SCA31 is one of the neurodegenerative disorders and caused by the insertion of expanded 5 bases repeat sequence d(TGGAA)_n in the region of the BEAN1 and TK2 genes. r(UGGAA)_n repeat forms RNA foci with various RNA binding proteins and impairs the original functions of proteins. We have recently reported NCD (Fig. 1A), a small molecule developed by our group, binds to r(UGGAA) repeat and alleviates RNA toxicity in the Drosophila model (T. Shibata et al., Nat. Comm., 2021). NMR structural analysis of the complex of r(UGGAA)_n-derived RNA (hereafter SCA31-hpRNA, Fig. 1B) and NCD revealed that two NCD molecules bind to the UGGAA sequence, and the naphthyridine moiety of NCD forms complementary hydrogen bonds to guanine (Fig. 1C).
Although physiochemical experiments are typically performed in a dilute buffer, the inside of the cells is crowded with various kinds of molecules. It is still unclear whether the complex of NCD and r(UGGAA)_n-repeat are maintained in the living cells. Thus, we aimed at investigating the behavior of the NCD and r(UGGAA)_n repeat complex by using in-cell NMR.
To obtain NMR signals in the cells, we have synthesized ¹³C-labeled NCD (¹³C-NCD, Fig. 1a (* = ¹³C)), which has carbon-13 in the methyl group at the 7-position. It is known that the methyl group gives signals with high sensitivity even in highly viscous cells, so we expected to obtain structural information by tracking the methyl groups. In ¹³C-¹H HSQC measurement of the methyl group in ¹³C-NCD, while ¹³C-NCD showed only a single cross peak (Fig. D(a)), the complex of ¹³C-NCD and SCA31-hpRNA showed two split peaks (Fig. D(b)). This suggests that we can monitor the behavior of the ligand with ease. Even after the addition of cell lysate, these two split peaks were maintained, meaning the complex is stable in the presence of other biomolecules (Fig. D(c)).
In this presentation, we will present the introduction of RNA into the cells and in-cell NMR measurement of SCA31-hpRNA and ¹³C-NCD complex.

<b>Figure 1</b>. (A) Chemical structure of NCD and <sup>13</sup>C-NCD (* = <sup>13</sup>C). (B) Sequence and of the RNA containing r(UGGAA/UGGAA) pentad (SCA31-hpRNA). (C) Schematic illustration of the binding mode of NCD to SCA31 hpRNA determined by NMR structural analysis. (D) <sup>13</sup>C-<sup>1</sup>H HSQC spectra of the methyl group of <sup>13</sup>C-NCD. (a) <sup>13</sup>C-NCD in buffer solution. (b) The complex of <sup>13</sup>C-NCD and SCA31-hpRNA in buffer solution. (c) The complex of <sup>13</sup>C-NCD and SCA31-hpRNA in the presence of cellular lysate.

Figure 1. (A) Chemical structure of NCD and ¹³C-NCD (* = ¹³C). (B) Sequence and of the RNA containing r(UGGAA/UGGAA) pentad (SCA31-hpRNA). (C) Schematic illustration of the binding mode of NCD to SCA31 hpRNA determined by NMR structural analysis. (D) ¹³C-¹H HSQC spectra of the methyl group of ¹³C-NCD. (a) ¹³C-NCD in buffer solution. (b) The complex of ¹³C-NCD and SCA31-hpRNA in buffer solution. (c) The complex of ¹³C-NCD and SCA31-hpRNA in the presence of cellular lysate.

Shuhei Sakurabayashi

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