Functional characterization of the SOFAdeltaribozyme

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Abstract
Molecular engineering has led to the development of a novel target-dependent riboswitch that increases δribozyme fidelity. This δ ribozyme possesses a specificon/offadapter (SOFA) that switches the cleavage activity fromoff(a “safety lock”) toonsolely in the presence of the desired RNA substrate. In this report, we investigate the influence of both the structure and the sequence of each domain of the SOFA module. Analysis of the cleavage activity, using a large collection of substrates and SOFA-ribozyme mutants, together with RNase H probing provided several insights into the nature of the sequence and the optimal design of each domain of the SOFA module. For example, we determined that (1) the optimal size of the blocker sequence, which keeps the ribozymeoffin the absence of the substrate, is 4 nucleotides (nt); (2) a single nucleotide difference between the substrate and the biosensor domain, which is responsible for the initial binding of the substrate that subsequently switches the SOFA-ribozymeon, is sufficient to cause nonrecognition of the appropriate substrate; (3) the stabilizer, which joins the 5′ and 3′ ends of the SOFA-ribozyme, plays only a structural role; and (4) the optimal spacer sequence, which serves to separate the binding regions of the biosensor and catalytic domain of the ribozyme on the substrate, is from 1 to 5 nt long. Together, these data should facilitate the design of more efficient SOFA-ribozymes with significant potential for many applications in gene-inactivation systems.

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