Hydrated electrons induce the formation of interstrand cross-links in DNA modified by cisplatin adducts.

Double-stranded oligonucleotides containing cisplatin adducts, with and without a mismatched region, were exposed to hydrated electrons generated by gamma-rays. Gel electrophoresis analysis demonstrates the formation of cisplatin-interstrand crosslinks from the cisplatin-intrastrand species. The rate constant per base for the reaction between hydrated electrons and the double-stranded oligonucleotides with and without cisplatin containing a mismatched region was determined by pulse radiolysis to be 7 × 109 and 2 × 109 M-1 s-1, respectively. These results provide a better understanding of the radiosensitizing effect of cisplatin adducts in hypoxic tumors and of the formation of interstrand crosslinks, which are difficult for cells to repair.

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2'-fluoro-ANA/RNA and DNA/RNA hybrids.

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'-fluoro-ANA analog (2'F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2'F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3'-endo (north, A-form) conformation, whereas those of the ANA strand adopt a 'rigid' O4'-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2'F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2'F-ANA/RNA and DNA/RNA helices is 9.0 +/- 0.5 A, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2'F-ANA/RNA hybrids to elicit RNase H activity.

Effect of substituting arabinonucleosides for deoxynucleotides in the DNA priming strand on the polymerase action of HIV-1 RT.

The ability of 5'-DNA-araN-3' chimeras to serve as primers during HIV-1 RT-catalyzed DNA synthesis was assessed. It is shown that while the structural changes imparted by the arabinose units are minimal, the biological outcome is significant. For example, a DNA strand with arabinocytidine (araC) at the 3'-terminus was found to serve as a primer of DNA synthesis but significant pausing of HIV-RT was observed after the addition of 4 dNTP's. This phenomenon was not observed for the analogous DNA primer containing a riboC unit or an all-DNA strand.

Syntheses of DNA duplexes containing a C-C interstrand cross-link.

Short DNA duplexes that contain a N4C-ethyl-N4C interstrand cross-link were prepared on controlled pore glass supports using a DNA synthesizer. The C-C cross-link was introduced via a convertible nucleoside on the support or by using a protected C-C cross-link phosphoramidite. An orthogonal protection scheme allowed selective chain growth in either a 3'-->5' or 5'-->3' direction. The cross-linked duplexes were purified by HPLC and characterized by MALDI-TOF mass spectrometry and/or by enzymatic digestion.

Properties of arabinonucleic acids (ANA & 20'F-ANA): implications for the design of antisense therapeutics that invoke RNase H cleavage of RNA.

Inversion of configuration of the C2' position of RNA leads to a very unique nucleic acid structure: arabinonucleic acid (ANA). ANA, and its 2'-fluoro derivative (2'F-ANA) from hybrids with RNA that are capable of activating RNase H, resulting in cleavage of the RNA strand. In this paper, we review the properties of duplexes formed between ANA (or 2'F-ANA) and its RNA complement. These studies support the notion that RNase H is sensitive to the minor groove dimensions of the hybrid substrate.

Synthesis and characterization of DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link.

Short DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link, C-C, were synthesized on controlled pore glass supports. Duplexes having two, three, or four A/T base pairs on either side of the C-C cross-link and terminating with a C(4) overhang at their 5'-ends were prepared. The cross-link was introduced using a convertible nucleoside approach. Thus, an oligonucleotide terminating at its 5'-end with O(4)-triazoyl-2'-deoxyuridine was first prepared on the support. The triazole group of support-bound oligomer was displaced by the aminoethyl group of 5'-dimethoxytrityl-3'-O-tert-butyldimethylsilyl-N(4)-(2-aminoethyl)deoxycytidine to give the cross-link. The dimethoxytrityl group was removed, and the upper and lower strands of the duplex were extended from two 5'-hydroxyl groups of the cross-link using protected nucleoside 3'-phosphoramidites. The tert-butyldimethylsilyl group of the resulting partial duplex was then removed, and the chain was extended in the 3'-direction from the resulting 3'-hydroxyl of the cross-link using protected nucleoside 5'-phosphoramidites. The cross-linked duplexes were purified by HPLC and characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Duplexes with three or four A/T base pairs on either side of the C-C cross-link gave sigmoidal shaped A(260) profiles when heated, a behavior consistent with cooperative denaturation of the A/T base pairs. Each cross-linked duplex could be ligated to an acceptor duplex using T4 DNA ligase, a result that suggests that the C-C cross-link does not interfere with the ligation reaction, even when it is located only two base pairs from the site of ligation. The ability to synthesize duplexes with a defined interstrand cross-link and to incorporate these duplexes into longer pieces of DNA should enable preparation of substrates that can be used for a variety of biophysical and biochemical experiments, including studies of DNA repair.

Synthesis and biophysical properties of arabinonucleic acids (ANA): circular dichroic spectra, melting temperatures, and ribonuclease H susceptibility of ANA.RNA hybrid duplexes.

Arabinonucleic acid (ANA), the 2'-epimer of RNA, was synthesized from arabinonucleoside building blocks by conventional solid-phase phosphoramidite synthesis. In addition, the biochemical and physicochemical properties of ANA strands of mixed base composition were evaluated for the first time. ANA exhibit certain characteristics desirable for use as antisense agents. They form duplexes with complementary RNA, direct RNase H degradation of target RNA molecules, and display resistance to 3'-exonucleases. Since RNA does not elicit RNase H activity, our findings establish that the stereochemistry at C2' (ANA versus RNA) is a key determinant in the activation of the enzyme RNase H. Inversion of stereochemistry at C2' is most likely accompanied by a conformational change in the furanose sugar pucker from C3'-endo (RNA) to C2'-endo ("DNA-like") pucker (ANA) [Noronha and Damha (1998) Nucleic Acids Res. 26, 2665-2671; Venkateswarlu and Ferguson (1999) J. Am. Chem. Soc. 121, 5609-5610]. This produces ANA/RNA hybrids whose CD spectra (i.e., helical conformation) are more similar to the native DNA/RNA substrates than to those of the pure RNA/RNA duplex. These features, combined with the fact that ara-2'OH groups project into the major groove of the helix (where they should not interfere with RNase H binding), help to explain the RNase H activity of ANA/RNA hybrids.

Carrageenan-induced acute inflammatory response in chicks.

Carrageenan from two commercial sources was used in varying concentrations to evoke an acute inflammatory response in the foot pad of chickens. Oedema formation and increased vascular permeability were less severe using this experimental model than with turpentine-induced injury. Inflammation was monophasic, of short duration and apparently mediated by histamine and serotonin as their respective antagonists diminished the response. Prostaglandins seemed to play only a minor role as indomethacin blocked only the later phase of oedema. Bothrops jararaca venom did not potentiate the inflammatory response, implying that bradykinin was not a significant mediator.