Protein-DNA footprinting by endcapped duplex oligodeoxyribonucleotides

Oligodeoxyribonucleotides (5'-phosphorylated) of varying lengths were capped using a polyamide linker to form thermodynamically stable, endcapped DNA duplexes containing 8-14 bp. We have employed these endcapped DNA duplexes as tools to determine the DNA footprint of T4 DNA ligase. By high-performance liquid chromatography and PAGE analysis of the ligation mixtures of the endcapped DNA duplexes, we have found that by varying the lengths and the position of the nick, we can determine the minimal DNA-binding site as well as the mode of binding (symmetrical or asymmetrical binding) by the enzyme. The results of the study revealed that a 11 bp endcapped duplex was the shortest duplex effectively ligated. Dependence of ligation efficiency on nick position demonstrates that T4 DNA ligase bound asymmetrically to its DNA substrate. The use of a set of thermodynamically stable endcapped deoxyribonucleoside duplexes as a tool to elucidate the DNA footprint provides an efficient strategy for footprinting, which avoids ambiguities associated with chemical and biochemical footprinting methods.     

Convenient synthesis of oligodeoxyribonucleotides bearing arabinofuranosyl pyrimidine derivatives and its duplex formation with complementary DNA

The oligodeoxyribonucleotides bearing 2,2'-anhydro-beta-D-arabinofuranosyluracil derivatives were synthesized and the modified residue was converted to beta-D-arabinofuranosyluracil derivatives or beta-D-arabinofuranosylisocytosine derivatives by post-synthetic modification method. The melting profiles of their ODNs with complementary DNA were studied.     

Protein–DNA footprinting by endcapped duplex oligodeoxyribonucleotides

Oligodeoxyribonucleotides (5′-phosphorylated) of varying lengths were capped using a polyamide linker to form thermodynamically stable, endcapped DNA duplexes containing 8–14 bp. We have employed these endcapped DNA duplexes as tools to determine the DNA footprint of T4 DNA ligase. By high-performance liquid chromatography and PAGE analysis of the ligation mixtures of the endcapped DNA duplexes, we have found that by varying the lengths and the position of the nick, we can determine the minimal DNA-binding site as well as the mode of binding (symmetrical or asymmetrical binding) by the enzyme. The results of the study revealed that a 11 bp endcapped duplex was the shortest duplex effectively ligated. Dependence of ligation efficiency on nick position demonstrates that T4 DNA ligase bound asymmetrically to its DNA substrate. The use of a set of thermodynamically stable endcapped deoxyribonucleoside duplexes as a tool to elucidate the DNA footprint provides an efficient strategy for footprinting, which avoids ambiguities associated with chemical and biochemical footprinting methods.     

Automated 3'-H-phosphonate synthesis of oligodeoxyribonucleotides using amidine protected purine synthons

Side reaction during phosphitylation of 5'-O-dimethoxytrityl-N2-isobutyryl-2'-deoxyguanosine was observed. As a suitable dG synthon, 5'-O-dimethoxytrityl-N2-dimethylaminomethyl-ene-3'-H-phosphonate was developed and used, along with 3'-H-phosphonates of 5'-O-dimethoxytrityl derivatives of dT, N4--benzoyl-dC and N6-dimethylaminoethylidene-dA, in automated synthesis of several oligodeoxyribonucleotides containing 30-40 bases.     

Unusual DNA duplex and hairpin motifs

Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of ligands. Several principles for the formation of those unusual structures have been established through the observation of a number of recurring structural motifs associated with different sequences. These include: (i) internal loops of consecutive mismatches can occur in a B-DNA duplex when sheared base pairs are adjacent to each other to confer extensive cross- and intra-strand base stacking; (ii) interdigitated (zipper-like) duplex structures form instead when sheared G·A base pairs are separated by one or two pairs of purine·purine mismatches; (iii) stacking is not restricted to base, deoxyribose also exhibits the potential to do so; (iv) canonical G·C or A·T base pairs are flexible enough to exhibit considerable changes from the regular H-bonded conformation. The paired bases become stacked when bracketed by sheared G·A base pairs, or become extruded out and perpendicular to their neighboring bases in the presence of interacting drugs; (v) the purine-rich and pyrimidine-rich loop structures are notably different in nature. The purine-rich loops form compact triloop structures closed by a sheared G·A, A·A, A·C or sheared-like G_anti·C_syn base pair that is stacked by a single residue. On the other hand, the pyrimidine-rich loops with a thymidine in the first position exhibit no base pairing but are characterized by the folding of the thymidine residue into the minor groove to form a compact loop structure. Identification of such diverse duplex or hairpin motifs greatly enlarges the repertoire for unusual DNA structural formation.     

