Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost, robust, scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined, xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally, PAS were scaled up to large culture-vessel formats. Synthetic, xeno-free, scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for both research purposes and development of cell therapies.

The development of ultraminiaturized identification tags has applications in fields ranging from advanced biotechnology to security. This paper describes micrometer-sized glass barcodes containing a pattern of different fluorescent materials that are easily identified by using a UV lamp and an optical microscope. A model DNA hybridization assay using these ''microbarcodes'' is described. Rare earth-doped glasses were chosen because of their narrow emission bands, high quantum efficiencies, noninterference with common fluorescent labels, and inertness to most organic and aqueous solvents. These properties and the large number (>1 million) of possible combinations of these microbarcodes make them attractive for use in multiplexed bioassays and general encoding. Encoded bead bioassays are emerging as an attractive alternative to traditional slide-based microarrays because beadbased bioassays offer multiplexing of both probes and samples (the ''analyte''), and they have significantly fewer drawbacks related to mass transport-limited binding of analytes to the immobilized probes. Several approaches have been described for the fabrication of encoded beads: those in which the coding material is randomly distributed in the bead (1, 2) and those in which the coding material is present in a defined pattern on the bead (3). Because different patterns of the same coding materials (e.g., position and thickness of metal stripes on cylindrical particles) result in distinguishable beads (3), a larger number of uniquely encoded beads can be obtained relative to beads with randomly distributed coding materials (e.g., polymer beads infused with mixtures of quantum dots) (2).Current methods for fabricating encoded beads are limited in terms of either the number of possible codes or the compatibility of the beads with bioassays and fluorescence detection. The most widely used method for making encoded beads, infusing polymer microspheres with mixtures of fluorescent dyes in predefined ratios, is not well suited for the fabrication of large (Ͼ10 5 ) numbers of uniquely distinguishable beads. Trau and coworkers have used silica microspheres containing fluorescent dyes for encoding polymer beads by using split-pool methods, and have also described the formation of dye-doped concentric silica layers around core silica particles (4). There are only a limited number of spectrally well-resolved dyes that do not also interfere with commonly used biological labels. Moreover, measurements of intensities and their ratios are inherently difficult, which limits the number of levels at which a dye can be incorporated to give distinguishable beads. Mixtures of quantum dots embedded in polymer microspheres offer significant advantages over conventional fluorescent dyes because they are relatively more photostable and have narrow emission linewidths (2). However, quantum dots are made of toxic materials (e.g., CdS, CdSe, CdTe) (5), and difficulties distinguishing between codes based on different amounts of the same quantum dots are similar to those ...

This paper describes a method for the detection of single-base mismatches using DNA microarrays in a format that does not require labeling of the sample ("target") DNA. The method is based on disrupting fluorescence energy transfer (FRET) between a fluorophore attached to an immobilized DNA strand ("probe") and a quencher-containing sequence that is complementary except for an artificial mismatch (e.g. 5-nitroindole, 3-nitropyrole, or abasic site) at the site of interrogation. As the displacement of the FRET acceptor and hybridization of the unlabeled probe are bimolecular, the term "bimolecular beacons" is used to describe this approach. The analysis of a mismatch was based on differences in the amount of disruption in FRET upon hybridization of perfectly matched DNA targets and those containing single-base mismatches. Using this method and an oligonucleotide model system, A/C single-base mismatches were successfully discriminated at levels greater than that observed using surface-immobilized molecular beacons. The amount of discrimination was dependent on the identity of the artificial mismatch; greater discrimination was observed with 5-nitroindole (a "universal" base) than with an abasic site. G/T mismatches, considered to be particularly difficult to detect, were also successfully discriminated when quencher sequences containing 5-nitroindole were used.

Synthetic derivatives of the calicheamicin oligosaccharide were designed to probe the molecular basis for pyrimidine recognition. Binding affinities of the oligosaccharide derivatives for a range of DNA sequences were determined using capillary electrophoresis. The results show that having an iodo-substituted C-ring is neither necessary nor sufficient for pyrimidine-selective binding. Pyrimidine-selective binding depends on maintaining the overall shape and rigidity of the calicheamicin oligosaccharide. These experiments support the proposal that the novel pyrimidine selectivity of calicheamicin is the result of a shape-dependent inducedfit interaction with DNA sequences.

A study of calicheamicin γ1 I complexed to seven different recognition sites is presented. The recognition sites encompass a range of oligopyrimidine sites that present different topological features in the minor groove. Intermolecular NOE networks for the different calicheamicin-DNA complexes show that the drug binds in the same mode to each recognition site. Calicheamicin binding also induces a set of characteristic conformational changes in the DNA in each complex that maximize the complementarity of the fit between calicheamicin and the DNA. Based on an analysis of the different complexes as well as biochemical information on cleavage preferences, we propose that calicheamicin displays a shape-selective preference for pyrimidine tracts through an induced-fit mechanism. We predict that any carbohydrate that maintains the overall shape of the calicheamicin oligosaccharide will exhibit similar sequence selectivity. This hypothesis is supported by experiments on calicheamicin oligosaccharide analogues reported in the following contribution.