons tructs had defined initiation and termination sequences to minimize tr anscript size and for message stability. In vitro the addition of T7 o r U1 terminator sequences had variable effects on catalytic activity, presumably due to structural interactions between the ribozyme and the added sequence. The ribozyme-encoding plasmids were cotransfected wit h an expression plasmid containing a rat ANF cDNA into COS-1 cells usi ng a liposome method, which provided high level transfection efficienc y. Quantitation of ANF mRNA by RNase protection showed marked decrease s in ANF transcript levels with both the U1- and the T7-expressed ribo zymes directed at three of the four sites on ANF mRNA. With all constr ucts, target accessibility, determined in vitro, was a more important determinant of intracellular ANF mRNA cleavage than catalytic activity per se. ANF mRNA cleavage was not merely due to an antisense effect, since a mutant construct that was catalytically inactive but could sti ll bind produced less cleavage than the corresponding wild-type ribozy me construct. These findings indicate that both U1 and T7 vector syste ms provide efficient ribozyme expression for the intracellular cleavag e of target mRNA.