Design, synthesis, and analysis of conjugated activators for modulation of alternative splicing

Organic chemistry;Alternative splicing;Spinal muscular atrophy;Peptide;Oligonucleotide

Langenfeld, Adam

关键词: Organic chemistry;    Alternative splicing;    Spinal muscular atrophy;    Peptide;    Oligonucleotide;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/34265/Langenfeld_Adam.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Recent genome wide analysis has estimated that >95% of human genes are alternatively spliced, allowing for the production of many protein isoforms from a single gene. Splicing is controlled by SR proteins, factors which bind to exonic splicing enhancers (ESEs) through RNA binding domains and recruit the splicosome via protein-protein interactions mediated by their RS domains, protein domains rich in highly charged arginine-serine dipeptides. Defects in alternative splicing have been linked to several diseases, including Cystic Fibrosis, breast cancer, and Spinal Muscular Atrophy (SMA). SMA is a neurodegenerative disorder caused by the loss of survival of motor neuron 1 (SMN1) gene. A nearly identical copy of the gene, SMN2, encodes an identical protein but contains a C-->T transition on the ESE of exon 7, disrupting SR protein binding and resulting in substantial exon 7 skipping. Significant research efforts have focused on redirecting the splicing pattern of SMN2 to produce a full-length SMN protein normally generated by SMN1. Splicing modulators have ranged from small molecule activators such as sodium phenylbutyrate and valproic acid, to viral vectors encoding antisense oligonucleotides. In particular, a synthetic peptide-oligonucleotide conjugate containing a short chain of arginine-serine dipeptides to mimic the RS domain has been shown to redirect splicing patterns. However, little work has been done to explore the potential flexibility of the design of this synthetic RS domain.This dissertation reports efforts toward the design and synthesis of several synthetic SR protein mimics and the experimental design to determine their ability to increase inclusion of exon 7 in SMN2. The synthetic RS domains consist of three motifs – α-peptide, β-peptide, and peptide dendrimer – attached to antisense oligonucleotides targeted to SMN2 exon 7. The synthesis and conjugation of these mimics is straightforward, allowing for easy derivatization.The efficacy of these molecules was examined using an in vitro splicing assay.Analysis of these molecules using in vitro splicing assays indicates that neither synthetic RS domains nor antisense oligonucleotides alone affect splicing patterns, but conjugated molecules may play a beneficial role.