Locked Nucleic Acid and Aptamers

Abstract

Aptamers are short single-stranded DNA or RNA oligonucleotides capable of adopting well-defined 3-dimensional shapes, which enables targeting of peptides, proteins, small molecules, and live cells (MAYER, 2009; THIEL, 2009, Keefe et al., 2010). Aptamers are typically generated by in vitro selection using the process known as SELEX (systematic evolution of ligands by exponential enrichment) (Ellington and Szostak, 1990; Tuerk and Gold, 1990). SELEX involves iterative rounds of selection and polymerase-catalyzed enrichment using polymerase chain reaction (PCR) of bound aptamers from a large library of typically ∼80-nucleotide-long nucleic acid sequences composed of a central randomized region flanked by two primer-binding regions. Aptamers with nanomolar binding constants against their targets have frequently been evolved, and aptamers can therefore be considered “the antibodies of the nucleic acid world.” Even though the use of aptamers as therapeutic agents is promising, there are challenges that need to be met. One is that the size and polyanionic character of aptamers limits intracellular delivery. However, recent in vivo results on aptamer-mediated specific delivery of an anti-cancer small interfering RNA (siRNA) to prostate cancer cells (Dassie et al., 2009) have not only underlined the potential applicability of aptamers as drug delivery constructs but also give hope for intracellular applications of therapeutic aptamers. Importantly, aptamers, contrary to siRNA and antisense constructs, obviously can be applied against extracellular targets. Size is not only an obstacle for intracellular delivery, but also poses a challenge in manufacturing, as current production technology relies on DNA synthesizers for automated solid support-based synthesis. Another challenge is biodistribution, as unconjugated oligonucleotides are rapidly excreted via the kidneys upon intravenous administration. Conjugation with polyethyene glycol (PEG) units (Veronese and Pasut, 2005) is an approach that has been applied to improve biodistribution of aptamers. The SELEX procedure also can be limiting, as it is time consuming if performed in the standard manual fashion. Automation may in some cases alleviate this challenge (Eulberg et al., 2005; Wochner et al., 2007), and alternative selection/evolution strategies are being developed (Drabovich et al., 2006; Nitsche et al., 2007; Aquino-Jarquin and Toscano-Garibay, 2011; Arnold et al., 2012). One aptamer has been approved as drug (Pegaptanib) to treat age-related macular degeneration, and others are, or have been, in various stages of clinical development (FAMULOK, 2009; Keefe et al., 2010). In general, the therapeutic candidates have been obtained by post-SELEX modification of aptamers evolved by a full SELEX procedure including 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 rather few modified nucleoside triphosphates, for example, 2′-fluoro-RNA, 2′-amino-RNA and 5-substituted pyrimidine nucleoside triphosphates (MAYER, 2009; Lauridsen et al., 2012), are substrates for the polymerase-catalyzed reactions required for efficient SELEX procedures. Post-SELEX modifications are performed in iterative rounds of synthesis and 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 locked nucleic acid (LNA, Fig. 1) (Imanishi and Obika, 2002; Jepsen and Wengel, 2004; Veedu and Wengel, 2010). Incorporation of LNA nucleotides into DNA or RNA strands induces unrivalled increases in duplex thermal stabilities, and LNA phosphorothioate oligonucleotides have shown unique characteristics as single-stranded antisense molecule targeting RNA, for example, messenger RNA or microRNA (Jepsen and Wengel, 2004; Lanford et al., 2010). LNA nucleotides increase nucleolytic stability of oligonucleotides, and their pronounced duplex-stabilizing effect furthermore renders LNA-modified siRNA duplexes highly biostable (Glud et al., 2009). FIG. 1. Chemical structure of DNA, RNA, and locked nucleic acids (LNA). In our research group, we are currently working on development and evolution of LNA-containing aptamers (Veedu and Wengel, 2009). We want to explore LNA toward a breakthrough within the aptamer field. As mentioned above, despite obvious benefits and two decades of research and development, only relatively few aptamers are currently undergoing clinical development. We therefore think that the need for new approaches and methods to obtain short aptamers with increased biostability, structural stability, and biological function is evident. Post-SELEX LNA modification of aptamers as summarized below have been reported to yield some of these desired properties, but we simultaneously focus on enabling efficient and reliable de novo evolution of LNA aptamers.