High-Resolution Structure of RNA G-Quadruplex Containing Unique Structural Motifs Originating from the 5′-UTR of Human Tyrosine Kinase 2 (TYK2)

Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor-associated tyrosine kinases together with highly homologous JAK1, JAK2, and JAK3 paralogues. Overexpression of TYK2 is associated with several inflammatory diseases, including severe complications during the COVID-19 infection. Since the downregulation of JAK paralogues could lead to serious health consequences or even death, it is critical to avoid it when designing drugs to suppress TYK2. To achieve the required specificity only for TYK2, researchers have recently selectively targeted TYK2 mRNA by developing antisense oligonucleotides. In this work, we expand the target space of TYK2 mRNA by showing that the mRNA adopts tetra-helical noncanonical structures called G-quadruplexes. We identified a TYKwt RNA oligonucleotide from the 5′-UTR of TYK2 mRNA, which adopts multiple different parallel G-quadruplexes that exist at equilibrium. Using NMR spectroscopy, we showed that some of the G-quadruplexes adopt unique structural motifs, mainly due to the formation of a stable GA bulge. Using guanine to uridine substitutions, we prepared the oligonucleotide TYK3_U6, which serves as an excellent model for the bulged G-quadruplexes formed by the TYKwt oligonucleotide. NMR structural analysis, including data on the residual coupling constants (RDC) of the loop regions, unveiled that the studied three-quartet parallel G-quadruplex contains many unusual structural features such as a G(U)A bulge, a guanine residue in the syn conformation, A and U residues stacked on the top G-quartet, and a well-defined adenine from a three-residue long propeller loop oriented in the groove, all of which could be valuable targets for future drug design.


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Figure S1. 1 H NMR spectra of wild type G-rich oligonucleotide with different length of overhangs in the presence of 50 mM KCl, 10 mM KPi buffer (pH 7.0) at 25 °C on 600 MHz spectrometer.Oligonucleotide concentrations were 0.15-0.3mM per strand.

Figure S2 .
Figure S2.CD spectra of L2TYK at 25 and 50 °C in the presence of 50 mM KCl, 10 mM KPi buffer (pH 7.0).Oligonucleotide concentrations were 0.025 mM per strand.

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Figure S3.Native PAGE of L2TYK and mutated oligonucleotides L2TYK1-4, TYK3_U6, TYK3 in the presence of 50 mM KCl at 25 °C.Oligonucleotide concentrations were 0.1 mM per strand.Single stranded DNA was used as reference ladder.

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Figure S4.Imino-regions of 1D 1 H NMR spectra of wild type G-rich oligonucleotide L2TYK and four Gto-U mutants L2TYK1-4 in the presence of 50 mM KCl, 10 mM KPi buffer (pH 7.0) at 25 °C on 600 MHz spectrometer.Oligonucleotide concentrations were 0.2-0.3mM per strand.

Figure S5 .
Figure S5.Integral intensities of imino signals in 1 H NMR spectra of L2TYK1-4 oligonucleotides in the presence of 50 mM KCl, 10 mM KPi buffer at pH 7.0 recorded at 50 °C with a 600 MHz spectrometer.All oligonucleotide concentrations were 0.2-0.3mM per strand.

Figure S6 .
Figure S6.CD spectra of oligonucleotides L2TYK1-4 at 25 °C in the presence of 50 mM KCl and 10 mM KPi buffer (pH 7.0).Oligonucleotide concentrations were 0.025 mM per strand.

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Figure S9.CD spectra of TYK3, TYK3_U6 and TYK3_U8 in the presence of 10 mM KCl, 10 mM KPi buffer (pH 7.0) at 25 °C.Oligonucleotide concentrations were 0.025 mM per strand.

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Figure S10. 1 H NMR spectra of TYK3 at different temperatures in the presence of 10 mM KCl, 10 mM KPi buffer (pH 7.0) on 600 MHz spectrometer.Oligonucleotide concentration was 0.3 mM per strand.

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Figure S11.Temperature-dependent UV experiment for TYK3 (a), TYK3_U6 (b) and TYK3_U8 (c) in the presence of 10 mM KCl, 10 mM KPi buffer (pH 7.0).Oligonucleotide concentrations were 0.01 mM per strand.The unfolding/refolding process was monitored between 5 and 95 °C by measuring absorbance at 295 nm with a scanning rate of 0.5 °C min -1 .

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Figure S12.Imino-regions of 1D 1 H spectra and 1D 15 N-edited HMQC spectra of L2TYK1, L2TYK2 and L2TYK4 oligonucleotides with partially (15%) 15 N1 labelled guanine residues at positions G6 and G8 on 600 MHz spectrometer.Samples were prepared in the presence of 50 mM KCl, 10 mM KPi buffer (pH 7.0) and recorded at 50 °C.Oligonucleotide concentrations were 0.1 mM per strand.

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Figure S15.Anomeric-aromatic region of 2D NOESY spectrum (mixing time 300 ms) of TYK3_U6 recorded at 0.5 mM oligonucleotide concentration per strand in presence of 10 mM KCl, 10 mM KPi buffer (pH 7.0) at 25 °C on 600MHz spectrometer.

Figure S17 .
Figure S17.Aromatic region of 2D 1 H-13 C HSQC spectrum of TYK3_U6 recorded at 0.5 mM oligonucleotide concentration per strand in presence of 10 mM KCl, 10 mM KPi buffer (pH 7.0) at 25 °C on 600MHz spectrometer.