Site-Specific Synthesis of N4-Acetylcytidine in RNA Reveals Physiological Duplex Stabilization

Abstract

N⁴-Acetylcytidine (ac4C) is a post-transcriptional modification of RNA that is conserved across all domains of life. All characterized sites of ac4C in eukaryotic RNA occur in the central nucleotide of a 5'-CCG-3' consensus sequence. However, the thermodynamic consequences of cytidine acetylation in this context have never been assessed due to its challenging synthesis. Here, we report the synthesis and biophysical characterization of ac4C in its endogenous eukaryotic sequence context. First, we develop a synthetic route to homogeneous RNAs containing electrophilic acetyl groups. Next, we use thermal denaturation to interrogate the biochemical effects of ac4C on duplex stability and mismatch discrimination in a native sequence found in human rRNA. Finally, we demonstrate the ability of this chemistry to incorporate ac4C into the complex modification landscape of human tRNA and use duplex melting to highlight an enforcing role for ac4C in this unique sequence context. By enabling ex vivo biophysical analyses of nucleic acid acetylation in its physiological sequence context, these studies establish a chemical foundation for understanding the function of a universally conserved nucleobase in biology and disease.

Figure 1.

(a) N⁴-Acetylcytidine (ac4C). (b) Schematic of the ac4C–G base pair. (c) Sites of ac4C that have been identified using nucleotide resolution methods in human RNA.

Figure 2.

(a) Strategy for site-specific synthesis of ac4C RNA. Retention of ac4C requires a solid support and protected building blocks that are labile to non-nucleophilic conditions (center), thus avoiding nucleophilic deprotection which cleaves ac4C (right). (b) Model substrates used in ac4C-DBU compatibility studies. (c) HPLC traces of model ceoc-C (top) and ac4C (bottom) substrates following exposure to DBU for 0.4 or 4 h. Extended HPLC traces are provided in the Supporting Information.

Figure 3.

Synthesis of building blocks for site-specific ac4C synthesis. Top: synthesis of N-ceoc-protected phosphoramidites. a) (i) (O-tBu)₂Si(OTf)₂, DMF, 0 °C, (ii) TBS-Cl, imidazole, DMF, 60 °C; b) ceoc-carbonyl-N-methylimidazolium chloride, DCM, 23 °C; c) HF-pyridine, pyridine, DCM, 0 °C; d) DMTr-Cl, pyridine, 4 °C; e) (OCH₂CH₂CN)P(iPr2N)₂, tetrazole, ACN, 23 °C; f) (O-tBu)₂Si(OTf)₂, TfOH, DMF, 0 °C, (ii) TBS-Cl, imidazole, DMF, 60 °C; g) H₂ (1 atm), Rh/alumina, MeOH, 23 °C; h) (OCH₂CH₂CN)(iPr₂N)PCl, iPr₂NEt, THF, 23 °C; j) triphenylphosphine, DIAD, (4-nitrophenyl)ethanol, dioxane, 100 °C; k) ceoc-chloroformate, DCM, 23 °C. Bottom: synthesis of photocleavable solid support. a) Methyl (4-bromobutyrate), K_zCO₃, DMF, 23 °C; b) TFA, KNO₃, THF, 23 °C; c) NaBH₄, MeOH, 23 °C, d) DMTr-Cl, pyridine, 4 °C; e) (i) aq. LiOH, THF, 23 °C, (ii) HATU, iPr₂NEt, LCAA-CPG, ACN, 23 °C, (iii) Piv-Cl, N-methylimidazole, 2,6-lutidine, THF, 23 °C. Right: graphical abbreviations for monomers used in this study. Full NMR and mass spectra are provided in the Supporting Information.

Figure 4.

(a) Solid-phase synthesis of ac4C-containing RNA oligonucleotides. Standard phosphoramidite synthesis conditions were employed, with the exception that no 5′-capping step was used. Optimizations critical for ac4C synthesis included on-column deprotection (#1), buffered photolytic cleavage (#2), and buffered desilylation condition (#3) to minimize alkylation and ac4C cleavage byproducts during release of the deprotected oligomer. (b) Ac4C synthesis is improved by the use of unprotected G monomer (#4). (c) Schematic for polyacrylamide gel electrophoresis (PAGE) purification and UV image of full-length and truncation products formed by optimized synthesis. (d) MALDI-TOF mass spectra of purified ac4C-containing 10-mer RNA. Full gels and MALDI-TOF spectra are provided in the Supporting Information.

Figure 5.

(a) Sequence alignment of the D-arm of serine and leucine tRNAs. D = dihydrouridine, yellow = ac4C, green = Gm, red = purine–purine pair. See Table 2 for the secondary structure. (c) PAGE analysis of the crude product. (c) MALDI-TOF analysis of the purified tRNA^Ser D-arm.

Figure 6.

Model for regulation of tRNA stability by ac4C-dependent D-stem stabilization. Yeast growth illustration made using Biorender.com. Primary data demonstrating reduced levels of tRNA^Ser and reduced cell fitness in ac4C-deficient Δtan1 strains are provided in ref 39.