Pan-selective aptamers for the family of small GTPases.

The members of the large family of small-GTP-binding proteins (GTPases) function as molecular switches and are involved in regulation of a wide variety of cell processing events through signal cascades. Their molecular masses typically range between 20–40 kDa. Small GTPases cycle between an inactive GDP-bound conformation and an active GTP-bound state. Only in their active conformation can GTPases interact with effector proteins to induce downstream signalling events. The cycling is regulated by guanine nucleotide exchange factors (GEFs) that induce the release of bound GDP to be replaced by GTP, and by GTPase-activating proteins (GAPs) that accelerate the GTPase activity of small GTPases. The Ras superfamily is structurally classified into at least five families : Ras, Rho, Rab, Sar1/Arf and Ran. The 36 Ras family members are key mediators of extracellular signal transduction and regulate multiple downstream effects, mainly by modulation of gene expression. They also directly or indirectly regulate cell proliferation, differentiation, and survival. Rho proteins, the second branch of the Ras superfamily comprising at least 20 members, also serve as key regulators of extracellular-stimuli-transducers that mainly direct actin reorganisation, cell-cycle progression and gene expression and thus are involved in cancer progression. The Rab family consists of 61 members and is known to regulate intracellular vesicle transport and the trafficking of proteins between different organelles of the endocytic and secretory pathways. They are involved in vesicle formation, budding, transport, fusion and release events. In humans, 27 genes for Arf family proteins have been found. The Arf family is involved mainly in vesicle budding, but also in endocytic recycling and cytoskeletal reorganization. Ran is the most frequent small GTPase in the cell, with only one protein in this family in humans. It is better understood for its role in nucleo-cytoplasmatic transport of both RNA and protein. Furthermore, it regulates mitotic spindle formation, DNA replication and nuclear envelope assembly. Many human diseases are related to small GTPases such as some cancers and immune and neurological ailments. All small GTPases share an ~20 kDa conserved G domain that contains a set of GDP/GTP-binding motifs (G1– G5). Although there has been much progress in understanding the biological roles of small GTPases, many unsolved questions remain, especially regarding their control and downstream effector functions. This highlights the necessity for specific inhibitory molecules targeting small GTPases and their application as tools for investigating and understanding the molecular mechanism of small GTPases in greater detail. GTPase-binding small molecules have been described, among them the compounds NSC23766, EHT1864, “arabinose-derived compound 2”, 6-thio-GTP, or the Secin series, which inhibit GEF activity and thereby effector functions of the small GTPases. An alternative strategy is the generation of aptamers that target small GTPases directly. This strategy provides rapid access to molecular inhibitors based on folded nucleic acids (“intramers”) that can be expressed intracellularly to analyze the consequences of target inhibition in an in vivo context. So far, aptamers have been described that either target specific regions of the GTPase or that bind to mutants representing a stabilized conformation of the molecule. Recently, an aptamer that targets active GTP bound Cdc42 has been reported. Less closely related GTPases have been targeted by RNA aptamers equally infrequently : we have described RNA aptamers that bind the bacterial elongation factor SelB, and one example describes the isolation of an aptamer for a G protein-coupled receptor. Given the huge number of family members of the GTPase superfamily, the lack of identified aptamers is surprising and efforts to find new sequences able to bind GTPases are desirable. Here we describe the identification of two RNA aptamers that bind different family members of the large Ras superfamily of small GTPases. We show that these two related sequences selectively bind to a huge variety of small GTPases without targeting non-GTPase proteins, thereby exhibiting pan-selectivity towards small-GTP-binding proteins. These RNA-aptamers were identified serendipitously during a selection experiment that targeted the RhoGEF domain of the GEF Vav1. In addition to Vav1-binding aptamers that will be described elsewhere, the enriched RNA library contained at least two orphan sequences that did not bind Vav1, but exhibited an unexpected specificity for members of the Ras superfamily of small GTPases. We termed these sequences V63 and V88, respectively. As shown in Figure 1A, V63 and V88 share some conserved sequences (shadowed in magenta), but based on their calculated mfold secondary structures, a common folding motif does not become apparent. Due to their interesting binding behaviour, we decided to further analyse these aptamers in greater detail. In an initial analysis we conducted a series of filter-retention analyses using at least one member of the five known subfamilies Ras (KRas, RalA), Ran, Rab (Rab1, Rab5c), Rho (Rac1, RhoA, Cdc42) and Arf (Arf1, Arf6) as binding partners for radiolabelled V63 and V88, respectively. In addition, several non-GTPase proteins were used as negative con[a] M. Hons, Dr. B. Niebel, N. Karnowski, B. Weiche, Prof. Dr. M. Famulok Life and Medical Sciences (LIMES) Institute Chemical Biology & Medicinal Chemistry Unit, University of Bonn Gerhard-Domagk-Strasse 1,53121 Bonn (Germany) E-mail : [email protected] [b] M. Hons Present address: Research Group of 3D Electron Cryomicroscopy Max Planck Institute of Biophysical Chemistry 37077 Gçttingen (Germany) [] These authors contributed equally to this work