Modified nucleotides may have enhanced early RNA catalysis.

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.

Crystal structure of interleukin-6 in complex with a modified nucleic acid ligand.

IL-6 is a secreted cytokine that functions through binding two cell surface receptors, IL-6Rα and gp130. Because of its involvement in the progression of several chronic inflammatory diseases, IL-6 is a target of pharmacologic interest. We have recently identified a novel class of ligands called SOMAmers (S low Off-rate Modified Aptamers) that bind IL-6 and inhibit its biologic activity. SOMAmers exploit the chemical diversity of protein-like side chains assembled on flexible nucleic acid scaffolds, resulting in an expanded repertoire of intra- and intermolecular interactions not achievable with conventional aptamers. Here, we report the co-crystal structure of a high affinity SOMAmer (Kd = 0.20 nm) modified at the 5-position of deoxyuridine in a complex with IL-6. The SOMAmer, comprised of a G-quartet domain and a stem-loop domain, engages IL-6 in a clamp-like manner over an extended surface exhibiting close shape complementarity with the protein. The interface is characterized by substantial hydrophobic interactions overlapping the binding surfaces of the IL-6Rα and gp130 receptors. The G-quartet domain retains considerable binding activity as a disconnected autonomous fragment (Kd = 270 nm). A single substitution from our diversely modified nucleotide library leads to a 37-fold enhancement in binding affinity of the G-quartet fragment (Kd = 7.4 nm). The ability to probe ligand surfaces in this manner is a powerful tool in the development of new therapeutic reagents with improved pharmacologic properties. The SOMAmer·IL-6 structure also expands our understanding of the diverse structural motifs achievable with modified nucleic acid libraries and elucidates the nature with which these unique ligands interact with their protein targets.

Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets.

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 protein-binding 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.

Broadly neutralizing aptamers to SARS-CoV-2: A diverse panel of modified DNA antiviral agents.

Since its discovery, COVID-19 has rapidly spread across the globe and has had a massive toll on human health, with infection mortality rates as high as 10%, and a crippling impact on the world economy. Despite numerous advances, there remains an urgent need for accurate and rapid point-of-care diagnostic tests and better therapeutic treatment options. To contribute chemically distinct, non-protein-based affinity reagents, we report here the identification of modified DNA-based aptamers that selectively bind to the S1, S2, or receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Several aptamers inhibit the binding of the spike protein to its cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and neutralize authentic SARS-CoV-2 virus in vitro, including all variants of concern. With a high degree of nuclease resistance imparted by the base modifications, these reagents represent a new class of molecules with potential for further development as diagnostics or therapeutics.

Telomere capping proteins are structurally related to RPA with an additional telomere-specific domain.

Telomeres must be capped to preserve chromosomal stability. The conserved Stn1 and Ten1 proteins are required for proper capping of the telomere, although the mechanistic details of how they contribute to telomere maintenance are unclear. Here, we report the crystal structures of the C-terminal domain of the Saccharomyces cerevisiae Stn1 and the Schizosaccharomyces pombe Ten1 proteins. These structures reveal striking similarities to corresponding subunits in the replication protein A complex, further supporting an evolutionary link between telomere maintenance proteins and DNA repair complexes. Our structural and in vivo data of Stn1 identify a new domain that has evolved to support a telomere-specific role in chromosome maintenance. These findings endorse a model of an evolutionarily conserved mechanism of DNA maintenance that has developed as a result of increased chromosomal structural complexity.

Elucidating mechanisms of genetic cross-disease associations at the PROCR vascular disease locus.

Many individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte-endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.

Sequence-specific binding to telomeric DNA is not a conserved property of the Cdc13 DNA binding domain.

In the budding yeast Saccharomyces cerevisiae, chromosome end protection is provided by a heterotrimeric complex composed of Cdc13 in association with the RPA-like proteins Stn1 and Ten1. We report here that the high affinity and specificity of the S. cerevisiae Cdc13 DNA binding domain for single-stranded telomeric DNA are not widely shared by other fungal Cdc13 proteins, suggesting that restriction of this complex to telomeres may be limited to the Saccharomyces clade. We propose that the evolutionarily conserved task of Stn1 and Ten1 (and their associated large subunit) is a genome-wide role in DNA replication rather than a telomere-dedicated activity.

Evolving A RIG-I Antagonist: A Modified DNA Aptamer Mimics Viral RNA.

Vertebrate organisms express a diversity of protein receptors that recognize and respond to the presence of pathogenic molecules, functioning as an early warning system for infection. As a result of mutation or dysregulated metabolism, these same innate immune receptors can be inappropriately activated, leading to inflammation and disease. One of the most important receptors for detection and response to RNA viruses is called RIG-I, and dysregulation of this protein is linked with a variety of disease states. Despite its central role in inflammatory responses, antagonists for RIG-I are underdeveloped. In this study, we use invitro selection from a pool of modified DNA aptamers to create a high affinity RIG-I antagonist. A high resolution crystal structure of the complex reveals molecular mimicry between the aptamer and the 5'-triphosphate terminus of viral ligands, which bind to the same amino acids within the CTD recognition platform of the RIG-I receptor. Our study suggests a powerful, generalizable strategy for generating immunomodulatory drugs and mechanistic tool compounds.

