Application of disposable plastic microfluidic device arrays with customized chemistries to multiplexed biochemical assays.

Plastic microfluidic array platforms and synergistic multiplexed assay chemistries are under development for a variety of applications, including assays of gene expression, proteomics, genotyping, DNA sequencing and fragment analysis, sample preparation and high-throughput pharmaceutical discovery. The low production costs of plastic substrates makes possible economical single-use device arrays, eliminating cleaning and sample-to-sample carryover contamination. Hundreds of microchannels and reservoirs are readily included on a single microtitre-plate-size substrate, enabling the manufacture of highly parallel fluidic array systems to increase throughput and speed.

Telomerase inhibitors--oligonucleotide phosphoramidates as potential therapeutic agents.

We have designed, synthesized, and evaluated using physical, chemical and biochemical assays various oligonucleotide N3'-->P5' phosphoramidates, as potential telomerase inhibitors. Among the prepared compounds were 2'-deoxy, 2'-hydroxy, 2'-methoxy, 2'-ribo-fluoro, and 2'-arabino-fluoro oligonucleotide phosphoramidates, as well as novel N3'-->P5' thio-phosphoramidates. The compounds demonstrated sequence specific and dose dependent activity with IC50 values in the sub-nM to pM concentration range.

Binding enhancement by tertiary interactions and suicide inhibition of a Candida albicans group I intron by phosphoramidate and 2'-O-methyl hexanucleotides.

Candida albicans is one of many infectious pathogens that are evolving resistance to current treatments. RNAs provide a large class of targets for new therapeutics for fighting these organisms. One strategy for targeting RNAs uses short oligonucleotides that exhibit binding enhancement by tertiary interactions in addition to Watson-Crick pairing. A potential RNA target in C. albicans is the self-splicing group I intron in the LSU rRNA precursor. The recognition elements that align the 5' exon splice site for a ribozyme derived from this precursor are complex [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. These recognition elements have been used to guide design of hexanucleotide mimics of the 5' exon that have backbones modified for nuclease stability. These hexanucleotides bind as much as 100000-fold more tightly to a ribozyme derived from the intron than to a hexanucleotide mimic of the intron's internal guide sequence, r(GGAGGC). Several of these oligonucleotides inhibit precursor self-splicing via a suicide inhibition mechanism. The most promising suicide inhibitor is the ribophosphoramidate rn(GCCUC)rU, which forms more trans-spliced than cis-spliced product at oligonucleotide concentrations of >100 nM at 1 mM Mg(2+). The results indicate that short oligonucleotides modified for nuclease stability can target catalytic RNAs when the elements of tertiary interactions are complex.

A remarkable stabilization of complexes formed by 2,6-diaminopurine oligonucleotide N3'-->P5' phophoramidates.

2'-Deoxyribo- and ribo-oligonucleotide N3'-->P5'phosphoramidates containing 2,6-diaminopurine nucleosides were synthesized. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed by these compounds with DNA and RNA complementary strands, relative to adenosine-containing phosphoramidate counterparts. The increase in melting temperature of the complexes reached up to 6.9 degrees C per substitution. The observed stabilization was attributed to the apparent synergistic effects of N-type sugar puckering of the oligonucleotide N3'-->P5' phosphoramidate backbone, and the ability of 2,6-diaminopurine bases to form three hydrogen bonds.

Synthesis and properties of RNA analogs-oligoribonucleotide N3'-->P5' phosphoramidates.

