טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentCherniavsky Marina
SubjectDevelopment of Reporter System for High Throughput Screening
of Synthetic Libraries for Evaluation of in-vitro
Stop Codon Readthrough Activity
DepartmentDepartment of Biotechnology
Supervisor Professor Timor Baasov
Full Thesis textFull thesis text - English Version


Abstract

Aminoglycoside antibiotics are clinically used drugs for the treatment of bacterial infections. They selectively bind to the A-site of the bacterial 16S ribosomal RNA (rRNA), and interfere with translational fidelity during protein synthesis.

 Nonsense mutations are single-point alterations in the DNA, where one of the three stop codons (TAG, TGA or TAA) replaces an amino acid-coding codon, leading to a premature termination of the translation process, producing a shortened, nonfunctional protein. In the last several years, certain aminoglycosides were suggested as a possible treatment for genetic diseases resulting from nonsense mutations due to their ability to induce eukaryotic ribosomes to readthrough nonsense mutations. The stop codon readthrough process appears to be a complex interplay of a number of factors including the type of nonsense mutation, the genomic contexts surrounding the nonsense mutation and the structure of aminoglycoside used to induce the readthrough. 

In our laboratory, a new generation of aminoglycoside-based drugs, targeting the mammalian ribosome, is currently being developed. Among the purposes of this research are improvement of stop codon readthrough potential of aminoglycosides and reduction of their toxicity in humans.

In order to evaluate the readthrough activity of a large number of substances we calibrated a high-throughput screening assay. The system consists of two plasmids, each carrying two luciferase genes (Renilla and Firefly) separated by a short linker carrying either a stop codon or the corresponding wild type codon respectively. The Firefly to Renilla luciferase activities ratio of the mutant and the wild type represents the readthrough level. The main advantage of this system is the ability to estimate the aminoglycoside-induced readthrough efficiency by direct monitoring of enzymatic activity of the translated proteins.

Using this assay, we found that addition of an AHB moiety (amino-hydroxy-butyric acid) provides an improvement in its stop codon readthrough potential. In order to test the effect of the nonsense mutation flanking sequence on stop codon readthrough, we cloned DNA fragments derived from several mutant genes carrying nonsense mutations and representing underlying causes for the genetic diseases Usher syndrome, cystic fibrosis, Duchenne muscular dystrophy and Hurler syndrome in frame between the Renilla and the Firefly luciferase genes. We demonstrated that genomic context of the nonsense mutation has a significant impact on the stop codon readthrough efficiency promoted by different aminoglycosides. Most importantly, we discovered that in all the cases tested our novel semisynthetic lead compound NB54 showed the highest level of stop codon readthrough in all concentrations tested.