Drug discovery based on small nucleic acid components has been an active research field during the last more than 20 years. Focus has primarily been on single-stranded antisense oligonucleotides and short interfering RNA duplexes (siRNAs) which can be designed to silence the biosynthesis of a disease-related protein or otherwise interfere with gene expression by Watson-Crick hybridization to an RNA target. The current status is one approved antisense drug (formivirsen; to treat cytomegallo virusinfection in the eye) and a number of compounds in various phases of clinical trials. A large variety of chemical modifications has been employed to improve the therapeutic properties of standard DNA or RNA oligonucleotides. Prominent among these modifications are phosphorothioate-DNA, 2’-O-alkyl-RNA and LNA (Figure 1, right), but poor tissue distribution, limited cell entry and low intracellular escape are challenging factors for the general applicability of gene silencing technologies.
Aptamers, which herein are defined as short single-stranded DNA or RNA oligonucleotides, are promising oligonucleotide constructs for drug development. Aptamers adopt well-defined three-dimensional shapes which enable targeting of peptides, proteins, small molecules and live cells. Aptamers are typically generated by evolution of specific sequences against a given target by in vitro evolution using the process known as SELEX (systematic evolution of ligands by exponential enrichment). SELEX involves iterative rounds of selection and polymerase-catalyzed enrichment (PCR) of bound aptamers selected from a pool of nucleic acid components, i.e. from a large library of typically ~80 nucleotide long sequences involving a central variable (sequence randomized) region flanked by two primer-binding regions. It is frequently possible to evolve aptamers with nanomolar affinity against their targets, and aptamers can therefore be considered “the antibodies of the nucleic acid world”.
Aptamers are versatile drug candidates as they can be evolved against extracellular targets like receptors or certain signaling molecules. Aptamers themselves have recently been applied in vivo to specifically deliver an anti-cancer siRNA to prostate cancer cells. Intracellular application of aptamers is a more challenging task due to poor intracellular delivery caused by their size and polyanionic character. Another challenge is biodistribution as unconjugated oligonucleotides are excreted rapidly via the kidneys upon i.v. administration. Pegylation and lipid formulation are approaches that has been applied to improve biodistribution. Size poses a challenge also for manufacture as current production technology relies on robotic solid phase synthesis. The above mentioned SELEX procedure is time consuming if performed in the standard manual fashion, but automation enables rather high-throughput evolution of aptamers whereas alternative selection/evolution strategies yet have to prove their general value and robustness.
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Figure 1. Structural representation (left) of a nucleic acid aptamer bound to its specific ligand (the exemplified structure shows an RNA aptamer complexed with HIV-1 REV peptide shown as coloured spheres model (data was obtained from Protein Data Bank - pdb ID: 484D - and remodelled using the software UCSF Chimera version 1); Structures (right) of typical modifications for therapeutic oligonucleotides, including a selection of those to be included in EVOLNA.
One aptamer has been approved as drug (pegaptanib; to treat age-related macular degeneration upon local administration), and others are in various stages of clinical development towards various diseases. In general, these therapeutic candidates have been obtained by so-called post-SELEX modification of aptamers evolved by a full SELEX procedure with 10-20 rounds of selection and enrichment. Post-SELEX modification typically involves truncation into shorter aptamer candidates, conjugation (e.g. pegylation) for improved biodistribution, and incorporation of chemically modified nucleotides for improved biostability. Post-SELEX chemical modification is necessary as only rather few modified nucleoside triphosphates, for example 2’-fluoro-RNA, 2’-amino-RNA and 5-substituted pyrimidine nucleoside triphosphates (Figure 1, right), are substrates for the polymerase-catalyzed reactions required for efficient SELEX procedures. Post-SELEX modifications are performed in iterative rounds of synthesis and binding assays/biological evaluation to ensure that modifications are compatible with the desired aptamer properties.
A prominent nucleotide modification in relation to nucleic acid drug discovery is LNA (locked nucleic acid, Figure 1). Incorporation of LNA nucleotides into DNA or RNA strands induces unrivalled increases in duplex thermodynamic stabilities, and LNA phosphorothioate oligonucleotides have shown unique characteristics as single stranded antisense molecule targeting RNA, e.g. mRNA or microRNA. LNA nucleotides increase nucleolytic stability of oligonucleotides, and their pronounced duplex-stabilizing effect furthermore add biostability to structured LNA-modified complexes. A few reports on post-SELEX modification of aptamers by chemical synthesis of LNA-modified variants have been published, and incorporation of LNA into a known Tenascin-C binding aptamer markedly improved plasma stability while maintaining target binding.
No reports exist on generation of LNA aptamers by in vitro selection. This is likely due to lack of compatibility of LNA nucleotides with the polymerase-catalyzed reactions involved in the necessary SELEX processes. Such an LNA-including SELEX process could as one example involve the steps illustrated (Figure 2) and described below:
Figure 2. Schematic illustration of an example LNA aptamer generation by SELEX.
Step 1 – Library generation: Random pools of LNA oligonucleotides are synthesised by automated procedures using standard phosphoramidite chemistry on a DNA synthesizer. Automated LNA synthesis is known to be compatible with DNA or RNA nucleotides as well as labels and modifiers.
Step 2 – Selection and partition: The LNA oligonucleotide library is incubated with the specific target of interest and the most strongly bound sequences are separated. This step is expected to be functional for LNA-based libraries.
Step 3 – Amplification: The selected LNA oligonucleotide sequences are amplified by PCR, either directly or indirectly which may include e.g. PCR to furnish LNA-DNA libraries, or PCR to furnish DNA-DNA libraries followed by assymmetric PCR to regenerate LNA-DNA libraries, or reverse transcription followed by transciption when using LNA-RNA libraries. These steps require compatibility of LNA nucleoside triphosphates and LNA-containing templates with polymerase activity (primer extension, PCR, reverse transcription and/or in vitro transcription).
Step 4 – Isolation and characterization: Cloning followed by sequencing after the last round of selection and amplification will allow characterization of the selected aptamer population, i.e. identification of consensus sequence fragments.
To set the stage for direct evolution of LNA aptamers we have at the Nucleic Acid Centre initiated a program aiming at development of efficient methods for enzymatic synthesis of LNA oligonucleotides using commercially available polymerases and LNA nucleoside 5’-triphosphates. We have identified the following DNA polymerases as able to incorporate LNA nucleotides into DNA strands opposite to DNA and LNA nucleotides of a template strand: Phusion High Fidelity DNA polymearase, 9o North DNA polymerase, KOD DNA polymerase, and KOD XL DNA polymerase. Primer extension reactions are effective using LNA nucleotides and LNA-containing templates. PCR with LNA-containing DNA duplexes as substrates has been successfully accomplished, though sequence effects and the number of LNA nucleotides seem to affect the efficiency if the goal is to amplify directly the LNA-containing PCR substrate. We have furthermore shown that transcription using T7 RNA polymerase is compatible with LNA nucleotides while we still have to explore reverse transcription with LNA substrates.