Detection of single amyloid beta-protein aggregates in the cerebrospinal fluid of Alzheimer's patients by fluorescence correlation spectroscopy.

Alzheimer's disease is associated with the intraparenchymal growth of plaque-like amyloid deposits. Amyloid plaques are formed by the progressive deposition and transformation of soluble amyloid beta-protein monomers into insoluble and fibrillar aggregates that contain amyloid beta-protein in a beta-pleated sheet conformation. This process is described as 'seeded polymerization' of the monomers with slow-nucleation and fast-growth kinetics. Soluble amyloid beta-protein monomers are present in the cortical extracellular space and in the cerebrospinal fluid, whereas insoluble aggregates so far can be found only by the examination of brain tissue by biopsy or autopsy. Here we present a biophysical method that uses the principle of seeded polymerization in combination with fluorescence correlation spectroscopy, which allowed us to detect single amyloid beta-peptide aggregates in the cerebrospinal fluid samples from Alzheimer's patients. All of 15 Alzheimer's samples but none of the 19 age-matched control samples produced large peaks with fluorescence correlation spectroscopy indicating the rapid aggregation of the fluorescent labelled synthetic amyloid beta-protein probe onto the amyloid beta-protein 'seeds' present in the cerebrospinal fluid. Our method could enable easy in vivo detection of the cerebral amyloid beta-protein pathology of Alzheimer's disease and might be of potential value to facilitate its routine diagnosis.

Inhibition of cytotoxicity and amyloid fibril formation by a D-amino acid peptide that specifically binds to Alzheimer's disease amyloid peptide.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. The 'amyloid cascade hypothesis' assigns the amyloid-beta-peptide (Abeta) a central role in the pathogenesis of AD. Although it is not yet established, whether the resulting Abeta aggregates are the causative agent or just a result of the disease progression, polymerization of Abeta has been identified as a major feature during AD pathogenesis. Inhibition of the Abeta polymer formation, thus, has emerged as a potential therapeutic approach. In this context, we identified peptides consisting of d-enantiomeric amino acid peptides (d-peptides) that bind to Abeta. D-peptides are known to be more protease resistant and less immunogenic than the respective L-enantiomers. Previously, we have shown that a 12mer D-peptide specifically binds to Abeta amyloid plaques in brain tissue sections from former AD patients. In vitro obtained binding affinities to synthetic Abeta revealed a K(d) value in the submicromolar range. The aim of the present study was to investigate the influence of this d-peptide to Abeta polymerization and toxicity. Using cell toxicity assays, thioflavin fluorescence, fluorescence correlation spectroscopy and electron microscopy, we found a significant effect of the d-peptide on both. Presence of D-peptides (dpep) reduces the average size of Abeta aggregates, but increases their number. In addition, Abeta cytotoxicity on PC12 cells is reduced in the presence of dpep.

Transcription of potato spindle tuber viroid by RNA polymerase II starts predominantly at two specific sites.

Pospiviroidae, with their main representative potato spindle tuber viroid (PSTVd), are replicated via a rolling circle mechanism by the host-encoded DNA-dependent RNA polymerase II (pol II). In the first step, the (+)-strand circular viroid is transcribed into a (-)-strand oligomer intermediate. As yet it is not known whether transcription is initiated by promotors at specific start sites or is distributed non-specifically over the whole circle. An in vitro transcription extract was prepared from a non-infected potato cell culture which exhibited transcriptional activity using added circular PSTVd (+)-strand RNA as template. In accordance with pol II activity, transcription could be inhibited by alpha-amanitin. RT-PCR revealed the existence of at least two different start sites and primer extension identified these as nucleotides A(111) and A(325). The sequences of the first 7 nt transcribed are very similar, (105)GGAGCGA(111) and (319)GGGGCGA(325). GC-boxes are located at a distance of 15 and 16 nt upstream, respectively, in the native viroid structure, which may act to facilitate initiation. The GC-boxes may have a similar function to the GC-rich hairpin II in the (-)-strand intermediate, as described previously. The results are compared with the corresponding features of avocado sunblotch viroid, which belongs to a different family of viroids and exhibits different transcription initiation properties.

A mini-RNA containing the tetraloop, wobble-pair and loop E motifs of the central conserved region of potato spindle tuber viroid is processed into a minicircle.

