Post-assembly Modification of Tetrazine-Edged Fe^II₄L₆ Tetrahedra

Post-assembly modification (PAM) is a powerful tool for the modular functionalization of self-assembled structures. We report a new family of tetrazine-edged Fe^II₄L₆ tetrahedral cages, prepared using different aniline subcomponents, which undergo rapid and efficient PAM by inverse electron-demand Diels–Alder (IEDDA) reactions. Remarkably, the electron-donating or -withdrawing ability of the <i>para</i>-substituent on the aniline moiety influences the IEDDA reactivity of the tetrazine ring 11 bonds away. This effect manifests as a linear free energy relationship, quantified using the Hammett equation, between σ_<i>para</i> and the rate of the IEDDA reaction. The rate of PAM can thus be adjusted by varying the aniline subcomponent

Survival and activity are compromised in <i>cln3</i> ATG MO morphant zebrafish.

<p>(A-B) <i>cln3</i> ATG MO morphants die prematurely. Progression of <i>cln3</i> ATG morphants demonstrated by monitoring cohorts of fish injected with 1.6 ng or 2.9 ng of <i>cln3</i> ATG MO and uninjected WT siblings. (A) The Kaplan-Meier survival curve shows that all WT larvae survive beyond 6 dpf, whereas the majority of morphants injected with 1.6 ng <i>cln3</i> ATG MO die between 3 and 6 dpf (median survival 5 dpf). Log rank (Mantel-Cox) test <i>p</i><0.0001 (<i>n</i> = 46 WT and <i>n</i> = 23 morphants). (B) The Kaplan-Meier survival curve shows that all WT larvae survive beyond 4 dpf, whereas the morphants injected with 2.9 ng <i>cln3</i> ATG MO die between 1 and 4 dpf (median survival 1 dpf). Log rank (Mantel-Cox) test <i>p</i><0.0001 (<i>n</i> = 16 WT and <i>n</i> = 31 morphants). Error bars indicate ± SE. (C-E) Increased activity was observed in <i>cln3</i> ATG MO morphants at 36 hpf. Analysis of number of spontaneous coils or tail flicks within a 3 minute time period comparing 1.6 ng <i>cln3</i> ATG MO morphants with age matched WT siblings was carried out at 24 hpf (C), 36 hpf (D) and 48 hpf (E). At 24 and 48 hpf the data show no significant difference in the number of coils or flicks. At 36 hpf the data show a significant difference in the number of flicks; <i>p</i><0.0001 (<i>n</i> = 10 zebrafish per treatment group). Data represent mean ± SD; results were evaluated using a 2-tailed unpaired Student’s <i>t</i>-test. (F-G) The <i>cln3</i> ATG MO morphant escape response is diminished. (F) The WT (control) fish aged 4 dpf respond to touch with a C-bend, a turn away from the stimulus and rapid swimming (0.03 second intervals). (G) 1.6 ng c<i>ln3</i> ATG MO morphants aged 4 dpf display a greatly attenuated escape response (0.06 second intervals).</p

Identification and morpholino targeting of the zebrafish Cln3 gene.

<p>(A) CLUSTAL alignment indicates that zebrafish Cln3 amino acid sequence (second row) is 62% similar and 49% identical to human CLN3 (first row). Black background indicates identical residues shared between human and zebrafish, grey background indicates similarity, ‘-’ indicates a gap in the alignment. (B) RT-PCR using primer pairs AF1-AR1 and AF1-AR2 (see C), followed by sequencing, confirmed that the <i>cln3</i> SPL MO affected mRNA splicing. Lanes one and two show the PCR product from cDNA made from <i>cln3</i> SPL MO injected embryos and WT embryos respectively. Arrow in lane three points to the positive control, which is the PCR product generated using plasmid containing <i>cln3</i> mRNA as a template. (C) i) the gene structure of WT <i>cln3</i> showing the relative position of the PCR primers used. ii) The WT mature mRNA. iii) Splice variants caused by injection of <i>cln3</i> SPL MO. Key: lines, introns; boxes, exons; white boxes, untranslated regions; red asterisk, stop codon.</p

<i>cln3</i> is expressed in the developing WT zebrafish.

