Stretched DNA structures observed with atomic force microscopy.

Double-stranded DNA molecules are occasionally found that appear to be straightened and stretched in atomic force microscope (AFM) images. Usually pBS+ plasmid and lambda DNA show relaxed structures with bends and kinks along the strands and have measured contour lengths consistent to about 5-7%; they also appear not to cross over each other, except in very high concentrations. The anomalous molecules observed here, compared with the majority of molecules in the preparation, show contour lengths increased by as much as 80% and have measured heights of about half that of normal relaxed DNA. Some molecules also appear to be in transition between stretched and relaxed forms. These observations are consistent with an uncoiling of the DNA helix without breakage of the covalent bonds in the deoxyribose-phosphate backbone.     

Restrained refinement of the monoclinic form of yeast phenylalanine transfer RNA. Temperature factors and dynamics, coordinated waters, and base-pair propeller twist angles

The structure of yeast phenylalanine transfer RNA in the monoclinic form has been further refined by using the restrained least-squares method of Hendrickson and Konnert. For the 4019 reflections between 10 and 3 A, with magnitudes at least 3 times their standard deviations, the R factor is 16.8%. The variation of the atomic temperature factors along the sequence indicates that the major flexibility regions are the amino acid and anticodon stems. The two strands of the amino acid helix exhibit large differential temperature factors, suggesting partial uncoiling or melting of the helix. In this work, the occupancy of all atoms was also varied. Residues D16 and D17 of the dihydrouridine loop as well as U33 and G37 of the anticodon loop have occupancies around 70%, indicating some local disorder or large-scale mobility at these positions. One hundred fifteen solvent molecules, including five magnesium ions, were found in difference maps. The role of several water molecules is clearly related to the stabilization of the secondary and tertiary interactions. The gold sites, which were not previously discussed, are described and show an energetically favored binding mode similar to that of cobalt and nickel complexes with nucleotides.     

Thread Cells from the Slime Glands of Hagfish (Myxinidae)

Scanning electron microscopy and light microscopy demonstrate that the mature thread cells in Eptatretus deani and Myxine glutinosa consist of a single, coiled thread up to 10 cm long. Mature thread cells apparently loose the cell membrane within the slime gland before expulsion. Thus the old idea that rupture of the cell membrane causes the uncoiling in sea water is no longer tenable. If the thread cells are transferred without additional fluid to a microscope slide, no uncoiling occurs until sea water or distilled water is added when the process occurs rapidly; however, in double strength sea water uncoiling is slowed down. In tetrahydrofuran or glycerin there is no uncoiling in 100 % or 50 % solutions; however in 5 % solutions uncoiling occurs slowly. Thus the availability of adequate amounts of water seems to be necessary for uncoiling. Presumably, water acts directly on the thread, causing the organelle to straighten and thus uncoil. The defensive value of the slime production is discussed.     

The properties of native and denatured DNA in buoyant rubidium trichloroacetate at neutral pH

Aqueous RbTCA is generally suitable as a buoyant solvent for both native and denatured DNA at neutral pH and room temperature. Native PM-2 DNA II, for example, is buoyant at 3.29 M salt, 25 degrees C; whereas the denatured strands band together at 4.52 M. Two properties of the solvent make this system uniquely useful for separations based upon the extent of secondary structure. First, the melting transition temperature for chemically unaltered DNA is depressed to room temperature or below. Second, the buoyant density increase accompanying denaturation is extraordinarily large, 174 mg/ml for PM-2 DNA II. This value is three times that found in aqueous NaI and ten times that for CsCl. The properties of the RbTCA buoyant solvent presented here include the compositional and buoyant density gradients and the buoyant density dependence upon base composition. The DNA remains chemically unaltered after exposure to RbTCA as shown by the absence of strand scissions for closed circular DNA and by the unimpaired biological activity in transformation assays. Intact virion DNA may be isolated by direct banding of whole virions in RbTCA gradients without prior phenol extraction. Strongly complexed or covalently bound proteins may be detected by their association with the buoyant polymer in the denaturing density gradient.     

Parental DNA strands segregate randomly during embryonic development of Caenorhabditis elegans

The fate of gamete DNA was followed in the next generation embryos of the nematode C. elegans. Either male worms or spermless hermaphrodites were grown on bromodeoxyuridine-containing E. coli in order to label germ-line DNA. Matings then produced embryos in which only the DNA strands provided by the gametes contained label. This original gamete DNA could be detected during embryonic development by using a fluorescently labeled monoclonal antibody specific to bromodeoxyuridine. Both the number and position of fluorescent spots in the embryo indicate that gamete DNA strands segregate randomly during development. Random segregation of parental DNA strands rules out models of development that invoke chromosome imprinting or immortal DNA strands.     

Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences

Formamide is the preferred solvent to lower the melting point and annealing temperature of nucleic acid strands in in situ hybridization (ISH). A key benefit of formamide is better preservation of morphology due to a lower incubation temperature. However, in fluorescence in situ hybridization (FISH), against unique DNA targets in tissue sections, an overnight hybridization is required to obtain sufficient signal intensity. Here, we identified alternative solvents and developed a new hybridization buffer that reduces the required hybridization time to one hour (IQFISH method). Remarkably, denaturation and blocking against repetitive DNA sequences to prevent non-specific binding is not required. Furthermore, the new hybridization buffer is less hazardous than formamide containing buffers. The results demonstrate a significant increased hybridization rate at a lowered denaturation and hybridization temperature for both DNA and PNA (peptide nucleic acid) probes. We anticipate that these formamide substituting solvents will become the foundation for changes in the understanding and performance of denaturation and hybridization of nucleic acids. For example, the process time for tissue-based ISH for gene aberration tests in cancer diagnostics can be reduced from days to a few hours. Furthermore, the understanding of the interactions and duplex formation of nucleic acid strands may benefit from the properties of these solvents.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

Secondary structure of the r(CUUCGG) tetraloop.

The oligonucleotide r(GGACUUCGGUCC) has been observed to adopt a hairpin conformation by solution NMR and a double helical conformation by X-ray diffraction. In order to understand this apparent conflict, we used time-resolved fluorescence depolarization and 19fluorine NMR to follow the secondary structure of this dodecamer as the solution composition was changed stepwise from the NMR experimental conditions to those used for crystallization. Calculation of the dodecamer concentration in the crystal (180 mM strands) and the cation concentration needed for neutrality (>2 M) prompted investigation of a tethered species, in which two dodecamers are connected by a string of 4 nt, geometrically equivalent to approximately 100 mM strands, in 2.5 M NaCl. The RNA tetraloop and its DNA analog maintain a single-strand hairpin conformation in solution, even under the conditions used to grow the crystal. Under high salt conditions, the tethered RNA and DNA analogs of this sequence yield secondary components which could be the double helical conformation. Crystal contacts in addition to solvent changes and high RNA concentrations are needed to obtain the double helix as the predominant species.     

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94- and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA liked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent DNA synthesis with dexamethasone. In this case, multiple, discrete subgenomic DNA plus strands were not found when the same methods applied to avian sarcoma virus DNA were used; instead, the plus strands present in the linear DNA of mouse mammary tumor virus fell mainly into two classes: (i) strands of ca. 1.3 kb which appeared early in synthesis and were similar in size and genetic content to the terminally repeated sequence in linear DNA; and (ii) plus strands of the same length as linear DNA. A heterogeneous population of other strands diminished with time, was not found in completed molecules, and was probably composed of strands undergoing elongation. These two retroviruses thus appear to differ with respect to both the number of priming sites used for the synthesis of plus strands and the abundance of full-length plus strands. On the other hand the major subgenomic plus strand of mouse mammary tumor virus DNA (1.3 kb) is probably the functional homolog of a major subgenomic plus strand of avian sarcoma virus DNA (0.35 kb). The significance of this plus strand species is discussed in the context of current models which hold that it is used as a template for the completion of the minus strand, thereby generating the long terminal redundancy.     

Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity

The number of strand-specific nicking endonucleases that are currently available for laboratory procedures and applications in vivo is limited, and none is sufficiently specific to nick single target sites within complex genomes. The extreme target specificity of homing endonucleases makes them attractive candidates for engineering high-specificity nicking endonucleases. I-SceI is a monomeric homing enzyme that recognizes an 18 bp asymmetric target sequence, and cleaves both DNA strands to leave 3′-overhangs of 4 bp. In single turnover experiments using plasmid substrates, I-SceI generates transient open circle intermediates during the conversion of supercoiled to linear DNA, indicating that the enzyme cleaves the two DNA strands sequentially. A novel hairpin substrate was used to demonstrate that although wild-type I-SceI cleaves either the top or bottom DNA strand first to generate two nicked DNA intermediates, the enzyme has a preference for cleaving the bottom strand. The kinetics data are consistent with a parallel sequential reaction mechanism. Substitution of two pseudo-symmetric residues, Lys122 and Lys223, markedly reduces top and bottom-strand cleavage, respectively, to generate enzymes with significant strand- and sequence-specific nicking activity. The two active sites are partially interdependent, since alterations to one site affect the second. The kinetics analysis is consistent with X-ray crystal structures of I-SceI/DNA complexes that reveal a role for the lysines in establishing important solvent networks that include nucleophilic water molecules thought to attack the scissile phosphodiester bonds.     

