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Using typical experimental parameters (tip radius good quantitative agreement suggests that tip convolution effects (i.e., the effective tip radius) are smaller than expected for tip radii extracted from calibration measurements as described in Materials and Methods. Taken Selleckchem Alpelisib together, the results of AFM measurements confirmed and complemented the conclusions drawn from the gel electrophoretic analysis. While smaller fragments are dominating for CRZ-2, a tendency toward larger constructs is seen for PBD1, and for PBD4 a majority of rather complex see more structures is detected (Fig. 6). Comparing the outcome of the AFM analysis for PBD4 with the gel shown in Figure 2, these complex structures are either cyclic species of varying lengths or folded concatemers, since they most likely correspond to the smear of bands in the area of d (Fig. 2, lane 4, cf. also Fig. 3D; Supplemental Fig. S4d), and thus show an anomalous migration behavior as typically observed for cyclic or folded RNA species (Grabowski et al. 1984; Sigurdsson and Eckstein 1996). We obtain clear results from contour-length measurements counting relative amounts of monomers, dimers, and trimers (Table 3). Regardless of whether we compare ratios of theoretically expected peaks only, or include the peaks corresponding to partly unresolved molecules, we observe more dimeric and trimeric species for our newly designed species PBD1 and PBD4, which is in agreement with our design objective. Furthermore, PBD1 tends to form dimer species, again ADAMTS12 in agreement with our design goal, while PBD4, which was theoretically optimized to form cyclic monomers, shows the highest multimer variety both on PAA gel electrophoresis and AFM imaging. To summarize, imaging ribozymes on the single-molecule level using AFM provides information that complements results obtained from the gel electrophoretic analysis and the computation analysis (and vice versa), making a combination of these techniques promising and very powerful. Our study revealed that (i) self-processing activity can be programmed into RNA structures, (ii) self-processing activity can be predicted and optimized by computer-aided design, and (iii) AFM turned out to be a powerful technique to image the reaction products at the single molecule level, even for short RNAs (