<p>A-N) <i>In situ</i> hybridisation for <i>cln3</i> (antisense) shows its expression from the one cell stage through to 4 dpf. From 1 dpf expression is increasingly restricted to the central nervous system. At 2 dpf <i>cln3</i> expression is particularly strong in the mid-hindbrain boundary (arrow) where the cerebellum is developing (G, H, M, N). (A’-L’) Experiments conducted using a <i>cln3</i> sense probe (sense) revealed the specificity of the antisense signal. Abbreviations: mhb, mid-hindbrain boundary.</p

Neurons and glia are disrupted in <i>cln3</i> ATG MO morphants.

<p>(A-A”, B-B”) Immunohistochemical staining for axons (acetylated α-tubulin) at 4 dpf. (A-A”) normal development of axons in WT larvae. (B-B”) 1.6 ng <i>cln3</i> ATG MO morphants have a complete absence of axonal organisation throughout the brain, with axonal accumulation (B, dashed arrows), loss of the optic tectum and a narrowing of the optic nerve (B'', dashed arrow). (A-A”, B-B”), anterior to the left; A, B, lateral view, dorsal up; A’, B’, dorsal view; A”, B” ventral view. (C-C’, D-D’) Immunohistochemistry using antibodies to glia (glial fibrillary acidic protein, GFAP) at 4 dpf. (C-C”) Normal staining in WT larvae. (D-D’) Ectopic GFAP is observed in the notochord in 1.6 ng <i>cln3</i> ATG MO morphants (dashed arrow). Lateral view. Anterior to the left. Dorsal up. (E-E’, F-F’, G-G’) Transgenic zebrafish expressing GFP under the control of the <i>HuC</i> promoter in neurons were injected with 1.6 ng <i>cln3</i> ATG MO and observed at 3 dpf. (E-E’) In WT zebrafish, the normal structure of the developing brain and retina can be observed. (F-F’) In morphants, there appear to be fewer neurons and the normal brain structure is lost. Many GFP-positive cells were enlarged and found nearer the surface of the brain (F’, dashed arrows). (G-G’) When the morphology of these enlarged cells was examined further, they lacked typical neuronal morphology. Lateral view. Anterior to the left. Dorsal up. Abbreviations: cb, cerebellum, fb, forebrain; hb, hindbrain; ot, optic tectum; on, optic nerve. A-G” (all images) Z projection. Scale bars: 100 μm. <i>n</i> = 4 per group.</p

Cellular proliferation is abnormal in <i>cln3</i> ATG MO morphant zebrafish.

<p>(A, A’) Proliferation, assayed at 4 dpf using anti-PH3 (a marker of proliferative cells in mitotic M phase), is observed throughout the 4 dpf WT retina, jaw and the brain. (B, B’) A marked reduction in the amount of cellular proliferation throughout the retina can be seen in the 1.6 ng <i>cln3</i> ATG MO morphant. Although not quantified, it appears that proliferation in the morphant brain (B) is increased compared to WT. Confocal images are Z-projections. Scale bar: 100 μm (A, A’, B) and 50 μm (B’). Lateral views. Anterior is to the left. Dorsal is up. (C) Quantification of these data show that the number of proliferating cells in the morphant retina is significantly reduced from 100.3 cells in WT to 50.3 cells in morphants; ***<i>p</i><0.0006 (<i>n</i> = 3 zebrafish per group). (D) Quantification demonstrating a significantly reduced mean retinal area in the morphants (0.0566 mm² for WT retinae compared to 0.0135 mm² for morphant retinae; ****<i>p</i><0.0001 (n = 10 zebrafish per group)). (C, D) Data represent mean ±SD; results were evaluated using a 2-tailed unpaired Student’s <i>t</i>-test.</p

Cellular proliferation is abnormal in <i>cln3</i> ATG MO morphant zebrafish.

<p>(A, A’) Proliferation, assayed at 4 dpf using anti-PH3 (a marker of proliferative cells in mitotic M phase), is observed throughout the 4 dpf WT retina, jaw and the brain. (B, B’) A marked reduction in the amount of cellular proliferation throughout the retina can be seen in the 1.6 ng <i>cln3</i> ATG MO morphant. Although not quantified, it appears that proliferation in the morphant brain (B) is increased compared to WT. Confocal images are Z-projections. Scale bar: 100 μm (A, A’, B) and 50 μm (B’). Lateral views. Anterior is to the left. Dorsal is up. (C) Quantification of these data show that the number of proliferating cells in the morphant retina is significantly reduced from 100.3 cells in WT to 50.3 cells in morphants; ***<i>p</i><0.0006 (<i>n</i> = 3 zebrafish per group). (D) Quantification demonstrating a significantly reduced mean retinal area in the morphants (0.0566 mm² for WT retinae compared to 0.0135 mm² for morphant retinae; ****<i>p</i><0.0001 (n = 10 zebrafish per group)). (C, D) Data represent mean ±SD; results were evaluated using a 2-tailed unpaired Student’s <i>t</i>-test.</p

Neurons and glia are disrupted in <i>cln3</i> ATG MO morphants.