Intramolecular triplex formation of the purine.purine.pyrimidine type

Six octadecamers with hairpin motifs have been synthesized and investigated for possible intramolecular triplex formation. Electrophoretic, hypochromic, and CD evidence suggest that d(CCCCTTTGGGGTTTGGGG) and d(GGGGTTTGGGGTTTCCCC) can form G.G.C intramolecular triplexes via double hairpin formation in neutral solutions, presumably with the terminal G tract folding back along the groove of the hairpin duplex. In contrast, d(GGGGTTTCCCCTTTGGGG) and the three corresponding 18-mers containing one G and two C tracts each forms a single hairpin duplex with a dangling single strand. The design of the sequences has led to the conclusion that the two G tracts are antiparallel to each other in such a triplex. Magnesium chloride titrations indicate that Mg2+ is not essential for such an intramolecular triplex formation. The main advantage of our constructs when compared to the intermolecular triplex formation is that the shorter triplex stem can be formed in a much lower DNA concentration. The merit of G.G.C triplex, in contrast to that of C+.G.C, lies in the fact that acidic condition is not required in its formation and will, thus, greatly expand our repertoire in the triplex strategy for the recognition and cleavage of duplex DNA. Spectral binding studies with actinomycin D (ACTD) and chromomycin A3 (CHR) as well as fluorescence lifetime measurements with ethidium bromide (EB) suggest that although hairpin duplexes bind these drugs quite well, the intramolecular triplexes bind poorly. Interestingly, the binding densities for the strong-binding hairpins obtained from Scatchard plots are about one ACTD molecule per oligomeric strand, whereas more than two drug molecules are found in the case of CHR, in agreement with the recent NMR studies indicating that CHR binds to DNA in the form of a dimer.     

Mixed-phase hybridization of short oligodeoxyribonucleotides on microscopic polymer particles: effect of one-base mismatches on duplex stability

Hybridization of short oligonucleotides (10- and 11-mers) to complementary probes immobilized to microscopic polymer particles was quantified by a sandwich type mixed-phase hybridization assay based on a time-resolved fluorometric measurement of a photoluminescent europium(III) chelate from the surface of a single particle. Among the 54 sequences that were studied, 21 were fully complementary to the particle-bound probes, while 33 contained an internal one-base mismatch. The observed affinities were compared to those predicted by the nearest-neighbor model. In addition, various factors, such as the pore size of the particle, the linker structure, the charge type of the probe, and the efficiency of agitation, that might be expected to affect the kinetics of mixed-phase hybridization have been examined.     

Liposome formation with wool lipid extracts rich in ceramides

Internal wool lipids (IWLs) are rich in cholesterol, free fatty acids, cholesteryl sulfate, and, mainly, ceramides. The repairing effect of these lipids structured as liposomes was demonstrated by reinforcing the skin-barrier integrity and increasing the water-holding capacity when applied onto the skin. This work was focused on the formation of liposomes with IWLs rich in ceramides, obtained at pilot plant level with organic solvent extraction by using methanol and acetone. The lipid composition of the two extracts was quantitatively analyzed. IWL extracts containing different amounts of sterol sulfate were used to form liposomes at physiologic p^H. Vesicle size distribution, polydispersity index, and zeta potential of all liposomes were determined to characterize them and to study their stability. The results obtained showed that IWL extract composition, which was different depending on the extraction methodologies used, greatly influences the characteristics of the liposomes formed. Vesicular size and polydispersity index liposomes were smaller when the extract composition contained a higher proportion of either free fatty acids or sterol sulfate. Moreover, liposome stability was improved when some amount of sterol sulfate was added to the composition of methanol and acetone extracts. This natural mixture with keratinaceous origin could have a special interest for cosmetic or dermopharmaceutical companies.     

Unusual olefin formation by PhSe-F trans-elimination

A new approach to the synthesis of 2',3'-didehydro-2',3-dideoxynucleosides was described in excellent yield through unusual olefin formation by PhSe-F trans-elimination.     