A Mini-RNA from potato spindle tuber viroid (PSTVd) was constructed specifically for cleavage and ligation to circles in vitro. It contains the C-domain with the so-called central conserved region (CCR) of PSTVd with a 17 nt duplication in the upper strand and hairpin structures at the left and rights ends of the secondary structure. The CCR was previously shown to be essential for processing of in vitro transcripts. When folded under conditions which favor formation of a kinetically controlled conformation and incubated in a potato nuclear extract, the Mini-RNA is cleaved correctly at the 5'- and the 3'-end and ligated to a circle. Thus, the CCR obviously contains all structural and functional requirements for correct processing and therefore may be regarded as 'processing domain' of PSTVd. Using the Mini-RNA as a model substrate, the structural and functional relevance of its conserved non-canonical motifs GAAA tetraloop, loop E and G:U wobble base pair were studied by mutational analysis. It was found that (i) the conserved GAAA tetraloop is essential for processing by favoring the kinetically controlled conformation, (ii) a G:U wobble base pair at the 5'-cleavage site contributes to its correct recognition and (iii) an unpaired nucleotide in loop E, which is different from the corresponding nucleotide in the conserved loop E motif, is essential for ligation of the 5'- with the 3'-end. Hence all three structural motifs are functional elements for processing in a potato nuclear extract.

Helix-coil transitions in double-stranded viral RNA. Fine resolution melting and ionic strength dependence.

Helix-coil transitions of double-stranded RNA from reovirus and infectious bursal disease virus were measured optically in aqueous medium of different ionic strengths. In RNA from reovirus four transitions and in RNA from infectious bursal disease virus two transitions were resolved and evaluated quantitatively. The ionic strength dependence of their midpoint temperatures dTm/dlog[Na+] were 13.5 +/- 0.3 degrees C for reovirus RNA and 14.9 +/-0.7 degrees C for infectious bursal disease virus RNA. The midpoint temperatures extrapolated to 1 M ionic strength were 102.9, 104.3, 105.6, and 108.8 +/- 0.3 degrees C for reovirus RNA, and 108.8 +/- 1.8 and 109.6 +/- 1.0 degrees C for infectious bursal disease virus RNA. The G + C content of the regions in reovirus RNA melting in the different transitions were determined from the spectrum of the hypochromicity. The quantitative interpretation of the data is carried out on the basis of the ion condensation theory. It is estimated for double-stranded RNA of 100% G + C, that dTm/dlog[na+] = 8.4 degrees C. The two-dimensional dependence Tm = Tm (ionic strength, G + C content) is given. The ionic strength dependence in different double-stranded RNAs is correlated to the spacing of the phosphate backbone, secondary structure, and tertiary structure.

Thermodynamic prediction of conserved secondary structure: application to the RRE element of HIV, the tRNA-like element of CMV and the mRNA of prion protein.

An algorithm for prediction of conserved secondary structure of single-stranded RNA is presented. For each RNA of a set of homologous RNAs optimal and suboptimal secondary structures are calculated and stored in a base-pair probability matrix. A multiple sequence alignment is performed for the set of RNAs. The resulting gaps are introduced into the individual probability matrices. These homologous probability matrices are summed to give a consensus probability matrix emphasizing the conserved secondary structure elements of the RNA set. Thus the algorithm combines the advantages of thermodynamic structure prediction by energy minimization with the information obtained from phylogenetic alignment of sequences. The algorithm is applied to three examples. The REV-responsive element of HIV, the structure of which is well known from the literature, was chosen to test the algorithm. The second example is the 3' terminal segment of genomic single-stranded RNAs of cucumber mosaic viruses; a structure similar to that of the related brome mosaic virus was expected and was confirmed. The third example is the prion-protein mRNA from different organisms; the structure of this mRNA is not known. By application of the algorithm highly conserved hairpins were found in the prion-protein mRNA.

Structural requirements for viroid processing by RNase T1.

