A Simple Route for Synthesis of 4-Phospho-D-Erythronate.

4-Phospho-D-erythronate is an intermediate in synthesis of pyridoxal 5'-phosphate in some bacteria and an inhibitor of ribose 5-phosphate isomerase. Previous synthetic schemes for the preparation of 4-phospho-D-erythronate required expensive precursors and typically gave low yields. We report a straightforward synthesis of 4-phospho-D-erythronate from the inexpensive precursor D-erythronolactone in 5 steps with a preparatively useful yield of 22%.

Aptamer-based multiplexed proteomic technology for biomarker discovery.

BACKGROUND: The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. METHODOLOGY/PRINCIPAL FINDINGS: We 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/SIGNIFICANCE: We 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.

Expanding the chemistry of DNA for in vitro selection.

Six new 5-position modified dUTP derivatives connected by a unique amide linkage were synthesized and tested for compatibility with the enzymatic steps of in vitro selection. Six commercially available DNA polymerases were tested for their ability to efficiently incorporate each of these dUTP derivatives during PCR. It was not possible to perform PCR under standard conditions using any of the modified dUTP derivatives studied. In contrast, primer extension reactions of random templates, as well as defined sequence templates, were successful. KOD XL and D. Vent DNA polymerases were found to be the most efficient at synthesizing full-length primer extension product, with all of the dUTP derivatives tested giving yields similar to those obtained with TTP. Several of these modified dUTPs were then used in an in vitro selection experiment comparing the use of modified dUTP derivatives with TTP for selecting aptamers to a protein target (necrosis factor receptor superfamily member 9, TNFRSF9) that had previously been found to be refractory to in vitro selection using DNA. Remarkably, selections employing modified DNA libraries resulted in the first successful isolation of DNA aptamers able to bind TNFRSF9 with high affinity.

Scanning probe-based fabrication of 3D nanostructures via affinity templates, functional RNA, and meniscus-mediated surface remodeling.

Developing generic platforms to organize discrete molecular elements and nanostructures into deterministic patterns on surfaces is one of the central challenges in the field of nanotechnology. Here we review three applications of the atomic force microscope (AFM) that address this challenge. In the first, we use two-step nanografting to create patterns of self-assembled monolayers (SAMs) to drive the organization of virus particles that have been either genetically or chemically modified to bind to the SAMs. Virus-SAM chemistries are described that provide irreversible and reversible binding, respectively. In the second, we use similar SAM patterns as affinity templates that have been designed to covalently bind oligonucleotides engineered to bind to the SAMs and selected for their ability to mediate the subsequent growth of metallic nanocrystals. In the final application, the liquid meniscus that condenses at the AFM tip-substrate contact is used as a physical tool to both modulate the surface topography of a water soluble substrate and guide the hierarchical assembly of Au nanoparticles into nanowires. All three approaches can be generalized to meet the requirements of a wide variety of materials systems and thereby provide a potential route toward development of a generic platform for molecular and materials organization.

Selection of biomolecules capable of mediating the formation of nanocrystals.

Biopolymers in the biosphere are well known to mediate the formation of a wide array of inorganic materials, such as bone, shells, lenses, and magnetic particles to name a few. Recently, in vitro experiments with biopolymers such as peptides, RNA, and DNA have shown that templating by these macromolecules can yield a variety of materials under mild reaction conditions. The primary sequence of the biopolymer can be viewed as a proteomic or genomic signature for the templating of an inorganic material from defined metal precursors and reaction conditions. Together with the rapid advances in inorganic particle synthesis by other combinatorial methods, these bioinspired in vitro materials experiments may provide additional insights into possible inorganic materials yet to be discovered and subsequently synthesized by conventional methods. Some of the concepts important to understanding the crystallization phenomena occurring during biopolymer mediation are discussed. A simple kinetic model is provided in the context of known biopolymer-mediated inorganic crystallizations.

RNA-mediated synthesis of palladium nanoparticles on Au surfaces.

RNA catalysts for the shape-controlled synthesis of Pd particles from the precursor complex trisdibenzylideneacetone dipalladium ([Pd2(DBA)3] were recently discovered in our laboratory (J. Am. Chem. Soc. 2005, 127, 17814-17818). In the work described here, RNA codes for hexagonal Pd platelets and Pd cubes were covalently immobilized on gold surfaces and evaluated for their activity toward particle synthesis. When coupled to gold via oligoethylene glycol linkers, both RNA sequences were able to catalyze the formation of Pd particles with the same shape control previously observed in solution. For low surface coverages, the average distance between RNA molecules on the surface was estimated at ca. 300 nm, yet large (e.g., dimensions of hundreds of nanometers) Pd hexagons and cubes still formed. This surprising result suggests that a single RNA molecule may be sufficient for nucleating and controlling the shapes of these particles. Finally, the use of surface-bound RNA as a tool for directing the orthogonal synthesis of materials on surfaces was demonstrated. Patterning the RNA code for Pd hexagons next to the code for Pd cubes, followed by incubation in a solution containing [Pd2(DBA)3], resulted in the spontaneous formation of spatially distinct spots of hexagonal and cubic particles.

RNA-mediated control of metal nanoparticle shape.

