Practical Synthesis of Cap-4 RNA

Abstract

Eukaryotic mRNAs possess 5' caps that are determinants for their function. A structural characteristic of 5' caps is methylation, with this feature already present in early eukaryotes such as Trypanosoma. While the common cap-0 (m⁷ GpppN) shows a rather simple methylation pattern, the Trypanosoma cap-4 displays seven distinguished additional methylations within the first four nucleotides. The study of essential biological functions mediated by these unique structural features of the cap-4 and thereby of the metabolism of an important class of human pathogenic parasites is hindered by the lack of reliable preparation methods. Herein we describe the synthesis of custom-made nucleoside phosphoramidite building blocks for m⁶₂ Am and m³ Um, their incorporation into short RNAs, the efficient construction of the 5'-to-5' triphosphate bridge to guanosine by using a solid-phase approach, the selective enzymatic methylation at position N7 of the inverted guanosine, and enzymatic ligation to generate trypanosomatid mRNAs of up to 40 nucleotides in length. This study introduces a reliable synthetic strategy to the much-needed cap-4 RNA probes for integrated structural biology studies, using a combination of chemical and enzymatic steps.

Keywords: RNA modifications; chemoenzymatic synthesis; mRNA caps; oligonucleotides; solid-phase synthesis.

Figure 1

Structure of the hypermethylated cap‐4 of Trypanosoma mRNAs.

Scheme 1

Three‐step synthesis of m³Um phosphoramidite for RNA solid‐phase synthesis.

Scheme 2

Five‐step synthesis of m⁶Am phosphoramidite for RNA solid‐phase synthesis.

Figure 2

Preparation of short cap‐4 RNAs on solid phase. A) Schematics of the individual steps involved. B) Reaction control of the individual steps based on small portions of RNA assembled on solid‐support (I to IV) that were withdrawn and individually deprotected and analyzed by anion‐exchange chromatography (Dionex DNAPac PA‐100 (4×250 mm) column; temperature: 40 °C; flow rate: 1 mL min⁻¹; eluent A: 25 mm Tris⋅HCl (pH 8.0), 6 m urea; eluent B: 25 mm Tris⋅HCl (pH 8.0), 6 m urea, 500 mm NaClO₄; gradient: 0–60 % B in A within 45 min; UV detection at 260 nm.

Figure 3

Enzymatic methylation of cap‐4 RNA using Ecm1 methyltransferase. A) RNA sequences and reaction scheme. B) Anion‐exchange HPLC analysis of a typical methylation reaction (start and after 45 min; see the Experimental Section for reaction conditions), inset shows methylated product after purification. C) LC–ESI mass spectrum of Gppp‐RNA 10 and purified m⁷GpppRNA 11.

Figure 4

Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μm RNA 10, 12.5 μm RNA 11, 12.5 μm splint; 0.5 mm ATP, 40 mm Tris⋅HCl (pH 7.8), 10 mm MgCl₂, 10 mm DTT, 5 % (w/v) PEG 4000, 0.5 U μL⁻¹ T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.