Viroids are replicated via a rolling circle-like mechanism in which (+) strand oligomeric intermediates have to be cleaved enzymatically to unit-length molecules followed by ligation to mature circles. A transcript of potato spindle tuber viroid, which is still infectious, consists of a monomeric molecule with only 22 additional nucleotides, thus doubling part of the central conserved region of viroids. It was shown that this transcript can be cleaved and ligated in vitro to circles by RNase T1. To elucidate the site and mechanism of processing, 16 different site-specific mutants of this longer-than-unit-length transcript were constructed and analyzed by in vitro processing with RNase T1, infectivity studies, temperature-gradient gel electrophoresis, and structure calculations. The wild-type sequence and several mutated transcripts are able to adopt a particular secondary structure which is the prerequisite for enzymatic cleavage and ligation by RNase T1. This "processing structure" exposes both potential cleavage sites in the nearest spatial neighborhood, thus favoring the subsequent ligation to circles. Those mutated sequences for which the formation of the processing structure is impossible or thermodynamically highly unfavored are not processed. The results demonstrate that the particular structural features of viroids enable them to be cleaved and ligated by one and the same enzyme, RNase T1. The in vitro mechanism may serve as a mechanistic model for cellular processing of viroids.

Co-existing structures of an mRNA stability determinant. The 5' region of the Escherichia coli and Serratia marcescens ompA mRNA.

The structure of untranslated regions of mRNA is thought to play a key role in the degradation of mRNAs by specific RNases. As a model system, in vitro transcripts of the stability determining 5' non-coding region of bacterial ompA mRNA were investigated by calculation of secondary structure models and by experiments applying the temperature-gradient gel electrophoresis (TGGE). For the theoretical prediction of secondary structures an algorithm was used, which yields the structure of lowest free energy as well as a large set of suboptimal structures. Three structures were predicted to co-exist in similar concentrations under native conditions. They denature in a low temperature transition leading to a unique structure which denatures in a high temperature transition. The prediction of three structures and two transitions could be confirmed experimentally by TGGE. Due to the use of transcripts of different length the conformational transitions could be attributed to distinct parts of the molecules. A pseudoknot structural motif was predicted theoretically, but could not be confirmed experimentally. Comparing ompA transcripts of E. coli and S. marcescens, a conservation of structural features could be shown in spite of a sequence homology of only 63%. Regarding the sequential folding of the transcript after synthesis, a metastable structure is formed first and is converted slowly into structures of lower free energy. The biological implications for in vivo degradation are discussed.

The structural transition of the prion protein into its pathogenic conformation is induced by unmasking hydrophobic sites.

A series of structural intermediates in the putative pathway from the cellular prion protein PrP(C) to the pathogenic form PrP(Sc) was established by systematic variation of low concentrations (<0.1%) of the detergent sodium dodecyl sulfate (SDS) or by the interaction with the bacterial chaperonin GroEL. Most extended studies were carried out with recombinant PrP (90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30. Similar results were obtained with full-length recombinant PrP, hamster PrP 27-30 and PrP(C) isolated from transgenic, non-infected CHO cells. Varying the incubation conditions, i.e. the concentration of SDS, the GroEL and GroEL/ES, but always at neutral pH and room temperature, different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and to molecular mass, as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation: alpha-helical monomers, soluble alpha-helical dimers, soluble but beta-structured oligomers of a minimal size of 12-14 PrP molecules, and insoluble multimers were observed. A high activation barrier was found between the alpha-helical dimers and beta-structured oligomers. The numbers of SDS-molecules bound to PrP in different conformations were determined: Partially denatured, alpha-helical monomers bind 31 SDS molecules per PrP molecule, alpha-helical dimers 21, beta-structured oligomers 19-20, and beta-structured multimers show very strong binding of five SDS molecules per PrP molecule. Binding of only five molecules of SDS per molecule of PrP leads to fast formation of beta-structures followed by irreversible aggregation. It is discussed that strongest binding of SDS has an effect identical with or similar to the interaction with GroEL thereby inducing identical or very similar transitions. The interaction with GroEL/ES stabilizes the soluble, alpha-helical conformation. The structure and their stabilities and particularly the induction of transitions by interaction of hydrophobic sites of PrP are discussed in respect to their biological relevance.

Analysis of RNA structures by temperature-gradient gel electrophoresis: viroid replication and processing.

