RESUMO
Anchoring DNA via hydrophobic units into the membrane of vesicles allows tagging of these nanocontainers with sequence information. Moreover, the hybridization of DNA on the surface of liposomes enables sequence specific functionalization, vesicle aggregation, and vesicle fusion. Specifically, DNA-hybridization-based approaches for fusion employing oligonucleotides terminally modified with one or two anchoring units were hindered by a limited degree of full fusion or by significant leakage during fusion. The current work deals with a new strategy for anchoring oligonucleotides on a membrane by lipid-modified nucleobases rather than by attaching hydrophobic units to the 3'- or 5'-termini. The lipid anchors were incorporated into the DNA sequence via phosphoramidite nucleotide building blocks during automated solid-phase synthesis allowing variation of the number and position of hydrophobic units along the DNA backbone. Single-stranded DNA functionalized with four lipid-modified nucleobases was stably grafted onto the membrane of lipid vesicles. It was found that the orientation of DNA hybridization and the number of anchoring units play a crucial role in liposomal fusion, which in the most efficient system reached remarkable 29 % content mixing without notable leakage.
Assuntos
DNA de Cadeia Simples/química , Lipossomos/química , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Transferência Ressonante de Energia de Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Hibridização de Ácido NucleicoRESUMO
Correction for 'Assessing the potential of photosensitizing flavoproteins as tags for correlative microscopy' by Alberto Rodríguez-Pulido et al., Chem. Commun., 2016, 52, 8405-8408.
RESUMO
Photosensitizing flavoproteins have great potential as tags for correlative light and electron microscopy (CLEM). We examine the photostability of miniSOG mutants and their ability to photo-oxidize diaminobenzidine, both key aspects for CLEM. Our experiments reveal a complex relation between these parameters and the production of different reactive oxygen species.
Assuntos
Flavoproteínas/metabolismo , Luz , Microscopia Eletrônica , Fármacos Fotossensibilizantes/metabolismo , Oxigênio Singlete/metabolismoRESUMO
Site-specific derivatization of chemically equivalent functional groups has recently been facilitated by the introduction of high-affinity aptamers as non-covalent protective groups. More specifically, a series of RNA aptamers have proven to be highly efficient in enhancing the regioselectivity of reactions with the aminoglycoside antibiotic neomycinâ B, which carries several chemically indistinguishable amino and hydroxy groups. Since small-molecule targets tend to exhibit multiple modes of binding with a single aptamer, the impact of secondary binding sites on the regioselectivity should be considered. To address this issue, we investigated a series of well-characterized RNA aptamers that bind neomycinâ B and propose a mechanism that accounts for the regioselective outcome of these transformations. We further demonstrate that the regioselectivity induced by non-covalent aptamer protective groups is determined by the number of binding sites, their affinity, and the mode of interaction with the guest molecule.
Assuntos
Aptâmeros de Nucleotídeos/química , Antibacterianos/química , Sequência de Bases , Sítios de Ligação , Framicetina/química , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Lipoplexes constituted by calf-thymus DNA (CT-DNA) and mixed cationic liposomes consisting of varying proportions of the cationic lipid 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) have been analyzed by means of electrophoretic mobility, SAXS, and fluorescence anisotropy experiments, as well as by theoretically calculated phase diagrams. Both experimental and theoretical studies have been run at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction, α, and either the mass or charge ratios of the lipoplex, respectively. The experimental electrochemical results indicate that DC-Chol/DOPE liposomes, with a mean hydrodynamic diameter of around (120 ± 10) nm, compact and condense DNA fragments at their cationic surfaces by means of a strong entropically driven electrostatic interaction. Furthermore, the positive charges of cationic liposomes are compensated by the negative charges of DNA phosphate groups at the isoneutrality L/D ratio, (L/D)(Ï), which decreases with the cationic lipid content of the mixed liposome, for a given DNA concentration. This inversion of sign process has been also studied by means of the phase diagrams calculated with the theoretical model, which confirms all the experimental results. SAXS diffractograms, run at several lipoplex compositions, reveal that, irrespectively of the lipoplex charge ratio, DC-Chol/DOPE-DNA lipoplexes show a lamellar structure, L(α), when the cationic lipid content on the mixed liposomes α ≥ 0.4, while for a lower content (α = 0.2) the lipoplexes show an inverted hexagonal structure, H(II), usually related with improved cell transfection efficiency. A similar conclusion is reached from fluorescence anisotropy results, which indicate that the fluidity on liposome and lipoplexes membrane, also related with better transfection results, increases as long as the cationic lipid content decreases.
