[Synthesis and properties of methylene carboxamide mimetics of nucleic acids based on morpholine nucleosides].

Uracyl and adenine containing oligocarboxamide mimetics of nucleic acids based on morpholine nucleosides (MorGly) are synthesized using peptide chemistry methods. Conditions for an analysis of homogeneity of protonated at physiological pH oligomers using a capillary electrophoresis are proposed. Studies of thermostability of complementary complexes formed by MorGly oligomers revealed that melting temperature dramatically depends on heterocyclic base composition (uracyl or adenine). Cooperative interactions realized at junctions in tandem complexes give more contribution to the thermostability in the case of complexes formed by modified oligomers than native oligodeoxyriboadenilates. Adenine containing MorGly oligomers form more stable complexes with poly(U) than native oligodeoxyriboadenilate of the same length. Complexes formed by modified oligomers with polyribonucleotides are more stable in compare with polydeoxyribonucleotide.

Development of salt-tolerance interface for an high performance liquid chromatography/inductively coupled plasma mass spectrometry system and its application to accurate quantification of DNA samples.

Accurate quantification of DNA is highly important in various fields. Determination of phosphorus by ICP-MS is one of the most effective methods for accurate quantification of DNA due to the fixed stoichiometry of phosphate to this molecule. In this paper, a smart and reliable method for accurate quantification of DNA fragments and oligodeoxythymidilic acids by hyphenated HPLC/ICP-MS equipped with a highly efficient interface device is presented. The interface was constructed of a home-made capillary-attached micronebulizer and temperature-controllable cyclonic spray chamber (IsoMist). As a separation column for DNA samples, home-made methacrylate-based weak anion-exchange monolith was employed. Some parameters, which include composition of mobile phase, gradient program, inner and outer diameters of capillary, temperature of spray chamber etc., were optimized to find the best performance for separation and accurate quantification of DNA samples. The proposed system could achieve many advantages, such as total consumption for small amount sample analysis, salt-tolerance for hyphenated analysis, high accuracy and precision for quantitative analysis. Using this proposed system, the samples of 20 bp DNA ladder (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 base pairs) and oligodeoxythymidilic acids (dT(12-18)) were rapidly separated and accurately quantified.

Self-assembled monolayers of peptide nucleic acids on gold surfaces: a spectroscopic study.

We have characterized self-assembled monolayers (SAMs) of thiol-derivatized peptide nucleic acid (PNA) chains adsorbed on gold surfaces by using reflection absorption infrared spectroscopy (RAIRS) and X-ray photoemission spectroscopy (XPS) techniques. We have found that the molecular orientation of PNAs strongly depends on surface coverage. At low coverage, PNA chains lie flat on the surface, while at high coverage, PNA molecules realign their molecular axes with the surface normal and form SAMs without the need of co-immobilization of spacers or other adjuvant molecules. The change in the molecular orientation has been studied by infrared spectroscopy and it has been confirmed by atomic force microscopy (AFM). PNA immobilization has been followed by analyzing the N(1s) XPS core-level peak. We show that the fine line shape of the N(1s) core-level peak at optimal concentration for biosensing is due to a chemical shift. A combination of the above-mentioned techniques allow us to affirm that the structure of the SAMs is stabilized by molecule-molecule interactions through noncomplementary adjacent nucleic bases.

Unconventional helical phasing of repetitive DNA motifs reveals their relative bending contributions.

A novel, multiple DNA phasing analysis is described in which three sequence motifs associated with bent DNA are clustered together in oligomers of identical base composition, but with different phasing relationships of these motifs to each other. Synthetic oligonucleotides containing different combinations of AAAAA(A), GGGCCC and GAGAG sequence motifs were ligated and analyzed by gel mobility and cyclization experiments to determine their global curvature. These assays were used to obtain relative bending contributions of the analyzed sequence motifs. The experimental results also provide a rigorous test of predictive models for DNA bending. We report, using molecular modeling, that none of the most widely used dinucleotide (nearest neighbor) models can accurately describe the conformational properties of these DNA sequences and that more complex models, at least at the trinucleotide level, are required.

Rainbow Universal CPG: a versatile solid support for oligonucleotide synthesis.

