Optical trapping force reduction and manipulation of nanoporous beads.

We studied the interaction of infrared optical traps with controlled-pore glass (CPG) beads in aqueous medium. The lateral optical trapping force and stiffness were experimentally found considerably smaller than those of their solid counterparts. The simulation using an average refractive index revealed significant losses of effective trapping efficiency, which quantitatively agreed well with experimentally fitted curves. This effect was ascribed to the reduced relative refractive index of medium-filled CPG beads with respect to the medium. Combining optical trapping with mechanical confinements, we demonstrated a microfluidic platform allowing for the synthesis of multiple DNA oligonucleotide sequences on individual beads of interest.

Localization of multiple DNA sequences on nanopatterns.

DNA oligonucleotides of different sequences were patterned at the nanoscale. Areas of positive charge were generated by exposure of insulating substrates, spin-on hydrogen silsesquioxane or vapor-deposited SiO(2) on Si, with ionizing radiation sources used in electron beam and extreme ultraviolet lithography. Au nanoparticles (NPs) with a diameter of 15 nm, carrying covalently bound negatively charged single-stranded DNA oligonucleotides, were site specifically immobilized directly on the exposed regions and presented oligonucleotides for subsequent hybridization. Repeated exposure and deposition of NPs allowed for patterning multiple DNA sequences. Patterns with dimensions as small as 15 nm were fabricated using electron beam lithography. The use of DNA-functionalized NPs rather than just DNA facilitates metrology in scanning electron microscopy and improves the hybridization efficiency of the oligonucleotides on the surface.

SHADOW3: a new version of the synchrotron X-ray optics modelling package.

A new version of the popular X-ray tracing code SHADOW is presented. An important step has been made in restructuring the code following new computer engineering standards, ending with a modular Fortran 2003 structure and an application programming interface (API). The new code has been designed to be compatible with the original file-oriented SHADOW philosophy, but simplifying the compilation, installation and use. In addition, users can now become programmers using the newly designed SHADOW3 API for creating scripts, macros and programs; being able to deal with optical system optimization, image simulation, and also low transmission calculations requiring a large number of rays (>10(6)). Plans for future development and questions on how to accomplish them are also discussed.

Optical tweezers directed one-bead one-sequence synthesis of oligonucleotides.

An optical tweezers directed parallel DNA oligonucleotide synthesis methodology is described in which controlled pore glass (CPG) beads act as solid substrates in a two-stream microfluidic reactor. The reactor contains two parallel sets of physical confinement features that retain beads in the reagent stream for synthetic reaction but allow the beads to be optically trapped and transferred between the reagent and the inert streams for sequence programming. As a demonstration, we synthesized oligonucleotides of target sequence 25-nt, one deletion and one substitution using dimethoxytrityl (DMT) nucleoside phosphoramidite chemistry. In detecting single-nucleotide mismatches, fluorescence in situ hybridization of the bead-conjugated probes showed high specificity and signal-to-noise ratios. These preliminary results suggest further possibilities of creating a novel type of versatile, sensitive and multifunctional reconfigurable one-bead one-compound (OBOC) bead array.

Specificity landscapes of DNA binding molecules elucidate biological function.

Evaluating the specificity spectra of DNA binding molecules is a nontrivial challenge that hinders the ability to decipher gene regulatory networks or engineer molecules that act on genomes. Here we compare the DNA sequence specificities for different classes of proteins and engineered DNA binding molecules across the entire sequence space. These high-content data are visualized and interpreted using an interactive "specificity landscape" which simultaneously displays the affinity and specificity of a million-plus DNA sequences. Contrary to expectation, specificity landscapes reveal that synthetic DNA ligands match, and often surpass, the specificities of eukaryotic DNA binding proteins. The landscapes also identify differential specificity constraints imposed by diverse structural folds of natural and synthetic DNA binders. Importantly, the sequence context of a binding site significantly influences binding energetics, and utilizing the full contextual information permits greater accuracy in annotating regulatory elements within a given genome. Assigning such context-dependent binding values to every DNA sequence across the genome yields predictive genome-wide binding landscapes (genomescapes). A genomescape of a synthetic DNA binding molecule provided insight into its differential regulatory activity in cultured cells. The approach we describe will accelerate the creation of precision-tailored DNA therapeutics and uncover principles that govern sequence-specificity of DNA binding molecules.

Acetal levulinyl ester (ALE) groups for 2'-hydroxyl protection of ribonucleosides in the synthesis of oligoribonucleotides on glass and microarrays.

