Michael Famulok

All you wanted to know about SELEX

In vitro selection, or SELEX, is a technique that allows the simultaneous screening of highly diverse pools of different RNA or DNA (dsDNA or ssDNA) molecules for a particular feature. Different examples from a great variety of applications ofin vitro selection experiments are described and a detailed overview of the method and its variations will be given. Some especially conclusivein vitro selection experiments are discussed in detail to illustrate the potential power and diversity of this method. Potential restrictions of the methods and possible ways to overcome them are pointed out.

Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance.

G proteins are an important class of regulatory switches in all living systems. They are activated by guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP. This activity makes GEFs attractive targets for modulating disease-relevant G-protein-controlled signalling networks. GEF inhibitors are therefore of interest as tools for elucidating the function of these proteins and for therapeutic intervention; however, only one small molecule GEF inhibitor, brefeldin A (BFA), is currently available. Here we used an aptamer displacement screen to identify SecinH3, a small molecule antagonist of cytohesins. The cytohesins are a class of BFA-resistant small GEFs for ADP-ribosylation factors (ARFs), which regulate cytoskeletal organization, integrin activation or integrin signalling. The application of SecinH3 in human liver cells showed that insulin-receptor-complex-associated cytohesins are required for insulin signalling. SecinH3-treated mice show increased expression of gluconeogenic genes, reduced expression of glycolytic, fatty acid and ketone body metabolism genes in the liver, reduced liver glycogen stores, and a compensatory increase in plasma insulin. Thus, cytohesin inhibition results in hepatic insulin resistance. Because insulin resistance is among the earliest pathological changes in type 2 diabetes, our results show the potential of chemical biology for dissecting the molecular pathogenesis of this disease.

The nuclear receptor PPARγ selectively inhibits Th17 differentiation in a T cell–intrinsic fashion and suppresses CNS autoimmunity

T helper cells secreting interleukin (IL)-17 (Th17 cells) play a crucial role in autoimmune diseases like multiple sclerosis (MS). Th17 differentiation, which is induced by a combination of transforming growth factor (TGF)-β/IL-6 or IL-21, requires expression of the transcription factor retinoic acid receptor–related orphan receptor γt (RORγt). We identify the nuclear receptor peroxisome proliferator–activated receptor γ (PPARγ) as a key negative regulator of human and mouse Th17 differentiation. PPARγ activation in CD4+ T cells selectively suppressed Th17 differentiation, but not differentiation into Th1, Th2, or regulatory T cells. Control of Th17 differentiation by PPARγ involved inhibition of TGF-β/IL-6–induced expression of RORγt in T cells. Pharmacologic activation of PPARγ prevented removal of the silencing mediator for retinoid and thyroid hormone receptors corepressor from the RORγt promoter in T cells, thus interfering with RORγt transcription. Both T cell–specific PPARγ knockout and endogenous ligand activation revealed the physiological role of PPARγ for continuous T cell–intrinsic control of Th17 differentiation and development of autoimmunity. Importantly, human CD4+ T cells from healthy controls and MS patients were strongly susceptible to PPARγ-mediated suppression of Th17 differentiation. In summary, we report a PPARγ-mediated T cell–intrinsic molecular mechanism that selectively controls Th17 differentiation in mice and in humans and that is amenable to pharmacologic modulation. We therefore propose that PPARγ represents a promising molecular target for specific immunointervention in Th17-mediated autoimmune diseases such as MS.

A novel RNA motif for neomycin recognition

BACKGROUND Antibiotics can interfere with RNA activity. Translation of RNA by the prokaryotic ribosome, self-splicing of group I introns, HIV replication and hammerhead ribozyme cleavage are inhibited by the aminoglycoside neomycin B. To explore the molecular basis by which small molecules such as antibiotics inhibit RNA function, we undertook an in vitro selection to obtain a variety of RNA molecules with the capacity to recognize neomycin. RESULTS The majority of the RNA molecules selected to specifically bind neomycin share a region of nucleotide sequence homology. From chemical probing and covariations among different clones we show that in all sequences this region folds into a hairpin structure, which from footprinting and partial alkaline hydrolysis experiments is shown to be the neomycin-binding site. Neomycin is recognized with high affinity (Kd approximately equal to 100 nM) and high specificity (> 100-fold higher affinity for neomycin than for paromomycin). CONCLUSIONS The fact that RNAs containing the consensus sequence, as well as sequences that display variations within this region, specifically recognize neomycin suggests that a structural motif rather than a particular nucleotide sequence is required for neomycin recognition. We propose that a hairpin stem-loop structural motif, which might feature a widened major groove, may be a prerequisite for neomycin recognition. This structural pattern can be extrapolated to other natural neomycin-responsive RNAs.

