RNA oligonucleotide synthesis

Contents

Introduction

The primary structures of RNA and DNA are similar, with two important differences: RNA has a ribose sugar rather than 2′-deoxyribose; and uracil replaces thymine as one of the heterocyclic bases. Not surprisingly, solid phase synthesis of RNA is based on solid-phase DNA synthesis, though considerable attention has to be given to the choice of protecting group for the 2′-hydroxyl function of ribose.

Solid-phase synthesis of RNA

The 2′-hydroxyl protecting group must be retained throughout the solid-phase synthesis cycle and then be removed cleanly at the end of the synthesis. It is imperative that there is no migration of the protecting group from 2′-OH to 3′-OH during synthesis of the phosphoramidite monomer, and no 3' to 2' phosphate migration during oligonucleotide synthesis or deprotection (see Figure 4).

A further important consideration in choosing appropriate protection for the 2′-OH is the influence that this group exerts on the phosphoramidite group in the 3′-position. Bulky protecting groups give reduced coupling efficiency, owing to steric hindrance. In practice, the choice of RNA synthesis methodology is dictated by the availability of RNA phosphoramidite monomers of high quality and reasonable cost. The most common method of 2′-OH protection for RNA synthesis is the tert-butyldimethylsilyl (TBS) method. During synthesis of the phosphoramidite monomers, the TBS group is added to the 2′-OH of the 5′-DMT-protected nucleoside, to afford the desired 2′-TBS protected nucleoside (Figure 1). However, this reaction is not regiospecific and a mixture of 2' and 3′-TBS-protected nucleosides are obtained that require careful separation by silica gel column chromatography. The desired 2′-TBS isomer is converted to the phosphoramidite monomer by reaction with 2-cyanoethyl diisopropylaminophosphorochloridite in the presence of the non-nucleophilic base N,N-diisopropylethylamine (DIPEA).

TBS-protected RNA monomer synthesis

Figure 1 | TBS-protected RNA monomer synthesisSynthesis of 2′-tert-butyldimethylsilyl (TBS)-protected phosphoramidite monomers for use in RNA synthesis.

After oligonucleotide synthesis and deprotection with ammonia, the 2′-TBS groups are removed from the oligoribonucleotide using a source of fluoride ion (e.g. tetrabutylammonium fluoride or Et3N · 3 HF). Removal of TBS protecting groups proceeds smoothly without cleavage or isomerization of the phosphodiester linkage.

In RNA synthesis, different heterocyclic base protecting groups are used from those employed in DNA synthesis. The tert-butylphenoxyacetyl group is used to protect the exocyclic amino groups of A, G and C. This group is useful for two reasons: (i) it is quickly removed in ammonia under conditions that do not cleave the 2′-TBS group (the carbonyl group of the amide is not sterically hindered), and (ii) the tert-butyl moiety increases the solubility of the monomer in organic solvents such as acetonitrile.

TBS-protected RNA monomers

Figure 2 | TBS-protected RNA monomersStructures of 2′-tert-butyldimethylsilyl (TBS)-protected phosphoramidite monomers used in solid-phase RNA synthesis.

The synthesis of oligoribonucleotides using 2′-TBS phosphoramidite monomers follows a similar synthesis cycle to that of standard solid-phase DNA synthesis. The differences are:

  • phosphoramidite coupling times are longer in RNA synthesis (up to 6 min) to allow for the steric bulk of the TBS group;
  • the capping reagent in RNA synthesis is tert-butylphenoxyacetic anhydride, rather than acetic anhydride, to negate the effect of acyl exchange at the protected amino groups of the bases.

After synthesis, the protected oligoribonucleotide is released from the solid support using concentrated aqueous ammonia/ethanol (3:1), and the protecting groups are then removed from the bases by heating at 55 °C in the same mixture for one hour. Finally, the 2′-TBS groups are removed using tetrabutylammonium fluoride in tetrahydrofuran.

Deprotection of the TBS group

Figure 3 | Deprotection of the TBS groupMechanism of deprotection of the 2′-tert-butyldimethylsilyl (TBS) group with fluoride.

The preparation of synthetic RNA has a number of limitations. The TBS group is not 100% stable to the basic conditions used to removed the tert-butylphenoxyacetyl protecting groups, and loss of the TBS group at this stage can lead to phosphodiester chain cleavage and (more seriously) 3′- to 2′-phosphate migration (Figure 4). This is a particularly severe problem for long oligoribonucleotides as the probability of any given RNA chain containing at least one 5′-2' linkage increases with oligonucleotide length.

