No description available.

It is widely accepted that alternative splicing is often a catalyst for the development and progression of disease. Recent advances in oligonucleotide therapeutics have seen these alternative splicing patterns forcibly changed, providing a specific and versatile attack against such diseases. This study has focused on the use of bifunctional oligonucleotides for the treatment of spinal muscular atrophy (SMA) and cancer. In SMA the bifunctional oligonucleotide is targeted to inhibit exon 7 skipping during splicing of SMN2. The current study has confirmed that this oligonucleotide is able to stimulate SMN2 exon 7 splicing, as has been seen previously (Skordis et al., 2003; Owen et al., 2011). Analysis of the action of this oligonucleotide indicated that it specifically recruits spliceosomal factors to the 3’ splice site of SMN2 intron 6. The tail domain was seen to enhance binding of U2 snRNP to this region and stabilize protein complex formation, while the annealing domain was seen to enhance U2AF65-RNA binding. A greater understanding of the mechanism of action of the SMN2 bifunctional oligonucleotide also highlighted flaws in the design of the previously tested RON exon 11 bifunctional oligonucleotide (Ghigna et al., 2010). This oligonucleotide was designed to combat the production of RON Δ165 mRNA, in which RON exon 11 is skipped. Expression of this RON isoform has been linked with the onset of metastasis in some epithelial cell cancers. A systematic analysis of the splicing characteristics of this region in RON pre-mRNA, suggested that weak 3’ splice site sequences limit RON exon 11 splicing. hnRNP H was also shown to bind RON introns 10 and 11 and may play a role in the regulation of RON exon 11 splicing. A new bifunctional oligonucleotide targeted combat RON Δ165 mRNA production, through the stimulation of RON exon 11 splicing, was designed with the weakness in RON exon 11 splicing and the mechanism of action of the SMN2 bifunctional oligonucleotide in mind.

Understanding the molecular mechanisms that allow splice site selection is of fundamental importance to the study of alternative splicing. The study presented here has focused on the interactions of two spliceosomal trans-acting factors with pre-mRNA. The U1 snRNP is very important in 5’ splice site selection. However, how binding of U1 snRNP to pre-mRNAs containing multiple 5’ splice sites leads to 5’ splice site selection is unknown. To investigate the role of U1 snRNP in 5’splice site selection, single molecule microscopy was used to determine the number of U1 snRNPs bound to a single molecule of pre-mRNA. In cases where multiple strong 5’ splice sites are competing the furthest downstream 5’ss is used. In these cases, as many U1 snRNPs were observed bound to the pre-mRNA at an early stage of the reaction as there were 5’ splice sites, demonstrating that multiple sites were occupied. However, later pre-spliceosomal complexes contained only one U1 snRNP, indicating that the surplus U1 snRNP had been removed. A novel model of 5’ splice site selection is presented, in which U1snRNP stimulates the binding of SR proteins to the pre-mRNA and thus increases exon rigidity, enforcing use of the downstream 5’ splice site. The U2 Auxiliary Factor (U2AF), binds to the polypyrimidine tract and 3’ splice site of an intron and is a hetrodimer of the U2AF65 and U2AF35 subunits. The length of the polypyrimidine tract has been shown to influence the efficiency with which a 3’splice site is spliced. To investigate the role of polypyrimidine tract length on U2AF binding, experiments have been conducted in which the binding of U2AF65 to pre-mRNAs has been analysed using single molecule microscopy. Through the analysis of constructs derived from the Globin C pre-mRNA, the mechanisms of U2AF65 association with pre-mRNA have been shown to comprise; (i) a non-specific association that occurs with all RNA tested in the absence of ATP, (ii) a U1 snRNP-dependent association with pre-mRNAs in a polypyrimidine tract-independent manner, and (iii) an association with pre-mRNA in a polypyrimidine tract-dependent manner.

Exonic mutations can result in altered protein function by affecting exon recognition during splicing. To understand this mechanism I have extensively evaluated the exonic regulatory elements affected by the disease G to T mutation at position +6 of BRCAl exon 18. This substitution induces the exclusion of the exon and it has been suggested that it disrupts an ASF/SF2-dependent enhancer. Using a pulldown assay with an internal standard, I show that WT and T6 sequences bind ASF/SF2 with similar efficiency, which is significantly lower compared to the binding to a typical enhancer derived from the fibronectin EDA exon. Consistent with the absence of an ASF/SF2 enhancing effect, siRNA depletion of ASF/SF2 did not induce exon WT exclusion indicating that ASF/SF2 is not essential for BRCAl exon 18 splicing.

TDP-43 is an RNA-binding protein with particular predilection for UG repeats that was shown to regulate transcription, to mediate mRNA stability and to inhibit splicing in the CFTR and Apo All genes.Recently, TDP-43 has been found to be the major disease protein in Amyotrophic Lateral Sclerosis (ALS) and Fronto-temporal Lobar dementia [ftld]. Objective of this thesis was to characterize the function of TDP-43 with in-vitro and in vivostudies.

