Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional proteinbinding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets.ince the advent of SELEX (Systematic Evolution of Ligands by EXponential enrichment) 22 years ago (1, 2), aptamers have been described that bind specifically and with high affinity to many different types of targets, including proteins, peptides, and small molecules (3). Binding interactions between aptamers and their targets are characterized by shape complementarity, polar contacts, hydrogen bonding interactions, and chargecharge interactions (3-5). Other than base stacking interactions, hydrophobic contacts, which are known to make key contributions to protein-protein interactions (6-8), have been notably limited, reflecting the lack of such moieties in nucleic acid libraries typically used in SELEX.We have recently shown that augmenting the diversity of randomized libraries with functional groups absent in natural nucleic acids can dramatically improve the success rate of SELEX, especially against difficult protein targets (9, 10). We have named this unique class of binding reagents SOMAmers (Slow Off-rate Modified Aptamers), to account for their distinct composition and binding properties. Among the different types of modifications we have tested, functional groups with hydrophobic character have typically yielded SOMAmers with the highest binding affinity. Although the contribution of such functional groups to the outcome of SELEX experiments has been quite apparent (9), the structural basis for the effect of these "side chains" on folding and binding has been unclear. Here, we report two cocrystal structures of related SOMAmers bound to a protein target, platelet-derived growth factor B (PDGF-BB), solved at a resolution of 2.2 Å and 2.3 Å. The structures elucidate the striking impact of the hydrophobic aromatic functional groups in creating novel intramolecular motifs and their extensive participation in shaping the contact surface with the native protein. By combining nucleic acid secondary st...

Discerning the structural building blocks of macromolecules is essential for understanding their folding and function. For a new generation of modified nucleic acid ligands (called slow off-rate modified aptamers or SOMAmers), we previously observed essential functions of hydrophobic aromatic side chains in the context of well-known nucleic acid motifs. Here we report a 2.45-Å resolution crystal structure of a SOMAmer complexed with nerve growth factor that lacks any known nucleic acid motifs, instead adopting a configuration akin to a triangular prism. The SOMAmer utilizes extensive hydrophobic stacking interactions, non-canonical base pairing and irregular purine glycosidic bond angles to adopt a completely non-helical, compact S-shaped structure. Aromatic side chains contribute to folding by creating an unprecedented intercalating zipper-like motif and a prominent hydrophobic core. The structure provides compelling rationale for potent inhibitory activity of the SOMAmer and adds entirely novel motifs to the repertoire of structural elements uniquely available to SOMAmers.

The addition of novel side chains at the 5-position of uracil is an effective means to increase chemical diversity of aptamers and hence the success rate for discovery of high-affinity ligands to protein targets. Such modifications also increase nuclease resistance, which is useful in a range of applications, especially for therapeutics. In this study, we assess the impact of these side chains on plasma pharmacokinetics of modified aptamers conjugated to a 40 kDa polyethylene glycol. We show that clearance from plasma depends on relative hydrophobicity: side chains with a negative cLogP (more hydrophilic) result in slower plasma clearance compared with side chains with a positive cLogP (more hydrophobic). We show that clearance increases with the number of side chains in sequences of ≥28 synthons, but this effect is dramatically diminished in shorter sequences. These results serve as a guide for the design of new therapeutic aptamers with diversity-enhancing side chains.

The modern version of the RNA World Hypothesis begins with activated ribonucleotides condensing (nonenzymatically) to make RNA molecules, some of which possess (perhaps slight) catalytic activity. We propose that noncanonical ribonucleotides, which would have been inevitable under prebiotic conditions, might decrease the RNA length required to have useful catalytic function by allowing short RNAs to possess a more versatile collection of folded motifs. We argue that modified versions of the standard bases, some with features that resemble cofactors, could have facilitated that first moment in which early RNA molecules with catalytic capability began their evolutionary path toward self-replication.