Nucleotides

Example 3

We tested the ability of mouse embryonic stem cells to tolerate s⁴U metabolic RNA-labeling after 12 h or 24 h at varying s⁴U concentrations (FIG. 6A). As reported previously, high concentrations of s⁴U compromised cell viability with an EC₅₀ of 3.1 mM or 380 μM after 12 h or 24 h labeling, respectively (FIG. 6A). Hence, we employed labeling conditions of 100 μM s⁴U, which did not severely affect cell viability. Under these conditions, we detected a steady increase in s⁴U-incorporation in total RNA preparation 3 h, 6 h, 12 h, and 24 h post labeling, as well as a steady decrease 3 h, 6 h, 12 h, and 24 h after uridine chase (FIG. 6B). As expected, the incorporation follows a single exponential kinetics, with a maximum average incorporation of 1.78% s⁴U, corresponding to one s⁴U incorporation in every 56 uridines in total RNA (FIG. 6C). These experiments establish s⁴U-labeling conditions in mES cells, which can be employed to measure RNA biogenesis and turnover rates under unperturbed conditions.

To test the ability of the method to uncover s⁴U incorporation events in high throughput sequencing datasets we generated mRNA 3′ end libraries (employing Lexogen's QuantSeq, 3′ mRNA-sequencing library preparation kit) using total RNA prepared from cultured cells following s⁴U-metabolic RNA labeling for 24 h (FIG. 7) (Moll et al., supra). Quant-seq 3′ mRNA-Seq Library Prep Kit generates Illumina-compatible libraries of the sequences close to the 3′end of the polyadenylated RNA, as exemplified for the gene Trim28 (FIG. 8A). In contrast to other mRNA-sequencing protocols, only one fragment per transcript is generated and therefore no normalization of reads to gene length is needed. This results in accurate gene expression values with high strand-specificity.

Furthermore, sequencing-ready libraries can be generated within only 4.5 h, with ˜2 h hands-on time. When combined with the invention, Quant-seq facilitates the accurate determination of mutation rates across transcript-specific regions because libraries exhibit a low degree of sequence-heterogeneity. Indeed, upon generating libraries of U-modified RNA through the Quant-seq protocol from total RNA of mES cells 24 h after s⁴U metabolic labeling we observed a strong accumulation of T>C conversions when compared to libraries prepared from total RNA of unlabeled mES cells (FIG. 8B). In order to confirm this observation transcriptome-wide, we aligned reads to annotated 3′ UTRs and inspected the occurrence of any given mutation per UTR (FIG. 9). In the absence of s⁴U metabolic labeling, we observed a median mutation rate of 0.1% or less for any given mutation, a rate that is consistent with Illumina-reported sequencing error rates. After 24 h of s⁴U metabolic labeling, we observed a statistically significant (p<10⁻⁴, Mann-Whitney test), 25-fold increase in T>C mutation rates, while all other mutations rates remained below expected sequencing error rates (FIG. 9). More specifically, we measured a median s⁴U-incorporation of 2.56% after 24 h labeling, corresponding to one s⁴U incorporation in every 39 uridines. (Note, that median incorporation frequency for mRNA are higher than estimated by HPLC in total RNA [FIG. 6C], most certainly because stable non-coding RNA species, such as rRNA, are strongly overrepresented in total RNA.) These analyses confirm that the new method uncovers s⁴U-incorporation events in mRNA following s⁴U-metabolic RNA labeling in cultured cells.

We expect the same incorporation results of other modified nucleotides, such as s⁶G or 5-ethynyluridine, as reported previously (Eidinoff et al., Science. 129, 1550-1551 (1959); Jao et al. PNAS 105, 15779-15784 (2008); Melvin et al. Eur. J. Biochem. 92, 373-379 (1978); Woodford et al. Anal. Biochem. 171, 166-172 (1988)).

Example 57

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¹H NMR (400 MHz, CDCl₃) δ 1.14-1.29 (m, 6H), 1.31-1.43 (m, 3H), 3.83-4.07 (m, 2H), 4.15-4.54 (m, 3H), 4.91-5.11 (m, 1H), 5.61-5.74 (m, 1H), 5.81-5.97 (m, 1H), 7.14-7.24 (m, 3H), 7.27-7.44 (m, 211), 7.48-7.51 (m, 1H), 7.80 (t, J=7.96, 7.96 Hz, OH), 9.30 (s, 1H). LC-MS m/z 516.3 (M+1+)

Example 122

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To a stirred solution of crude 325 in MeCN (100 mL) under nitrogen at rt, was added dropwise a solution of 2,6-dimethylphenol (1.22 g, 10.0 mmol), triethylamine (4.18 mL, 30.0 mmol), and DABCO (0.112 g, 1.00 mmol) in MeCN over 30 min. The mixture immediately turned deep red at the beginning of addition, and was stirred an additional 90 min after addition was completed. The reaction mixture was concentrated by rotary evaporation, and the residue was redissolved in CHCl₃ (300 mL). The solution was washed sequentially with sat. aq. NaHCO₃ (1×300 mL) and brine (2×300 mL), dried over Na₂SO₄, filtered, and concentrated by rotary evaporation to give a crude red oil. Flash chromatography on the Combiflash (330 g column, 5 to 20% EtOAc in hexanes gradient), gave 326 (5.02 g, 85% yield over 2 steps) as an off-white solid foam.

¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, J=7.4 Hz, 1H), 7.06 (s, 3H), 6.08 (d, J=7.4 Hz, 1H), 5.94 (d, J=15.9 Hz, 1H), 5.02 (dd, J=52.1 Hz, 3.1 Hz, 1H), 4.31 (d, J=13.8 Hz, 1H), 4.32-4.18 (m, 2H), 4.03 (dd, J=13.6 Hz, 2.0 Hz, 1H), 2.13 (s, 6H), 1.15-0.97 (m, 28H).