Structural basis of ligand discrimination by two related RNA aptamers resolved by NMR spectroscopy.

In a previous study, an RNA aptamer for the specific recognition of arginine was evolved from a parent sequence that bound citrulline specifically. The two RNAs differ at only 3 positions out of 44. The solution structures of the two aptamers complexed to their cognate amino acids have now been determined by two-dimensional nuclear magnetic resonance spectroscopy. Both aptamers contain two asymmetrical internal loops that are not well ordered in the free RNA but that fold into a compact structure upon ligand binding. Those nucleotides common to both RNAs include a conserved cluster of purine residues, three of which form an uneven plane containing a G:G pair, and two other residues nearly perpendicular to that surface. Two of the three variant nucleotides are stacked on the cluster of purines and form a triple contact to the amino acid side chain, whereas the edge of the third variant nucleotide is capping the binding pocket.

Slit2-Robo4 signalling promotes vascular stability by blocking Arf6 activity.

Slit–Roundabout (Robo) signalling has a well-understood role in axon guidance. Unlike in the nervous system, however, Slit-dependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4–paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2–Robo4–paxillin–GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system.

Protein-dependent ribozymes report molecular interactions in real time

Most approaches to monitoring interactions between biological macromolecules require large amounts of material, rely upon the covalent modification of an interaction partner, or are not amenable to real-time detection. We have developed a generalizable assay system based on interactions between proteins and reporter ribozymes. The assay can be configured in a modular fashion to monitor the presence and concentration of a protein or of molecules that modulate protein function. We report two applications of the assay: screening for a small molecule that disrupts protein binding to its nucleic acid target and screening for protein–protein interactions. We screened a structurally diverse library of antibiotics for small molecules that modulate the activity of HIV-1 Rev-responsive ribozymes by binding to Rev. We identified an inhibitor that subsequently inhibited HIV-1 replication in cells. A simple format switch allowed reliable monitoring of domain-specific interactions between the blood-clotting factor thrombin and its protein partners. The rapid identification of interactions between proteins or of compounds that disrupt such interactions should have substantial utility for the drug-discovery process.

Prion-Protein-Specific Aptamer Reduces PrPSc Formation

The critical initial event in the pathophysiology of transmissible spongiform encephalopathies (TSEs) appears to be the conversion of the cellular prion protein (PrPC) into the abnormal isoform PrPSc. This isoform forms high‐molecular‐weight protease K (PK) resistant aggregates that accumulate in the central nervous system of affected individuals. We have selected nuclease‐resistant 2′‐amino‐2′‐deoxypyrimidine‐modified RNA aptamers which recognize a peptide comprising amino acid residues 90–129 of the human prion protein with high specificity. This domain of prion proteins is thought to be functionally important for the conversion of PrPC into its pathogenic isoform PrPSc and is highly homologous among prion proteins of various species including mouse, hamster, and man. Consequently, aptamer DP7 binds to the full‐length human, mouse, and hamster prion protein. At low concentrations in the growth medium of persistently prion‐infected neuroblastoma cells, aptamer DP7 significantly reduced the relative proportion of de novo synthesized PK‐resistant PrPSc within only 16 h. These findings may open the door towards a rational development of a new class of drugs for the therapy or prophylaxis of prion diseases.

Aptamers for allosteric regulation

Aptamers are useful for allosteric regulation because they are nucleic acid-based structures in which ligand binding induces conformational changes that may alter the function of a connected oligonucleotide at a distant site. Through this approach, a specific input is efficiently converted into an altered output. This property makes these biomolecules ideally suited to function as sensors or switches in biochemical assays or inside living cells. The ability to select oligonucleotide-based recognition elements in vitro in combination with the availability of nucleic acids with enzymatic activity has led to the development of a wide range of engineered allosteric aptasensors and aptazymes. Here, we discuss recent progress in the screening, design and diversity of these conformational switching oligonucleotides. We cover their application in vitro and for regulating gene expression in both prokaryotes and eukaryotes.

Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures

Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19+ Burkitts lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library.