Combined Approaches to Site-Specific Modification of RNA

Abstract

Both natural and unnatural modifications in RNA are of interest to biologists and chemists. More than 100 different analogues of the four standard RNA nucleosides have been identified in nature. Unnatural modifications are useful for structure and mechanistic studies of RNA. This Review highlights chemical, enzymatic, and combined (semisynthesis) approaches to generate site specifically modified RNAs. The availability of these methods for site-specific modifications of RNAs of all sizes is important in order to study the relationships between RNA chemical composition, structure, and function.

Figure 1

a) Four major types of natural modification are shown: isomerization of uridine to pseudouridine (Ψ) base modification of adenosine to N⁶, N⁶-dimethyladenosine (m₂⁶A), 2′-O-methylation of cytidine to 2′-O-methylcytidine (Cm), and multiple modification of pseudouridine to 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (m¹acp³Ψ). b) Four examples of unnatural modifications are shown: 2-aminopurine, 2′-O-(2-nitrobenzyl)adenine, 2′-Se-methyluridine, and 5-iodocytidine.

Figure 2

The general scheme for solid-phase RNA synthesis is shown. A number of protective groups used at the 5′ (blue squares), 2′ (open triangles), and 3′ (black circles) positions are summarized in Table 1. The solid support is represented with a red circle.

Figure 3

Three types of nucleoside phosphoramidites are shown: a) 5′-O-DMT–2′-O-TBDMS–3′-O-(2-cyanoethyl-NN, -diisopropyl), b) 5′-O-DMT–2′-O-TOM–3′-O-(2-cyanoethyl-NN, -diisopropyl), and c) 5′-O-DOD–2′-O-ACE–3′-O-(methyl-NN, -diisopropyl) phosphoramidites.

Figure 4

A schematic diagram shows the generation of a site-specifically modified RNA. a) The enzymatic synthesis of an unmodified RNA segment (in red) using standard ribonucleotide triphosphates, a DNA template, and T7 RNA polymerase is represented. b) Chemical synthesis can be employed to generate an RNA segment (in blue) containing modified nucleotides (in green) at specific locations using the corresponding phosphoramidites. c) DNA-dependent RNA ligation of the two RNA segments using T4 DNA ligase generates a long segment of RNA representing a natural, functional RNA from the ribosome. d) An alternative strategy shows DNA-templated RNA ligation of two RNA segments with single-stranded regions using T4 RNA ligase.