Parallel stranded DNA under the scanning tunnelling microscope.

Using scanning tunnelling microscopy, we have directly observed parallel stranded DNA helixes of 43 nucleotides in length. The double helix is right-handed and has an average spacing, 17.43 A (+/- 1 S.D.: 2.30 A), and an average apparent depth, 4.79 A (+/- 1 S.D.: 1.04 A) for each groove. The average pitch of the helical turn is 34 A (+/- 1 S.D.: 3.35 A) and consists of no more than ten base pairs. The diameter of the helix is approx. 17-20 A. Our results provide direct evidence for the existence of a parallel structure of DNA in vitro and some details of its fine structure.     

Parallel stranded duplex DNA.

Three linear 21-nt oligonucleotides (C2, C3, C7) have been synthesized with different sequences of A and T residues. One pairwise combination, (C3, C7), hybridizes to form a conventional antiparallel duplex (aps-C3.C7), whereas the pair C2, C3 forms a duplex (ps-C2.C3) in which the two strands are in a parallel orientation and the A.T base-pairs in a reverse Watson-Crick configuration. The existence of the novel ps helical structure was established from the following criteria: (i) The electrophoretic mobilities of the ps and aps duplexes in native and denaturing polyacrylamide gels are similar. (ii) The ps duplex is not a substrate for T4 DNA ligase. (iii) Salt-dependent thermal transitions are observed for the two duplexes, but the melting temperatures of the ps molecules are 15 degrees C lower. (iv) The ultraviolet absorption and circular dichroism spectra of the ps duplex are indicative of a base-paired structure, but differ systematically from that of the aps helix. (v) Based on fluorescent measurements, the bis-benzimidazole drug BBI-258 shows a lower affinity for the ps compared to the aps duplex, whereas the opposite preference holds for the intercalator ethidium bromide. We conclude from the present study that parallel stranded DNA is a stable conformation which can arise by interaction between two conventional strands with appropriate sequence homology.     

Dynamics of double stranded DNA reptation from bacteriophage.

The dynamics of dsDNA release process from a phage head has been analyzed theoretically. The process was considered as dsDNA reptation through the phage tail. The driving force is assumed to be the decrease of the DNA globule free energy on its releasing from the head in the surrounding medium. The results of the equilibrium theory on an intraphage DNA globule were applied. Three possible sources of friction were examined. The first one is in the inner channel of the tail. The second is the friction of DNA segments in the whole globule volume, which is essential when the globule decondensation is a collective process, at simultaneous moving of all the turns (mechanism 1). The third is the globule friction with the capsid inner surface, that is most important when decondensation proceeds via the globule rotation as a whole spool (mechanism 2). Mechanism 1 would require a lot of time for ejection. Mechanism 2 would lead to different ejection dynamics of short- and long-tailed phages. Comparison of the theoretical results with the published experimental data argues in favor of mechanism 2.     

Biotinylation of double stranded DNA after transamination

The bisulfite catalyzed transamination of cytidine and cytosine has been reported to be single strand specific, but local thermal instabilities of the DNA double helix, coupled with the extreme sensitivity of the Biotin-Avidin revelation methods, allows the random labelling of cytosines in d.s. DNA to detectable levels for those purposes where the overall label can be very low. We have evaluated the use of this reaction to prepare double stranded DNA molecules containing N4-aminoethyl-cytosine (4-aeC). After this step 4-aeC residues can be conjugated to biotinyl-n-hydroxysuccinimide ester yielding biotinylated DNA. This reaction allows the massive production of biotinylated probes. Labelled DNA can serve as molecular weight marker and positive control in Southern-blots. Moreover it can be useful in the study of DNA-protein interaction and in the isolation of d.s. DNA-binding proteins through chromatographic procedures.     

Strong bending of the DNA double helix

During the past decade, the issue of strong bending of the double helix has attracted a lot of attention. Here, we overview the major experimental and theoretical developments in the field sorting out reliably established facts from speculations and unsubstantiated claims. Theoretical analysis shows that sharp bends or kinks have to facilitate strong bending of the double helix. It remains to be determined what is the critical curvature of DNA that prompts the appearance of the kinks. Different experimental and computational approaches to the problem are analyzed. We conclude that there is no reliable evidence that any anomalous behavior of the double helix happens when DNA fragments in the range of 100 bp are circularized without torsional stress. The anomaly starts at the fragment length of about 70 bp when sharp bends or kinks emerge in essentially every molecule. Experimental data and theoretical analysis suggest that kinks may represent openings of isolated base pairs, which had been experimentally detected in linear DNA molecules. The calculation suggests that although the probability of these openings in unstressed DNA is close to 10⁻⁵, it increases sharply in small DNA circles reaching 1 open bp per circle of 70 bp.     

Parallel double stranded helices and the tertiary structure of nucleic acids

Thermal denaturation of four oligonucleotides, viz. 3'-d(AT)5pO(CH2)6Opd(AT)5-3'(par(AT], 3'-d(AT)5pO(CH2)6Opd(AT)5-5'(anti(AT],3'-d(A)10pO(CH2) 6Op(T)10-3'(par(A-T], and 3'-d(A)10pO(CH2)6Opd(T)10-5' (anti(A-T], was studied in 0.01 M phosphate buffer, pH 7, in the presence of 0.1, 0.25, 0.5 and 1.0 M NaCl. All the oligomers were found to exist at a lower temperature (0 to 20 degrees C) as complexes composed either of two oligomer molecules (a canonical duplex) or of more oligomer molecules whereas, at a higher temperature (30 to 70 degrees C), they formed hairpins with a parallel (par(AT) and par(A-T] or antiparallel (anti(AT) and anti(A-T) orientation of the chains. Melting curves (A260(T] were used to calculate thermodynamic parameters for the formation of hairpins and "low-temperature" duplexes. Experiments on ethidium bromide binding to the oligonucleotides have shown that the oligomer anti(A-T) exists, at a low ionic strength, as a four stranded complex ("quadruplex") contains two antiparallel helices, d(A).d(T), which have a parallel orientation and are bound to one another owing to the formation of additional hydrogen bonds between nucleic acid bases. The possible biological function of quadruplexes is discussed.     

