Reporter ribozymes for real-time analysis of domain-specific interactions in biomolecules: HIV-1 reverse transcriptase and the primer-template complex.

The major task in the postgenome-era is to decipher the function of thousands of new proteins, their involvement in regulatory networks and to check their suitability as pharmaceutical drug targets. For this reason, novel methods are required that facilitate rapid and reliable identification of molecular interactions of complex biological systems compatible with high-throughput screening protocols. Knowledge about the interaction partners, binding affinity, and interacting domains represent the basis for identification of the biological function and discovery of novel inhibitors, modulators, and drug leads of a given protein.[1] Although there are several powerful methods available for detection and quantification of molecular interactions,[2±7] they are often not generally applicable or compatible with high-throughput screening protocols, in real time. Hence, the development of novel, broadly applicable methods, independent of target protein function, is of fundamental importance. We are interested in using ribozymes for developing functional assays that allow the analysis of interactions of biologically relevant molecules in real-time.We have reported a novel system for rapid and reliable measurement of the catalytic activity of the hammerhead ribozyme (HHR) by using substrate oligonucleotides labeled with two fluorescent dyes.[8] The spatial proximity of the two dyes results in fluorescence quenching of the donor fluorophore by fluorescence resonance energy transfer (FRET). Ribozyme cleavage activity can be then monitored by a time-dependent increase of fluorescence in real-time. By using these reporter ribozymes we have identified novel inhibitors of the hammerhead ribozyme[9] and of the HIV-1 Rev protein,[10] which were also able to inhibit the biological function of the target molecule in vivo. Herein, we report the rational design of a reporter ribozyme, which is specifically regulated by HIV-1 reverse transcriptase (HIV-1 RT). We demonstrate that the HIV-1 RT dependent reporter ribozyme is not only capable of selectively detecting the presence of HIV-1 RT but also of sensing the domain-specific interaction of other HIV-1 RT binders such as the primer±template complex. For the construction of the reporter ribozyme we have chosen a strategy similar to that used for previous systems that are regulated by small organic molecules, by inserting an aptamer sequence into stem II of the HHR. Proper folding of stem III is essential for cleavage activity of the HHR.[11±14] We have chosen an aptamer which was selected by Tuerk et al. from a combinatorial RNA-library and which binds HIV-1 RT with an affinity of 25 pm.[15] The crystal structure of the RNA±protein complex shows that the anti-HIV-1 RTaptamer in the complex with HIV-1 RT forms a pseudoknot structure, in which the 5’and 3’-ends of the aptamers are spatially separated.[16] We have deliberately chosen an aptamer with a pseudoknot structure because this motif is often used as a regulatory element in nature. For example, formation of a pseudoknot induces a frameshift in some viral mRNA sequences.[17] In some eukaryotic transcripts, a pseudoknot structure in the 5’untranslated region leads to activation of a regulatory protein, which then locally controls translation of the transcript.[18] Owing to these known structural and regulatory features of pseudoknot motifs the anti-HIV-1 RT aptamer seemed to be well suited as a regulatory element of a hammerhead ribozyme. The aptamer was inserted into stem II of the HHR, as shown in Figure 1a, resulting in a fusion construct FK-1 with competing folds of the ribozyme and the pseudoknot structures. The simultaneous folding of both domains is impossible in this design, because in the absence of HIV-1 RT the inserted aptamer sequence folds into a hairpin loop structure (see Figure 1a, left). As shown in Figure 1b, the reporter ribozyme FK-1 is active in the absence of HIV-1 RT due to the folding of the hairpin loop, forming stem II in FK-1. The presence of an unpaired loop was proven by digestion with ribonucleases specific for single-stranded RNA (Figure 2). In the presence of HIV-1 RT, the catalytic activity of FK-1 is inhibited (Figure 1b) due to the induction of the pseudoknot fold by the protein. This leads to disruption of stem II and, hence, to the inhibition of cleavage activity. To further characterize and verify the influence of the stability of stem II on the capability of structural changes of FK-1, two variants of FK-1 were generated, one with weakened and one with stabilized stem II structures. Deletion of the GC base pair highlighted in gray in Figure la yields construct FK-2 with a destabilized stem II (Figure 1c). The complete absence of catalytic activity of FK-2 (Figure 1d) indicates that the lack of the stabilizing GC base pair diminishes formation of the catalytically active conformation in favor of the pseudoknot fold. Indeed, nuclease digest reactions of FK-2 shown in Figure 2 resulted in cleavage patterns, which are in accordance with the kinetic data in Figure 1d, thus supporting the exclusive formation of the pseudoknot. For further validation of this hypothesis we constructed a third version of the reporter ribozyme, which is only capable of forming the catalytically active fold, but not the pseudoknot structure. Starting from FK-1, an additional GC base pair was inserted, which leads to stabilization of stem II in the construct FK-3 (Figure 1e). Figure 1 f shows, as expected, that FK-3 is catalytically active, with the cleavage activity remaining unchanged even in the presence of HIV-1 RT. Owing to the increased stability of stem II, the protein is no longer able to induce the folding of the pseudoknot. Indeed, the nuclease COMMUNICATIONS