Phosphorodiamidate morpholino oligonucleotides (PMOs) constitute 3 out of the 11 FDA-approved oligonucleotide-based drugs in the last 6 years. PMOs can effectively silence disease-causing genes and modify splicing. However, PMO synthesis has remained challenging for a variety of reasons: inefficient deprotection and coupling methods and instability of monomers. Here, we report the development of a suitable combination of resin supports, deblocking and coupling reagents for synthesizing PMOs using either trityl or Fmoc-protected chlorophosphoramidate monomers. The synthesized PMOs using both the methods on a solid support have been validated for gene silencing in a zebrafish model. The protocol was successfully transferred into an automated DNA synthesizer to make several sequences of PMOs, demonstrating for the first time the adaptation of regular PMOs in a commercial DNA synthesizer. Moreover, PMOs with longer than 20-mer sequences, including FDA-approved Eteplirsen (30-mer), were achieved in >20% overall yield that is superior to previous reports. Hybridization study shows that PMOs exhibit a higher binding affinity toward complementary DNA relative to the DNA/DNA duplex (>6 °C). Additionally, the introduction of Fmoc chemistry into PMOs opens up the possibility for PMO synthesis in commercial peptide synthesizers for future development.

Dynamic co-regulation of the actin and microtubule subsystems enables the highly precise and adaptive remodelling of the cytoskeleton necessary for critical cellular processes, like axonal pathfinding. The modes and mediators of this interpolymer crosstalk, however, are inadequately understood. We identify Fmn2, a non-diaphanous related formin associated with cognitive disabilities, as a novel regulator of cooperative actin-microtubule remodelling in growth cones. We show that Fmn2 stabilizes microtubules in the growth cones of cultured spinal neurons and also in vivo. Superresolution imaging revealed that Fmn2 facilitates guidance of exploratory microtubules along actin bundles into the chemosensory filopodia. Using live imaging, biochemistry and single-molecule assays we show that a C-terminal domain in Fmn2 is necessary for the dynamic association between microtubules and actin filaments. In the absence of the cross- bridging function of Fmn2, filopodial capture of microtubules is compromised resulting in de-stabilized filopodial protrusions and deficits in growth cone chemotaxis. Our results uncover a critical function for Fmn2 in actin-microtubule crosstalk in neurons and demonstrate that modulating microtubule dynamics via associations with F-actin is central to directional motility.

22Dynamic co-regulation of the actin and microtubule subsystems enables the highly 23 precise and adaptive remodeling of the cytoskeleton necessary for critical cellular 24 processes, like axonal pathfinding. The modes and mediators of this interpolymer 25 crosstalk, however, are inadequately understood. 26 We identify Fmn2, a non-diaphanous related formin associated with cognitive 27 disabilities, as a novel regulator of cooperative actin-microtubule remodeling in 28 growth cones. We show that Fmn2 stabilizes microtubules in the growth cones of 29 cultured spinal neurons and also in vivo. Superresolution imaging revealed that 30 Fmn2 facilitates guidance of exploratory microtubules along actin bundles into the 31 chemosensory filopodia. Using live imaging, biochemistry and single-molecule 32 assays we show that a C-terminal domain in Fmn2 is necessary for the dynamic 33 association between microtubules and actin filaments. In the absence of the cross-34 bridging function of Fmn2, filopodial capture of microtubules is compromised 35 resulting in de-stabilized filopodial protrusions and deficits in growth cone 36 chemotaxis. 37 Our results uncover a critical function for Fmn2 in actin-microtubule crosstalk in 38 neurons and demonstrate that modulating microtubule dynamics via associations 39 with F-actin is central to directional motility. 40 41 42 Key words: Actin -Microtubule crosstalk; Formin-2 (Fmn2); axon guidance; growth 43 cone; filopodia 44 3 SIGNIFICANCE STATEMENT 45The formin family member, Fmn2, is associated with cognitive impairment and 46 neurodegenerative conditions though its function in neurons is poorly characterized. 47 We report a novel actin-microtubule cross-bridging activity for Fmn2 that facilitates 48 efficient targeting and capture of microtubules in growth cone filopodia. This activity 49 is necessary for accurate pathfinding of axons and may contribute to Fmn2-50 associated neuropathologies. 51The precision and adaptability of cytoskeleton-driven processes are intimately 52 dependent on the coupled activities of its component systems. Our study identifies a 53 novel modality of co-regulated remodelling of the actin and microtubule 54 cytoskeletons that facilitate critical cellular behaviour like neuronal chemotaxis. 56Formin 2 (Fmn2) is a non-DRF formin whose expression is enriched in developing 106

The formin family member, Fmn2, is a neuronally enriched cytoskeletal remodelling protein conserved across vertebrates. Recent studies have implicated Fmn2 in neurodevelopmental disorders, including sensory processing dysfunction and intellectual disability in humans. Cellular characterization of Fmn2 in primary neuronal cultures has identified its function in the regulation of cell-substrate adhesion and consequently growth cone translocation. However, the role of Fmn2 in the development of neural circuits in vivo, and its impact on associated behaviours have not been tested.Using automated analysis of behaviour and systematic investigation of the associated circuitry, we uncover the role of Fmn2 in zebrafish neural circuit development. As reported in other vertebrates, the zebrafish ortholog of Fmn2 is also enriched in the developing zebrafish nervous system. We find that Fmn2 is required for the development of an excitatory interneuron pathway, the spiral fiber neuron, which is an essential circuit component in the regulation of the Mauthner cell-mediated acoustic startle response. Consistent with the loss of the spiral fiber neurons tracts, high-speed video recording revealed a reduction in the short latency escape events while responsiveness to the stimuli was unaffected.Taken together, this study provides evidence for a circuit-specific requirement of Fmn2 in eliciting an essential behaviour in zebrafish. Our findings underscore the importance of Fmn2 in neural development across vertebrate lineages and highlight zebrafish models in understanding neurodevelopmental disorders.SIGNIFICANCE STATEMENTFmn2 is a neuronally enriched cytoskeletal remodelling protein linked to neurodevelopment and cognitive disorders in humans. Recent reports have characterized its function in growth cone motility and chemotaxis in cultured primary neurons. However, the role of Fmn2 in the development of neural circuits in vivo and its implications in associated behaviours remain unexplored. This study shows that Fmn2 is required for the development of neuronal processes in the acoustic startle circuit to ensure robust escape responses to aversive stimuli in zebrafish. Our study underscores the crucial role of the non-diaphanous formin, Fmn2, in establishing neuronal connectivity and related behaviour in zebrafish.

The formin Fmn2b is required for the development of an excitatory interneuron module in the zebrafish acoustic startle circuit