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The current toolkit for genetic manipulation in the model animal Drosophila melanogasteris extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogasterusing the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesexusing scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3′ fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes.Techniques for genetic manipulation in Drosophila melanogasterare powerful but not without limitations. Here, the authors detail a novel approach to achieve strong RNA knockdown in Drosophilausing self-cleaving ribozymes. Ribozyme insertion via CRISPR/Cas9 into both nuclear and cytoplasmic noncoding RNAs and a male-specific splice variant of doublesexresulted in cleavage and efficient degradation of 3’ RNA fragments without observable off-target effects. This work demonstrates the potential of self-cleaving ribozymes for gene knockdown in Drosophila.

The factors affecting the formation and crystal structures of unusual 6d1Th(III) square planar aryloxide complexes, as exemplified by [Th(OArMe)4]1–(OArMe= OC6H2tBu2-2,6-Me-4), were explored by synthetic and reduction studies of a series of related Th(IV) tetrakis(aryloxide) complexes, Th(OArR)4(OArR= OC6H2tBu2-2,6-R-4). Specifically, electronic, steric, and countercation effects were explored by varying the aryloxide ligand, the alkali metal reducing agent, and the alkali metal chelating agent. Salt metathesis reactions between ThBr4(DME)2(DME = 1,2-dimethoxyethane) and 4 equiv of the appropriate potassium aryloxide salt were used to prepare a series of Th(IV) aryloxide complexes in high yields: Th(OArH)4(OArH= OC6H3tBu2-2,6), Th(OArtBu)4(OArtBu= OC6H2tBu3-2,4,6), Th(OArOMe)4(OArOMe= OC6H2tBu2-2,6-OMe-4), and Th(OArPh)4(OArPh= OC6H2tBu2-2,6-Ph-4). Th(OArH)4can be reduced by KC8, Na, or Li in the absence or presence of 2.2.2-cryptand (crypt) or 18-crown-6 (crown) to form dark purple solutions that have EPR and UV–visible spectra similar to those of the square planar Th(III) complex, [Th(OArMe)4]1–. Hence, the paraposition of the aryloxide ligand does not have to be alkylated to obtain the Th(III) complexes. Furthermore, reduction of Th(OArOMe)4, Th(OArtBu)4, and Th(OArPh)4with KC8in THF generated purple solutions with EPR and UV–visible spectra that are similar to those of the previously reported Th(III) anion, [Th(OArMe)4]1–. Although many of these reduction reactions did not produce single crystals suitable for study by X-ray diffraction, reduction of Th(OArH)4, Th(OArtBu)4, and Th(OArOMe)4with Li provided X-ray quality crystals whose structures had square planar coordination geometries. Reduction of Th(OArPh)4with Li also gave a product with EPR and UV–visible spectra that matched those of [Th(OArMe)4]1–, but X-ray quality crystals of the reduction product were too unstable to provide data. Neither Th(Odipp)4(THF)2(Odipp = OC6H3iPr2-2,6) nor Th(Odmp)4(THF)2(Odmp = OC6H3Me2-2,6) could be reduced to Th(III) products under similar conditions. Reduction of U(OArH)3(THF) with KC8in the presence of 2.2.2-cryptand (crypt) was examined for comparison and formed [K(crypt)][U(OArH)4], which has a tetrahedral arrangement of the aryloxide ligands. Moreover, no further reduction was observed when either [K(crypt)][U(OArH)4] or [K(crown)(THF)2][U(OArH)4] were treated with KC8or Li.

The importance of the specific trialkylsilyl substituent in the cyclopentadienyl chemistry of C5H4SiR3ligands has been demonstrated by the synthesis of low oxidation-state thorium complexes. Although the structure of the disilyl-substituted cyclopentadienyl Th(III) complex, [C5H3(SiMe3)2]3ThIII(Cp″3ThIII), was reported in 1986, no monosilyl-substituted analogues, (C5H4SiR3)3ThIII(R = alkyl, aryl), have been isolated to date, even though analogues are well known in U(III) chemistry. We now report that crystalline tris(monosilyl-substituted cyclopentadienyl) Th(III) and Th(II) complexes can be isolated when R = isopropyl, i.e., using the (triisopropylsilyl)cyclopentadienyl ligand, C5H4SiiPr3(CpTIPS). The salt metathesis reaction between three equiv of KCpTIPSand ThIVBr4(DME)2(DME = 1,2-dimethoxyethane) afforded the colorless Th(IV) complex, CpTIPS3ThIVBr, 1, which was identified spectroscopically and crystallographically. KC8reduction of 1in THF produced dark blue CpTIPS3ThIII, 2, in crystalline form. The complex was identified by X-ray crystallography, EPR, and UV–visible spectroscopy in contrast to ″(C5H4SiMe3)3ThIII,″ which has never been isolated due to its instability. This Th(III) complex can be reduced further with KC8in the presence of 2.2.2-cryptand (crypt) to make [K(crypt)][CpTIPS3ThII], 3, which is only the second crystallographically characterized Th(II) complex isolated since (Cp″3ThII)1–was discovered in 2014. Spectroscopic, crystallographic, and density functional theory (DFT) analyses are consistent with 6d1and 6d2electron configurations for the Th(III) and Th(II) complexes, respectively. The importance of the triisopropylsilyl substituent and the role that steric factors play in the successful isolation of Th(III) and Th(II) complexes were evaluated by Guzei solid angle calculations and electrochemical studies. The results suggest that both electronic and steric effects should be considered in the isolation of Th(III) and Th(II) complexes.