Crossing the Halfway Point: Aptamer-Based, Highly Multiplexed Assay for the Assessment of the Proteome.

Measuring responses in the proteome to various perturbations improves our understanding of biological systems. The value of information gained from such studies is directly proportional to the number of proteins measured. To overcome technical challenges associated with highly multiplexed measurements, we developed an affinity reagent-based method that uses aptamers with protein-like side chains along with an assay that takes advantage of their unique properties. As hybrid affinity reagents, modified aptamers are fully comparable to antibodies in terms of binding characteristics toward proteins, including epitope size, shape complementarity, affinity and specificity. Our assay combines these intrinsic binding properties with serial kinetic proofreading steps to allow highly effective partitioning of stable specific complexes from unstable nonspecific complexes. The use of these orthogonal methods to enhance specificity effectively overcomes the severe limitation to multiplexing inherent to the use of sandwich-based methods. Our assay currently measures half of the unique proteins encoded in the human genome with femtomolar sensitivity, broad dynamic range and exceptionally high reproducibility. Using machine learning to identify patterns of change, we have developed tests based on measurement of multiple proteins predictive of current health states and future disease risk to guide a holistic approach to precision medicine.

Genomic atlas of the human plasma proteome.

Although plasma proteins have important roles in biological processes and are the direct targets of many drugs, the genetic factors that control inter-individual variation in plasma protein levels are not well understood. Here we characterize the genetic architecture of the human plasma proteome in healthy blood donors from the INTERVAL study. We identify 1,927 genetic associations with 1,478 proteins, a fourfold increase on existing knowledge, including trans associations for 1,104 proteins. To understand the consequences of perturbations in plasma protein levels, we apply an integrated approach that links genetic variation with biological pathway, disease, and drug databases. We show that protein quantitative trait loci overlap with gene expression quantitative trait loci, as well as with disease-associated loci, and find evidence that protein biomarkers have causal roles in disease using Mendelian randomization analysis. By linking genetic factors to diseases via specific proteins, our analyses highlight potential therapeutic targets, opportunities for matching existing drugs with new disease indications, and potential safety concerns for drugs under development.

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.

Modified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imaging.

Although current implementations of super-resolution microscopy are technically approaching true molecular-scale resolution, this has not translated to imaging of biological specimens, because of the large size of conventional affinity reagents. Here we introduce slow off-rate modified aptamers (SOMAmers) as small and specific labeling reagents for use with DNA points accumulation in nanoscale topography (DNA-PAINT). To demonstrate the achievable resolution, specificity, and multiplexing capability of SOMAmers, we labeled and imaged both transmembrane and intracellular targets in fixed and live cells.

Vascular dysfunction following breath-hold diving.

The pathogenesis of predominantly neurological decompression sickness (DCS) is multifactorial. In SCUBA diving, besides gas bubbles, DCS has been linked to microparticle release, impaired endothelial function, and platelet activation. This study focused on vascular damage and its potential role in the genesis of DCS in breath-hold diving. Eleven breath-hold divers participated in a field study comprising eight deep breath-hold dives with short surface periods and repetitive breath-hold dives lasting for 6 h. Endothelium-dependent vasodilation of the brachial artery, via flow-mediated dilation (FMD), and the number of microparticles (MPs) were assessed before and after each protocol. All measures were analyzed by two-way within-subject ANOVA (2 × 2 ANOVA; factors: time and protocol). Absolute FMD was reduced following both diving protocols (p < 0.001), with no interaction (p = 0.288) or main effect of protocol (p = 0.151). There was a significant difference in the total number of circulating MPs between protocols (p = 0.007), where both increased post-dive (p = 0.012). The number of CD31+/CD41- and CD66b+ MP subtypes, although different between protocols (p < 0.001), also increased by 41.0% ± 56.6% (p = 0.050) and 60.0% ± 53.2% (p = 0.045) following deep and repetitive breath-hold dives, respectively. Both deep and repetitive breath-hold diving lead to endothelial dysfunction that may play an important role in the genesis of neurological DCS.