Synthetic biology aims to engineer biological systems and components, ultimately for applications as diverse as medical therapeutics and energy production. A major focus in synthetic biology has been the design and construction of relatively simple synthetic gene networks which display interesting dynamics, or functions. Many synthetic gene networks have been designed, and in some cases constructed in vivo, and can display a variety of dynamics, such as oscillations and switching. The most studied network has been the oscillator, and a number of different designs now exist. This thesis first reviews the oscillator field, the review forming the motivation for designing synthetic gene networks that are capable of either using the output from oscillators, or responding to other sources of oscillations. Two novel, theoretical synthetic gene networks are then presented which are designed to use oscillations as an input. The first network is a frequency multiplier, designed to use oscillations of the type produced by the current synthetic oscillators. Further analysis of this network shows that it possesses the property of multi-functionality, and can additionally function as a switch or an oscillator depending on the nature of the input it receives. The second network is a pulse generator, designed to use temperature as an input. In addition to a theoretical analysis, the pulse generator was constructed in vivo in E. coli. Experimental testing of the pulse generator suggested that the degradation conferred by ssrA tags, used to ensure rapid degradation of the network's proteins, is highly temperature dependent. Subsequent experiments confirmed this, highlighting an important weakness in using ssrA tags, the standard means of achieving rapid protein degradation in synthetic gene networks.

Phosphoramidite derivatives of a nucleoside analogue bearing photoswitchable ortho-, meta- and para azobenzene moieties were prepared and used to incorporate the azobenzene groups into DNA.8mers. The photochemical E-Z isomerisation of the azobenzene-appended 8mers was investigated by UV/vis spectroscopy and RP-HPLC. In order to investigate the stabilities of the irradiated-8mers towards thermal Z - E isomerisation, Arrhenius and Eyring parameters for the photoisomerisation were determined. The meta-isomer was found to be the most thermally stable. An initial investigation into the stability of duplexes containing a para-azobenzene-modified 8mer was carried out using melting studies. The duplex-forming activity of the oligonucleotide was modulated by the E- Z photoisomerisation of the para-azobenzene residue. A divergent methodology for the preparation of a novel structural class of photoswitchable oligonucleotide has been described. A novel anthracene methyl phosphoramidite derivative suitable for the preparation of end- labelled oligonucleotides under solid-phase directed-Arbusov conditions was prepared and its reactivity investigated. A comparison of the utility of this anthracene methyl phosphorarnidite with a related benzyl phosphorarnidite in a model reaction with the 5'-hydroxyl of support-bound decathymidylate was made. Directed Michaelis-Arbusov reactions of the putative phosphite triester intermediates with primary and secondary amines in the presence of 0.01 M iodine gave the corresponding phosphoramidate diesters in high yields. This reactivity was also demonstrated using commercially available phosphoramidites for the preparation of inter-nucleotide phosphoramidates bearing terminal primary amines. Derivatisation of these primary amine-functionalised oligomers was accomplished in solution-phase following treatment with the meta-azobenzene NHS ester.

The external environment and internal cellular processes generate DNA double strand breaks (DSBs), a particularly toxic form of DNA damage that can result in chromosome fragmentation, replication failure, mutagenesis and cell death. Cells have evolved effective mechanisms to preserve the mtegrity of the genome including DNA damage signalling, cell cycle checkpoint activation and DNA repair.

No description available.

The cell lysis step is one of the most critical operations in large-scale plasmid DNA processing as fragments of chromosomal DNA and degraded plasmids remaining in solution have a detrimental effect on downstream operations. In the present thesis the effect of the engineering environment on the bacterial cell suspension used in the lysis step and on solutions containing gene vectors of up to 242 kb in size was investigated. An integral aspect of the thesis was to evaluate current analytic techniques relating to the characterisation of process streams containing plasmid vectors and to identify and develop characterisation techniques suitable for process development and manufacturing. Cell pastes derived from laboratory- and 450 litre-scale fermentations of plasmid-containing bacterial cells were characterised using a variety of analytical techniques. The impact of pilot scale continuous centrifugation equipment used for cell harvesting was then investigated. It was observed that equipment design and operating conditions had an impact on product yield and the molecular weight of contaminant DNA in the process stream. It was found that direct measurement of released nucleic acids at the cell resuspension stage can provide a simple analytical technology to characterise cells before the lysis stage. After cell lysis, the downstream options depend on the susceptibility of the product to chemical and mechanical damage in solution. In the case of circular DNA molecules, it is of particular importance to characterise the backbone integrity. A microwell-based high throughput method to evaluate this for pure double stranded DNA solutions was developed. The method was then validated for reproducibility, accuracy and sensitivity and was successfully adapted for use on a robotic liquid handling system. The method was utilised to investigate the response to hydrodynamic stresses of bacterial artificial chromosomes. It was observed that for a 116 kb vector, average shear rates 104 s-1 (comparable with standard process equipment), caused a 5 fold decrease in the integrity of the molecules in the sample. Finally, the thesis ends with a look towards future process analytical technologies and assay development within the context of DNA-based pharmaceuticals.