There are now several well-documented SSBs from both prokaryotes and eukaryotes that function in replication, recombination, and repair; however, no "consensus" view of their interactions with ssDNA has emerged. Although these proteins all bind preferentially and with high affinity to ssDNA, their modes of binding to ssDNA in vitro, including whether they bind with cooperativity, often differ dramatically. This point is most clear upon comparing the properties of the phage T4 gene 32 protein and the E. coli SSB protein. Depending on the solution conditions, Eco SSB can bind ssDNA in several different modes, which display quite different properties, including cooperativity. The wide range of interactions with ssDNA observed for Eco SSB is due principally to its tetrameric structure and the fact that each SSB protomer (subunit) can bind ssDNA. This reflects a major difference between Eco SSB and the T4 gene 32 protein, which binds DNA as a monomer and displays "unlimited" positive cooperativity in its binding to ssDNA. The Eco SSB tetramer can bind ssDNA with at least two different types of nearest-neighbor positive cooperativity ("limited" and "unlimited"), as well as negative cooperativity among the subunits within an individual tetramer. In fact, this latter property, which is dependent upon salt concentration and nucleotide base composition, is a major factor influencing whether ssDNA interacts with all four or only two SSB subunits, which in turn determines the type of intertetramer positive cooperativity. Hence, it is clear that the interactions of Eco SSB with ssDNA are quite different from those of T4 gene 32 protein, and the idea that all SSBs bind to ssDNA as does the T4 gene 32 protein must be amended. Although it is not yet known which of the Eco SSB-binding modes is functionally important in vivo, it is possible that some of the modes are used preferentially in different DNA metabolic processes. In any event, the vastly different properties of the Eco SSB-binding modes must be considered in studies of DNA replication, recombination, and repair in vitro. Since eukaryotic mitochondrial SSBs as well as SSBs encoded by prokaryotic conjugative plasmids are highly similar to Eco SSB, these proteins are likely to show similar complexities. However, based on their heterotrimeric subunit composition, the eukaryotic nuclear SSBs (RP-A proteins) are significantly different from either Eco SSB or T4 gene 32 proteins. Further subclassification of these proteins must await more detailed biochemical and biophysical studies.

Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic lipid-DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride, N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl)ammonium bromide) and polycationic (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate) lipid-based assemblies, with or without neutral lipid (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, cholesterol) were used to prepare lipoplexes of different L(+)/DNA(-) charge ratios. Circular dichroism, cryogenic-transmission electron microscopy, and static light scattering were used for lipoplex characterization, whereas expression of human growth hormone or green fluorescent protein was used to quantify transfection efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, cholesterol) induced appearance of Psi(-) DNA, a chiral tertiary DNA structure. The formation of Psi(-) DNA was also dependent on cationic lipid-DNA charge ratio. On the other hand, monocationic lipids either alone or with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine as helper lipid, or polycationic 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate-based assemblies, neither of which promotes a lipid-DNA hexagonal phase, did not induce the formation of Psi(-) DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of Psi(-) DNA structure, correlate with optimal transfection.

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All types of amyloidosis are structurally characterized by the cross beta-pleated sheet conformation of the fibrils, irrespective of their biochemical composition. The clinical observation that the anthracycline 4'-iodo-4'-deoxy-doxorubicin (IDOX) can induce amyloid resorption in patients with immunoglobulin light chain amyloidosis was the starting point for this investigation of its possible mechanism of action. IDOX binds strongly to all five types of natural amyloid fibrils tested: immunoglobulin light chains, amyloid A, transthyretin (methionine-30 variant), beta-protein (Alzheimer), and beta 2-microglobulin. Quantitative binding studies showed that IDOX, but not doxorubicin, binds strongly to amyloid fibrils. This binding is saturable and involves two apparently distinct binding sites with Kd values of 5.9 x 10(-11) M and 3.4 x 10(-9) M. IDOX inhibited in vitro insulin amyloid fibrillogenesis. In vivo studies using the experimental amyloid murine model confirmed the specific targeting of IDOX to amyloid deposits. Preincubation of amyloid enhancing factor with IDOX significantly reduced the formation of amyloid deposits. It is hypothesized that IDOX exerts its beneficial effects through the inhibition of fibril growth, thus increasing the solubility of existing amyloid deposits and facilitating their clearance. IDOX may represent the progenitor of a class of amyloid-binding agents that could have both diagnostic and therapeutic potential in all types of amyloidoses.

