RNA Aptamers Recognizing Murine CCL17 Inhibit T Cell Chemotaxis and Reduce Contact Hypersensitivity In Vivo.

The chemokine CCL17, mainly produced by dendritic cells (DCs) in the immune system, is involved in the pathogenesis of various inflammatory diseases. As a ligand of CCR4, CCL17 induces chemotaxis and facilitates T cell-DC interactions. We report the identification of two novel RNA aptamers, which were validated in vitro and in vivo for their capability to neutralize CCL17. Both aptamers efficiently inhibited the directed migration of the CCR4+ lymphoma line BW5147.3 toward CCL17 in a dose-dependent manner. To study the effect of these aptamers in vivo, we used a murine model of contact hypersensitivity. Systemic application of the aptamers significantly prevented ear swelling and T cell infiltration into the ears of sensitized mice after challenge with the contact sensitizer. The results of this proof-of-principle study establish aptamers as potent inhibitors of CCL17-mediated chemotaxis. Potentially, CCL17-specific aptamers may be used therapeutically in humans to treat or prevent allergic and inflammatory diseases.

Hydrolysis of chlorogenic acid in apple juice using a p-coumaryl esterase of Rhizoctonia solani.

BACKGROUND: Apple juice is rich in polyphenolic compounds, especially in chlorogenic acid. A sour and bitter taste has been attributed to the compound. Chlorogenic acid in coffee powder was quickly hydrolysed by a p-coumaryl esterase of Rhizoctonia solani (RspCAE) at its optimal pH of 6.0. It was unknown, however, if RspCAE would also degrade chlorogenic acid under the strongly acidic conditions (pH 3.3) present in apple juice. RESULTS: Treatment of apple juice with RspCAE led to a chlorogenic acid degradation from 53.38 ± 0.94 mg L-1 to 21.02 ± 1.47 mg L-1 . Simultaneously, the caffeic acid content increased from 6.72 ± 0.69 mg L-1 to 19.33 ± 1.86 mg/L-1 . The aroma profile of the enzymatically treated sample and a control sample differed in only one volatile. Vitispirane had a higher flavour dilution factor in the treated juice. Sensory analysis showed no significant difference in the taste profile ( p < 0.05). CONCLUSION: These results demonstrated a high stability and substrate specificity of RspCAE. An increase in caffeic acid and a concurrent decrease in chlorogenic acid concentration may exert a beneficial effect on human health. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A Receptor-Guided Design Strategy for Ligand Identification.

Biomedical sciences require effective tools to manipulate, detect, and study biological phenomena. Oligo(deoxy)nucleotide ligands represent such tools, but the current strategies to generate them are restricted. Their limited availability is insufficient to address the broad range of targets related to biomedical research. Exemplified by targeting the hydrophobic molecule (-)-Δ9 -tetrahydrocannabinol (THC), we report a receptor-guided design (RGD) strategy to generate chemically modified oligodeoxynucleotide libraries for the tailored selection of clickmers.

Click Reaction on Solid Phase Enables High Fidelity Synthesis of Nucleobase-Modified DNA.

The post-synthetic functionalization of nucleic acids via click chemistry (CuAAC) has seen tremendous implementation, extending the applicability of nucleobase-modified nucleic acids in fields like fluorescent labeling, nanotechnology, and in vitro selection. However, the production of large quantities of high-density functionalized material via solid phase synthesis has been hampered by oxidative by-product formation associated with the alkaline workup conditions. Herein, we describe a rapid and cost-effective protocol for the high fidelity large-scale production of nucleobase-modified nucleic acids, exemplified with a recently described nucleobase-modified aptamer.

Copper-Catalyzed Alkyne-Azide Cycloaddition on the Solid Phase for the Preparation of Fully Click-Modified Nucleic Acids.

Click chemistry has become a widely used method to insert modifications into DNA. Due to its commercial availability, 5'-ethynyl-deoxyuridine (EdU) is commonly incorporated into the DNA for subsequent modification by click reaction. However, it is partially oxidized during deprotection during solid-phase synthesis, resulting in a ketone that is no longer accessible for click modification. To enable the high-fidelity solid-phase synthesis of EdU-containing DNA, this protocol describes a procedure to perform the click reaction on the solid phase before deprotection. Afterwards, the DNA can be deprotected and purified according to standard procedures, and the full modification of EdU with the azide of choice can be analyzed by HPLC and HPLC/MS.

Identification and characterization of nucleobase-modified aptamers by click-SELEX.