The synthesis and characterization of RNA mimetics, uniformly modified oligoribonucleotide N3'-->P5' phosphoramidates containing all four natural bases (uracil, cytosine, adenine and guanine) as well as thymidine and 2,6-diaminopurine, are described. These RNA analogs contain N3'-->P5' phosphoramidate internucleotide linkages which replaced natural RNA O3'-->P5' phosphodiester groups. These oligonucleo-tides were constructed from novel monomeric units (2'- t -butyldimethylsilyl)-3'-(monomethoxyltrityl)-amino-nucleoside-5'- phos phoramidites, the preparation of which is also presented. Several mixed base 9-13mer oligoribonucleotide phosphoramidates were synthesized with step-wise coupling yields of 96-98%. Thermal denaturation experiments demonstrated that ribo-N3'-->P5' phosphoramidates form stable duplexes with a complementary RNA strand. Thus, the melting temperature ( T (m)) of a duplex formed by a 13mer ribo-N3'-->P5' phosphoramidate (84 degrees C) was higher than that observed for the isosequential natural RNA oligomer (64.0 degrees C), or for the 2'-deoxy-N3'-->P5' phosphoramidate counterpart (71.7 degrees C). Moreover, substitution of adenine by 2, 6-diaminopurine in an oligoribophosphoramidate pentamer resulted in a very significant increase in the duplex melting temperature ( approximately 7 degrees C per base substitution). The RNA phosphoramidates also showed similar rates of hydrolysis by both RNase A and RNase T(1)as compared to natural RNA oligomers. The data presented indicate that this class of RNA analogs may be used as structural and functional RNA mimetics.

A specific partner for abasic damage in DNA.

In most models of DNA replication, Watson-Crick hydrogen bonding drives the incorporation of nucleotides into the new strand of DNA and maintains the complementarity of bases with the template strand. Studies with nonpolar analogues of thymine and adenine, however, have shown that replication is still efficient in the absence of hydrogen bonds. The replication of base pairs might also be influenced by steric exclusion, whereby inserted nucleotides need to be the correct size and shape to fit the active site against a template base. A simple steric-exclusion model may not require Watson-Crick hydrogen bonding to explain the fidelity of replication, nor should canonical purine and pyrimidine shapes be necessary for enzymatic synthesis of a base pair if each can fit into the DNA double helix without steric strain. Here we test this idea by using a pyrene nucleoside triphosphate (dPTP) in which the fluorescent 'base' is nearly as large as an entire Watson-Crick base pair. We show that the non-hydrogen-bonding dPTP is efficiently and specifically inserted by DNA polymerases opposite sites that lack DNA bases. The efficiency of this process approaches that of a natural base pair and the specificity is 10(2)-10(4)-fold. We use these properties to sequence abasic lesions in DNA, which are a common form of DNA damage in vivo. In addition to their application in identifying such genetic lesions, our results show that neither hydrogen bonds nor purine and pyrimidine structures are required to form a base pair with high efficiency and selectivity. These findings confirm that steric complementarity is an important factor in the fidelity of DNA synthesis.

The ring fragmentation product of thymidine C5-hydrate when present in DNA is repaired by the Escherichia coli Fpg and Nth proteins.

Various forms of oxidative stress, including gamma-radiolysis and UV irradiation, result in the formation of damaged bases. (5R)-Thymidine C5-hydrate is one of several modified nucleosides produced from thymidine under these conditions. N-(2-Deoxy-beta-D-erythro-pentofuranosyl)-N-3-[(2R)-hydroxyisobutyric acid]urea or alphaRT is the respective fragmentation product formed from (5R)-thymidine C5-hydrate upon hydrolysis. This modified nucleoside has potential mutagenic or lethal properties. No enzymatic activity responsible for the removal of alphaRT has been identified. We report here that when present in DNA, alphaRT is a substrate for two purified enzymes from Escherichia coli involved in the repair of oxidized bases: the Nth and the Fpg proteins. The Fpg protein removes the alphaRT lesion more efficiently than the Nth protein. This is the first example of efficient excision of a ring-opened form of a pyrimidine by the Fpg protein. The high efficacy of the Fpg protein suggests that it is likely to be involved in vivo in the excision of alphaRT. The kinetics of the reaction of the Fpg protein with DNA containing alphaRT suggest substrate inhibition. Duplex oligodeoxynucleotides containing alphaRT positioned opposite T, dG, dC, and dA were cleaved efficiently by both enzymes, although the profiles of activity of the two enzymes were different. The Nth enzyme preferentially excises alphaRT when opposite a dG, followed by alphaRT.dA, alphaRT. T, and alphaRT.dC. For the Fpg protein, the order is alphaRT.dC >/= alphaRT.dG approximately alphaRT.T > alphaRT.dA. Moreover, we show that human cell extract exhibits an activity that excises alphaRT from an oligonucleotide, suggesting that human homologues of the Nth and/or Fpg proteins could be involved in repair of this lesion in human cells.

