RESUMO
6-mercapto hexyl-oligonucleotides bind to a gold surface strongly enough to permit imaging by a scanning tunneling microscope (STM). STM images showed worm-like chains that were approximately 12-(A-wide for single-stranded DNA and 20-(A-wide for double-stranded DNA. The chain lengths corresponded to 3.4 +/- 0.4 A per basepair for double-stranded DNA and 2.2 +/- 0.4 A per base for single-stranded DNA. This unexpectedly short length for single-stranded DNA was confirmed using oligomers with both single- and double-stranded regions. When the attachment of the samples was weakened (by imaging in water or scraping with the STM tip) the images changed to pairs of "blobs," apparently reflecting the attachment points of the molecules to the gold surface. Given this interpretation, images of DNA containing a five-base bulge imply that the bulge bends the oligomer by 90 degrees +/- 20 degrees.
Assuntos
DNA/química , DNA/ultraestrutura , Ouro , Oligodesoxirribonucleotídeos/química , Artefatos , Composição de Bases , Sequência de Bases , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Microscopia de Tunelamento/métodos , Oligodesoxirribonucleotídeos/síntese química , Eletricidade Estática , Compostos de SulfidrilaRESUMO
Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20-21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60-90 degrees. Rotation of the hairpin position in the three-way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60-90 degrees between the arms. Computer modeling of the three-way junction with 60 degrees pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Polidesoxirribonucleotídeos/química , Sequência de Bases , DNA Circular/química , Eletroforese em Gel Bidimensional , Dados de Sequência Molecular , Polidesoxirribonucleotídeos/síntese químicaRESUMO
Scanning tunneling microscopy (STM) reveals nanometer scale details of hydrated DNA but the interpretation of the images is controversial because of substrate artifacts and the lack of a theory for image contrast. We demonstrate that we have overcome these problems by identifying five DNA samples by their STM images alone in a blinded trial. The samples were single-stranded and double-stranded DNA with and without covalent modification by the anti-tumor drug cisplatin. The cisplatin adducts were distinguished by substantial kinking at the drug binding site. The oligomers were 20 bases in length, which was too short to permit the kinking angle to be determined with precision. However, models with a 45 degree kink gave a better fit to the images of the duplex adducts than models with a 90 degrees kink. A variety of structures was observed for the single-stranded adducts.
Assuntos
Cisplatino/análise , Adutos de DNA , DNA/análise , Sequência de Bases , Processamento de Imagem Assistida por Computador , Microscopia , Modelos Moleculares , Dados de Sequência MolecularRESUMO
We have used the scanning tunneling microscope (STM) to image several synthetic oligonucleotides adsorbed onto a positively charged Au(111) electrode. The molecules were deposited and imaged in aqueous electrolyte under potential control, a procedure that eliminated the problem of the substrate artifacts that had limited some previous STM studies. Experiments were carried out with two types of single-stranded molecules (11 and 20 bases long) and three types of double-stranded molecules (20 and 61 base pairs and 31 bases with 25 bases paired and 6-base "sticky" ends). The molecules lie along symmetry directions on the reconstructed (23 x square root of 3) gold surface, and length measurements indicate that they adopt simple base-stacked structures. The base stacking distances are, within experimental uncertainty, equal to the 0.33 nm measured for polymeric aggregates of stacked purines by direct imaging in identical conditions. The images show features consistent with helical structures. Double helices have a major-groove periodicity that is consistent with a 36 degrees twist. The single helices appear to be more tightly twisted. A simple tunneling model of STM contrast generates good agreement between measured and calculated images.
Assuntos
DNA/ultraestrutura , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Microscopia de Tunelamento/métodos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese químicaRESUMO
We have performed a conformational analysis of DNA double helices with parallel directed backbone strands. The calculations were made for homopolymers poly(dG).poly(dC). All possible models of base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of parallel double polynucleotides were calculated. The dependences of conformational energy on the base pair structure were studied. Possible structure of parallel helices with various nucleotide composition are discussed.