Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications

High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.

Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding small molecules in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although additional highly fluorescent RNA–fluorophore complexes would extend the utility of this technology, the identification of novel RNA–fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Additionally, aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here we describe a platform for rapid generation of highly fluorescent RNA–fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in Escherichia coli. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, we identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concentrations. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells.

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.