Genetic code expansion, an emerging tool in the Ca2+ ion channel field.

Various methods for characterizing binding forces as well as for monitoring and remote control of ion channels are still emerging. A recent innovation is the direct incorporation of unnatural amino acids (UAAs) with corresponding biophysical or biochemical properties, which are integrated using genetic code expansion technology. Minimal changes to natural amino acids, which are achieved by chemical synthesis of corresponding UAAs, are valuable tools to provide insight into the contributions of physicochemical properties of side chains in binding events. To gain unique control over the conformational changes or function of ion channels, a series of light-sensitive, chemically reactive and posttranslationally modified UAAs have been developed and utilized. Here, we present the existing UAA tools, their mode of action, their potential and limitations as well as their previous applications to Ca2+-permeable ion channels.

Insights into the dynamics of the Ca2+ release-activated Ca2+ channel pore-forming complex Orai1.

An important calcium (Ca2+) entry pathway into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel, which controls a series of downstream signaling events such as gene transcription, secretion and proliferation. It is composed of a Ca2+ sensor in the endoplasmic reticulum (ER), the stromal interaction molecule (STIM), and the Ca2+ ion channel Orai in the plasma membrane (PM). Their activation is initiated by receptor-ligand binding at the PM, which triggers a signaling cascade within the cell that ultimately causes store depletion. The decrease in ER-luminal Ca2+ is sensed by STIM1, which undergoes structural rearrangements that lead to coupling with Orai1 and its activation. In this review, we highlight the current understanding of the Orai1 pore opening mechanism. In this context, we also point out the questions that remain unanswered and how these can be addressed by the currently emerging genetic code expansion (GCE) technology. GCE enables the incorporation of non-canonical amino acids with novel properties, such as light-sensitivity, and has the potential to provide novel insights into the structure/function relationship of CRAC channels at a single amino acid level in the living cell.

Identification of Broadly Applicable Adeno-Associated Virus Vectors by Systematic Comparison of Commonly Used Capsid Variants In Vitro.

Adeno-associated viruses (AAVs) represent highly attractive gene therapy vectors and potent research tools for the modulation of gene expression in animal models or difficult-to-transfect cell cultures. Engineered variants, comprising chimeric, mutated, or peptide-inserted capsids, have strongly broadened the utility of AAVs by altering cellular tropism, enabling immune evasion, or increasing transduction efficiency. In this work, the performance of 50 of the most used, predominantly published, AAVs was compared on several primary cells, cell lines, and induced pluripotent stem cell-derived models from different organs, including the adipose tissue, liver, lung, brain, and eyes. To identify the most efficient capsids for each cell type, self-complementary AAVs were standardized by digital polymerase chain reaction, arrayed on 96-well plates, and screened using high-content imaging. To enable best use of the data, all results are also provided in a web app. The utility of one selected AAV variant is further exemplified in a liver fibrosis assay based on primary hepatic stellate cells, where it successfully reversed a small interfering RNA (siRNA)-induced phenotype. Most importantly, our comparative analysis revealed that a subselection of only five AAV variants (AAV2.NN, AAV9-SLRSPPS, AAV6.2, AAV6TM, and AAV1P5) enabled efficient transduction of all tested cell types and markedly outperformed other well-established capsids, such as AAV2-7m8. These findings suggest that a core panel comprising these five capsid variants is a universally applicable and sufficient tool to identify potent AAVs for gene expression modulation in cellular systems.

Exploiting orthology and de novo transcriptome assembly to refine target sequence information.

BACKGROUND: The ability to generate recombinant drug target proteins is important for drug discovery research as it facilitates the investigation of drug-target-interactions in vitro. To accomplish this, the target's exact protein sequence is required. Public databases, such as Ensembl, UniProt and RefSeq, are extensive protein and nucleotide sequence repositories. However, many sequences for non-human organisms are predicted by computational pipelines and may thus be incomplete or incorrect. This could lead to misinterpreted experimental outcomes due to gaps or errors in orthologous drug target sequences. Transcriptome analysis by RNA-Seq has been established as a standard method for gene expression analysis. Apart from this common application, paired-end RNA-Seq data can also be used to obtain full coverage cDNA sequences via de novo transcriptome assembly. METHODS: To assess whether de novo transcriptome assemblies can be used to determine a protein's sequence by searching the assembly for a known orthologous sequence, we generated 3 × 6 = 18 tissue specific assemblies (three organs: brain, kidney and liver; six species: human, mouse, rat, dog, pig and cynomolgus monkey). These assemblies and the manually curated human protein sequences from UniProtKB/Swiss-Prot were used in a reciprocal BLAST search to identify best matching hits. We automated and generalised our approach and present the a&o-tool, a workflow which exploits de novo assemblies of paired-end RNA-Seq data and orthology information for target sequence validation and refinement across related species. Furthermore, the a&o-tool extracts best hits' sequences from a reciprocal BLAST search, translates them into protein sequences, computes a multiple sequence alignment and quantifies the refinement. RESULTS: For the three human assemblies we observed a hit rate greater than 60% with 100% sequence coverage and identity. For assemblies from the other species we observed similar hit rates and coverage with highest identities for cynomolgus monkey. CONCLUSIONS: In summary, we show how to refine protein sequences using RNA-Seq data and sequence information from closely related species. With the a&o-tool we provide a fully automated pipeline to perform refinement including cDNA translation and multiple sequence alignment for visual inspection. The major prerequisite for applying the a&o-tool is high quality sequencing data.

An RNA-Seq atlas of gene expression in mouse and rat normal tissues.

Gene functionality is closely connected to its expression specificity across tissues and cell types. RNA-Seq is a powerful quantitative tool to explore genome wide expression. The aim of this study is to provide a comprehensive RNA-Seq dataset across the same 13 tissues for mouse and rat, two of the most relevant species for biomedical research. The dataset provides the transcriptome across tissues from three male C57BL6 mice and three male Han Wistar rats. We also describe our bioinformatics pipeline to process and technically validate the data. Principal component analysis shows that tissue samples from both species cluster similarly. We show by comparative genomics that many genes with high sequence identity with respect to their human orthologues also have a highly correlated tissue distribution profile and are in agreement with manually curated literature data for human. In summary, the present study provides a unique resource for comparative genomics and will facilitate the analysis of tissue specificity and cross-species conservation in higher organisms.