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1.
Biophys J ; 116(10): 1836-1844, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31076102

RESUMO

Compaction of T4 phage DNA (166 kbp) by short oligopeptide octamers composed of two types of amino acids, four cationic lysine (K), and four polar nonionic serine (S) having different sequence order was studied by single-molecule fluorescent microscopy. We found that efficient DNA compaction by oligopeptide octamers depends on the geometrical match between phosphate groups of DNA and cationic amines. The amino acid sequence order in octamers dramatically affects the mechanism of DNA compaction, which changes from a discrete all-or-nothing coil-globule transition induced by a less efficient (K4S4) octamer to a continuous compaction transition induced by a (KS)4 octamer with a stronger DNA-binding character. This difference in the DNA compaction mechanism dramatically changes the packaging density, and the morphology of T4 DNA condensates: DNA is folded into ordered toroidal or rod morphologies during all-or-nothing compaction, whereas disordered DNA condensates are formed as a result of the continuous DNA compaction. Furthermore, the difference in DNA compaction mechanism has a certain effect on the inhibition scenario of the DNA transcription activity, which is gradual for the continuous DNA compaction and abrupt for the all-or-nothing DNA collapse.


Assuntos
DNA Viral/química , DNA Viral/genética , Conformação de Ácido Nucleico , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Bacteriófago T4/genética , DNA Viral/metabolismo , Modelos Moleculares , Conformação Proteica
2.
Int J Pharm ; 269(1): 71-80, 2004 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-14698578

RESUMO

Plasmid DNA is known to form complexes with a variety of cationic peptides and lipids, which have been explored as possible carriers for DNA transfection in mammalian cells. We synthesized oligopeptides consisting of nine amino acid residues including lysine (K), tryptophan (W), and cysteine (C), and also their symmetrical dimmers with a disulfide bond as possible carriers. The pDNA(pGL3)/oligopeptide complexes generally showed poor transfection efficiencies but little cytotoxicity for HeLa S3. The ternary system of pDNA/oligopeptide/liposome containing cationic liposomes formulated from the cholesterol derivative (DMB-Chol) and dioleoylphosphatidylethanolamine (DOPE) showed 10(4)-10(5)-fold greater effective gene expression (10(8)-10(9) level, RLU/min/mg protein) than those of the corresponding pDNA/oligopeptide complexes. In the presence of 10% serum, the ternary complexes were maintained at 10(7) levels. The ethidium bromide exclusion studies showed the ternary complexes have much greater affinity to pDNA than the corresponding pDNA/oligopeptide complexes. Plasmid sensitivity against DNase I degradation showed that the ternary complexes were well protected from the digestion. Synthetic oligopeptides are active as potential enhancers for DOPE-containing cationic liposome-mediated transfection. These findings have implications for successful in vivo transfection.


Assuntos
DNA/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Oligopeptídeos/farmacologia , Butanóis/química , Colesterol/análogos & derivados , DNA/química , Desoxirribonuclease I/química , Eletroforese em Gel de Ágar , Etídio/química , Genes Reporter , Células HeLa , Humanos , Lipossomos , Luciferases/genética , Luciferases/metabolismo , Oligopeptídeos/química , Tamanho da Partícula , Fosfatidiletanolaminas , Plasmídeos , Transfecção
3.
Biomacromolecules ; 8(1): 273-8, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17206817

RESUMO

A large-scale conformational change in genomic DNA is an essential feature of gene activation in living cells. Considerable effort has been applied to explain the mechanism in terms of key-lock interaction between sequence-specific regulatory proteins and DNA, in addition to the modification of DNA and histones such as methylation and acetylation. However, it is still unclear whether these mechanisms can explain the ON/OFF switching of a large number of genes that accompanies differentiation, carcinogenesis, etc. In this study, using single-molecule observation of DNA molecules by fluorescence microscopy with the addition of poly-L-lysine with different numbers of monomer units (n = 3, 5, 9, and 92), we found that an ON/OFF discrete transition in the higher-order structure of long duplex DNA is induced by short poly-L-lysine, whereas a continuous gradual change is induced by long poly-L-lysine. On the other hand, polycations with a lower positive charge have less potential to induce DNA compaction. Such a drastic difference in the conformational transition of a giant DNA between short and large oligomers is discussed in relation to the mechanisms of gene regulation in a living cell.


Assuntos
Físico-Química/métodos , DNA/química , Polilisina/química , Biofísica/métodos , Íons , Cinética , Substâncias Macromoleculares , Microscopia de Força Atômica , Microscopia de Fluorescência , Modelos Químicos , Conformação Molecular , Conformação de Ácido Nucleico
4.
Chem Pharm Bull (Tokyo) ; 53(8): 893-8, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16079515

RESUMO

Gabexate mesilate (GM) and camostat mesilate (CM) are protease inhibitors used for the treatment of pancreatitis, and have been reported to show anticancer effects in vivo. Lipid emulsions (20% fractionated soybean oil) were investigated in terms of physicochemical interaction between the drugs and lipid emulsions as a possible drug carrier. The result showed that the drugs did not distribute in the oil phase but were adsorbed at the phospholipid interface of oil droplets. With increasing concentration of the drugs, the adsorption amount at the interface rose steeply to around 2.2x10(-11) mol/cm2 for GM and 1.2x10(-11) mol/cm2 for CM, respectively, followed by further adsorption deviated from the Langmuir adsorption manner after the inflection. To interpret this two-stage adsorption of the drugs, surface potential and fluorescence changes were examined in addition to thermodynamics for their interaction with the interfacial lipid layer. The primary adsorption was exothermic and was due to electrostatic interaction and van der Waals interaction between drug molecules and phospholipid molecules. Both acidic and neutral phospholipids in the lipid were involved in the adsorption of GM, while acidic phospholipids were mainly involved in the adsorption of CM. On the other hand, the secondary adsorption was endothermic and was entropy-driven most probably due to hydrophobic interaction for GM and CM in common, including peripheral penetration of drug molecules into the interfacial lipid layer.


Assuntos
Gabexato/análogos & derivados , Gabexato/química , Lipídeos/química , Inibidores de Proteases/química , Emulsões , Ésteres , Guanidinas , Espectrometria de Fluorescência , Termodinâmica
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