BackgroundThe interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.Methodology/Principal FindingsWe present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.Conclusions/SignificanceWe describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

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...

The polymerization of e-caprolactone, (e-CL) using porcine pancreatic lipase (PPL) as the catalyst was studied. Polymerization reactions (4 days, 65 °C) of e-CL at ~10% (w/v) concentrations in dioxane, toluene, and heptane using butanol as an initiating species (monomer/butanol ratio = 14.7) gave poly(e-caprolactone) (PCL) with Mn values (by GPC) of 313, 753, and 1600, respectively. Monomer conversion to PCL for these polymerizations was 33, 55, and 100%, respectively. Mn measurements of PCL products by NMR end group analyses were slightly lower (by a factor of -0.9) than the values obtained by GPC. Polymerizations conducted in heptane at 37, 45, 55, and 65 °C showed the highest extent of monomer conversion at 65 °C. Therefore, subsequent studies were conducted at 65 °C in heptane. For a polymerization carried out with a 15/1 monomer/butanol ratio and ~0.29 mmol of water, ~70 and ~100% of the monomer had been converted to PCL by reaction times of 24 and 96 h, respectively. Polymer molecular weight increased slowly with conversion, suggesting that this is a chain polymerization with rapid initiation and slow propagation. Increases in the e-CL/butanol ratio from 15/1 up to where no butanol was added showed only a modest increase in product molecular weight from 1600 to 2700. This was explained by the fact that the water present in polymerizations was active in chain initiation. Variation in the monomer/butanol ratio at constant water concentration resulted in PCL chains with 0-0.65 mol fraction of butyl ester and 0.33-0.86 mol fraction of carboxylic acid chain end groups (by NMR analyses). The presence of water concentrations in polymerization reactions above that which is strongly enzyme bound is believed to be an important factor which limited the formation of PCL chains of significantly higher molecular weight.

It is well-known that thermal treatment of aspartic acid produces poly(anhydr0aspartic acid) (polysuccinimide), which can then be hydrolyzed to sodium polyaspartate. Both polysuccinimide and sodium polyaspartate have been analyzed by a variety of one-and two-dimensional NMR techniques. 'H NMR indicates that sodium polyaspartate invariably contains a 3:l ratio of j3:a linkages, under a variety of synthesis and hydrolysis conditions. NMR and lW3C HMBC data show that the sequencing is random. Residual succinimide levels in sodium polyaspartate are detectable down to about 1% by lH NMR. COSY data of polysuccinimide and lW16N HMQC-TOCSY of polysuccinimide synthesized from 100% 15N monomer indicate that phosphoric acid catalysis produces linear polymers, while uncatalyzed materials are branched.

The structures and hydrogen exchange properties of the mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2 have been studied by high-resolution nuclear magnetic resonance. Both the adenine-adenine and thymine-thymine mismatches are intercalated in the duplexes. The structures of these self-complementary duplexes are symmetric, with the two strands in equivalent positions. The evidence indicates that these mismatches are not stably hydrogen bonded. The mismatched bases in both duplexes are in the anti conformation. The mismatched thymine nucleotide in d(CCCTGGG)2 is intercalated in the duplex with very little distortion of the bases or sugar-phosphate backbone. In contrast, the bases of the adenine-adenine mismatch in d(CCCAGGG)2 must tilt and push apart to reduce the overlap of the amino groups. The thermodynamic data show that the T-T mismatch is less destabilizing than the A-A mismatch when flanked by C-G base pairs in this sequence, in contrast to their approximately equal stabilities when flanked by A-T base pairs in the sequence d(CAAAXAAAG.CTTTYTTTG) where X and Y = A, C, G, and T [Aboul-ela, F., Koh, D., & Tinoco, I., Jr. (1985) Nucleic Acids Res. 13, 4811]. Although the mechanism cannot be determined conclusively from the limited data obtained, exchange of the imino protons with solvent in these destabilized heteroduplexes appears to occur by a cooperative mechanism in which half the helix dissociates.