Inhibition of klenow fragment (exo-) catalyzed DNA polymerization by (5R)-5,6-dihydro-5-hydroxythymidine and structural analogue 5,6-dihydro-5-methylthymidine.

Oligonucleotides containing 5R-5,6-dihydro-5-hydroxythymidine (5R-3) and structural analogue 5,6-dihydro-5-methylthymidine (9) at defined sites were chemically synthesized via a method that obviates the use of NH4OH. Oligonucleotides prepared by this method were used to examine the effects of 5R-3 and 9 on the fidelity of Klenow (exo-) in vitro. The presence of lesions 5R-3 and 9 in DNA templates was shown to inhibit polymerization of primers hybridized to these templates. Inhibition was observed for both translesional synthesis and extension one nucleotide past the lesion, with the latter being more pronounced. The fidelity of Klenow (exo-) was reduced only slightly when utilizing substrates containing either dihyropyrimidine nucleotide. These results provide the first experimental verification of computational studies carried out on the effects of 3 on DNA templates, and are consistent with a structural model in which the C5-methyl group of 5R-3 adopts a pseudoaxial orientation resulting in a disruption in base stacking.

Synthesis of oligonucleotides containing 3'-alkylcarboxylic acids using a palladium labile oligonucleotide solid phase synthesis support.

Oligonucleotides containing a 3'-alkylcarboxylic acid are isolated using a Pd(0)-catalyzed cleavage reaction, in yields that are in most cases within experimental error of those isolated using standard oligonucleotide cleavage conditions (concentrated NH4OH). In contrast to results obtained with photolabile solid phase synthesis supports, no reduction in isolated yields of the oligonucleotides is observed when their length is increased from 20 to 40 nucleotides. The oligonucleotides are characterized by anion exchange HPLC, electrospray mass spectrometry, and enzymatic digestion. When methyl phosphoramidites are employed in the synthesis of the biopolymers, 3 serves as an orthogonal solid phase oligonucleotide synthesis support.

The effects of the ring fragmentation product of thymidine C5-hydrate on phosphodiesterases and klenow (exo-) fragment.

N-(2-Deoxy-beta-D-erythro-pentofuranosyl)-N-3-(2R-hydroxyisobutyric acid)urea (alpha-R-hydroxy-beta-ureidoisobutyric acid, 8) was site specifically incorporated into a series of oligonucleotides via the ammonolysis of biopolymers containing 5R-thymidine C5-hydrate (3). alpha-R-hydroxy-beta-ureidoisobutyric acid (8) inhibits snake venom phosphodiesterase, lambda exonuclease and Klenow (exo-) fragment. Kinetic measurements for insertion of nucleotides opposite 8 by Klenow (exo-) fragment indicate that this lesion is instructive.

Subsecond grating growth in a photorefractive polymer.

We survey the dynamics of the photorefractive effect in a methyl methacrylate copolymer with the nonlinear chromophore p-nitroaniline in a pendant side group doped with a charge-transport agent, diethylaminobenzaldehyde diphenylhydrazone, a material that represents a new class of photorefractive polymer. The grating growth times are several orders of magnitude smaller than that for the previous epoxy-based photorefractive polymers and fall below 1 s at the highest intensities used. Grating competition and revelation effects suggest that charge carriers other than photogenerated holes are mobile. A sublinear dependence of growth rate on writing intensity implies that shallow traps may also be present.