The structure and structural transitions of single-stranded RNA were investigated by energy calculations and temperature-gradient gel electrophoresis. Most experiments have been carried out on RNA of mature viroids and their replication intermediates, which are RNA (-) strand oligomers and RNA (+) strand oligomers. The technique of temperature-gradient gel electrophoresis proved to be particularly useful for analysing co-existing structures. The secondary structure of lowest free energy for unit length and oligomeric replication intermediates is an extended rod-like structure similar to that of the mature circular viroid. When this structure is used as a model for calculations, there is a large degree of agreement between theoretical and experimental curves. Under particular solution conditions, however, (+) strand oligomers undergo a rearrangement from the extended structure to a branched structure, in which every two units form a region of three helices, together 28 bp long. This structure is called the tri-helical structure. The process of structure formation during the synthesis of oligomers could be followed: at first, a transient multi-branched structure is formed which is then transformed into the extended and the tri-helical structures. The region of the three stable helices serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes, and be processed into circular mature viroids. Co-transcription of complementary (+) and (-) strands shows that energetically favored double-strand formation may at least partially be prohibited by stable secondary structures of the single strands. Natural replication intermediates have been analysed in respect to their subcellular location and their size distribution. They are associated with the nucleoli as was found earlier for mature viroids. Natural (-) strand oligomers are larger than (+) strand oligomers; both types show a periodicity in the size distribution of two units. The models of the structures, which are involved in viroid processing, are in accordance with recent infectivity data and with the results on natural replication intermediates.

Cyclosporin synthetase is a 1.4 MDa multienzyme polypeptide. Re-evaluation of the molecular mass of various peptide synthetases.

The earlier determined molecular mass of 0.8 MDa for the multifunctional polypeptide, cyclosporin synthetase, was re-evaluated by SDS-PAGE and CsCl density gradient centrifugation. In SDS-PAGE, new molecular mass values as standards were available from sequencing data. In the CsCl density gradient extremely low protein concentrations, such as 10-50 nM could be analysed due to the fluorescence detection system of the analytical ultracentrifuge. Both methods yielded approximately the same value of about 1.4 MDa. Using this molecular mass of cyclosporin synthetase as a reference the molecular masses of various related enzymes could be re-evaluated in SDS-PAGE. The sedimentation coefficient of 26.3 S for cyclosporin synthetase indicates an oblate overall shape of the enzyme.

Enzymatic biosynthesis of cyclosporin A and analogues.

The final assembly of the undecapeptide chain of cyclosporin A and its cyclization is accomplished in Beauveria nivea by cyclosporin synthetase. This multienzyme is the largest integrated enzyme structure so far reported. Its size has been estimated at approximately 1,400 kDa by two different methods: 1), by 3% SDS-PAGE using the related multienzymes ACV synthetase and gramicidin S synthetase 2 as references (420 and 556 kDa, respectively); and 2), by CsCl density gradient centrifugation experiments using fluorescence-labeled cyclosporin synthetase. Besides cyclosporin A and a number of cyclosporins known from fermentation studies cyclosporin synthetase is capable of synthesizing some new cyclosporins which are so far unobtainable by fermentation. So, for example the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid1]CyA, dihydro-CyA, [L-norvaline2,5, N-methyl-L-norvaline11]CyA, [L-allo-isoleucine5, N-methyl-L-allo-isoleucine11]CyA, [D-2-aminobutyric acid8]CyA, [beta-chloro-D-alanine8]CyA and some related compounds could be established. By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide [L-threonine2, L-leucine5,10, D-2-hydroxyisovaleric acid8]CyA could be synthesized in vitro. We were able to synthesize these cyclosporins in sufficient quantities to examine their structure by FAB mass spectroscopy and explore their immunosuppressivity. It was found that all new cyclosporins so far synthesized in the in vitro system are immunosuppressive.

Inhibition of potato spindle tuber viroid (PSTVd) infection with DNA oligonucleotides.

The effects of DNA oligonucleotides complementary to potato spindle tuber viroid (PSTVd) on viroid infection were investigated. The oligonucleotides were used to form hybrids with PSTVd in the infection mixture. A 75% reduction of viroid infection was found when an oligonucleotide complementary to nucleotides 79-110 of PSTVd was hybridized with PSTVd at a molar ratio of approximately 5000:1, respectively. A total inhibition of PSTVd infection was observed using an oligonucleotide complementary to nucleotides 42-78 at the same molar excess of DNA over PSTVd, although a 200-fold molar excess was found to be sufficient for the complete blocking of infection by PSTVd. This oligonucleotide caused a significant reduction (about 83%) of viroid infection even if the hybridization was done at a low (30 degrees C) temperature. Shorter oligonucleotides containing 22 and 15 bases corresponding to position 42-62 and 63-78, respectively, exhibited a significant effect only at a high (80 degrees C) initial temperature of molecular hybridization. Heteroduplexes formed between PSTVd RNA and antisense DNA were found to be less stable in a crude nuclease extract from tomato leaves as compared with PSTVd RNA alone. RNase H was demonstrated to cleave the molecular hybrids in vitro.