Selection of DNA aptamers with two modified bases.

The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid-like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.

Single-Molecule Super-Resolution Microscopy Reveals Heteromeric Complexes of MET and EGFR upon Ligand Activation.

Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors.

Targeting Unique Epitopes on Highly Similar Proteins GDF-11 and GDF-8 with Modified DNA Aptamers.

The mature forms of the TGF-ß family members GDF-11 and GDF-8 are highly similar 25 kDa homodimers with 90% amino acid sequence identity and 99% similarity. Cross-reactivity of GDF-11 and GDF-8 binding reagents is common, making it difficult to attribute distinct roles of these two proteins in biology. We report the selection of GDF-11 and GDF-8 specific SOMAmer (Slow Off-rate Modified Aptamer) reagents aided by a combination of positive selection for one protein coupled with counter-selection against the other. We identified GDF-11 specific SOMAmer reagents from four modified DNA libraries that showed a high affinity (Kd range 0.05-1.2 nM) for GDF-11 but did not bind to GDF-8 (Kd > 1 µM). Conversely, we identified one SOMAmer reagent for GDF-8 from one of the modified libraries that demonstrated excellent affinity (Kd = 0.23 nM) and specificity. In contrast, standard protocols that utilized only positive selection produced binding reagents with similar affinity for both proteins. High affinity and specificity were efficiently encoded in minimal sequences of 21 nucleotides for GDF-11 and 24 nucleotides for GDF-8. Further characterization in pull-down, competition, sandwich-binding, and kinetic studies revealed robust binding under a wide range of buffer and assay conditions. For highly similar proteins like GDF-11 and GDF-8, our method of selection coupled with counter-selection was essential for identification of high-affinity, specific reagents that have the potential to elucidate the fundamental distinction of these growth factors in biology.

Correction: Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study.

[This corrects the article DOI: 10.1371/journal.pmed.1002781.].

Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study.

BACKGROUND: A nonsputum blood test capable of predicting progression of healthy individuals to active tuberculosis (TB) before clinical symptoms manifest would allow targeted treatment to curb transmission. We aimed to develop a proteomic biomarker of risk of TB progression for ultimate translation into a point-of-care diagnostic. METHODS AND FINDINGS: Proteomic TB risk signatures were discovered in a longitudinal cohort of 6,363 Mycobacterium tuberculosis-infected, HIV-negative South African adolescents aged 12-18 years (68% female) who participated in the Adolescent Cohort Study (ACS) between July 6, 2005 and April 23, 2007, through either active (every 6 months) or passive follow-up over 2 years. Forty-six individuals developed microbiologically confirmed TB disease within 2 years of follow-up and were selected as progressors; 106 nonprogressors, who remained healthy, were matched to progressors. Over 3,000 human proteins were quantified in plasma with a highly multiplexed proteomic assay (SOMAscan). Three hundred sixty-one proteins of differential abundance between progressors and nonprogressors were identified. A 5-protein signature, TB Risk Model 5 (TRM5), was discovered in the ACS training set and verified by blind prediction in the ACS test set. Poor performance on samples 13-24 months before TB diagnosis motivated discovery of a second 3-protein signature, 3-protein pair-ratio (3PR) developed using an orthogonal strategy on the full ACS subcohort. Prognostic performance of both signatures was validated in an independent cohort of 1,948 HIV-negative household TB contacts from The Gambia (aged 15-60 years, 66% female), longitudinally followed up for 2 years between March 5, 2007 and October 21, 2010, sampled at baseline, month 6, and month 18. Amongst these contacts, 34 individuals progressed to microbiologically confirmed TB disease and were included as progressors, and 115 nonprogressors were included as controls. Prognostic performance of the TRM5 signature in the ACS training set was excellent within 6 months of TB diagnosis (area under the receiver operating characteristic curve [AUC] 0.96 [95% confidence interval, 0.93-0.99]) and 6-12 months (AUC 0.76 [0.65-0.87]) before TB diagnosis. TRM5 validated with an AUC of 0.66 (0.56-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. The 3PR signature yielded an AUC of 0.89 (0.84-0.95) within 6 months of TB diagnosis and 0.72 (0.64-0.81) 7-12 months before TB diagnosis in the entire South African discovery cohort and validated with an AUC of 0.65 (0.55-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. Signature validation may have been limited by a systematic shift in signal magnitudes generated by differences between the validation assay when compared to the discovery assay. Further validation, especially in cohorts from non-African countries, is necessary to determine how generalizable signature performance is. CONCLUSIONS: Both proteomic TB risk signatures predicted progression to incident TB within a year of diagnosis. To our knowledge, these are the first validated prognostic proteomic signatures. Neither meet the minimum criteria as defined in the WHO Target Product Profile for a progression test. More work is required to develop such a test for practical identification of individuals for investigation of incipient, subclinical, or active TB disease for appropriate treatment and care.

