Diamond-like carbon (DLC) is an amorphous form of carbon with a significant fraction of sp3 bonds. In general, it enjoys high hardness, chemical inertness, and optical transparency. It can be ...deposited by various chemical (CVD) and physical (PVD) vapor deposition methods, including sputtering, which is an industrially important PVD method. Here we investigate the reactivity of carbon deposited by conventional direct current magnetron sputtering (DCMS) and by the newer high power impulse magnetron sputtering (HiPIMS). Scanning electron microscopy (SEM) showed that HiPIMS produced more compact films. Both types of carbon films were very smooth by atomic force microscopy (AFM): for HiPIMS: 1.20 ± 0.55 nm and for DCMS: 1.55 ± 0.27 nm. X-ray photoelectron spectroscopy (XPS) revealed chemical differences between the two types of carbon surfaces. Two functionalization schemes were investigated. The first consisted of amidation of the surfaces by activation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy sulfosuccinamide (sulfo-NHS) followed by reaction with a range of water-soluble and insoluble amines, and the second consisted of activation via halogenation with PCl5 or PBr5 followed by subsequent amination. The resulting surfaces were characterized by XPS and wetting. HiPIMS-deposited carbon showed higher levels of amidation. On the other hand, the DCMS surface showed greater functionalization in the halogenation/amination route. These results are consistent with the higher level of oxidized carbon initially found on the HiPIMS surface.
•The reactivities of two types of sputtered carbon (DCMS and HiPIMS) were evaluated vis-à-vis two synthetic organic routes.•The carbon surfaces are stabile under heated, aqueous conditions.•DCMS and HiPIMS carbon have different surface chemistries - the HiPIMS surface is richer in oxygen by XPS.•The degree of amine reaction is greater with the HiPIMS carbon in an activation/amidation route.•The degree of amine reaction is greater with the DCMS carbon in a halogenation/amination route.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
An mRNA‐protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate ...(the PROfusion™ process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA‐protein fusion to create an addressable protein microarray that self‐assembles via hybridization to surface‐bound DNA capture probes. The nucleic acid component not only directs the mRNA‐protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA‐protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub‐attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
An oligonucleotide substrate containing a 5‘-bridging phosphorothioate linkage adjacent to a ribonucleotide has been used to investigate the cleavage mechanism of the hammerhead ribozyme and to probe ...the catalytic role of the metal cofactor(s). Specifically, we tested the hypothesis that a second metal interacts with the 5‘-leaving group to facilitate the cleavage event. To this end, we have examined the ribozyme-mediated cleavage activity of the phosphorothioate substrate at pH 7.5 with a series of divalent metals in both the presence and absence of the polycation spermine. The cleavage products are found to be the same as for the native sequence under a variety of reaction conditions. The influence of divalent metal ion concentration, temperature, and pH on the cleavage rate has also been examined for both the oxo linkage and the thio analogue. Spermine (but not spermidine or NaCl) is shown to support efficient cleavage of the thio analogue in the absence 5 mM ethylenediaminetetraacetic acid (EDTA) of a divalent metal cofactor. The cleavage of the oxo linkage exhibits a solvent deuterium isotope effect of 3.6, but a similar effect is not observed with the thio analogue. The pseudo-first-order rate constants for cleavage of the thio analogue in the presence of 10 mM Mg2+ or Mn2+ at pH 7.5 are 65 and 82 × 10-3 min-1, respectively. The native oxo linkage is cleaved at essentially the same rate as the thio analogue (35 and 97 × 10-3 min-1 for Mg2+ and Mn2+, respectively). The absence of an appreciable thio effect and the lack of a preference for either Mg2+ or Mn2+ provides compelling evidence that the metal cofactor does not interact with the 5‘-thioanion (or oxyanion) leaving group in the transition state. These rate comparisons additionally reveal that the departure of the 5‘-leaving group is not the rate-limiting step of the cleavage reaction catalyzed by the hammerhead ribozyme.
