Dynamics of Co-Transcriptional Pre-mRNA Folding Influences the Induction of Dystrophin Exon Skipping by Antisense Oligonucleotides

Antisense oligonucleotides (AONs) mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a “window of analysis” that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered “engaged” if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency) of 94% of 176 previously reported AONs. Four novel insights are inferred: (1) the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2) engaged nucleotides at 3′ or 5′ ends of the target site attenuate AON performance more than at other sites; (3) the performance of longer AONs is less attenuated by engaged nucleotides at 3′ or 5′ ends of the target site compared to shorter AONs; (4) engaged nucleotides at 3′ end of a short target site attenuates AON efficiency more than at 5′ end

Spontaneously opening GABA receptors play a significant role in neuronal signal filtering and integration

Acknowledgements This work was supported by The Rosetrees Trust Research Grant A1066, RS MacDonald Seedcorn Award and Wellcome Trust—UoE ISSF Award to S.S. The authors thank Prof. David Wyllie (University of Edinburgh) and Prof. Dmitri Rusakov (UCL) for their valuable suggestions on paper preparation.Peer reviewedPublisher PD

Kinetic changes and modulation by carbamazepine on voltage-gated sodium channels in rat CA1 neurons after epilepsy

AIM: To study whether the functional properties of sodium channels, and subsequently the channel modulation by carbamazepine (CBZ) in hippocampal CA1 neurons can be changed after epileptic seizures. METHODS: We used the acutely dissociated hippocampal CA1 pyramidal cells from epilepsy model rats 3 weeks and 3 months respectively after kainate injection, and whole-cell voltage-clamp techniques. RESULTS: After long-term epileptic seizures, both sodium channel voltage-dependence of activation and steady-state inactivation shifted to more hyperpolarizing potentials, which resulted in the enlarged window current; the membrane density of sodium current decreased and the time constant of recovery from inactivation increased. CBZ displayed unchanged efficacy on sodium channels, with a similar binding rate to them, except that at higher concentrations, the voltage shift of inactivation was reduced. For the short-term kainate model rats, no differences were detected between the control and epilepsy groups. CONCLUSION: These results indicate that the properties of sodium channels in acutely dissociated hippocampal neurons could be changed following long-term epilepsy, but the alternation might not be enough to induce the channel resistance to CBZ