Assuntos
Colesterol/análogos & derivados , DNA/química , Lipídeos/análise , Lipossomos/química , Transição de Fase , Fosfatidiletanolaminas/química , Colesterol/química , Lipídeos/química , Fluidez de Membrana , Transfecção/métodos , Transfecção/normasRESUMO
The mixed system consisting of two anionic surfactants of identical headgroups but with 10 and 12 carbon atoms on the hydrophobic tail, sodium decanoate (C(10)Na) and sodium dodecanoate (C(12)Na), has been studied in aqueous solution at 298.15 K by means of conductivity and fluorescence spectroscopy experiments and from a theoretical point of view. The monomeric and micellar phases of the mixed aggregates were analyzed through the experimental determination of the total critical micelle concentration, cmc*, the degree of ionization of the mixed micelle, beta, and the total aggregation number, N*. Results indicate that, compared to the ideal behavior, the mixed system with two anionic surfactants differing only in two methylenes in the hydrophobic tail shows a negative deviation in the cmc* and a positive one in N*. Pure surfactants (C(10)Na and C(12)Na) form spherical micelles, but mixed micelles must aggregate with a rodlike shape to allow more surfactant molecules than expected. In addition, rodlike micelles result in more compacted aggregation (i.e., less area per polar head). From the experimental data in this work, several theoretical models for mixed surfactant systems have been checked: Rubingh's model predicts lower deviations from ideality than Motomura's model. The stability of the micelles has been analyzed by computing the standard Gibbs energy of micelle formation, Delta G(mic,0), of pure and mixed micelles. Results of this work reinforce the feature that mixed systems formed by alkylsurfactants with the same polar head that differ in the hydrocarbon length, usually admitted as roughly ideal systems, may show nonideal behavior. This deviation, being mostly related to the difference in the chain length, Delta n(c), between surfactants can be analyzed only when very accurate experimental techniques as well as adequate theoretical models are used.
Assuntos
Decanoatos/química , Modelos Teóricos , Tensoativos/química , Micelas , Soluções/química , TermodinâmicaRESUMO
The compaction of DNA by cationic liposomes constituted by a mixture of a cationic lipid, dioctadecyldimethylammonium bromide (DODAB), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), has been evaluated by means of experimental studies (electrophoretic mobility, conductometry, cryogenic electron transmission microscopy or cryo-TEM, and fluorescence spectroscopy) as well as theoretical calculations. This information reveals that DODAB/DOPE and DODAB/DLPC liposomes are mostly spherical and unilamellar, with a mean diameter of around 70 and 61 nm, respectively, a bilayer thickness of 4.5 nm, and gel-to-fluid transition temperatures, T(m), of around 19 and 28 degrees C, respectively. Their positively charged surfaces efficiently compact the negatively charged DNA by means of a strong entropically driven surface interaction that yields DODAB/DOPE-DNA and DODAB/DLPC-DNA lipoplexes as confirmed by zeta potential and ethidium bromide fluorescence intercalation assays. These experiments have permitted as well the evaluation of the different microenvironments of varying polarity of the DNA helix, liposomes, and/or lipoplexes. DODAB/DOPE-DNA and DODAB/DLPC-DNA lipoplexes have been characterized by isoneutrality ratios (L/D)(phi) of around 4.7 and 4.8, respectively, a more fluid membrane than that of the parent liposomes, and T(m) around 24 and 28 degrees C, respectively, as revealed by fluorescence anisotropy. Cryo-TEM micrographs reveal a rich scenario of nanostructures and morphologies, from unilamellar DNA-coated liposomes to multilamellar lipoplexes passing through cluster-like structures. Phase diagrams (aggregation and re-entrant condensation phenomena), calculated by means of a phenomenological theory, have confirmed the experimental concentration domains and the isoneutrality conditions. The influence of helper lipid in the compaction process, as well as the optimum choice among those herein chosen, has been analyzed.