Analysis of nucleic acids in purified scrapie prion preparations.

Amount, type, and size of nucleic acid molecules associated with purified prion preparations were analyzed. Return refocusing gel electrophoresis (RRGE) was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. With this method all types of nucleic acids in the size range from 13 to several thousand nucleotides could be analyzed. The recovery of all nucleic acids, after deproteinization and two-phase extraction was higher than 90%. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics for such a hypothetical molecule. If it was homogeneous in size, then it would be < 80 nt in length at a particle-to-infectivity ratio (P/I) near unity; if the other extreme, i.e. totally heterogeneous scrapie-specific nucleic acids were assumed, then scrapie-specific nucleic acids would have to include molecules smaller than 240 nt. In order to exclude the possibility that unspecific background nucleic acid is entrapped in prion-rods, infectious material has to be prepared without a proteolysis and rod formation, and the analysis of nucleic acids performed with those preparations.

Neurotoxicity but not infectivity of prion proteins can be induced reversibly in vitro.

Prion diseases include Creutzfeldt-Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy. The hallmark of prion diseases is the accumulation of an abnormal isoform (PrP(Sc)) of the cellular prion protein accompanied by neuronal cell death and astroglial proliferation. To characterize the correlation between PrP secondary and quarternary structure and their biological effects we assayed soluble and aggregated forms of PrP 27-30, the N-terminal truncated form of PrP(Sc), as well as the corresponding recombinant PrP(90-231) for their neurotoxicity and infectivity. PrP was kept soluble in 0.2% SDS and subsequently re-aggregated either by diluting the SDS or by adding acetonitril. The neurotoxicity of the re-aggregated states were comparable to that of prion rods (PrP 27-30) whereas the soluble forms had no neurotoxic effects. The solubilized PrP 27-30 showed no significant infection upon re-aggregation as determined by bioassays in Syrian golden hamsters. The recombinant PrP did not exhibit infectivity in any state.

Biolistic inoculation of plants with viroid nucleic acids.

Parameters for biolistic transfer of viroid nucleic acids using a Helios Gene Gun device were assayed. The main achievement of this method is high efficiency of inoculation with linear monomeric viroid cDNAs and RNAs. This greatly facilitates the study of mutated sequence variants, viroid libraries and mixed populations. The lower limits for efficient inoculation of monomeric cDNA fragments with the sequence of potato spindle tuber viroid (PSTVd) and native PSTVd RNA as detected 21 days p.i. are in the range of 50 ng and 200 pg per tomato plant, respectively. At a higher dose, i.e. 2 ng of native RNA per plant, biolistic transfer causes drastic stunting compared to conventional mechanical inoculation, which points to higher PSTVd titers after the biolistic transfer. Infection is readily achieved with exact length monomeric RNA transcripts having 5'-triphosphate and 3'-OH termini in amounts ranging from 2 to 20 ng per plant, suggesting no need for any supplementary modifications of ends or RNA circularization. The biolistic transfer is efficient for viroid "thermomutants", which exhibit low or no infectivity with conventional mechanical inoculation with Carborundum. The biolistic inoculation is also efficient for two other members of the Pospiviroidae family, hop stunt and hop latent viroid.

Temperature-gradient gel electrophoresis. Thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extracts.