Modified aptamers as reagents to characterize recombinant human erythropoietin products.

Reliable and reproducible monitoring of the conformational state of therapeutic protein products remains an unmet technological need. This need is amplified by the increasing number of biosimilars entering the drug development pipeline as many branded biologics are reaching the end of their market exclusivity period. Availability of methods to better characterize protein conformation may improve detection of counterfit and unlicensed therapeutic proteins. In this study, we report the use of a set of modified DNA aptamers with enhanced chemical diversity to probe the conformational state of 12 recombinant human erythropoietin (rHuEPO) therapeutic protein products; one FDA-licensed rHuEPO originator biological product, three rHuEPO products that are approved for marketing in the US or EU as biosimilars, and eight rHuEPO products that are not approved for marketing in the US or EU. We show that several of these modified aptamers are able to distinguish rHuEPO reference products or approved biosimilars from non-licensed rHuEPO products on the basis of differences in binding kinetics and equilibrium affinity constants. These reagents exhibit sensitivity to the conformational integrity of various forms of rHuEPO and as such represent powerful, simple-to-use analytical tools to monitor the conformational integrity of therapeutic-proteins during manufacture and to screen for and identify both substandard and counterfeit products.

Structural basis for IL-1α recognition by a modified DNA aptamer that specifically inhibits IL-1α signaling.

IL-1α is an essential cytokine that contributes to inflammatory responses and is implicated in various forms of pathogenesis and cancer. Here we report a naphthyl modified DNA aptamer that specifically binds IL-1α and inhibits its signaling pathway. By solving the crystal structure of the IL-1α/aptamer, we provide a high-resolution structure of this critical cytokine and we reveal its functional interaction interface with high-affinity ligands. The non-helical aptamer, which represents a highly compact nucleic acid structure, contains a wealth of new conformational features, including an unknown form of G-quadruplex. The IL-1α/aptamer interface is composed of unusual polar and hydrophobic elements, along with an elaborate hydrogen bonding network that is mediated by sodium ion. IL-1α uses the same interface to interact with both the aptamer and its cognate receptor IL-1RI, thereby suggesting a novel route to immunomodulatory therapeutics.The cytokine interleukin 1α (IL-1α) plays an important role in inflammatory processes. Here the authors use SELEX to generate a modified DNA aptamer which specifically binds IL-1α, present the structure of the IL-1α/aptamer complex and show that this aptamer inhibits the IL-1α signaling pathway.

Embracing proteins: structural themes in aptamer-protein complexes.

Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer-protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer-protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity.

A structure-based interpretation of E.coli GrpE thermodynamic properties.

GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the prokaryotic homologue of Hsp70. Thermodynamic properties of GrpE structural domains were characterized by examining a number of structural and point mutants using circular dichroism, differential scanning calorimetry and analytical ultracentrifugation. These structural domains are the long paired N-terminal helices, the central four-helix bundle, and the C-terminal compact beta-domains. We show that the central four-helix bundle (t(m) approximately 75 degrees C) provides a stable platform for the association of the long paired N-terminal helices (t(m) approximately 50 degrees C), which can then function as a temperature sensor. The stability of the N-terminal helices is linked to the presence of the C-terminal compact beta-domains of GrpE, providing a potential mechanism for coupling of DnaK-binding activity of GrpE with temperature. On the basis of our thermodynamic analysis of E.coli GrpE, we present a structure-based model for the melting properties of the nucleotide exchange factor, wherein the long paired helices function as a molecular thermocouple.

Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation.

DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.

Non-helical DNA Triplex Forms a Unique Aptamer Scaffold for High Affinity Recognition of Nerve Growth Factor.

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.

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

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.

Thermodynamic linkage in the GrpE nucleotide exchange factor, a molecular thermosensor.

GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the bacterial homologue of Hsp70. In the temperature range of the bacterial heat shock response, the long helices of GrpE undergo a helix-to-coil transition, and GrpE exhibits non-Arrhenius behavior with respect to its nucleotide exchange function. It is hypothesized that GrpE acts as a thermosensor and that unwinding of the long helices of E. coli GrpE reduces its activity as a nucleotide exchange factor. In turn, it was proposed that temperature-dependent down-regulation of the activity of GrpE may increase the time in which DnaK binds its substrates at higher temperatures. A combination of thermodynamic and hydrodynamic techniques, in concert with the luciferase refolding assay, were used to characterize a molecular mechanism in which the long helices of GrpE are thermodynamically linked with the beta-domains via an intramolecular contact between Phe86 and Arg183. These "thermosensing" long helices were found to be necessary for full activity as a nucleotide exchange factor in the luciferase refolding assay. Point mutations in the beta-domains and in the long helices of GrpE destabilized the beta-domains. Engineered disulfide bonds in the long helices alternately stabilized the long helices and the four-helix bundle. This allowed the previously reported 75 degrees C thermal transition seen in the excess heat capacity function as monitored by differential scanning calorimetry to be further characterized. The observed thermal transition represents the unfolding of the four-helix bundle and the beta-domains. The thermal transitions for these two domains are superimposed but are not thermodynamically linked.