Functional Characterization of Ribozymes Expressed Using U1 and T7 Vectors for the Intracellular Cleavage of ANF mRNA

Abstract

Hammerhead ribozymes targeted to various GUC or GUA sites on rat atrial 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. In vitro, ribozyme-mediated cleavage was highly Mg(2+)-dependent, and at concentrations approaching those found intracellularly, the rate but not the extent of cleavage was markedly reduced. To test for cellular activity, synthetic genes encoding the ribozymes were cloned between the initiation and termination sequences of the U1snRNA gene or between the T7RNA polymerase promoter and terminator sequences in pSP64. Both constructs had defined initiation and termination sequences to minimize transcript size and for message stability. In vitro the addition of T7 or 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 with an expression plasmid containing a rat ANF cDNA into COS-1 cells using a liposome method, which provided high-level transfection efficiency. Quantitation of ANF mRNA by RNase protection showed marked decreases in ANF transcript levels with both the U1- and the T7-expressed ribozymes directed at three of the four sites on ANF mRNA. With all constructs, 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 still bind produced less cleavage than the corresponding wild-type ribozyme construct. These findings indicate that both U1 and T7 vector systems provide efficient ribozyme expression for the intracellular cleavage of target mRNA.