Project description:Here, we report an ssDNA aptamer with high specificity and affinity towards Salmonella paratyphi A generated using the whole-cell SELEX process. The aptamers generated against an organism show salient features, such as higher affinity than existing antibodies, and are highly specific towards the targeted organism. Thus, the generated aptamer sequences can serve as potential biomarkers for the onsite detection of pathogens with high specificity and sensitivity. Molecular dynamics simulation was used to model the linear chain of the aptamers to a three-dimensional conformation, and the binding mechanism against DNA gyrase was established.
Project description:We created mutliple sequence mutants of several RNA binding proteins. We thenmeasured the in-vivo RNA target specificity of them and the corresponidng (and more) wild type strains using a RNA tagging approach
Project description:Metazoan genomes encode hundreds of RNA binding proteins (RBPs) but relatively few have well-defined RNA-binding preferences. Current techniques for determining RNA targets, including those involving in vitro selection and RNA co-immunoprecipitation, require significant time and labour investment. Here we introduce RNAcompete, a new method for the systematic analysis of RNA-binding specificities that employs a single binding reaction to determine the relative preferences of RBPs for short RNAs that containing a complete range of k-mers in structured and unstructured RNA contexts. We tested RNAcompete by analyzing nine diverse RBPs (HuR, Vts1, FUSIP1, PTB, U1A, SF2/ASF, SLM2, RBM4, and YB1). RNAcompete identified both expected and previously unknown RNA binding preferences. Using in vitro and in vivo binding data, we demonstrate that preferences for individual 7-mers identified by RNAcompete are a more accurate representation of binding activity than conventional motif models. We anticipate that RNAcompete will be a valuable tool for the large-scale study of RNA-protein interactions. The bound RNA from each RNA binding protein pulldown assay is analyzed on a custom Agilent microarray using a pool RNA control as a reference.
Project description:Metazoan genomes encode hundreds of RNA binding proteins (RBPs) but relatively few have well-defined RNA-binding preferences. Current techniques for determining RNA targets, including those involving in vitro selection and RNA co-immunoprecipitation, require significant time and labour investment. Here we introduce RNAcompete, a new method for the systematic analysis of RNA-binding specificities that employs a single binding reaction to determine the relative preferences of RBPs for short RNAs that containing a complete range of k-mers in structured and unstructured RNA contexts. We tested RNAcompete by analyzing nine diverse RBPs (HuR, Vts1, FUSIP1, PTB, U1A, SF2/ASF, SLM2, RBM4, and YB1). RNAcompete identified both expected and previously unknown RNA binding preferences. Using in vitro and in vivo binding data, we demonstrate that preferences for individual 7-mers identified by RNAcompete are a more accurate representation of binding activity than conventional motif models. We anticipate that RNAcompete will be a valuable tool for the large-scale study of RNA-protein interactions.
Project description:Intracellular pH (pHi) dynamics regulates diverse cell processes such as proliferation, dysplasia, and differentiation, often mediated by the protonation state of a functionally critical histidine residue in endogenous pH sensing proteins. How pHi dynamics can directly regulate gene expression and whether transcription factors can function as pH sensors has received limited attention. We tested the prediction that transcription factors with a histidine in their DNA binding domain (DBD) that forms hydrogen bonds with nucleotides can have pH-regulated activity, which is relevant to more than 85 transcription factors in distinct families, including FOX, KLF, SOX and MITF/Myc. Focusing on FOX family transcription factors, we used unbiased SELEX-seq to identify pH-dependent DNA binding motif preferences, then confirm pH-regulated binding affinities for FOXC2, FOXM1, and FOXN1 to a canonical FkhP DNA motif that are 2.5 to 7.5 greater at pH 7.0 compared with pH 7.5. For FOXC2, we also find greater activity for an FkhP motif at lower pHi in cells and that pH-regulated binding and activity are dependent on a conserved histidine (His122) in the DBD. RNA-seq with FOXC2 also reveals pH-dependent differences in enriched promoter motifs. Our findings identify pH-regulated transcription factor-DNA binding selectivity with relevance to how pHi dynamics can regulate gene expression for myriad cell behaviours.