Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents

Abstract

Limited chemical diversity of nucleic acid libraries has long been suspected to be a major constraining factor in the overall success of SELEX (Systematic Evolution of Ligands by EXponential enrichment). Despite this constraint, SELEX has enjoyed considerable success over the past quarter of a century as a result of the enormous size of starting libraries and conformational richness of nucleic acids. With judicious introduction of functional groups absent in natural nucleic acids, the “diversity gap” between nucleic acid–based ligands and protein-based ligands can be substantially bridged, to generate a new class of ligands that represent the best of both worlds. We have explored the effect of various functional groups at the 5-position of uracil and found that hydrophobic aromatic side chains have the most profound influence on the success rate of SELEX and allow the identification of ligands with very low dissociation rate constants (named Slow Off-rate Modified Aptamers or SOMAmers). Such modified nucleotides create unique intramolecular motifs and make direct contacts with proteins. Importantly, SOMAmers engage their protein targets with surfaces that have significantly more hydrophobic character compared with conventional aptamers, thereby increasing the range of epitopes that are available for binding. These improvements have enabled us to build a collection of SOMAmers to over 3,000 human proteins encompassing major families such as growth factors, cytokines, enzymes, hormones, and receptors, with additional SOMAmers aimed at pathogen and rodent proteins. Such a large and growing collection of exquisite affinity reagents expands the scope of possible applications in diagnostics and therapeutics.

RAID3 - An interleukin-6 receptor-binding aptamer with post-selective modification-resistant affinity

“…This finding is in general agreement with experiences from other SELEX experiments and shows a bias for one target site in the selection of aptamers. [30][31][32] To circumvent these problems and probe new potential sites for aptamer binding on the surface of the sIL-6R, we set up a 2-tier RNA-SELEX. In the first 3 cycles of selection, immobilized sIL-6R was used to ensure enrichment of unmodified RNA aptamers that bind to the native soluble cytokine receptor.…”

Customised nucleic acid libraries for enhanced aptamer selection and performance

“…SOMAmers can also be further modified post-selectively, for example by replacing a modification at a specific nucleotide position with a slightly different chemical entity leading to SOMAmer variants with optimized properties [17][18][19]. For now, aromatic hydrophobic side chains have been found to be the most successful modifications in SELEX experiments [16].…”

“…Artificial nucleotides also enable the formation of novel three-dimensional structures of aptamers [16,20]. In comparison to conventional aptamers, the amount of polar contacts of SOMAmers to target molecules has been found to be decreased [20] and instead architectures with hydrophobic networks within a SOMAmer are formed, thus enabling the adoption of binding modalities that mimic those found in proteins.…”

Proteomics in Diagnosis: Past, Present and Future

“…The first is a versatile set of chemical modifications that endow SOMAmers with protein-like and other functional groups [47,48]. Synthesis and generation of SOMAmers are based on in vitro screening of large libraries of randomized sequences by a modified version of the process of systematic evolution of ligands by exponential (SELEX) enrichment [49].…”