Modification of Splicing Patterns using Artifical Enhancer Sequences within Tripartite Structure
thesisposted on 2013-09-03, 15:46 authored by Andrew Jonathan Perrett
Genes SMN1 and SNM2 encode SMN protein which is essential for motor neurone survival. In Spinal Muscular Atrophy patients, absence of functional SMN1 means functional SMN protein is encoded by SMN2 alone. SMN2 splicing products do not usually include exon 7, which is required for functional SMN production. Therefore, methods for increasing exon 7 inclusion levels of SMN2 spliced products were investigated. 2’OMe RNA annealer and enhancer sequences were synthesised based on Targeted Oligonucleotide Enhancers of Splicing (TOES) with the addition of HEG linkers and bioconjugation intermediates. Cu-catalysed click chemistry was used to conjugate these sequences together to form tripartite sequences. Tripartite sequences RNA3-RNA11 were synthesised along with phosphothioate (pS) versions of RNA6 and RNA7. Adding a bioconjugation group between enhancer and annealer strands increased exon 7 inclusion above basal levels in splicing experiments. pS tripartite sequences were less effective than non-pS, versions, possibly due pS backbones affecting the strength of binding between annealers and mRNA. RNA6 and RNA7, containing 1 and 2 HEG linkers respectively, achieved exon 7 inclusion in 40% and 35% of SMN2 spliced products, respectively. GGA-O only achieved 26% exon 7 inclusion, with basal levels at 11%. Less flexible abasic linkers equivalent in length to one HEG unit, gave lower levels of exon 7 inclusion than those achieved with HEG, suggesting the flexibility of HEG linkers is important for enhancing exon inclusion. Multivalent systems were constructed consisting of annealer and enhancer sequences polyconjugated to gold nanoparticles (GNPs). Splicing experiments showed exon 7 inclusion levels were dependent on GNP size, surface coating and directionality of strand attachment. GNP16 with an enhancer coating and 5nm diameter achieved exon 7 inclusion in 80% of spliced products, with GGA-O achieving 67% and basal levels at 44%. Addition of annealers to GNP16, significantly reduced exon 7 inclusion.
Alternative titleModification of Splicing Patterns using Artificial Enhancer Sequences within Tripartite Structure
Supervisor(s)Burley, Glen; Eperon, Ian C.
Date of award2013-06-15
Awarding institutionUniversity of Leicester