RNA sequences previously isolated by in vitro selection were further characterized for their ability to control palladium particle growth. Five pyridyl-modified RNA sequences (Pdases) representing each of the different evolved families were found to form hexagonal plates with a high degree of shape specificity. However, a sixth nonrelated pyridyl-modified RNA sequence was found to form exclusively cubic particles under identical conditions. Replacing pyridyl-modified RNA with native RNA resulted in a complete loss of RNA function. Removing the 3'-fixed sequence region from the Pdase had little effect on particle growth; however, further truncations into the variable region resulted in a significant loss of activity and particle shape control. These Pdases were selected using the organometallic precursor complex tris(dibenzylideneacetone) dipalladium(0) ([Pd2(DBA)3]). Changing the metal center and ligand of the group VIII organometallic precursor complex revealed a strong dependence of particle growth and shape on the DBA ligands. Changing the metal center from Pd to Pt while retaining the DBA ligands gave predominantly hexagonal Pt, but with a decrease in shape control. Taken together, the results of this study suggest that the full-length Pdases contain active sites capable of highly specific molecular recognition of organometallic complexes as particle formation reagents.

Charge-transfer excited state dynamics in DNA hairpins substituted with an ethylenylpyrenyl-dU electron source and halo-dU traps.

In this work nine DNA hairpins (HPs) are studied at room temperature to observe their pyrene(*+)/dU(*-) CT excited-state dynamics following photoexcitation at 355 nm with a 25 ps laser pulse. The HPs are 18-24 bases long, have a central tetra-T loop, and have a single U(PE) (5-(2-pyren-1-yl-ethylenyl)-2'-deoxyuridine) substitution in the central region of their stems. Three of the HPs are also substituted with 5-XdU traps, where X = Br or F, to learn about the effects of these traps on CT excited-state lifetimes and emission quantum yields in U(PE) substituted HPs. The combination of lengthened average CT lifetime and enhanced CT emission quantum yield in HPs with excess electron traps compared to HPs lacking traps strongly suggests that excess electrons are injected into the DNA stem at pyrimidine sites external to U(PE) as well via charge separation within U(PE) itself. Furthermore, the increased CT emission quantum yield in HPs with traps compared to HPs without traps implies that externally injected electrons can migrate to uracil in U(PE) (i.e., Py(*+)dU) and thus indirectly form the emissive Py(*+)dU(*-) CT state of U(PE).

The effect of mutation on RNA Diels-Alderases.

Chemical mutagenesis of a previously reported RNA Diels-Alderase (DA22) was followed by in vitro selection based on [4 + 2] catalysis. New mutated families of RNA Diels-Alderases closely related in sequence space were obtained. The mutated Diels-Alderases selected showed significant improvements in catalytic efficiency (k(cat)/K(m)) as compared to the original DA22. The improvement in catalytic activity was primarily due to a decrease in K(m), but modest increases in k(cat) were also observed. The increase in catalytic activity of these new Diels-Alderases was found not to negatively affect their dienophile specificity. Surprisingly, one of the most active Diels-Alderases (DAM 40), a subtle sequence mutant of DA22, was found to show a new metal dependence and could function with Ni(2+) as the only transition-metal ion. Truncation experiments of DA22 showed that the region shown to be hypervariable at the 3'-end of the structure could be deleted without a significant decrease in the relative rate of Diels-Alder catalysis.

T7 RNA polymerase transcription with 5-position modified UTP derivatives.

Seven UTP derivatives modified at the 5-position through an amide linkage were tested as substrates for T7 RNA polymerase (T7 RNAP) transcription. All UTP derivatives gave good yields of full-length transcript even from DNA templates that showed a significant number of abortive transcripts using unmodified UTP. A kinetic assay to determine the relative K(m) and V(max) for T7 RNAP transcription gave surprisingly similar values for UTP and the 5-position hydrophobic modifications phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl. The 5-position modifications imidazole and amino, which could both be positively charged, gave K(m) values significantly higher than UTP. All seven UTP derivatives gave relative V(max) values similar to UTP, indicating that insertion of these modified bases into the transcript did not impede its elongation.

RNA-mediated metal-metal bond formation in the synthesis of hexagonal palladium nanoparticles.

RNA sequences have been discovered that mediate the growth of hexagonal palladium nanoparticles. In vitro selection techniques were used to evolve an initial library of approximately 10(14) unique RNA sequences through eight cycles of selection to yield several active sequence families. Of the five families, all representative members could form crystalline hexagonal palladium platelets. The palladium particle growth occurred in aqueous solution at ambient temperature, without any endogenous reducing agent, and at low concentrations of metal precursor (100 micromolar). Relative to metal precursor, the RNA concentration was significantly lower (1 micromolar), yet micrometer-size crystalline hexagonal palladium particles were formed rapidly (7.5 to 1 minutes).

Synthesis of 5-(2,2'-bipyridinyl and 2,2'-bipyridinediiumyl)-2'-deoxyuridine nucleosides: precursors to metallo-DNA conjugates.

The synthesis of 2,2'-bipyridinyl-2'-deoxyuridine metal-chelator nucleosides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been accomplished. These new nucleosides will permit the construction of a number of corresponding metallo-DNA conjugates where many types of metals can be complexed to the 2,2'-bipyridinyl chelator group and the resulting metallo-dU conjugates incorporated into DNA oligonucleotides. Additionally this paper also reports the synthesis of a di-N-alkylated bipyridinediiumyl-2'-deoxyuridine nucleoside (Bipy(2+)-dU) with an ethylenyl linker. The Bipy(2+)-dU nucleoside was found to decompose under basic conditions precluding its use in standard automated DNA-synthesis by the phosphoramidite method. No such restrictions apply to the two Bipy-dU nucleosides reported here for use as metal chelators.