We have examined the linkage between salt concentration and temperature for the equilibrium binding of the tetrameric Escherichia coli single-stranded binding (SSB) protein to three single-stranded nucleic acids, poly(U), dA(pA)69, and dT(pT)69, by van't Hoff analysis and isothermal titration calorimetry (ITC). For SSB binding to poly(U) in its (SSB)65 mode, the equilibrium association constant, Kobs, decreases with increasing salt concentration at all temperatures examined, and binding is enthalpy-drive; however, the value of [symbol see text] log Kobs/ [symbol see text] log [NaCl] is highly temperature- dependent, varying from -9.3 +/- 0.3 at 10 degrees C to -5.1 +/- 0.4 at 37 degrees C. This indicates that delta Hobs for SSB-poly(U) binding is strongly dependent on [NaCl]; based on van't Hoff analyses, delta Hobs varies from -57 +/- 3 kcal/mol at 0.18 M NaCl to -34 +/- 3 kcal/mol at 042 M NaCl ([symbol see text] delta Hobs/[symbol see text] log [NaCl] = 60 +/- 5 kcal/mol). However, [symbol see text] delta Hobs/[symbol see text] log [NaF] is independent of temperature (25-37 degrees C), indicating that the effect of [NaCl] on delta Hobs is due primarily to Cl-. Similar effects were also observed for SSB binding to dA(pA)69. We also measured delta Hobs and its dependence on [NaCl] for SSB binding dT(pT)69 by ITC and find delta Hobs = -144 +/- 4 kcal/mol (0.175 M NaCl, pH 8.1, 25 degrees C) and [symbol see text] delta Hobs/ [symbol see text] log [NaCl] = 46 +/- 2 kcal/ mol (0.175-2.0 M NaCl). These large effects of [NaCl] on delta Hobs appear to result, at least partly, from the release of preferentially bound Cl- from SSB protein upon binding nucleic acid, with the release of Cl- being linked to a process with delta H > > 0. Effects of salt concentration on delta Hobs are not observed for processes in which only monovalent cations are released from the nucleic acid, presumably since Na+ of K+ are bound to linear nucleic acids as delocalized, fully hydrated cations. Such salt effects on delta Hobs may serve as a signature for differential ion-protein binding. These results underscore the need to examine the linkage of [salt] to delta Hobs, as well as delta Hobs degrees and delta S(obs) degrees, in order to understand the bases for stability and specificity of protein-nucleic acid interactions.

We have examined the effects of temperature on the equilibrium constant, Kobs, for Escherichia coli SSB tetramer binding to a series of single-stranded (ss) oligodeoxyribonucleotides, dT(pT)n, dC(pC)n, and dA(pA)n (n = 34, 55, and 69) in order to investigate the thermodynamic basis for the strong preference of E. coli SSB (and other SSB proteins) for binding polypyrimidine stretches of ss-DNA. In addition to the expected base-dependent differences in the magnitude of Kobs, we also observe qualitatively different temperature dependencies for the binding of the SSB tetramer to oligodeoxyadenylates. Linear van't Hoff plots are obtained for SSB tetramer binding to dT(pT)n and dC(pC)n, with delta H0obs ranging from -50 to -100 kcal/mol depending on the oligodeoxynucleotide length and salt concentration. In contrast, all van't Hoff plots for SSB tetramer binding to dA(pA)N are distinctly nonlinear with maxima in K(obs) occurring near 25 degrees C, indicative of an apparent large negative change in molar heat capacity (delta C0P,obs < 0). Thus for the SSB-dA(pA)n interaction, delta H0obs and delta S0obs are both highly temperature dependent, but compensate such that delta G0obs is relatively insensitive to temperature. These nonlinear nonlinear van't Hoff plots are not due to coupling of SSB assembly to dA(pA)n binding or to temperature-dependent shifts in the formation of other SSB-DNA binding modes. The nonlinear van't Hoff plots for SSB tetramer binding to dA(pA)n appear to result from the coupling of two processes: (1) the unstacking of the dA(pA)n bases (occurring with delta H0 > 0 and delta C0P = 0) and (2) the binding of SSB to the unstacked DNA (occurring with delta H0 < 0 and delta C0P = 0). Therefore, although each isolated equilibrium occurs with delta C0P approximately 0, the overall equilibrium displays an apparent delta C0P,obs < 0 due to the coupled equilibrium. The binding of SSB to dT(pT)n and dC(pC)n occurs with delta H0 < 0 and delta C0P,obs = 0, since the bases in these ss-DNA molecules do not stack appreciably. These results indicate that a nonspecific protein-DNA interaction can display a large negative apparent delta C0P; however, this effect appears not to be due to the hydrophobic effect, but rather to a temperature-dependent conformational transition in the DNA that is coupled to protein binding. Implications of these observations for other protein-nucleic acid systems are discussed.

DNA vaccines provide an attractive technology platform against bioterrorism agents due to their safety record in humans and ease of construction, testing, and manufacture. We have designed monovalent and bivalent anthrax plasmid DNA (pDNA) vaccines encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins and tested their immunogenicity and ability to protect rabbits from an aerosolized inhalation spore challenge. Immune responses after two or three injections of cationic lipidformulated PA, PA plus LF, or LF pDNAs were at least equivalent to two doses of anthrax vaccine adsorbed (AVA). High titers of anti-PA, anti-LF, and neutralizing antibody to lethal toxin (Letx) were achieved in all rabbits. Eight or nine animals in each group were challenged with 100؋ LD50 of aerosolized anthrax spores 5 or 9 weeks after vaccination. An additional 10 animals vaccinated with PA pDNA were challenged >7 months postvaccination. All animals receiving PA or PA plus LF pDNA vaccines were protected. In addition, 5 of 9 animals receiving LF pDNA survived, and the time to death was significantly delayed in the others. Groups receiving three immunizations with PA or PA plus LF pDNA showed no increase in anti-PA, anti-LF, or Letx neutralizing antibody titers postchallenge, suggesting little or no spore germination. In contrast, titer increases were seen in AVA animals, and in surviving animals vaccinated with LF pDNA alone. Preclinical evaluation of this cationic lipid-formulated bivalent PA and LF vaccine is complete, and the vaccine has received U.S. Food and Drug Administration Investigational New Drug allowance.

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