<p>(A-A”, B-B”) Immunohistochemical staining for axons (acetylated α-tubulin) at 4 dpf. (A-A”) normal development of axons in WT larvae. (B-B”) 1.6 ng <i>cln3</i> ATG MO morphants have a complete absence of axonal organisation throughout the brain, with axonal accumulation (B, dashed arrows), loss of the optic tectum and a narrowing of the optic nerve (B'', dashed arrow). (A-A”, B-B”), anterior to the left; A, B, lateral view, dorsal up; A’, B’, dorsal view; A”, B” ventral view. (C-C’, D-D’) Immunohistochemistry using antibodies to glia (glial fibrillary acidic protein, GFAP) at 4 dpf. (C-C”) Normal staining in WT larvae. (D-D’) Ectopic GFAP is observed in the notochord in 1.6 ng <i>cln3</i> ATG MO morphants (dashed arrow). Lateral view. Anterior to the left. Dorsal up. (E-E’, F-F’, G-G’) Transgenic zebrafish expressing GFP under the control of the <i>HuC</i> promoter in neurons were injected with 1.6 ng <i>cln3</i> ATG MO and observed at 3 dpf. (E-E’) In WT zebrafish, the normal structure of the developing brain and retina can be observed. (F-F’) In morphants, there appear to be fewer neurons and the normal brain structure is lost. Many GFP-positive cells were enlarged and found nearer the surface of the brain (F’, dashed arrows). (G-G’) When the morphology of these enlarged cells was examined further, they lacked typical neuronal morphology. Lateral view. Anterior to the left. Dorsal up. Abbreviations: cb, cerebellum, fb, forebrain; hb, hindbrain; ot, optic tectum; on, optic nerve. A-G” (all images) Z projection. Scale bars: 100 μm. <i>n</i> = 4 per group.</p

<i>cln3</i> ATG MO morphants display epileptiform activity in the 2–4 Hz range.

<p>Single channel surface EEG shows frequent high amplitude spiking in both 1.6 ng <i>cln3</i> ATG MO morphants (B) and 1.6 ng <i>cln3</i> ATG MO + 8 ng <i>p53</i> MO double morphants (C) compared to WT (A). Fast Fourier transformation (FFT) was performed on 20 s representative samples, 5 times per trace and an average produced per trace; an average FFT per genotype was then used to calculate the mean frequency spectrum. The greatest spiking amplitude was observed between 2–4 Hz in the 1.6 ng <i>cln3</i> ATG morphants (E) and 1.6 ng <i>cln3</i> ATG MO + 8 ng <i>p53</i> MO double morphants (F) compared to WT (D), and this was a statistically significant difference (G). WT <i>n</i> = 5, <i>cln3</i> ATG MO <i>n</i> = 6, <i>cln3</i> ATG MO + <i>p53</i> MO n = 6.</p

Subunit c accumulates in lysosomes, and mitochondria are compromised in <i>cln3</i> ATG MO morphants.

<p>(A-A", B-B") Immunohistochemical staining for lysosomal associated membrane protein 1 (LAMP1, green) and subunit c of the mitochondrial ATP synthase (subunit c, red) at 4 dpf. (A-A”) WT. (B-B”) 1.6 ng <i>cln3</i> ATG MO morphants. In morphants, lysosomes appear larger (B, dashed arrow), subunit c accumulates (B’, dashed arrow) and they co-localise (B”, dashed arrows). Abbreviations: nc, notochord. Z slice. Lateral view. Anterior to left. Dorsal up. Scale bars: 50 μm. (C-C’, D-D’) Mitotracker stain labelling mitochondria in superficial cells of the eye at 4 dpf. (C-C’) WT. (D-D’) 1.6 ng <i>cln3</i> ATG MO morphant. In WT cells, many individual mitochondria are observed, whereas in morphant cells the stain has not been accumulated by mitochondria suggesting loss of mitochondrial membrane potential. Z slice. Lateral view. Scale bars: C, D 25 μm; C’, D’ 10 μm.</p