Multiple-Ascending-Dose Phase 1 Clinical Study of the Safety, Tolerability, and Pharmacokinetics of CRS3123, a Narrow-Spectrum Agent with Minimal Disruption of Normal Gut Microbiota.

CRS3123 is a novel small molecule that potently inhibits methionyl-tRNA synthetase of Clostridioides difficile, inhibiting C. difficile toxin production and spore formation. CRS3123 has been evaluated in a multiple-ascending-dose placebo-controlled phase 1 trial. Thirty healthy subjects, ages 18 to 45 years, were randomized into three cohorts of 10 subjects each, receiving either 200, 400, or 600 mg of CRS3123 (8 subjects per cohort) or placebo (2 subjects per cohort) by oral administration twice daily for 10 days. CRS3123 was generally safe and well tolerated, with no serious adverse events (SAEs) or severe treatment-emergent adverse events (TEAEs) reported. All subjects completed their assigned treatment and follow-up visits, and there were no trends in systemic, vital sign, or laboratory TEAEs. There were no QTcF interval changes or any clinically significant changes in other electrocardiogram (ECG) intervals or morphology. CRS3123 showed limited but detectable systemic uptake; although absorption increased with increasing dose, the increase was less than dose proportional. Importantly, the bulk of the oral dose was not absorbed, and fecal concentrations were substantially above the MIC90 value of 1 µg/ml at all dosages tested. Subjects receiving either of the two lower doses of CRS3123 exhibited minimal disruption of normal gut microbiota after 10 days of twice-daily dosing. CRS3123 was inactive against important commensal anaerobes, including Bacteroides, bifidobacteria, and commensal clostridia. Microbiome data showed favorable differentiation compared to other CDI therapeutics. These results support further development of CRS3123 as an oral agent for the treatment of CDI. (This study has been registered at Clinicaltrials.gov under identifier NCT02106338.).

Structural model of the dimeric Parkinson's protein LRRK2 reveals a compact architecture involving distant interdomain contacts.

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.

Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study.

Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.).

Discovery and Validation of a Six-Marker Serum Protein Signature for the Diagnosis of Active Pulmonary Tuberculosis.

New non-sputum biomarker tests for active tuberculosis (TB) diagnostics are of the highest priority for global TB control. We performed in-depth proteomic analysis using the 4,000-plex SOMAscan assay on 1,470 serum samples from seven countries where TB is endemic. All samples were from patients with symptoms and signs suggestive of active pulmonary TB that were systematically confirmed or ruled out for TB by culture and clinical follow-up. HIV coinfection was present in 34% of samples, and 25% were sputum smear negative. Serum protein biomarkers were identified by stability selection using L1-regularized logistic regression and by Kolmogorov-Smirnov (KS) statistics. A naive Bayes classifier using six host response markers (HR6 model), including SYWC, kallistatin, complement C9, gelsolin, testican-2, and aldolase C, performed well in a training set (area under the sensitivity-specificity curve [AUC] of 0.94) and in a blinded verification set (AUC of 0.92) to distinguish TB and non-TB samples. Differential expression was also highly significant (P < 10-20) for previously described TB markers, such as IP-10, LBP, FCG3B, and TSP4, and for many novel proteins not previously associated with TB. Proteins with the largest median fold changes were SAA (serum amyloid protein A), NPS-PLA2 (secreted phospholipase A2), and CA6 (carbonic anhydrase 6). Target product profiles (TPPs) for a non-sputum biomarker test to diagnose active TB for treatment initiation (TPP#1) and for a community-based triage or referral test (TPP#2) have been published by the WHO. With 90% sensitivity and 80% specificity, the HR6 model fell short of TPP#1 but reached TPP#2 performance criteria. In conclusion, we identified and validated a six-marker signature for active TB that warrants diagnostic development on a patient-near platform.