A temperature-gradient gel electrophoresis technique and its application to the study of structural transitions of nucleic acids and protein-nucleic acid complexes are described. The temperature gradient is established in a slab gel by means of a simple ancillary device for a commercial horizontal gel apparatus. The gradient may be freely selected between 10 and 80 degrees C, and is highly reproducible and linear. In a normal application the biopolymers migrate perpendicular to the temperature gradient so that every individual molecule is at constant temperature throughout electrophoresis. The structural transition of a biopolymer is seen as a continuous band which is retarded or speeded up in the temperature range of the transition. Dissociation processes are mostly irreversible under the conditions of electrophoresis and, therefore, show up as discontinuous transitions from a slow-moving to fast-moving band. As examples the conformational transitions of viroids, double-stranded RNA from reovirus, double-stranded satellite RNA from cucumber mosaic virus and repressor-operator complexes have been studied. It could be shown that by this method dsRNA molecules may be differentiated which differ only in one base-pair, or proteins differing in one amino acid only. As a particular advantage, temperature-gradient gel electrophoresis allows the study of conformational transitions of biopolymers which have not been purified. The biopolymer may either be identified by silver staining as a specific band among many others or, if the study is carried out on nucleic acids, these may be recorded by hybridization with a radioactive probe.

Kinetics of conformational changes in tRNA Phe (yeast) as studied by the fluorescence of the Y-base and of formycin substituted for the 3'-terminal adenine.

The kinetics of the melting transitions of tRNA Phe (yeast) were followed by the fluorescence of the Y-base and of formycin substituted for the 3'-terminal adenine. As judged from differential UV absorbance melting curves the formycin label had virtually no influence on the conformation of the tRNA. A temperature jump apparatus was modified to allow the simultaneous observation of transmission and fluorescence intensities by two independent optical channels. The design of a temperature jump cell with an all quartz center piece is given. The cell is resistant to temperatures up to 90 degrees C; it provides high optical sensitivity, low stray light intensity and the possibility of measuring fluorescence polarization. The T-jump experiments allowed to discriminate between fast unspecific fluorescence quenching (r less than 5 musec) and slow cooperative conformational changes. In the central part of the temperature range of UV-melting (midpoint temperature 30 degrees C in 0.01 M Na+ and 39 degrees C in 0.03 M Na+, pH 6.8) two resolvable relaxation processes were observed. The corresponding relaxation times were 20 msec and 800 msec at 30 degrees C in 0.01 M Na+, and 4 msec and 120 msec at 39 degrees C in 0.03 M Na+. The Y-base fluorescence shows both of the relaxation effects, which almost cancel in equilibrium fluorescence melting, because their amplitudes have opposite signs. From this finding the existence of some residual tertiary structure is inferred which persists after the unfolding of the main part of tertiary structure during early melting (midpoint temperature 24 degrees C in 0.03 M Na+). In the fluorescence signal of the formycin also the two relaxation effects appear. Both of them are connected with a decrease of the fluorescence intensity. From the results a coupled opening of the anticodon and acceptor branches is concluded.

Influence of lipid membranes on the conformational transitions of nucleic acids.

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.

Interaction of nucleic acids with lipid membranes.

The thermodynamics of nucleic acids which were enclosed in reverse-phase evaporation vesicles was studied by thermal denaturation with optical recording. The denaturation curves were recorded with a dual wavelength spectrophotometer. The sum of the hypochromicity of the nucleic acid and of the change in turbidity of the vesicles was measured at 260 nm and was corrected for the change in turbidity at 320 nm. Cloned fragments of double-stranded DNA containing 180 base pairs and poly A:poly U were enclosed in REV with a yield up to every vesicle containing five nucleic acid molecules. Vesicles were prepared from egg-lecithin, and the surface charge of the vesicles was varied by addition of stearic acid, phosphatidyl-glycerol and phosphatidyl-serine. The helix-coil transition of the nucleic acid enclosed in the vesicle could be resolved from that of the free nucleic acid. Due to the enclosure into the egg-lecithin REV the transition is stabilized from 70.5 degrees to 74 degrees C, the transition is broadened from 0.7 degrees C to 2.7 degrees C. Varying the phosphatidyl-serine-lecithin-ratio from 0-100%, an optimum in the yield of enclosure at 20% was obtained, a further broadening of the transition to 5.5 degrees C and a decrease of the stabilization down to a small destabilization at 100% phosphatidyl serine was observed. Qualitatively, similar effects were observed with poly A:poly U. Variation of the ionic strength led to the conclusion that the replacement of the counterions of the phosphate backbone by the surface charge of the membrane, as well as a direct contact between the nucleic acid and the membrane have to be assumed.(ABSTRACT TRUNCATED AT 250 WORDS)