Nonalternating purine pyrimidine sequences can form stable left-handed DNA duplex by strong topological constraint

In vivo, left-handed DNA duplex (usually refers to Z-DNA) is mainly formed in the region of DNA with alternating purine pyrimidine (APP) sequence and plays significant biological roles. It is well known that d(CG)_n sequence can form Z-DNA most easily under negative supercoil conditions, but its essence has not been well clarified. The study on sequence dependence of Z-DNA stability is very difficult without modification or inducers. Here, by the strong topological constraint caused by hybridization of two complementary short circular ssDNAs, left-handed duplex part was generated for various sequences, and their characteristics were investigated by using gel-shift after binding to specific proteins, CD and T_m analysis, and restriction enzyme cleavage. Under the strong topological constraint, non-APP sequences can also form left-handed DNA duplex as stable as that of APP sequences. As compared with non-APP sequences, the thermal stability difference for APP sequences between Z-form and B-form is smaller, which may be the reason that Z-DNA forms preferentially for APP ones. This result can help us to understand why nature selected APP sequences to regulate gene expression by transient Z-DNA formation, as well as why polymer with chirality can usually form both duplexes with left- or right-handed helix.     

Effect of cations on purine.purine.pyrimidine triple helix formation in mixed-valence salt solutions.

The effect of various monovalent, divalent and oligovalent cations on the reaction of triplex formation by GT and AG motif triplex-forming oligonucleotides, designed to bind to biologically relevant polypurine-polypyrimidine sequences occurring in the promoters of the murine Ki-ras and human bcr genes, has been investigated by means of electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments. We found that in the presence of 10 mm MgCl2 the triple helices were progressively destabilized by adding increasing amounts of NaCl, from 20 to 140 mm, to the solution. We also observed that, while the total monovalent-ion concentration was constant at 100 mm, the exchange of sodium with potassium, but not lithium, results in a further destabilization of the triple helices, due to self-association equilibria involving the G-rich triplex-forming oligonucleotides. Potassium was found to destabilize triplex DNA even when the triple helices are preformed in the absence of K+. However, footprinting experiments also showed that the inhibitory effect of K+ on triplex DNA is partially compensated for by millimolar amounts of divalent transition metal ions such as Mn2+ and Ni2+, which upon coordinating to N7 of guanine are expected to enhance hydrogen-bond formation between the target and the third strand, and to reduce the assembly in quadruple structures of G-rich triplex-forming oligonucleotides. Triplex enhancement in the presence of potassium was also observed, but to a lesser extent, when spermine was added to the reaction mixture. Here, the ion effect on triplex DNA is rationalized in terms of competition among the different valence cations to bind to triplex DNA, and differential cation stabilization of unusual quadruplex structures formed by the triplex-forming oligonucleotides.     

Investigation of the formation and intracellular stability of purine.(purine/pyrimidine) triplexes.

In a previous work we showed that a short triple helix-forming oligonucleotide (TFO) targeted to the murine c-pim-1 proto-oncogene promoter gives a very stable triple helix under physiological conditions in vitro . Moreover, this triplex was stable inside cells when preformed in vitro . However, we failed to detect triplex formation for this sequence inside cells in DMS footprinting studies. In the present work, in order to determine whether our previous in vivo results are limited to this particular short triplex or can be generalized to other purine.(purine/pyrimidine) triplexes, we have tested three other DNA targets already described in the literature. All these purine.(purine/pyrimidine) triplexes are specific and stable at high temperature in vitro . In vivo studies have shown that the preformed triplexes are stable inside cells for at least 3 days. This clearly demonstrates that intracellular conditions are favourable for the existence of purine. (purine/pyrimidine) triplexes. The triplexes can also be formed in nuclei. However, for all the sequences tested, we were unable to detect any triple helix formation in vivo in intact cells by DMS footprinting. Our results show that neither (i) chromatinization of the DNA target, (ii) intracellular K+concentration nor (iii) cytoplasmic versus nuclear separation of the TFO and DNA target are responsible for the intracellular arrest of triplex formation. We suggest the existence of a cellular mechanism, based on a compartmentalization of TFOs and/or TFO trapping, which separates oligonucleotides from the DNA target. Further work is needed to find oligonucleotide derivatives and means for their delivery to overcome the problem of triplex formation inside cells.     

