Nonvolatile copolymer compositions for fabricating gel element microarrays.

By modifying polymer compositions and cross-linking reagents, we have developed a simple yet effective manufacturing strategy for copolymerized three-dimensional gel element arrays. A new gel-forming monomer, 2-(hydroxyethyl) methacrylamide (HEMAA), was used. HEMAA possesses low volatility and improves the stability of copolymerized gel element arrays to on-chip thermal cycling procedures relative to previously used monomers.

Unidirectional excitation of surface plasmons by slanted gratings.

Surface plasmon excitation by normally incident light on surface-relief metallic diffraction gratings is studied numerically. Predominantly unidirectional excitation is achieved with a grating of either a slanted lamellar or an inclined sinusoidal groove profile, both having shallow depths. Maps of Poynting vector illustrate that the energy flow turns from normal incidence in the far-field region to a pattern almost parallel to the grating surface in the required direction of excitation of a single SPP wave.

Suspension array analysis of 16S rRNA from Fe- and SO(4)2- reducing bacteria in uranium-contaminated sediments undergoing bioremediation.

A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence.

Evaluation of gel-pad oligonucleotide microarray technology by using artificial neural networks.

Past studies have suggested that thermal dissociation analysis of nucleic acids hybridized to DNA microarrays would improve discrimination among duplex types by scanning through a broad range of stringency conditions. To more fully constrain the utility of this approach using a previously described gel-pad microarray format, artificial neural networks (NNs) were trained to recognize noisy or low-quality data, as might derive from nonspecific fluorescence, poor hybridization, or compromised data collection. The NNs were trained to classify dissociation profiles (melts) into groups based on selected characteristics (e.

Differential theory: application to highly conducting gratings.

The recently developed fast Fourier factorization method, which has greatly improved the application range of the differential theory of gratings, suffers from numerical instability when applied to metallic gratings with very low losses. This occurs when the real part of the refractive index is small, in particular, smaller than 0.1-0.

SuUR protein binds to the boundary regions separating forum domains in Drosophila melanogaster.

Forum domains are 50-150 kb DNA fragments that are released during spontaneous fragmentation of chromosomes. They are separated by islands of putative heterochromatin boundary regions. The SuUR protein, which is involved in the control of chromosome organization, is localized exclusively in heterochromatin and often colocalizes on chromosomes with Polycomb group proteins.

Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization.

DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids.

RNA-binding properties of the 63 kDa protein encoded by the triple gene block of poa semilatent hordeivirus.

The 63 kDa '63K' movement protein encoded by the triple gene block of poa semilatent virus (PSLV) comprises the C-terminal NTPase/helicase domain and the N-terminal extension domain, which contains two positively charged sequence motifs, A and B. In this study, the in vitro RNA-binding properties of PSLV 63K and its mutants were analysed. Membrane-immobilized 63K and N-63K (isolated N-terminal extension domain) bound RNA at high NaCl concentrations.

Parallel-stranded DNA with mixed AT/GC composition: role of trans G.C base pairs in sequence dependent helical stability.

Parallel-stranded (ps) DNAs with mixed AT/GC content comprising G.C pairs in a varying sequence context have been investigated. Oligonucleotides were devised consisting of two 10-nt strands complementary either in a parallel or in an antiparallel orientation and joined via nonnucleotide linkers so as to form 10-bp ps or aps hairpins.

Gene-specific silencing by expression of parallel complementary RNA in Escherichia coli.

Gene-specific silencing refers to a phenomenon in which expression of an individual gene can be specifically repressed by different mechanisms on the levels of transcription, RNA splicing, transport, degradation in nuclei or cytoplasm, or blocking of translation. In different species gene-specific silencing was observed by expression or injections of antiparallel double-stranded RNA formed by a fragment of mRNA and antisense RNA. Here we show a potent and specific gene silencing in bacteria by expression of RNA, that is complementary in a parallel orientation to Escherichia coli lon mRNA.

[Generation of Kruppel phenocopies by injecting into Drosophila embryos RNA complementary to mRNA in parallel orientation].

RNA preparations synthesized in vitro were used to study the influence of RNA interference on the Kruppel gene activity in Drosophila embryos. RNA complementary in parallel orientation to the mRNA fragment proved to induce the development of Kruppel phenocopies. The data obtained indicate that mechanisms of specific regulation of gene activity exist in Drosophila cells, which are sensitive to the formation of both parallel and antiparallel RNA-RNA duplexes that include mRNA of the corresponding gene.

Advanced method for oligonucleotide deprotection.

A new procedure for rapid deprotection of synthetic oligodeoxynucleotides has been developed. While all known deprotection methods require purification to remove the residual protective groups (e.g.

DNA sequence recognition by bis-linked netropsin and distamycin derivatives.

We studied the interaction of cis-diammine Pt(II)-bridged bis-netropsin, cis-diammine Pt(II)-bridged bis-distamycin and oligomethylene-bridged bis-netropsin with synthetic DNA fragments containing pseudosymmetrical AT-rich nucleotide sequences and compared it with the interaction of the parent compounds netropsin and distamycin A. For fragments containing multiple blocks of (AIT)4 and (T/A)4 separated by zero, one, two and three GC-base pairs, DNase I footprinting and CD spectroscopy studies reveal that 5'-TTTTAAAA-3' is the strongest affinity binding site for cis-diammine Pt(II)-bridged bis-netropsin and bis-distamycin. They both bind less strongly to a DNA region containing the sequence 5'-AAAATTTT-3'.

Forum domain in Drosophila melanogaster cut locus possesses looped domains inside.

We have studied the relationship between chromosomal forum domains and looped domains in the cut locus of Drosophila melanogaster . Forum domains were earlier detected by separation in pulsed-field gels of 50-150 kb chromosomal DNA fragments obtained after spontaneous non-random degradation of chromosomes. We have localized the boundary region where cleavage sites are scattered between two forum domains in the regulatory region of the cut locus.

The role of phosphorylation of DNA-binding proteins in regulation of transcription of the human c-myc gene.

The goal of the present study was to identify transcriptional factors, to determine their specificity toward nucleotide sequences of binding sites, and to elucidate their regulatory effects on transcription of the human c-myc oncogene. Three novel phosphorylated proteins (transcriptional factors) participating in the regulation of transcription of the c-myc gene have been identified, and the following data have been obtained. A) The binding of the regulatory phosphoproteins activates in vitro transcription of the human c-myc gene.

DNA mobility in crystals of a nonspecific lambda cro/DNA complex.

DNA location in the crystal of the nonspecific lambda cro/(GT)4.(AC)4 complex has been studied by the isomorphous replacement method using iodinated and brominated oligonucleotides. The results of the search for heavy atom positions combined with previously obtained molecular replacement data suggest that the DNA octamer occupies two overlapping positions, each of the two duplexes (GTGTGTGT).

The formation of parallel RNA-RNA duplexes in vitro.

Over the years the structural properties of nucleic acids have been of interest, providing data that may be of importance for DNA and RNA organization and function in the cell. We have attempted to look for the formation of parallel RNA-RNA duplexes in vitro. RNA molecules comprising complementary in the same polarity alternating stretches of A and U of increasing length were enzymatically synthesized and annealed in physiological conditions.

Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA.

A mutant T7 RNA polymerase (T7 RNAP) having two amino-acid substitutions (Y639F and S641A) is altered in its specificity towards nucleotide substrates, but is not affected in the specificity of its interaction with promoter and terminator sequences. The mutant enzyme gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA. DNA synthesis can also be initiated from a single stranded template using a DNA primer.