Cyanine dyes for the detection of double stranded DNA

Twenty three novel cyanine dyes have been applied as fluorescent stains for the detection of nucleic acids in agarose gel electrophoresis. Significant fluorescence enhancement of these dyes in the presence of double stranded DNA was observed. Five dyes offered superior sensitivity in the detection and quantification of DNA, over Ethidium Bromide, the most commonly used nucleic acid stain.     

DNA self-fitting: the double helix directs the geometry of its supramolecular assembly.

Groove-backbone interaction is a natural and biologically relevant mechanism for the specific assembly of B-DNA double helices. Crystal engineering and crystal packing analysis of oligonucleotides of different sizes and sequences reveal that the sequence-dependent self-fitting of B-DNA helices is a dominant constraint for their ordered assembly. It can override the other intermolecular interactions and impose the overall geometry of the packing. Analysis of experimental examples of architectural motifs formed by the geometric combination of self-fitted DNA segments leads to general rules for DNA assembly. Like a directing piece for a supramolecular 'construction set', the double helix imposes a limited number of geometric solutions. These basic architectural constraints could direct, in a codified manner, the formation of higher-order structures. DNA architectural motifs exhibit new structural and electrostatic properties which could have some implications for their molecular recognition by proteins acting on DNA.     

Design and applications of single and double stranded DNA analogs.

Chemical ligation method for assembling modified DNA-duplexes as well as solid support method and postsynthetic modification in aqueous media was used to synthesize a variety of single and double stranded oligonucleotide derivatives. Novel oligodeoxynucleotides with cluster or alternating sugar modifications and probes resistant to cell nuclease degradation were obtained. The use of such probes for effective and regiospecific hybridase RNA-hydrolysis is provided. Effective synthesis of new DNA duplexes containing modified and chemical cleavable internucleotide bonds was developed. A novel type of affinity reagent was suggested for covalent attachment of substrate to active site nucleophiles in the restriction/modification systems.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Construction of a 42 base pair double stranded DNA microcircle.

A procedure for the construction of double stranded DNA microcircles is described that overcomes the natural limits of established circularization procedures. Starting with two synthetic oligonucleotides which are able to form dumbbell shaped structures, two subsequent ligation reactions yield a microcircle of double stranded DNA of 42 base pairs. This is by far the smallest circle of double stranded DNA yet described. These microcircles can be constructed in quantities required for high resolution structural analyses such as X-ray crystallography and NMR spectroscopy.     

Force-induced melting of the DNA double helix 1. Thermodynamic analysis

The highly cooperative elongation of a single B-DNA molecule to almost twice its contour length upon application of a stretching force is interpreted as force-induced DNA melting. This interpretation is based on the similarity between experimental and calculated stretching profiles, when the force-dependent free energy of melting is obtained directly from the experimental force versus extension curves of double- and single-stranded DNA. The high cooperativity of the overstretching transition is consistent with a melting interpretation. The ability of nicked DNA to withstand forces greater than that at the transition midpoint is explained as a result of the one-dimensional nature of the melting transition, which leads to alternating zones of melted and unmelted DNA even substantially above the melting midpoint. We discuss the relationship between force-induced melting and the B-to-S transition suggested by other authors. The recently measured effect on T7 DNA polymerase activity of the force applied to a ssDNA template is interpreted in terms of preferential stabilization of dsDNA by weak forces approximately equal to 7 pN.     

Parallel double helix and tertiary structure of nucleic acids

The thermal denaturation of four oligonucleotides, viz. 3'-d(AT)5pO(CH2)6Opd(AT)5-3' (parAT), 3'-d(AT)5pO(CH2)5Opd(AT)5-5' (antiAT), 3'-d(A)10pO(CH2)6Op(T)10-3' (parA-T) and 3'd(A)10pOX X (CH2)6Opd(T)10-5' (antiA-T) in 0.01 M phosphate buffer at pH 7 in presence 0.1, 0.25, 0.5 and 1.0 M NaCl have been studied. It was shown that at lower temperature (0-20 degrees C) all oligomeres exist as complexes of two (canonic duplex) or four (eight) molecules of oligonucleotides, but at higher temperature (30-70 degrees C)- as hairpins with parallel (parAT and parA-T) of antiparallel (antiAT and antiA-T) orientation of chains. Thermodinamic parameters of separated strands-hairpins and hairpins--"low temperature complexes" transition were computated from the melting curves [A260 (T)] by nonlinear regression. AntiA-T was shown by ethidium bromide binding to exist at low strength (0.01 M phosphate buffer without NaCl) as four-stranded complex from two antiparallel double stranded helices parallely oriented and bonded by satisfy hydrogen-bond of groups not involved in WC-pairing. At higher ionic strength the two of such tetramers was conjugated by hydrophobic interaction into octamers. We speculate that four-stranded complexes serves to bring together, and zipper up two antiparallel double stranded helices at replication of DNA, cross-over of gomologues chromosomes and other biochemically important processes.