A series of four hairpin deoxyoligonucleotides was synthesized with a four-nucleotide central loop (either C or G) flanked by the complementary sequences d(T)10 and d(A)10. Two of the molecules contain either a 3'-p-3' or 5'-p-5' linkage in the loop, so that the strands in the stem have the same, that is, parallel (ps) polarity. The pair of reference oligonucleotides have normal phosphodiester linkages throughout and antiparallel (aps) stem regions. All the molecules adopt a duplex helical structure in that (i) the electrophoretic mobilities in polyacrylamide gels of the ps and aps oligomers are similar. (ii) The ps hairpins are substrates for T4 polynucleotide kinase, T4 DNA ligase, and Escherichia coli exonuclease III. (iii) Salt-dependent thermal transitions are observed for all hairpins, but the ps molecules denature 10 degrees C lower than the corresponding aps oligomers. (iv) The ultraviolet absorption and circular dichroism spectra are indicative of a base-paired duplex in the stems of the ps hairpins but differ systematically from those of the aps counterparts. (v) The bis-benzimidazole drug Hoechst-33258, which binds in the minor groove of B-DNA, exhibits very little fluorescence in the presence of the ps hairpins but a normal, enhanced emission with the aps oligonucleotides. In contrast, the intercalator ethidium bromide forms a strongly fluorescent complex with all hairpins, the intensity of which is even higher for the ps species. (vi) The pattern of chemical methylation is the same for both the ps and aps hairpins. The combined results are consistent with the prediction from force field analysis of a parallel stranded right-handed helical form of d(A)n.d(T)n with a secondary structure involving reverse Watson-Crick base pairs and a stability not significantly different from that of the B-DNA double helix. Models of the various hairpins optimized with force field calculations are described.

SUMMARY A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica was cloned and sequenced, and is the first sea hare antimicrobial protein to be functionally expressed in E. coli. Sequence analysis suggested that this protein is a flavin-containing l-amino acid oxidase (LAAO), with one predicted potential glycosylation site, although the glycosylation could not be experimentally confirmed. This protein, which we call `escapin', has high sequence similarity to several other gastropod proteins. Escapin was verified by NMR, mass spectroscopy and HPLC to have FAD as its flavin cofactor. Escapin's antimicrobial effects, bacteriostasis and bactericidal, were determined using a combination of two assays: (1) incubation of bacteria on solid media followed by assessment of inhibition by direct observation of zones of inhibition or by turbidity measurements; and (2) incubation of bacteria in liquid media followed by counting viable colonies after growing on agar plates. Native escapin inhibited the growth of Gram-positive and Gram-negative bacteria, including marine bacteria (Vibrio harveyiiand Staphylococcus aureus) and pathogenic bacteria(Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa). Escapin also inhibited the growth of yeast and fungi, with different efficacies. Escapin's antimicrobial activity was concentration dependent and did not decrease when stored for more than 5 months at room temperature. Escapin was bacteriostatic and not bactericidal in minimal media (e.g. salt media) with glucose, yeast extract, and a mixture of 20 amino acids each at 50 μmol l-1, but was bactericidal in media enriched with Tryptone Peptone. Escapin was also strongly bactericidal in media with l-lysine at concentrations as low as 3 mmol l-1 and slightly bactericidal in 50 mmol l-1l-arginine, but not in most other amino acids even at 50 mmol l-1. Escapin had high oxidase activity (producing hydrogen peroxide) with either l-arginine or l-lysine as a substrate and little to no oxidase activity with other l-amino acids. Hydrogen peroxide alone (without escapin or amino acids) was strongly bacteriostatic but poorly bactericidal, similar in this respect to l-arginine but different from l-lysine in the presence of escapin. Together these results suggest that there are multiple mechanisms to escapin's antimicrobial effects, with bacteriostasis resulting largely or entirely from the effects of hydrogen peroxide produced by escapin's LAAO activity, but bactericidal effects resulting from lysine-dependent mechanisms not directly involving hydrogen peroxide. Recombinant escapin expressed in bacteria was also active against Gram-positive and Gram-negative bacteria,suggesting that glycosylation is not essential for antimicrobial activity.

Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA) n and (ARKKAAKA) n (n ‫؍‬ 1-6). Affinity coelectrophoresis revealed that low M r peptides (600 -1300) had no affinities for low M r heparin, but higher M r peptides (2000 -3500) exhibited significant affinities (K d Х 50 -150 nM), which increased with peptide M r . Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K d Х 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an ␣-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M r peptides exhibited high affinities for total endothelial cell proteoglycans (K d Х 300 nM), and ϳ4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices. Proteoglycans (PGs)1 are composed of a core protein to which are covalently attached one or more sulfated glycosaminoglycans (GAGs). PGs are ubiquitous components of cell surfaces and the extracellular matrix, and their GAG chains contribute to myriad biological functions, such as modulation of enzyme activities, regulation of cell growth, and control of assembly of the extracellular matrix (1). PGs are thus potential targets for therapeutic intervention. For example, heparin antagonists are needed to take the place of protamine, a heterogeneous, sometimes toxic protein mixture commonly used to neutralize the anticoagulant activity of heparin in humans (2, 3); in the design of drugs to be targeted to PG-rich tissues, such as cartilage (4); and to be used to promote cell adhesion in a variety of situations, e.g. by promoting binding of cells that express abundant amounts of PGs, such as endothelial cells (5), to synthetic vascular graft surfaces. To develop a rationale for the design of such reagents, it is useful to examine known features of protein structure required for high affinity interactions with GAGs. Thus, analysis of the structural features of many known heparin-and heparan sulfate (HS)-binding proteins has revealed the presence of conserved motifs, through which GAG binding has been postulated to occur. Cardin and Weintraub (6) identified two clusters of basic charge in known heparin-binding proteins in which amino acids tend to be arranged in the patterns XBBXBX or XBBBXXBX, where B represents an ...

The concept of using alumina nanoporous filters (AAO) modified with DNA for "label-free" detection and separation/purification of the target ss-DNA is demonstrated. The high surface density of DNA (4 x 10(12) cm(-2)) and high efficiency of hybridization (ca. 70%) in combination with increased effective surface area make this system very attractive for development of various ss-DNA (or RNA) detection methods. Moderate transparency of AAO in the UV and IR regions allows direct detection of DNA hybridization by optical and IR absorption. Close to the quantitative efficiency of binding the target ss-DNA from solution using a single pass through the modified filter is observed.