Michael Blind

Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers.

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine–nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine–nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

Aptamer Selection Technology and Recent Advances

Over the last decade, aptamers have begun to find their way from basic research to diverse commercial applications. The development of diagnostics is even more widespread than clinical applications because aptamers do not have to be extensively modified to enhance their in vivo stability and pharmacokinetics in diagnostic assays. The increasing attention has propelled the technical progress of the in vitro selection technology (SELEX) to enhance the efficiency of developing aptamers for commercially interesting targets. This review highlights recent progress in the technical steps of a SELEX experiment with a focus on high-throughput next-generation sequencing and bioinformatics. Achievements have been made in the optimization of aptamer libraries, separation schemes, amplification of the selected libraries and the identification of aptamer sequences from enriched libraries.

Aptamers that bind to the antibiotic moenomycin A

Nuclease-resistant moenomycin-binding aptamers with dissociation constants in the range of 300 to 400 nM have been selected. Competition experiments have demonstrated that these aptamers recognize a disaccharide analogue of moenomycin. The results offer the opportunity of setting up a selective and sensitive assay for identifying moenomycin biosynthetic precursors.

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

An RNA Aptamer that Induces Transcription

We identified an RNA aptamer that induces TetR-controlled gene expression in Escherichia coli when expressed in the cell. The aptamer was found by a combined approach of in vitro selection for TetR binding and in vivo screening for TetR induction. The smallest active aptamer folds into a stem-loop with an internal loop interrupting the stem. Mutational analysis in vivo and in-line probing in vitro reveal this loop to be the protein binding site. The TetR-inducing activity of the aptamer directly correlates with its stability and the best construct is as efficient as the natural inducer tetracycline. Because of its small size, high induction efficiency, and the stability of the TetR aptamer under in vivo conditions, it is well suited to be an alternative RNA-based inducer of TetR-controlled gene expression.

Using Aptamers as Capture Reagents in Bead-Based Assay Systems for Diagnostics and Hit Identification

Most applications of xMAP™ (Luminex®) bead-based assay technology in diagnostics and drug discovery use immobilized antigens or antibodies. Here the authors describe the development of novel assay systems in which synthetic oligonucleotides that specifically bind and inhibit other biomolecules—so-called aptamers—are directly immobilized on beads. The robustness, specificity, and sensitivity of aptamer-based assays were demonstrated in a test system that detected human α-thrombin in serum samples. xMAP technology was also adapted to competitive screening formats where an aptamer/protein complex was disrupted by a functionally analogous competitor. The results indicate that such assays are excellently suited for diagnostic applications or drug screening, where aptamers serve as competitive binding probes for the identification of small-molecule hits. These methods should be transferable to a large number of applications because specific aptamers can be rapidly generated for almost any protein target.

Kinetic Binding Analysis of Aptamers Targeting HIV-1 Proteins by a Combination of a Microbalance Array and Mass Spectrometry (MAMS)

An enhanced chip-based detection platform was developed by integrating a surface acoustic wave biosensor of the Love-wave type with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). The system was applied to characterize the interaction of aptamers with their cognate HIV-1 proteins. The aptamers, which target two proteins of HIV-1, were identified using an automated in vitro selection platform. For aptamers S66A-C6 and S68B-C5, which target the V3 loop of the HIV-1 envelope protein gp120, KD values of 406 and 791 nM, respectively, were measured. Aptamer S69A-C15 was shown to bind HIV-1 reverse transcriptase (HIV-1 RT) with a KD value of 637 nM when immobilized on the biosensor surface. HIV-1 RT was identified with high significance using MALDI-ToF MS even in crude protein mixtures. The V3-loop of gp120 could be directly identified when using chip-bound purified protein samples. From crude protein mixtures, it could be identified indirectly with high significance via its fusion-partner glutathione-S-transferase (GST). Our data show that the combination of the selectivity of aptamers with a sensitive detection by MS enables the reliable and quantitative analysis of kinetic binding events of protein solutions in real time.

From selection to caged aptamers: Identification of light-dependent ssDNA aptamers targeting cytohesin

Caged aptamers represent valuable tools for the spatiotemporal control of protein function by light. Here we describe a general route starting with the de novo selection process targeting cytohesin-1 and aiming at the synthesis of caged aptamers without the prior knowledge of detailed structural determinants of aptamer-target binding.

A RNA-based approach towards small molecule inhibitors

The identification of low-molecular-weight protein inhibitors for functional studies as well as for the generation of lead drug compounds is one of the most important tasks in chemical biology. We describe a generalized assay that utilizes inhibitory aptamers for the identification of small molecules with aptamer-inherited properties. These compounds share the functional profile of the aptamers and thus are considered as potent protein inhibitors.

Positive Modulation of the Glycine Receptor by Means of Glycine Receptor–Binding Aptamers

According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl− channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyRα1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyRα1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyRα1 and/or GlyRα1β. This aptamer potentiated whole-cell Cl− currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.