RESUMO
Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.
Assuntos
Enzimas de Restrição-Modificação do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Biologia Sintética/métodosRESUMO
DNA is increasingly employed as a programmable building block for nanoscale structures. Self-assembly via specific DNA base-pair recognition allows an unparalleled variety of structures to be formed. Subsequent stabilization of such structures may be desirable and can be accomplished by metal coordination bonds to substituted bases. We investigated the switching of the mechanics of dsDNA carrying salicylic aldehyde nucleosides upon copper complexation. We found the rupture force to increase by up to a factor of two. Furthermore we discovered that the strongly localized coordinative bond dominates the mechanics of this biomolecular hybrid for high loading rates, whereas at lower rates the broad binding potential of the DNA dominates the stability.
Assuntos
Aldeídos/química , Complexos de Coordenação/química , Cobre/química , DNA/química , Pareamento de Bases , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Nucleosídeos/química , TermodinâmicaRESUMO
All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains. Here, we analyze protein-lipid interactions for MscS by quenching of tryptophan fluorescence with brominated lipids. By this strategy, we define the limits of the bilayer for TM1, which is the most lipid exposed helix of this protein. In addition, we show that residues deep in the pockets, created by the oligomeric assembly, interact with lipid chains. On the cytoplasmic side, lipids penetrate as far as the pore-lining helices and lipid molecules can align along TM3b perpendicular to lipids in the bilayer. Cardiolipin, free fatty acids, and branched lipids can access the pockets where the latter have a distinct effect on function. Cholesterol is excluded from the pockets. We demonstrate that introduction of hydrophilic residues into TM3b severely impairs channel function and that even "conservative" hydrophobic substitutions can modulate the stability of the open pore. The data provide important insights into the interactions between phospholipids and MscS and are discussed in the light of recent developments in the study of Piezo1 and TrpV4.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fenômenos Bioquímicos , Transporte Biológico , Cardiolipinas , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/genética , Modelos Moleculares , Fosfolipídeos/química , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Triptofano/metabolismoRESUMO
Recent developments show encouraging results for the use of DNA as a construction material for nanometer-sized objects. Today, however, DNA-based molecular nanoarchitectures are constructed with mainly unmodified or at best end-modified oligonucleotides, thus shifting the development of functionalized DNA structures into the limelight. One of most recent developments in this direction is the substitution of the canonical Watson-Crick base pairs by metal complexes. In this way "metal-base pairs" are created, which could potentially impart magnetic or conductive properties to DNA-based nanostructures. This review summarizes research which started almost 45 years ago with the investigation of how metal ions interact with unmodified DNA and which recently culminated in the development of artificial ligand-like nucleobases so far able to coordinate up to ten metal ions inside a single DNA duplex in a programmable fashion.
Assuntos
Pareamento de Bases , DNA/química , Metais/química , Sequência de Bases , Dicroísmo Circular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The universal genetic code relies on two hydrogen-bonded Watson-Crick base pairs that can form 64 triplet codons. This places a limit on the number of amino acids that can be encoded, which has motivated efforts to create synthetic base pairs that are orthogonal to the natural ones. An additional base pair would result in another 61 triplet codons. Artificial organic base pairs have been described in enzymatic incorporation studies, and inorganic T-Hg-T and C-Ag-C base pairs have been reported to form in primer extension studies. Here, we demonstrate a metal base pair that is fully orthogonal and can be replicated, and can even be amplified by polymerase chain reaction in the presence of the canonical pairs dA:dT and dG:dC. Crystal structures of a dS-Cu-dS base pair inside a polymerase show that reversible chemistry is possible directly inside the polymerase, which enables the efficient copying of the inorganic crosslink. The results open up the possibility of replicating and amplifying artificial inorganic DNA nanostructures by extending the genetic alphabet.
Assuntos
Complexos de Coordenação/química , DNA/metabolismo , Etilenodiaminas/química , Pareamento de Bases , Sulfato de Cobre/química , Cristalografia por Raios X , DNA/química , DNA Polimerase I/metabolismo , Reação em Cadeia da Polimerase , Estrutura Terciária de ProteínaRESUMO
The ultimate bottom-up approach for the construction of functional nanosystems requires the precise arrangement of atoms and molecules in three dimensions. DNA is currently one of the most prominent molecules able to self-assemble into complex networks and is therefore regarded as the 'silicon of the nano-world'. Metals and metal ions, in contrast, are the atomic building-blocks needed in such materials to establish functions such as electrical conductivity or magnetism. Here we report a new concept, which efficiently combines metal ions and DNA. The DNA structure is used as a matrix to program robustly the complexation of different metal ions under precise control with regard to element, number and composition.