Synthetic oligonucleotides have become increasingly popular as a result of the recent discovery of ribonucleic acid interference (RNAi), a natural process for silencing gene expression. As biomedical researchers evaluate the use of antisense and small interfering RNAs (siRNAs) as potential therapies for the treatment of disease, the need for improved methods for the chromatographic separation and analysis of oligonucleotides has become apparent. This article presents a review of different liquid chromatography (LC) methods and strategies for the chromatographic separation of short RNA oligonucleotides.
There has been considerable interest recently in the use of synthetic oligonucleotides as potential therapeutic agents capable of suppressing the synthesis of specific proteins (1–3). Targeted "knockdown" of specific gene products using an antisense ribonucleic acid (RNA) strategy dates to the late 1990s (4). In this approach, a single-stranded oligonucleotide complementary to the messenger RNA (mRNA) encoding a targeted protein leads to disruption of ribosomal transcription and protein synthesis. In theory, antisense oligonucleotides can be applied to any disease in which protein overexpression is detrimental, and a number of antisense oligonucleotides have been evaluated as potential therapies (5). The need for long complementary oligonucleotides and the stoichiometric nature of mRNA inactivation (1 antisense molecule:1 mRNA inactivation) places considerable constraints on developing cost-effective antisense drugs.
The more recently discovered small interfering RNA (siRNA) mechanism for silencing gene expression involves a short double-stranded RNA molecule of about 21 base pair length, which activates the RNA interference (RNAi) silencing pathway (6,7), thereby achieving catalytic degradation of the target mRNA (one siRNA molecule inactivates multiple mRNAs). An overview of the RNAi pathway for targeted gene silencing is illustrated in Figure 1.
The discovery of siRNA gene silencing in animals (8) and human cells (9) has led to a surge of interest in the use of siRNA for biomedical and drug development research. Many biomedical and pharmaceutical companies have become involved in the exploration of the preparation and use of siRNAs as potential therapies for the treatment of diseases such as cancer, macular degeneration, and viral infections (10).
Figure 1: RNA interference mechanism. Long dsRNA in the cytoplasm is cleaved into 21-mer strands (siRNA) by the protein, Dicer. Small interfering RNA is incorporated into the RNA-induced silencing complex (RISC), where the passenger strand is unwound and degraded leaving the guide strand bound to RISC. The RISC-guide strand complex base-pairs with a complementary sequence of the mRNA and induces cleavage of the mRNA, thereby preventing protein translation. Synthetic siRNAs can be introduced into the cell and achieve the same action in the RNAi mechanism.
Oligonucleotide and siRNA Structure and Preparation
RNA is a biologically important molecule that consists of a long chain of nucleotide units. Each nucleotide contains a ribose sugar, a nitrogenous base, and a phosphate group. There are four bases in RNA: adenine (A), guanine (G), cytosine (C), and uracil (U) (Figure 2). Oligonucleotides are short, single-stranded RNA or deoxyribonucleic acid (DNA) molecules that can readily bind, in a sequence-specific manner, to their respective complementary oligonucleotides to form duplexes. Small interfering RNA is a small double-stranded RNA (usually 21 nucleotides) with two nucleotide overhangs on each 3'-end. Each strand has a 5'-phosphate group and a 3'-hydroxyl group (Figure 3).
Figure 2: Oligonucleotide structure. Different modifications include phosphothioate backbone modification where one oxygen atom on the phosphodiester backbone is replaced with a sulfur atom, and 2’-sugar modifications, such as 2’-F and 2’-O-Me. The four bases in RNA are adenine (A), guanine (G), cytosine (C), and uracil (U).
Various chemical modifications are often made to synthetic oligonucleotides to prevent attack by nucleases, which can lead to siRNA degradation and instability (11,12). Incorporation of either a fluoro or methoxy group into the 2' position of the sugar or the use of a phosphothioate linkage is commonly used to improve siRNA stability (Figure 2) (13). In the phosphothioate modification, oxygen in the phosphodiester linkage is replaced with a sulfur atom. This introduces an additional stereocenter into the molecule giving rise to two possible diastereomers for every phosphothioate linkage, and making the resulting oligonucleotide sample mixtures highly complex and very difficult to chromatographically resolve. All of these modifications help to improve oligonucleotide stability while retaining, and sometimes even increasing, their silencing activity. These modifications also tend to increase the hydrophobicity of the oligonucleotides, while also increasing the temperature at which the duplex melts (T
m) into its corresponding single strands.
Figure 3: Small interfering RNA (siRNA) structure. A 21-mer siRNA is shown with two nucleotide overhangs on each 3’-end. Each strand has a 5’-phosphate group and a 3’-hydroxyl group. The siRNA duplex consists of two complementary strands, the sense (or passenger) and antisense (or guide) strands. In RNA, adenine base-pairs with uracil by forming two intermolecular hydrogen bonds (A–U) and guanine base-pairs with cytosine by forming three intermolecular hydrogen bonds (G–C).
Oligonucleotides are readily synthesized via stepwise synthesis using phosphoramidite chemistry with automated solid-phase synthesizers (14). Although the individual synthetic process reactions can be very efficient and provide high yields, the total number of synthetic steps for making a 21-mer RNA can be more than 80 chemical steps (with about four chemical steps for each cycle). Consequently, because of the accumulation of many small errors, the final oligonucleotide product typically contains a variety of closely related impurities that can be very difficult to separate and remove during final product purification (15). Some of the most common impurities include sequence deletions, such as n–1, n–2, and so on, where one or more nucleotide fails to attach to the sequence during synthesis. Additionally, depurination, oxidation, and other chemical modification or degradation of the nucleotide bases can lead to a variety of closely related impurities that can be very challenging to resolve from the desired product. When dealing with double-stranded siRNAs, the sample mixtures can become even more complex with each strand introducing its own set of impurities. These impurities include mismatched sequences and noncomplementary single stranded sequences. The presence of these impurities in a therapeutic mixture can lead to unwanted, nontargeted gene silencing, while the presence of any nonhybridized single-stranded RNAs can also lead to a decrease in therapeutic potency (40). Therefore, when developing siRNA therapeutics, one of the major challenges is ensuring good purity to minimize off-target gene silencing effects. Consequently, developing good chromatographic techniques is often critically important in the oligonucleotide drug development process.