We employ a reporter assay and Selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Qβ, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wideswath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation.Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo. INTRODUCTIONThe regulation of gene expression is a process central to all biological life-forms. It is a process thought to be mediated largely by proteins, which interact with either chromatin or its RNA product.The best-known form of regulation is mediated by transcription factors, which control RNA levels by their sequence-specific interaction with DNA. Post transcriptional regulation based on protein-RNA interactions, however, is quite different, due to the nature of RNA. Unlike DNA which is a long, chromatinized, replicated, and for the most part exists as a double stranded molecule, RNA is a short, transient (i.e. constantly manufactured and degraded), exists in multiple copies, has particular modifications (1, 2), and folds into functional secondary and tertiary structures. RNA structure is

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT).

We employ a reporter assay and Selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Qβ, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wideswath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation.Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo. thought to be highly dynamic, and is dependent on many factors such as temperature, cellular RNAbinding protein (RBP) content, presence or absence of translating ribosomes, and interaction with other RNA molecules (3). Thus, a typical RBP-RNA interaction is likely to depend not only on the presence of a specific binding sequence, but also on many other factors.In bacteria, post-transcriptional regulation has been studied extensively in recent decades.There are well-documented examples of RBPs that either inhibit or directly compete with ribosome binding via a variety of mechanisms. These include direct competition with the 30S ribosomal subunit for binding via single stranded recognition (4), entrapment of the 30S subunit in an inactive complex via a nested pseudoknot structure (5) and ribosome assembly inhibition when the RBP is bound to a structured RBP binding site, or hairpin (6-9). These hairpins have been studied in three distinct positions: either immediately downstream and including the AUG (7), upstream of the Shine-Dalgarno sequence (8), or as structures that entrap Shine-Dalgarno motifs, as is the case for the PP7 and MS2 phage coat-protein binding sites. There is also a well-characterized example of translation stimulation:binding of the phage Com RBP was shown to destabilize a sequestered ribosome binding site (RBS) of the Mu phage mom gene, thereby facilitating translation (10, 11). While these studies indicate a richness of RBP-RNA-based regulatory mechanisms, a systematic understanding of the relationship between RBP binding, sequence specificity, the underlying secondary and tertiary RNA structure, and the resultant regulatory output is still lacking.In recent years, advances in next generation ...

The ability to control mammalian genes in a synergistic mode using synthetic transcription factors is highly desirable in fields of tissue engineering, stem cell reprogramming and fundamental research. In this study, we developed a standardized toolkit utilizing an engineered CRISPR/Cas9 system that enables customizable gene regulation in mammalian cells. The RNA-guided dCas9 protein was implemented as a programmable transcriptional activator or repressor device, including targeting of endogenous loci. For facile assembly of single or multiple CRISPR RNAs, our toolkit comprises a modular RNAimer plasmid, which encodes the required noncoding RNA components.

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Chemically triggered molecular switches for controlling the fate and function of biological systems are fundamental to the emergence of synthetic biology and the development of biomedical applications. We here present the first chemically triggered switch for controlling the infectivity of adeno-associated viral (AAV) vectors.

We present Triplex-seq, a deep-sequencing method that systematically maps the interaction space between an oligo library of ssDNA triplex-forming oligos (TFOs) and a particular dsDNA triplex target site (TTS). We demonstrate the method using a randomized oligo library comprising 67 million variants, with five TTSs that differ in guanine (G) content, at two different buffer conditions, denoted pH 5 and pH 7. Our results show that G-rich triplexes form at both pH 5 and pH 7, with the pH 5 set being more stable, indicating that there is a subset of TFOs that form triplexes only at pH 5. In addition, using information analysis, we identify triplex-forming motifs (TFMs), which correspond to minimal functional TFO sequences. We demonstrate, in single-variant verification experiments, that TFOs with these TFMs indeed form a triplex with G-rich TTSs, and that a single mutation in the TFM motif can alleviate binding. Our results show that deepsequencing platforms can substantially expand our understanding of triplex binding rules and aid in refining the DNA triplex code.

Whole cell bioreporters, such as bacterial cells, can be used for environmental and clinical sensing of specific analytes. However, the current methods implemented to observe such bioreporters in the form of chemotactic responses heavily rely on microscope analysis, fluorescent labels, and hard-to-scale microfluidic devices. Herein, we demonstrate that chemotaxis can be detected within minutes using intrinsic optical measurements of silicon femtoliter well arrays (FMAs). This is done via phase-shift reflectometric interference spectroscopic measurements (PRISM) of the wells, which act as silicon diffraction gratings, enabling label-free, real-time quantification of the number of trapped bacteria cells in the optical readout. By generating unsteady chemical gradients over the wells, we first demonstrate that chemotaxis toward attractants and away from repellents can be easily differentiated based on the signal response of PRISM. The lowest concentration of chemorepellent to elicit an observed bacterial response was 50 mM, whereas the lowest concentration of chemoattractant to elicit a response was 10 mM. Second, we employed PRISM, in combination with a computational approach, to rapidly scan for and identify a novel synthetic histamine chemoreceptor strain. Consequently, we show that by using a combined computational design approach, together with a quantitative, real-time, and label-free detection method, it is possible to manufacture and characterize novel synthetic chemoreceptors in Escherichia coli (E. coli).