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IJS, KILJ, NUK, PNG, UL, UM
An oligonucleotide has been synthesized that contains a single bridging 5'-phosphorothioate at an RNA linkage (5'-ApCpGpGpTpCpTprCpsApCpGpApGpC-3'). This new phosphodiester linkage is found to be ...particularly susceptible to cleavage when compared with the corresponding oxo, deoxy and thiodeoxy derivatives. Divalent metal cations were observed to dramatically increase the cleavage rate. The products of the cleavage under a variety of conditions are a 5'-thiol-containing fragment (6mer) and a 2',3'-cyclic phosphate-containing fragment (8mer). The pseudo-first order rate constant, kobs, for cleavage at pH 7.5 (50 mM Tris-HCI) in the presence of 5 mM EDTA is 1.5 x 10(-4)/min. In the presence of 5 mM metal dichloride and 50 mM Tris-HCI, pH 7.5, the relative cleavage rate enhancements are 10, 24, 71, 98, 370 and 3400 for Mg2+, Ca2+, Mn2+, Co2+, Zn2+ and Cd2+ respectively. The rate enhancements correlate well with Pearson's HSAB principle, suggesting that cleavage is mediated in part by coordination of the metal to the 5'-mercapto leaving group. RNA linkages containing bridging 5'-phosphorothioates should prove valuable for studying the mechanistic details of a variety of RNA cleaving agents, such as ribozymes.
A method is described for the incorporation of 2′-de-oxy2-thlourldlne (dS2U) and 2′-deoxy-2-thlothymldlne (dS2T) into oligodeoxynucleotides at predetermined positions. This requires N3 orO4-acylation ...of dS2U and d32T with toluoyl chloride. These base-protected thlo-pyrlmldlnes are completely stable toward the aqueous iodine oxidation reagent used in the phosphoramldlte DNA synthesis method. The toluoyl protecting group is removed during the standard post-synthetic ammonia treatment. This novel protection strategy allows dS2U and d82T to be efficiently incorporated into oligodeoxynucleotides at predetermined sites without the usual problem of desutfurization and decomposition. Several 14-mers containing the Eco-RI recognition site (dGGCGGAAXXCCGCC and dGGCGGAAXXCGCGG, where X represents dT, dS2U or dS2T) have been synthesized and characterized by base composition, thermal denaturatlon, CD spectroscopy and endonuclease substrate activity.
This paper describes the preparation and application of a chimeric DNA/RNA oligonucleotide that contains a single 5′-bridging phosphorothioate linkage adjacent to a ribonucleotide and embedded in an ...otherwise all-DNA sequence. The influence of pH, divalent metal cation, hybridization, and secondary structure on the susceptibility of the thio linkage towards transesterification is investigated in an effort to better understand the metal-phosphorothioate interactions and the basis for catalysis. In addition to the chemical cleavage, we have examined the hammerhead ribozyme mediated cleavage of the 5′-bridging phosphorothioate linkage specifically to test the hypothesis that the ribozyme employs a second metal cofactor, which functions as a Lewis acid, to catalyze transesterification. The results of our kinetics experiments do not support this double-metal model.
The synthesis and cleavage activity of a chimeric DNA/RNA oligonucleotide containing a 5′-bridging phosphorothioate linkage adjacent to a ribonucleotide in an otherwise all-DNA sequence is described.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We constructed a library of >10
12 unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (
...10Fn3). The antibody mimics that bound TNF-α were isolated from the library using mRNA display. Ten rounds of selection produced
10Fn3 variants that bound TNF-α with dissociation constants (K
d) between 1 and 24 nM. After affinity maturation, the lowest K
d measured was 20 pM. Selected antibody mimics were shown to capture TNF-α when immobilized in a protein microarray.
10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
HPLC is a powerful and popular method for analyzing and purifying biomolecules. Reversed-phase HPLC allows a high-capacity method for purification, and uses volatile buffer systems that simplify ...product recovery. Anion-exchange HPLC provides better resolution and a more predictable elution pattern. This unit presents protocols that are optimized for HPLC of oligonucleotides. Because of the resolution limits of both reversed-phase and anion-exchange HPLC, it can be used for oligonucleotides of up to approximately 50 nt in length.
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