We have developed a universal solid support, termed Rainbow Universal CPG, for use in automated oligonucleotide synthesis. The universal solid support allows any oligodeoxyribonucleotide sequence to be synthesized from a single type of controlled pore glass (CPG) support. Deprotection of oligodeoxyribonucleotides was optimized using 0.5 M LiCl in concentrated ammonium hydroxide. PCR experiments using three different sets of primers proved that the 3'-hydroxyl function of oligodeoxyribonucleotides synthesized from Rainbow Universal CPG was retained. This universal solid support shows promise for replacing the standard nucleoside CPG supports.

Affinity capillary electrophoresis using DNA conjugates.

An oligonucleotide ((dT)12)-immobilized capillary was prepared for the affinity capillary electrophoresis of DNA. Oligonucleotides ((dA)6 and (dT)6) which had the same chain length but had different base composition were separated in high resolution in the presence of Mg2+ ion. Whereas (dA)6 eluted slightly earlier than (dT)6 in a conventional polyacrylamide-coated capillary, the order of their mobilities was reversed in the (dT)12-modified system, suggesting the complementary hydrogen bondings between (dA)6 and the immobilized (dT)12.

Duplex to quadruplex equilibrium of the self-complementary oligonucleotide d(GGGGCCCC).

The structure of the deoxyoligonucleotide d(GGGGCCCC) has been monitored by 1H and 31P NMR, and by gel electrophoresis. In low-salt solution, this oligonucleotide forms a stable duplex structure. Upon titration with KCl, an equilibrium is established between duplex and quadruplex forms. The quadruplex form is the dominant one at physiological KCl concentrations, despite the fact that fewer hydrogen bonds are formed per strand in the quadruplex than in the duplex.

Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides.

Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA.

Isolation of the human peroxisome proliferator activated receptor gamma cDNA: expression in hematopoietic cells and chromosomal mapping.

The nuclear receptor superfamily of transcription factors, which includes the retinoic acid receptors and v-erb A, play important roles in the molecular control of hematopoiesis. To identify nuclear receptors expressed in hematopoietic cells, we screened a human bone marrow cDNA library using a degenerate oligonucleotide and isolated a 1.85-kb full-length cDNA encoding a new human member of this superfamily, the peroxisome proliferator activated receptor gamma (hPPAR gamma). Two different hPPAR gamma transcripts were expressed in hematopoietic cells: a 1.85-kb transcript, which corresponds to the full-length mRNA (PPAR gamma 1), and a 0.65-kb transcript (PPAR gamma 2), which cannot encode all of the nuclear receptor functional domains. Normal neutrophils and peripheral blood lymphocytes, as well as circulating leukemic cells from patients with AML, ALL, and CML, express only PPAR gamma 2 on Northern blot analysis. In contrast, only the PPAR gamma 1 transcript was detected in a variety of human leukemia cell lines and in cultured normal primary bone marrow stromal cells. Both transcripts were detected in various fetal and adult nonhematopoietic tissues. We mapped the location of the hPPAR gamma gene to human chromosome 3p25 by somatic cell hybridization and linkage analysis. PPARs have been shown to be activated by peroxisome proliferating agents, long-chain fatty acids and arachidonic acid. Human PPAR gamma, although homologous to the PPAR gamma s of other species, has unique sequence and amino acid differences. Identification of hPPAR gamma will allow further understanding of its role in human cellular leukotriene, prostaglandin, and peroxide degradative or synthetic pathways, as well as its role in lipid metabolism and regulation of adipocyte differentiation.

Synthesis and selective cleavage of an oligodeoxynucleotide containing a bridged internucleotide 5'-phosphorothioate linkage.

A self complementary oligodeoxynucleotide dodecamer containing an achiral bridged 5'-phosphorothioate linkage 3'-O-P-S-5' has been prepared using the solid phase phosphoramidite procedure. For the incorporation of the phosphorothioate link we have used a 5'-(S-trityl)-mercapto-5'-deoxythymidine-3'-phosphoramidite. After coupling this building block the S-trityl group was removed by silver ions. The free thiol moiety was then coupled with a standard phosphoramidite in the presence of tetrazole. After oxidation of the 5'-phosphorothioite with iodine/water the synthesis was continued with standard building blocks up to the desired dodecamer. This backbone modified dodecamer can be cleaved selectively and quantitatively at the P-S bond by silver or mercuric ions under very mild conditions.