DNA Display II. Genetic Manipulation of Combinatorial Chemistry Libraries for Small-Molecule Evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA “genes,” making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This “chemical translation” provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science “biology.

Casper Is a FADD- and Caspase-Related Inducer of Apoptosis

AbstractCaspases are cysteine proteases that play a central role in apoptosis. Caspase-8 may be the first enzyme of the proteolytic cascade activated by the Fas ligand and tumor necrosis factor (TNF). Caspase-8 is recruited to Fas and TNF receptor-1 (TNF-R1) through interaction of its prodomain with the death effector domain (DED) of the receptor-associating FADD. Here we describe a novel 55 kDa protein, Casper, that has sequence similarity to caspase-8 throughout its length. However, Casper is not a caspase since it lacks several conserved amino acids found in all caspases. Casper interacts with FADD, caspase-8, caspase-3, TRAF1, and TRAF2 through distinct domains. When overexpressed in mammalian cells, Casper potently induces apoptosis. A C-terminal deletion mutant of Casper inhibits TNF- and Fas-induced cell death, suggesting that Casper is involved in these apoptotic pathways

DNA Display III. Solid-Phase Organic Synthesis on Unprotected DNA

DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl–based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide–DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation

Long-term trends in Indigenous deaths from chronic diseases in the Northern Territory: a foot on the brake, a foot on the accelerator

Objective: To examine trends in Northern Territory Indigenous mortality from chronic diseases other than cancer. Design: A comparison of trends in rates of mortality from six chronic diseases (ischaemic heart disease [IHD], chronic obstructive pulmonary disease [COPD], cerebrovascular disease [CVD], diabetes mellitus [DM], renal failure [RF] and rheumatic heart disease [RHD]) in the NT Indigenous population with those of the total Australian population. Participants: NT Indigenous and total Australian populations, 1977–2001. Main outcome measures: Estimated average annual change in chronic disease mortality rates and in mortality rate ratios. Results: DeathratesfromIHDandDMamongNTIndigenouspeoplesincreased between 1977 and 2001, but this increase slowed after 1990. Death rates from COPD rose before 1990, but fell thereafter. There were non-significant declines in death rates from CVD and RHD. Mortality rates from RF rose in those aged 50 years. The ratios of mortality rates for NT Indigenous to total Australian populations from these chronic diseases increased throughout the period. Conclusions: Mortality rates from IHD and DM in the NT Indigenous population have been increasing since 1977, but there is evidence of a slower rise (or even a fall) in death rates in the 1990s. These early small changes give reason to hope that some improvements (possibly in medical care) have been putting the brakes on chronic disease mortality among Aboriginal and Torres Strait Islander peoples

Irreversibility, Mechanical Entanglement and Thermal Melting in Superconducting Vortex Crystals with Point Impurities

We discuss the onset of irreversibility and entanglement of vortex lines in high Tc superconductors due to point disorder and thermal fluctuations using a simplified cage model. A combination of Flory arguments, known results from directed polymers in random media, and a Lindemann criterion are used to estimate the field and temperature dependence of irreversibility, mechanical entanglement and thermal melting. The qualitative features of this dependence, including its nonmonotonicity when disorder is sufficiently strong, are in good agreement with recent experiments.Comment: 7 pages, uses RevTeX, multicol.sty and epsf.sty, 5 EPS figures include

B(H) Constitutive Relations Near H_c1 in Disordered Superconductors

We provide a self-contained account of the B vs. H constitutive relation near H_c1 in Type II superconductors with various types of quenched random disorder. The traditional Abrikosov result B ~ [ln (H - H_c1)]^{-2}, valid in the absence of disorder and thermal fluctuations, changes significantly in the presence of disorder. Moreover, the constitutive relations will depend strongly on the type of disorder. In the presence of point disorder, B ~ (H - H_c1)^{3/2} in three-dimensional (thick) superconductors, as shown by Nattermann and Lipowsky. In two-dimensional (thin film) superconductors with point disorder, B ~ (H - H_c1). In the presence of parallel columnar disorder, we find that B ~ exp[-C / (H - H_c1)] in three dimensions, while B ~ exp[-K / (H - H_c1)^{1/2}] in two dimensions. In the presence of nearly isotropically splayed disorder, we find that B ~ (H - H_c1)^{3/2} in both two and three dimensions.Comment: 37 pages, 12 figures included in text; submitted to Physica

DNA Display I. Sequence-Encoded Routing of DNA Populations

Recently reported technologies for DNA-directed organic synthesis and for DNA computing rely on routing DNA populations through complex networks. The reduction of these ideas to practice has been limited by a lack of practical experimental tools. Here we describe a modular design for DNA routing genes, and routing machinery made from oligonucleotides and commercially available chromatography resins. The routing machinery partitions nanomole quantities of DNA into physically distinct subpools based on sequence. Partitioning steps can be iterated indefinitely, with worst-case yields of 85% per step. These techniques facilitate DNA-programmed chemical synthesis, and thus enable a materials biology that could revolutionize drug discovery

Vortex wandering in a forest of splayed columnar defects

We investigate the scaling properties of single flux lines in a random pinning landscape consisting of splayed columnar defects. Such correlated defects can be injected into Type II superconductors by inducing nuclear fission or via direct heavy ion irradiation. The result is often very efficient pinning of the vortices which gives, e.g., a strongly enhanced critical current. The wandering exponent \zeta and the free energy exponent \omega of a single flux line in such a disordered environment are obtained analytically from scaling arguments combined with extreme-value statistics. In contrast to the case of point disorder, where these exponents are universal, we find a dependence of the exponents on details in the probability distribution of the low lying energies of the columnar defects. The analytical results show excellent agreement with numerical transfer matrix calculations in two and three dimensions.Comment: 11 pages, 9 figure