Physiological studies on pea tendrils. XII. Effects of temperature on contact coiling and uncoiling

When excised tendrils of pea ( Pisum sativum L. cv. Alaska 2B) are mechanically perturbed and allowed to coil at different constant temperatures, the greatest amount of coiling occurs between 27°C and 33°C. Coiling of tendrils continues for about 2 h after mechanical perturbation at which time uncoiling usually begins. The temperature at which the rate of uncoiling is greatest appears to be influenced, at least in part, by the temperature at which the tendrils coiled. For example, when tendrils coil at 20°C their rate of uncoiling at 20°C is less than if they had coiled at 23°C. Estimated activation energies for the uncoiling process are greater than for coiling, with 35 J/mol × s and 97 J/mol × s for uncoiling in the temperature ranges 18°C to 23°C and 10°C to 18°C, respectively. The estimated activation energy for coiling is 5.4 J/mol × s. It is suggested that the process of tendril uncoiling, as well as tendril coiling, might be an active, energy requiring process.
When mechanically perturbed tendrils are placed in the cold (5°C) they do not coil. But this interruption of the coiling process with a cold (5°C) treatment, either immediately after mechanical perturbation or after coiling has begun, does not prevent coiling from continuing after tendrils are again given a more suitable temperature. It is concluded that the cessation of coiling during the cold period may be due to a slowdown in metabolism. It is suggested that there may be a factor which is responsible for the motor response and which is retained during the cold treatment.     

Molecular simulation study of assembly of DNA-grafted nanoparticles: effect of bidispersity in DNA strand length

In this paper, we use molecular dynamics simulations to study the assembly of DNA-grafted nanoparticles to demonstrate specifically the effect of bidispersity in grafted DNA strand length on the thermodynamics and structure of nanoparticle assembly at varying number of grafted single-stranded DNA (ssDNA) strands and number of guanine/cytosine (G/C) bases per strand. At constant number of grafted ssDNA strands and G/C nucleotides per strand, as bidispersity in strand lengths increases, the number of nanoparticles that assemble as well as the number of neighbours per particle in the assembled cluster increases. When the number of G/C nucleotides per strand in short and long strands is equal, the long strands hybridise with the other long strands with higher frequency than the short strands hybridise with short/long strands. This dominance of the long strands leads to bidisperse systems having similar thermodynamics to that in corresponding systems with monodisperse long strands. Structurally, however, as a result of long–long, long–short and short–short strand hybridisation, bidispersity in DNA strand length leads to a broader inter-particle distance distribution within the assembled cluster than seen in systems with monodisperse short or monodisperse long strands. The effect of increasing the number of G/C bases per strand or increasing the number of grafted DNA strands on the thermodynamics of assembly is similar for bidisperse and monodisperse systems. The effect of increasing the number of grafted ssDNA strands on the structure of the assembled cluster is dependent on the extent of strand bidispersity because the presence of significantly shorter ssDNA strands among long ssDNA strands reduces the crowding among the strands at high grafting density. This relief in crowding leads to larger number of strands hybridised and as a result larger coordination number in the assembled cluster in systems with high bidispersity in strands than in corresponding monodisperse or low bidispersity systems.     

Exclusion of RNA strands from a purine motif triple helix.

Research concerning oligonucleotide-directed triple helix formation has mainly focused on the binding of DNA oligonucleotides to duplex DNA. The participation of RNA strands in triple helices is also of interest. For the pyrimidine motif (pyrimidine.purine.pyrimidine triplets), systematic substitution of RNA for DNA in one, two, or all three triplex strands has previously been reported. For the purine motif (purine.purine.pyrimidine triplets), studies have shown only that RNA cannot bind to duplex DNA. To extend this result, we created a DNA triple helix in the purine motif and systematically replaced one, two, or all three strands with RNA. In dramatic contrast to the general accommodation of RNA strands in the pyrimidine triple helix motif, a stable triplex forms in the purine motif only when all three of the substituent strands are DNA. The lack of triplex formation among any of the other seven possible strand combinations involving RNA suggests that: (i) duplex structures containing RNA cannot be targeted by DNA oligonucleotides in the purine motif; (ii) RNA strands cannot be employed to recognize duplex DNA in the purine motif; and (iii) RNA tertiary structures are likely to contain only isolated base triplets in the purine motif.     

