RESUMO
In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.
Assuntos
Ácidos Graxos Monoinsaturados/química , Lipossomos/química , Compostos de Amônio Quaternário/química , Anisotropia , Colesterol , Difenilexatrieno , Corantes Fluorescentes , Fosfatidiletanolaminas , Temperatura , Transfecção/métodosRESUMO
Redox-controlled covalent templating and macrocyclization underlie a novel scheme for stepwise exponential self-replication. This process has been demonstrated by using Tröger's base analogue 1 as the template.
RESUMO
Rapid cooling is the recipe for the self-assembly of nanostructures from trisoligonucleotidyls, a novel class of branched oligonucleotides whose 3'-termini are connected by a trifunctional linker. The topology of the smallest complex is formally equivalent to the topology of acetylene, if a DNA double strand is envisioned as a C-C bond. The model of nano-acetylene is shown.
RESUMO
The development of non-enzymatic self-replicating systems based on autocatalytic template-directed reactions is a current objective of bioorganic chemistry. Typically, a self-complementary template molecule AB is synthesized autocatalytically from two complementary template fragments A and B. Natural replication of nucleic acids, however, utilizes complementary rather than self-complementary strands. Here we report on a minimal implementation of this type of replication based on cross-catalytic template-directed syntheses of hexadeoxynucleotide derivatives from amino-trideoxynucleotides. In our experiments, two self-complementary and two complementary templates compete for their combinatorial synthesis from four common trimeric precursors. We provide kinetic evidence that cross-catalytic self-replication of complementary templates can proceed with an efficiency similar to that of autocatalytic self-replication of self-complementary templates. We observe selective stimulation of template synthesis, and thus information transfer, on seeding the reaction mixtures with one of four chemically labelled templates bearing the sequence of the reaction products. Our results bring a stage closer the development of schemes that might explain how replicating systems based on nucleic acids arose on the prebiotic Earth.
Assuntos
Desoxirribonucleotídeos/química , Evolução Biológica , Catálise , Cromatografia Líquida de Alta Pressão , Cinética , Modelos Químicos , Estrutura Molecular , Origem da Vida , Moldes GenéticosRESUMO
Self-replicating chemical systems have been designed and studied to identify the minimal requirements for molecular replication, to translate the principle into synthetic supramolecular systems and to derive a better understanding of the scope and limitations of self-organization processes that are believed to be relevant to the origin of life on Earth. Current implementations make use of oligonucleotide analogues, peptides, and other molecules as templates and are based either on autocatalytic, cross-catalytic, or collectively catalytic pathways for template formation. A common problem of these systems is product inhibition, leading to parabolic instead of exponential amplification. The latter is the dynamic prerequisite for selection in the darwinian sense. We here describe an iterative, stepwise procedure for chemical replication which permits an exponential increase in the concentration of oligonucleotide analogues. The procedure employs the surface of a solid support and is called SPREAD (surface-promoted replication and exponential amplification of DNA analogues). Copies are synthesized from precursor fragments by chemical ligation on immobilized templates, and then liberated and immobilized to become new templates. The process is repeated iteratively. The role of the support is to separate complementary templates which would form stable duplexes in solution. SPREAD combines the advantages of solid-phase chemistry with chemical replication, and can be further developed for the non-enzymatic and enzymatic amplification of RNA, peptides and other templates as well as for studies of in vitro evolution and competition in artificial chemical systems. Similar processes may also have played a role in the origin of life on Earth, because the earliest replication systems may have proliferated by spreading on mineral surfaces.
Assuntos
Replicação do DNA , DNA/química , Cromatografia Líquida de Alta Pressão , Oligonucleotídeos/química , Origem da Vida , Propriedades de Superfície , Moldes GenéticosRESUMO
Acylamino inhibitors of 14 kDa-PLA2 were synthesized which differ in the moiety that is not bound into the enzyme's active site but immersed in the lipid aggregate when a ternary inhibitory complex is formed. Our results indicate that this part of the inhibitors does not significantly influence inhibitory properties as long the amphiphilic character is retained. So, inhibitory and biophysical properties should be variable independently.
Assuntos
Acetamidas/síntese química , Acetamidas/farmacologia , Organofosfatos/síntese química , Organofosfatos/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2RESUMO
Electrostatic interactions between amidinium and carboxylates were used for the construction of interdigitated architectures at the air-solution interface. Spreading the water-insoluble amphiphile p-pentadecylbenzoic acid (A) on an aqueous solution of p-methylbenzamidinium (B) ions results in an intercalation of the water-soluble base between the acidic headgroups of the water-insoluble amphiphile to form an amorphousA-B-A-B monolayer according to grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity measurements. Upon compression the monolayer transforms into a crystalline film composed of three bilayers with interdigitated hydrocarbon chains, and a top layer whose chains are disordered. Water-insoluble p-heptadecylbenzamidinium spread on an aqueous solution of benzoic acid displays a surface pressure-area isotherm similar to that obtained from the above system. A mechanism that accounts for the formation of these films is presented. Deposition of p-heptadecylbenzamidinium and p-pentadecylbenzoic acid amphiphiles in a 1:1 ratio on pure water led to the formation of a crystalline monolayer phase but which is partially disordered. Over an aqueous solution containing a 1:1 mixture of benzamidinium and benzoic acid no measurable binding of these solute molecules to the polar headgroups of the 1:1 mixed monolayer could be detected by X-ray reflectivity or GIXD.