It is imperative that RNA is handled with care to avoid contamination with RNAse enzymes that might otherwise result in degradation. RNAses are ubiquitous and sterile conditions are necessary when handing RNA. Prification of RNA is carried out by anion-exchange HPLC and/or reversed-phase HPLC and RNA molecules can be analysed by capillary or gel electrophoresis.

RNA chain cleavage and phosphate migration

Figure 4 | RNA chain cleavage and phosphate migrationMechanisms of RNA chain cleavage (top) and 3′- to 2′-phosphate migration (bottom).

Alternative RNA protecting group strategies

Improvements in the synthesis, deprotection and purification of RNA have produced more robust methods that enhance the yield and purity of the final product.

2-O-Triisopropylsilyloxymethyl (TOM) protecting groups

The 2-O-triisopropylsilyloxymethyl protecting group (TOM, Figure 5) is a simple modification of the widely used 2′-O-tert-butyldimethylsilyl (TBDMS/TBS) group and therefore is compatible with the currently established RNA synthesis chemistries.

TOM-protected RNA monomers

Figure 5 | TOM-protected RNA monomersStructures of 2-O-triisopropylsilyloxymethyl (TOM)-protected phosphoramidite monomers, used in solid-phase RNA synthesis.

The spacer between the 2′-position of the nucleoside and the silyl group overcomes the problem of steric hindrance encountered during the phosphoramidite coupling step of TBS-based RNA synthesis. The TOM protecting group is stable to the conditions required to remove the protecting groups from the heterocyclic bases and phosphodiesters (10 M methylamine in ethanol/water (1:1)). The TOM group is cleaved using 1 M TBAF in THF, without any degradation of the RNA oligonucleotide, and is stable to both basic and weakly acidic conditions. Importantly, the acetal group prevents 2′- to 3′-silyl migration in the nucleoside, which can occur with TBS protection, leading (after phosphitylation) to isomeric RNA monomers (Figure 1). Using the TOM protecting strategy, high coupling yields are achieved, in shorter reaction times, facilitating the synthesis of long RNA molecules.

2′-bis(2-Acetoxyethoxy)methyl (ACE) protecting groups

By changing the 5′-O-DMT protecting group to a silyl ether, the 2′-bis(2-acetoxyethoxy)methyl (ACE) orthoester group has been used to protect the 2′-OH of RNA monomers (Figure 6). The 5′-O-silyl ether can be removed with fluoride under neutral conditions that are compatible with the acid-labile 2′-O-ACE group. The ACE esters are stable to the conditions of oligonucleotide synthesis but are deacylated during the base deprotection step, resulting in a 2′-bis(2-hydroxyethoxy)methyl orthoester, which can be removed using weakly acidic conditions (pH 3, 10 min, 55 °C). This method also produces very high coupling yields with short reaction times. A major advantage is that the RNA can be purified with the 2′-protecting group attached. The 2-ACE-protected RNA is resistant to RNase degradation, and so can be stored for extended periods and deprotected prior to use.

ACE-protected RNA monomers

Figure 6 | ACE-protected RNA monomersStructures of 2′-Bis(2-acetoxyethoxy)methyl (ACE)-protected phosphoramidite monomers, used in solid-phase RNA synthesis.

2′-Thiomorpholine-4-carbothioate (TC) protecting groups

The use of 2′-thiomorpholine-4-carbothioate (TC) protecting groups (Figure 7), which can be removed in the same conditions used in the deprotection of the nucleobase protecting groups, allows the one-step deprotection of all protecting groups. This is in contrast to the other methods described here, all of which require separate deprotection steps for the 2′-OH protecting groups and nucleobase protecting groups.

TC-protected RNA monomers

Figure 7 | TC-protected RNA monomersStructures of 2′-thiomorpholine-4-carbothioate (TC)-protected phosphoramidite monomers, used in solid-phase RNA synthesis.

A solution of anhydrous ethylenediamine in toluene is used for this global deprotection of TC and base protecting groups (Figure 8).

Deprotection of the TC group

Figure 8 | Deprotection of the TC groupMechanism of deprotection of the 2′-thiomorpholine-4-carbothioate (TC) protecting group.

See also

This article on RNA oligonucleotide synthesis is part of the Nucleic Acids Book.