We describe a synthetic strategy that permits both the growth and deprotection of RNA chains that remain attached to a solid polymer support or chip surface. The key synthons for RNA synthesis are novel 5'-O-DMTr 2'-acetal levulinyl ester (2'-O-ALE) ribonucleoside 3'-phosphoramidite derivatives. In the presence of 4,5-dicyanoimidazole (DCI) as the activator, these monomers coupled to Q-CPG solid support with excellent coupling efficiency (approximately 98.7%). The method was extended to the light directed synthesis of poly rU and poly rA on a microarray through the use of a 5'-O-(2-(2-nitrophenyl)propoxycarbonyl)-2'-O-ALE-3'-phosphoramidite derivative. A two-stage deprotection strategy was employed to fully deblock the RNA directly on the Q-CPG or microarray support without releasing it from the support's surface: phosphate group deblocking with NEt(3) in acetonitrile (ACN) (2:3 v/v; 1 h, r.t.) followed by removal of the 2'-O-ALE groups under mild hydrazinolysis conditions (0.5-4 h, r.t.). This last treatment also removed the levulinyl (Lv) group on adenine (N(6)) and cytosine (N(4)) and the dimethylformamidine (dmf) group on guanine (N(2)). The chemistry and methods described here pave the way to the fabrication of microarrays of immobilized RNA probes for analyzing molecular interactions of biological interest.

Controlling oligonucleotide surface density in light-directed DNA array fabrication.

Over the past two decades high-density DNA arrays have developed into a central technology for nucleic acid analyses. Important application areas include whole-genome gene expression studies, high throughput analyses of single nucleotide polymorphisms, and, most recently, the determination of binding site specificities for transcription factors and other critical elements involved in gene regulation. A key parameter in the performance of DNA arrays is the density of the surface-bound oligonucleotides, which strongly affects both thermodynamic and kinetic aspects of DNA hybridization. In this report, we describe an approach for the control of oligonucleotide density in photolithographically fabricated DNA arrays, based upon a controlled UV light deprotection procedure. Modulation of the UV exposure permits a desired degree of deprotection of surface synthesis sites; a subsequent capping reaction to inactivate the exposed sites leaves only a desired fraction of active sites remaining for synthesis, corresponding to a lower oligonucleotide density. It is shown that the procedure is reasonably general, in that it is readily transferable to alternative substrate materials with similar results.

Carbon-on-metal films for surface plasmon resonance detection of DNA arrays.

Surface plasmon resonance (SPR) imaging affords label-free monitoring of biomolecule interactions in an array format. A surface plasmon conducting metal thin film is required for SPR measurements. Gold thin films are traditionally used in SPR experiments as they are readily functionalized with thiol-containing molecules through formation of a gold-sulfur bond. The lability of this gold-thiol linkage upon exposure to oxidizing conditions and ultraviolet light renders these surfaces incompatible with light-directed synthetic methods for fabricating DNA arrays. It is shown here that applying a thin carbon overlayer to the gold surface yields a chemically robust substrate that permits light-directed synthesis and also supports surface plasmons. DNA arrays fabricated on these carbon-metal substrates are used to analyze two classes of biomolecular interactions: DNA-DNA and DNA-protein. This new strategy allows the combinatorial study of binding interactions directly from native, unmodified biomolecules of interest and offers the possibility of discovering new ligands in complex mixtures such as cell lysates.

4X reduction extreme ultraviolet interferometric lithography.

We report the initial results from a 4X reduction interferometric lithography technique using extreme ultraviolet (EUV) radiation from a new undulator on the Aladdin storage ring at the Synchrotron Radiation Center of the University of Wisconsin-Madison. We have extended traditional interferometric lithography by using 2(nd) diffraction orders instead of 1(st) orders. This change considerably simplifies mask fabrication by reducing the requirements for mask resolution. Interferometric fringes reduced by 4X (from 70 nm half-period grating to 17.5 nm) have been recorded in a 50 nm thick hydrogen silsesquioxane photoresist using 13.4 nm wavelength EUV radiation.

Vacuum ellipsometry as a method for probing glass transition in thin polymer films.

A vacuum ellipsometer has been designed for probing the glass transition in thin supported polymer films. The device is based on the optics of a commercial spectroscopic phase-modulated ellipsometer. A custom-made vacuum chamber evacuated by oil-free pumps, variable temperature optical table, and computer-based data acquisition system was described. The performance of the tool has been demonstrated using 20-200 nm thick poly(methyl methacrylate) and polystyrene films coated on silicon substrates at 10(-6)-10(-8) torr residual gas pressure. Both polymers show pronounced glass transitions. The difficulties in assigning in the glass transition temperature are discussed with respect to the experimental challenges of the measurements in thin polymer films. It is found that the experimental curves can be significantly affected by a residual gas. This effect manifests itself at lower temperatures as a decreased or even negative apparent thermal coefficient of expansion, and is related to the uptake and desorption of water by the samples during temperature scans. It is also found that an ionization gauge--the standard accessory of any high vacuum system--can cause a number of spurious phenomena including drift in the experimental data, roughening of the polymer surface, and film dewetting.

