Recoding Aminoacyl-tRNA Synthetases for Synthetic Biology by Rational Protein-RNA Engineering

We have taken a rational approach to redesigning the amino acid binding and aminoacyl–tRNA pairing specificities of bacterial glutaminyl–tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA^Gln by over 10⁵-fold compared to the wild-type enzyme. Better optimized designs of the protein–RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl–tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology

Additional file 1: of A methylation PCR method determines FMR1 activation ratios and differentiates premutation allele mosaicism in carrier siblings

Table S1. CGG repeat lengths and methylation analysis of a pooled positive control including 18, 30, 32, 56, 85, 116 and >200 CGG. Table S2. Cohort panel distribution of allele sizes and ARs determined using mPCR compared to the activation ratio from Southern blot analysis. (PDF 445 kb

Novel Insight into Mutational Landscape of Head and Neck Squamous Cell Carcinoma

<div><p>Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection.</p></div

Clinical characteristics of the cohort.

<p>Clinical characteristics of the cohort.</p

Complete list of point mutations among the 11 cancer genes sequenced in the 37 HNSCC tumor tissues.

<p>Complete list of point mutations among the 11 cancer genes sequenced in the 37 HNSCC tumor tissues.</p

The 51 cancer genes selected for targeted or whole exon sequencing.

<p>The 51 cancer genes selected for targeted or whole exon sequencing.</p

Genetic alterations in 37 HNSCC tumors.

<p>Heat-map representation of individual mutations present in a series of 37 HNSCC tumors, presented in columns. Mutation events are represented by black color. Left, Mutated genes, asterisks indicate genes characterized by whole-exon sequencing. Novel genes mutated in HNSCC are labeled by bold font. Right, mutation rate for each gene. Genes are ranked by mutation rate. Bottom, number of mutations per tumor sample. Note, that two patients have two mutations in the same gene TP53 gene: X16 and X27 (with number 2 on the heat-map). The smoking status of tumor patients is identified as: S – for smokers, NS – for never smoked patients, ND – not determined. The HPV status of tumor patients is identified as: “+” for HPV-positive patients and “-” for HPV-negative patients.</p