Potential of High-Affinity, Slow Off-Rate Modified Aptamer Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Applications.

Direct pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant (P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals (P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.

Highly Multiplexed Proteomic Analysis of Quantiferon Supernatants To Identify Biomarkers of Latent Tuberculosis Infection.

The tests for diagnosing latent tuberculosis infection (LTBI) are limited by a poor predictive value for identifying people at the highest risk for progressing to active tuberculosis (TB) and have various sensitivities and specificities in different populations. Identifying a more robust signature for LTBI is important for TB prevention and elimination. A pilot study was conducted with samples from immigrants to the United States that were screened for LTBI by the three commercially approved tests, namely, the tuberculin skin test (TST), the Quantiferon-TB Gold in-tube (QFT-GIT), and the T-SPOT.TB (T-SPOT). QFT-GIT supernatants from 13 people with concordant positive results and 26 people with concordant negative results were analyzed via the highly multiplexed SOMAscan proteomic assay. The proteins in the stimulated supernatants that distinguished LTBI from controls included interleukin-2 (IL-2), monocyte chemotactic protein 2 (MCP-2), interferon gamma inducible protein-10 (IP-10), interferon gamma (IFN-γ), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT), monokine induced by gamma interferon (MIG), and granzyme B (P <0.00001). In addition, antigen stimulation increased the expression of heparin-binding EGF-like growth factor (HB-EGF) and activin AB in LTBI samples. In nil tubes, LIGHT was the most significant marker (P <0.0001) and was elevated in LTBI subjects. Other prominent markers in nonstimulated QFT-GIT supernatants were the complement-3 components C3b, iC3b, and C3d, which were upregulated in LTBI and markedly decreased upon stimulation. We found known and novel proteins that warrant further studies for developing improved tests for LTBI, for predicting progression to active disease, and for discriminating LTBI from active TB.

Influence of 5-N-carboxamide modifications on the thermodynamic stability of oligonucleotides.

We have recently shown that the incorporation of modified nucleotides such as 5-N-carboxamide-deoxyuridines into random nucleic acid libraries improves success rates in SELEX experiments and facilitates the identification of ligands with slow off-rates. Here we report the impact of these modifications on the thermodynamic stability of both duplexes and intramolecular 'single-stranded' structures. Within duplexes, large, hydrophobic naphthyl groups were destabilizing relative to the all natural DNA duplex, while the hydrophilic groups exhibited somewhat improved duplex stability. All of the significant changes in stability were driven by opposing contributions from the enthalpic and entropic terms. In contrast, both benzyl and naphthyl modifications stabilized intramolecular single-stranded structures relative to their natural DNA analogs, consistent with the notion that intramolecular folding allows formation of novel, stabilizing hydrophobic interactions. Imino proton NMR data provided evidence that elements of the folded structure form at temperatures well below the Tm, with a melting transition that is distinctly less cooperative when compared to duplex DNA. Although there are no data to suggest that the unmodified DNA sequences fold into structures similar to their modified analogs, this still represents clear evidence that these modifications impart thermodynamic stability to the folded structure not achievable with unmodified DNA.

Chemically modified DNA aptamers bind interleukin-6 with high affinity and inhibit signaling by blocking its interaction with interleukin-6 receptor.

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2'-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2'-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases.

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.