beta-Amyloid protein induces the formation of purine dimers in cellular DNA

Experimental evidence implicates oxidative free radical reactions as central in the processes of neurodegenerative diseases. In particular, cellular interactions with the beta-amyloid protein have been linked to neuron cell death in Alzheimer's disease. Also, uncharacterized dimeric purine moieties have been detected in oxidized DNAs. It has been suggested that inadequate excision-repair of such products plays a functional role in the neurological degeneration observed in familial Alzheimer's disease, Down's syndrome, and xeroderma pigmentosum. Therefore, in order to obtain a reagent to monitor the presence of such products, the purine dimer 8-8-(2'-deoxyguanosyl)-2'-deoxyguanosine-5'-monophosphate was used as a hapten for elicitation of rabbit anti-purine dimer antiserum. This antiserum specifically recognizes various purified 8-8-bideoxyribonucleosides and 8-8-bideoxyribonucleotides. We found that DNA oxidized by the Fenton reaction is specifically recognized by this antiserum. This reagent can therefore be used to demonstrate formation and excision of DNA purine dimers. Moreover, incubation of cultured rat pheochromocytoma PC-12 cells with the beta-amyloid protein resulted in formation of these purine dimers in cellular DNA. These dimers were subsequently removed from cellular DNA. From these results we conclude that the free radicals generated by A beta cause oxidative DNA alterations including purine dimers. Deficient repair of this type of DNA damage might result in neural cell loss via apoptosis. Our findings suggest mechanisms for the roles of beta-amyloid and oxidative free radicals in neurodegenerative diseases and the role of DNA excision-repair in the prevention of lethal neurotoxicity.     

Reduced-stringency DNA reassociation: sequence specific duplex formation.

Reduced-stringency DNA reassociation conditions allow low stability duplexes to be detected in prokaryotic, plant, fish, avian, mammalian, and primate genomes. Highly diverged families of sequences can be detected in avian, mouse, and human unique sequence dNAs. Such a family has been described among twelve species of birds; based on species specific melting profiles and fractionation of sequences belonging to this family, it was concluded that permissive reassociation conditions did not artifactually produce low stability structures (1). We report S1 nuclease and optical melting experiments, and further fractionation of the diverged family to confirm sequence specific DNA reassociation at 50 degrees in 0.5 M phosphate buffer.     

DNA triple-helix formation at target sites containing duplex mismatches

We have studied the formation of DNA triple helices at target sites that contain mismatches in the duplex target. Fluorescence melting studies were used to examine a series of parallel triple helices that contain all 64 N.XZ triplet combinations at the centre (where N, X and Z are each of the four natural DNA bases in turn). Similar experiments were also performed with N=bis-amino-U (BAU) (for stable recognition of AT base pairs) and N=S (for recognition of TA inversions). We find that the introduction of a duplex mismatch destabilises the C+.GZ, T.AZ and G.TZ triplets. A similar effect is seen with BAU.AZ triplets. In contrast, other base combinations, based on non-standard triplets such as C.AZ, T.TZ, G.CZ and A.CZ are stabilised by the presence of a duplex mismatch. In each case S binds to sites containing duplex mismatches better than the corresponding Watson-Crick base pairs.     

Energetics of B-Z junction formation in a sixteen base-pair duplex DNA

We report analysis of the NaCl-induced B-Z transition in a 16 base-pair duplex DNA with sequence designed such that when NaCl is increased the left half of the molecule undergoes the B-Z transition while the right half remains in the B-form. An equilibrium thermodynamic model based on the body of available published experimental data and the recent theoretical work of Soumpasis, which indicate, in the salt range above approximately 0.9 M-NaCl, the transition free-energy of B-Z conversion in DNA is a linear function of the NaCl concentration, is employed. Analysis of the B-Z transition of the junction-containing molecule indicates the B-Z junction formed in this 16 base-pair DNA is composed of approximately three base-pairs and has a free energy of formation of approximately 4.7 kcal/mol junction. These values for the junction are in excellent agreement with published estimates of B-Z junction size and energy derived from much longer DNA pieces.     

Radical-induced purine lesion formation is dependent on DNA helical topology

Abstract

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO^?) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5′,8-cyclo-2′-deoxyguanosine (5′,8-cdG) and 5′,8-cyclo-2′-deoxyadenosine (5′,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]₄ (TG4T), d[AGGG(TTAGGG)₃] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)₃A] (mutTel24) were exposed to HO^? radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L?>?OC???SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (～50% solution folded) and mutTel24 (～90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33?Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.