In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication.

Glass has become the standard substrate for the preparation of DNA arrays. Typically, glass is modified using silane chemistries to provide an appropriate functional group for nucleic acid synthesis or oligonucleotide immobilization. We have found substantial issues with the stability of these surfaces as manifested in the unwanted release of oligomers from the surface when incubated in aqueous buffers at moderate temperatures. To address this issue, we have explored the use of carbon-based substrates. Here, we demonstrate in situ synthesis of oligonucleotide probes on carbon-based substrates using light-directed photolithographic phosphoramidite chemistry and evaluate the stabilities of the resultant DNA arrays compared to those fabricated on silanized glass slides. DNA arrays on carbon-based substrates are substantially more stable than arrays prepared on glass. This superior stability enables the use of high-density DNA arrays for applications involving high temperatures, basic conditions, or where serial hybridization and dehybridization is desired.

The con-focal method on verifying focal plane of MAS machine.

The Maskless DNA Array Synthesizer (MAS) is very efficient and flexible device on custom designed DNA array fabricating. To make the MAS's focal plane keep stable is important during the chip synthesizing process. In this paper we bring a creative idea using con-focal method to verify focal plane of MAS machine. That method is very sensitive on focal plane verification and could be used on other optical lithography system or the projecting system, which need real time focal plane precision control.

A scalable method for multiplex LED-controlled synthesis of DNA in capillaries.

As research in synthetic biology and genomic sciences becomes more widespread, the need for diverse oligonucleotide populations has increased. To limit reagent cost, it would be advantageous to obtain high quality populations in minute amounts. Towards that end, synthesis of DNA strands in capillaries utilizing photolabile 3-nitrophenylpropyloxycarbonyl (NPPOC) chemistry and ultraviolet-light emitting diodes (UV-LEDs) was examined. Multiple oligonucleotides were made in single capillaries and were characterized by hybridization, sequencing and gene synthesis. DNA synthesized in capillaries was capable of being hybridized and signal intensities correlated with microarray data. Sequencing demonstrated that the oligonucleotides were of high quality (up to 44% perfect sequences). Oligonucleotides were combined and used successfully for gene synthesis. This system offers a novel, scalable method to synthesize high quality oligonucleotides for biological applications.

Spatial photorelease of oligonucleotides, using a safety-catch photolabile linker.

[reaction: see text] We report the development of a safety-catch photolabile linker that allows the light-directed synthesis and spatially selective photorelease of oligonucleotides from microarrays. The linker remains stable to light during DNA synthesis, and is activated for photorelease after acidic hydrolysis. We demonstrate that the photoreleased oligonucleotides can be amplified by PCR to produce double stranded DNA. The advantages offered by this linker could aid the development of an automated gene synthesis platform.

Light-directed radial combinatorial chemistry: orthogonal safety-catch protecting groups for the synthesis of small molecule microarrays.

We describe the development of photolabile protecting groups based on the 3,4,5-trimethoxyphenacyl group (TMP). Orthogonal safety-catches were created by introducing an acid-activatible dimethyl ketal (AA-TMP) and an oxidatively activatible 1,3-dithiane (OA-TMP) into the photolabile TMP group. We demonstrate the application of these protecting groups in light-directed synthesis of small molecule microarrays with diversity elements radially attached to a hydroxyproline scaffold.

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis.

A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.

A comment on 'A new ray-tracing program RIGTRACE for X-ray optical systems' [J. Synchrotron Rad. (2001), 8, 1047-1050].

Some points concerning the characteristics of the X-ray simulation code SHADOW [Welnak et al. (1994). Nucl. Instrum. Methods, A347, 344-347] are clarified which are not correctly mentioned by Yamada et al. [J. Synchrotron Rad. (2001), 8, 1047-1050]. It is shown that, contrary to the Authors' statement, some functionality of their new program is not original. In particular, we show that SHADOW can deal correctly with crystal monochromators.

Gene expression analysis using oligonucleotide arrays produced by maskless photolithography.

Microarrays containing 195,000 in situ synthesized oligonucleotide features have been created using a benchtop, maskless photolithographic instrument. This instrument, the Maskless Array Synthesizer (MAS), uses a digital light processor (DLP) developed by Texas Instruments. The DLP creates the patterns of UV light used in the light-directed synthesis of oligonucleotides. This digital mask eliminates the need for expensive and time-consuming chromium masks. In this report, we describe experiments in which we tested this maskless technology for DNA synthesis on glass surfaces. Parameters examined included deprotection rates, repetitive yields, and oligonucleotide length. Custom gene expression arrays were manufactured and hybridized to Drosophila melanogaster and mouse samples. Quantitative PCR was used to validate the gene expression data from the mouse arrays.