Synthesis of 5'-Thiamine-Capped RNA

Abstract

RNA 5'-modifications are known to extend the functional spectrum of ribonucleotides. In recent years, numerous non-canonical 5'-modifications, including adenosine-containing cofactors from the group of B vitamins, have been confirmed in all kingdoms of life. The structural component of thiamine adenosine triphosphate (thiamine-ATP), a vitamin B1 derivative found to accumulate in Escherichia coli and other organisms in response to metabolic stress conditions, suggests an analogous function as a 5'-modification of RNA. Here, we report the synthesis of thiamine adenosine dinucleotides and the preparation of pure 5'-thiamine-capped RNAs based on phosphorimidazolide chemistry. Furthermore, we present the incorporation of thiamine-ATP and thiamine adenosine diphosphate (thiamine-ADP) as 5'-caps of RNA by T7 RNA polymerase. Transcripts containing the thiamine modification were modified specifically with biotin via a combination of thiazole ring opening, nucleophilic substitution and copper-catalyzed azide-alkyne cycloaddition. The highlighted methods provide easy access to 5'-thiamine RNA, which may be applied in the development of thiamine-specific RNA capture protocols as well as the discovery and confirmation of 5'-thiamine-capped RNAs in various organisms.

Keywords: RNA modification; chemical capture of thiamine-capped RNA; click chemistry; in vitro transcription; non-canonical initiating nucleotide (NCIN); thiamine (vitamin B1); thiamine adenosine triphosphate; thiamine-adenosine diphosphate; thiamine-capped RNA.

Figure 1

Synthesis and purification of thiamine-ATP (ThATP). (A) Synthesis scheme of ThATP via coupling of adenosine 5’-phosphoroimidazolide (ImpA) (method A) and thiamine diphosphate β-P‑imidazolide (ImppTh) (method B) to thiamine pyrophosphate (ThDP) and adenosine 5’-monophosphate (5′‑AMP), respectively. Method A: 1. ThDP, magnesium chloride (MgCl₂), dimethylformamide (DMF), room temperature (rt), 2. ImpA; method B: 1. 5′-AMP, MgCl₂, DMF, rt, 2. ImppTh. (B) High-performance liquid chromatography (HPLC, see Supplementary Figure S1) and high-resolution mass spectrometry (HR-MS) analysis confirm the formation of ThATP following both methods. The formation of the major side product P¹,P²-di(adenosine-5′)-diphosphate (AppA) by homodimerization is avoided in method B.

Figure 2

Synthesis of 5′-thiamine-capped RNA by thiamine diphosphate β-P‑imidazolide (ImppTh) coupling to 5′-monophosphate RNA (5’-pRNA). (A) Schematic illustration of the preparation of 5′-thiamine RNA from 5′-monophosphate RNA (20mer) via (a) ImppTh capping (ImppTh, magnesium chloride, H₂O, 50 °C) and (b) 5′-monophosphate-dependent exoribonuclease digest using Xrn1, removing unreacted 5′-monophosphate RNA. (B) Deconvoluted mass spectra from the high-resolution mass spectrometry (HR-MS, electrospray ionization (ESI), negative mode) analysis of 5′‑monophosphate RNA and 5′-thiamine-capped RNA prepared via ImppTh capping. (C) Analysis of varied reaction conditions for ImppTh capping and complete digest of 5′-monophosphate RNA (20mer) from 5′-thiamine-capped RNA by denaturing polyacrylamide gel electrophoresis (see Supplementary Figure S4).

Figure 3

Synthesis of 5′-thiamine-capped RNA (8mer) by in vitro transcription with T7 RNA polymerase using ThATP as a non-canonical initiating nucleotide. (A) Schematic illustration of the in vitro transcription of 5′-thiamine-capped RNA with T7 RNA polymerase (T7 RNAP) using thiamine-ATP (ThpppA) and a 20mer DNA template containing a T7 class II promoter (Φ2.5) (Supplementary Table S2). After non-canonical transcription initiation with thiamine-ATP, the elongation process using CTP (pppC), GTP (pppG) and UTP (pppU) in the absence of ATP terminates after passing the nucleotide at the +8 position. In this case, a maximum transcript length of eight nucleotides with the sequence Th‑ACGGCUGG is obtained, which is thiamine-modified at the 5′-end. (B) High-performance liquid chromatography (HPLC) analysis of a phenol-ether extracted in vitro transcription reaction with thiamine-ATP in the absence of ATP. (C) Assignment of thiamine-capped oligomers to the HPLC peaks via high-resolution mass spectrometry analysis (Supplementary Figure S5).

Figure 4

Biotinylation of 5′-thiamine-capped RNA. (A) Schematic illustration depicting a combination of thiazole ring opening at elevated pH (see Supplementary Figure S8), (a) nucleophilic substitution (S_N) at the halogenated benzylic position of the azide-labeled linker L01 molecule (see Supplementary Figure S9) and (b) copper-catalyzed azide-alkyne cycloaddition (CuAAC) with biotin alkyne leading to biotinylation of 5′-thiamine-capped RNA I. (B) High-performance liquid chromatography (HPLC) analysis of the nucleophilic substitution reaction with Th-4mer RNA performed at different pH values. High-resolution mass spectrometry (HR-MS) analysis confirmed the formation of the desired azide-labeled Th-RNA species. (C) HPLC analysis of the CuAAC reaction with azide-modified 5’-thiamine 4mer RNA (Th-4mer RNA) and biotin alkyne. HR‑MS analysis confirmed the formation of the desired biotinylated Th‑RNA species. (D) Analysis of a streptavidin shift assay with [³²P]-cytidine-labeled 5′-thiamine RNA I (107mer, ThATP‑ and ThADP‑capped) by denaturing polyacrylamide gel electrophoresis. A retardation is visible only for the Th-capped RNA I samples treated via both nucleophilic substitution and CuAAC reaction, whereas no shift is observed for non-fully treated samples or upon similar treatment of control samples of 5′-triphosphate RNA I (see Supplementary Figure S10). Indicators + and − describe the incubation under the respective reaction conditions of nucleophilic substitution or CuAAC in the presence and absence respectively, of linker L01, biotin alkyne and streptavidin.