Assuntos
DNA/química , Lipossomos/química , Compostos de Amônio Quaternário/química , Cátions/química , Etídio , Nanoestruturas/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Espectrometria de FluorescênciaRESUMO
The use of cationic micelles consisting of octadecyltrimethylammonium bromide (C18TAB) to compact calf thymus DNA has been investigated in aqueous buffered solution at 310.15 K by means of conductometry, electrophoretic mobility, and several fluorescence spectroscopy methods. The results indicate that C18TAB micelles, consisting of 44 monomers on average, may compact DNA molecule by an electrostatic interaction that takes place at the cationic spherical micelle surface. The surfoplexes thus formed show a surface density charge that goes from negative to positive values at a Lmic/D mass ratio of around 1.0 (where Lmic and D are the masses of micellized cationic surfactant and DNA), called the isoneutrality ratio (Lmic/D)phi. Values of this characteristic parameter, determined in this work not only from the electrochemical experimental data but also from spectroscopic measurements, are in very good agreement with those ones calculated from molecular parameters and some other properties also obtained in this work. The electrostatic character of the DNA-micelle interaction has been confirmed by analyzing the decrease in fluorescence emission of the fluorophore ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the surfoplexes are formed. Fluorescence anisotropy experiments have revealed that micelle packing becomes more rigid in the presence of DNA, but once the surfoplex is formed, the fluidity increases with the Lmic/D mass ratio, attaining its maximum when the isoneutrality ratio is exceeded. This fact, together with the net positive charge of the surfoplexes with the Lmic/D mass ratio over the isoneutrality ratio, makes this regimen of lipid and DNA content the optimum for efficiency in the transfection process.
Assuntos
Alcanos/química , DNA/química , Eletroquímica/métodos , Compostos de Amônio Quaternário/química , Espectrofotometria/métodos , Anisotropia , Bioquímica/métodos , Cátions , Coloides/química , Condutividade Elétrica , Concentração de Íons de Hidrogênio , Lipídeos/química , Micelas , Espectrometria de Fluorescência/métodos , Propriedades de Superfície , TemperaturaRESUMO
A 1:1 mixture of the cationic lipid 3beta-[ N-( N', N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl- sn-glycero-3-phosphoetanolamine (DOPE), has been used to compact calf-thymus DNA (CT-DNA) in aqueous buffered solution at 298.15 K. The formation process of this lipoplex has been analyzed by means of electrophoretic mobility, cryo-TEM, dynamic light scattering, and fluorescence spectroscopy techniques. The experimental results indicate that DC-Chol/DOPE liposomes are mostly spherical and unilamellar, with a mean diameter of around 99 +/- 10 nm and a bilayer with a thickness of 4.5 +/- 0.5 nm. In the presence of CT-DNA, DC-Chol/DOPE/CT-DNA lipoplexes are formed by means of a strong entropically driven surface electrostatic interaction, as confirmed by zeta potential and fluorescence results, as a consequence of which DNA is compacted and condensed at the surface of the cationic liposomes. The negative charges of DNA phosphate groups are neutralized by the positive charges of cationic liposomes at the isoneutrality L/ D ratio, ( L/ D) varphi around 4, obtained from electrophoretic, fluorescence, and DLS measurements. The decrease in the fluorescence emission intensity of ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the association between the biopolymer and the cationic liposomes takes place has permitted one to confirm its electrostatic character as well as to evaluate the different microenvironments of varying polarity of DNA-double helix, liposomes, and/or lipoplexes. Electronic microscopy reveals a rich scenario of possible nanostructures and morphologies for the lipoplexes, from unilamellar DNA-coated liposomes to multilamellar lipoplexes passing through cluster-like structures and several intermediate morphologies.
Assuntos
Colesterol/análogos & derivados , DNA/química , Lipossomos/química , Fosfatidiletanolaminas/química , Animais , Bovinos , Colesterol/química , Colesterol/metabolismo , DNA/metabolismo , Eletroforese , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Fosfatidiletanolaminas/metabolismo , Espectrometria de FluorescênciaRESUMO
The compaction of calf thymus DNA (CT-DNA) by cationic liposomes constituted by a 1:1 mixture of a cationic lipid, 1,2-distearoyl-3-(trimethylammonio)propane chloride (DSTAP), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE, null net charge at pH = 7.4), has been evaluated in aqueous buffered solution at 298.15 K by means of conductometry, electrophoretic mobility, cryo-TEM, and fluorescence spectroscopy techniques. The results reveal that DSTAP/DOPE liposomes are mostly spherical and unilamelar, with a mean diameter of around 77 +/- 20 nm and a positively charged surface with a charge density of sigmazeta = (21 +/- 1) x 10(-3) C m(-2). When CT-DNA is present, the genosomes DSTAP/DOPE/CT-DNA, formed by means of a surface electrostatic interaction, are generally smaller than the liposomes. Furthermore, they show a tendency to fuse forming cluster-type structures when approaching isoneutrality, which has been determined by the electrochemical methods at around (L/D)phi = 5.6. The analysis of the decrease on the fluorescence emission of the fluorophore ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the genosomes are formed has permitted us to confirm the electrostatic character of the DNA-liposome interaction.