Relaxation kinetics of the triple-stranded helix-coil transition at short chain length. A model including the staggering of chains.

The relaxation kinetics of a staggering zipper model are presented, in which the formation of helical nuclei is regarded to be rate-limiting and in which the sliding of strands along the helices is prohibited. Instead a realignment of staggered chains is only possible via complete uncoiling. While maintaining the third order of the initial reaction as in an all-or-none mechanism, the model predicts a large range of relaxation times, which contribute to the mean relaxation time according to the stability of the individual species and which are weakly coupled in the range of small amounts of helical content. The model can easily be compared to experimental results and agrees well with relaxation data obtained from a triple-helical peptide fragment of collagen. It may be readily expanded to other multistranded helix ? coil transitions with steady-state formation of the individual species and it suggests that the fraying of the helix ends is hidden by the fast-relaxation times due to the equilibration of the shortest helical species.     

Electron attachment to DNA single strands: gas phase and aqueous solution

The 2′-deoxyguanosine-3′,5′-diphosphate, 2′-deoxyadenosine-3′,5′-diphosphate, 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3′,5′-dTDP (0.17 eV) and 3′,5′-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3′,5′-dTDP > 3′,5′-dCDP > 3′,5′-dGDP > 3′,5′-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3′-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3′-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3′,5′-dADP⁻ (0.26 eV) and 3′,5′-dGDP⁻ (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers such as glycosidic bond breakage. However, the radical anions of the pyrimidine nucleotides with high VDE are expected to be electronically stable. Thus the base-centered radical anions of the pyrimidine nucleotides might be the possible intermediates for DNA single-strand breakage.     

Maintenance of genetic information in stem cells

The available data on DNA cosegregation in some stem cells are reviewed. Cairns was the first to assume cosegregation of template DNA strands for adult stem cells; i.e., all maternal DNA strands are preserved in one daughter cell, which remains a stem cell, while the newly synthesized DNA strands, which may contain errors, appear in the daughter cell that is committed to differentiation and passes to the transitory compartment of the cell population. The role of asymmetric mitosis in DNA cosegregation and maintenance of genetic information in stem cells is discussed.     

Maintenance of genetic information in stem cells

The available data on DNA cosegregation in some stem cells are reviewed. Cairns was the first to assume cosegregation of template DNA strands for adult stem cells; i.e., all maternal DNA strands are preserved in one daughter cell, which remains a stem cell, while the newly synthesized DNA strands, which may contain errors, appear in the daughter cell that is committed to differentiation and passes to the transitory compartment of the cell population. The role of asymmetric mitosis in DNA cosegregation and maintenance of genetic information in stem cells is discussed.     

The role of structural reorganization in charge carrier transfer in DNA molecule

A model of hole transfer in DNA molecules has been proposed, which takes into account changes in the reorganization energy and orbital coupling between the neighboring bases during the charge transfer in different molecular sequences. It is shown that the rate of hole transfer by the superexchange and hopping transfer mechanisms is limited by the relaxation of the geometries of nucleobases participating in charge migration and the dynamics of solvent molecules. The rate of charge transfer in the DNA molecule is found to be dependent on the height of the potential barriers between the nucleotide and the molecular sequences. The inclusion of the interchain charge transfer, which is characterized by weak coupling between the nucleotides located in opposite strands, does not affect the general charge transport in DNA. The increase in the number of the parallel components of the hopping mechanism leads to a rise in the charge transfer rate in the double helix.     

Development of an electrochemical polypyrrole-based DNA sensor and subsequent studies on the effects of probe and target length on performance

DNA sensors have a wide scope of applications in the present and emerging medical and scientific fields, such as medical diagnostics and forensic investigations. However, much research-to-date on DNA sensor development has focused on short target DNA strands as model genes. In this communication we study the effect of the length of oligonucleotide probe and target strands as a significant step towards real world applications for DNA detection. The sensor technology described uses the conducting polymer polypyrrole as both a sensing element and transducer of sensing events - namely the hybridization of complementary target oligonucleotide to probe oligonucleotide. Detection is performed using electrical impedance spectroscopy. Initially sensor development is performed, wherein we demonstrate an improvement in stability and sensitivity as well as show a reduction in non-specific DNA binding for fabricated sensors, through use of a specific dopant and post-growth treatment. Subsequently, we show that longer target DNA strands display increased response, as do sensors containing longer probe DNA strands. It is suggested that these results are a feature of the increase in negative charges associated with the longer DNA strands. The results of this comparative study are aimed to guide future design of analogous sensors.