Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme

Figure 2

Selection of APP-SOFA-HDV ribozyme with the greatest potential cleavage. The left portion illustrates the strategy that was used to identify potential cleavage sites. APP mRNA was preincubated in the presence of a 7 nt long randomized DNA oligonucleotide, and RNA/DNA heteroduplexes were hydrolyzed by RNase H. Accessible regions were then visualized by primer extension using one of the four 5′-end-labeled primers (in box) complementary to sequences retrieved in the first ~900 nucleotides of the APP mRNA. Once the most accessible sites were identified, the appropriate SOFA-HDV ribozymes were synthesized and the cleavage activity was tested in vitro using 5′-end-labeled APP mRNA. A typical autoradiogram of a resulting PAGE is indicated in the right panel. The number of each Rz indicates the cleavage position within the APP mRNA. The Rz-HBV, previously used for HBV RNA cleavage [15], served as an irrelevant Rz. “-” indicates a reaction without Rz.