Aptamers are single-stranded oligonucleotides that are in vitro-selected to recognize their target molecule with high affinity and specificity. As they consist of the four canonical nucleobases, their chemical diversity is limited, which in turn limits the addressable target spectrum. Introducing chemical modifications into nucleic acid libraries increases the interaction capabilities of the DNA and thereby the target spectrum. Here, we describe a protocol to select nucleobase-modified aptamers by using click chemistry (CuAAC) to introduce the preferred chemical modification. The use of click chemistry to modify the DNA library enables the introduction of a wide range of possible functionalities, which can be customized to the requirements of the target molecule and the desired application. This protocol yields modified DNA aptamers with extended interaction properties that are not accessible with the canonical set of nucleotides. After synthesis of the starting library containing a commercially available, alkyne-modified uridine (5-ethynyl-deoxyuridine (EdU)) instead of thymidine, the library is functionalized with the modification of choice by CuAAC. The thus-modified DNA is incubated with the target molecule and the best binding sequences are recovered. The chemical modification is removed during the amplification process. Therefore, this protocol is compatible with conventional amplification procedures and avoids enzymatic incompatibility problems associated with more extensive nucleobase modifications. After single-strand generation, the modification is reintroduced into the enriched library, which can then be subjected to the subsequent selection cycle. The duration of each selection cycle as outlined in the protocol is ∼1 d.

Systematic evaluation of error rates and causes in short samples in next-generation sequencing.

Next-generation sequencing (NGS) is the method of choice when large numbers of sequences have to be obtained. While the technique is widely applied, varying error rates have been observed. We analysed millions of reads obtained after sequencing of one single sequence on an Illumina sequencer. According to our analysis, the index-PCR for sample preparation has no effect on the observed error rate, even though PCR is traditionally seen as one of the major contributors to enhanced error rates in NGS. In addition, we observed very persistent pre-phasing effects although the base calling software corrects for these. Removal of shortened sequences abolished these effects and allowed analysis of the actual mutations. The average error rate determined was 0.24 ± 0.06% per base and the percentage of mutated sequences was found to be 6.4 ± 1.24%. Constant regions at the 5'- and 3'-end, e.g., primer binding sites used in in vitro selection procedures seem to have no effect on mutation rates and re-sequencing of samples obtains very reproducible results. As phasing effects and other sequencing problems vary between equipment and individual setups, we recommend evaluation of error rates and types to all NGS-users to improve the quality and analysis of NGS data.

Customised nucleic acid libraries for enhanced aptamer selection and performance.

Aptamers are short single-stranded oligo(deoxy)nucleotides that are selected to bind to target molecules with high affinity and specificity. Because of their sophisticated characteristics and versatile applicability, aptamers are thought to become universal molecular probes in biotechnological and therapeutic applications. However, the variety of possible interactions with a putative target molecule is limited by the chemical repertoire of the natural nucleobases. Consequently, many desired targets are not addressable by aptamers. This obstacle is overcome by broadening the chemical diversity of aptamers, mainly achieved by nucleobase-modifications and the introduction of novel bases or base pairs. We discuss these achievements and the characteristics of the respective modified aptamers, reflected by SOMAmers (slow off-rate modified aptamers), clickmers, and aptamers bearing an expanded genetic alphabet.

Selection and Biosensor Application of Aptamers for Small Molecules.

Small molecules play a major role in the human body and as drugs, toxins, and chemicals. Tools to detect and quantify them are therefore in high demand. This review will give an overview about aptamers interacting with small molecules and their selection. We discuss the current state of the field, including advantages as well as problems associated with their use and possible solutions to tackle these. We then discuss different kinds of small molecule aptamer-based sensors described in literature and their applications, ranging from detecting drinking water contaminations to RNA imaging.

Selection of Aptamers for Metabolite Sensing and Construction of Optical Nanosensors.

Optical nanosensors are based on particles with diameters from 20 to 200 nm containing sensory elements. The latter are comprised of one or more signaling molecules and one or more references, which allow measurements to be ratiometric and hence independent on the amount of sensor. The signaling molecules may range from simple ion-binding fluorophores, e.g., pH-sensitive dyes, to complex biochemical assays. Aptamers are ideal for use in nanosensors because they are relatively easy to modify chemically and hence to transform into signaling molecules, and their binding affinities may be fine-tuned to a desired measuring range in the selection process. Here we first describe the selection of metabolite binding aptamers, how they are transformed into signaling molecules using a molecular beacon construct and then how they are inserted into nanoparticles. Finally, we briefly describe how the sensors are calibrated before inserted into cells to measure metabolite concentration in real time. As examples we present aptamers binding to key metabolites in cells: ATP and fructose 1, 6-bisphosphate (FBP).