Small DNA circles as probes of DNA topology.

Small DNA circles can occur in Nature, for example as protein-constrained loops, and can be synthesized by a number of methods. Such small circles provide tractable systems for the study of the structure, thermodynamics and molecular dynamics of closed-circular DNA. In the present article, we review the occurrence and synthesis of small DNA circles, and examine their utility in studying the properties of DNA and DNA-protein interactions. In particular, we highlight the analysis of small circles using atomistic simulations.

Thermal stabilisation of RNA·RNA duplexes and G-quadruplexes by phosphorothiolate linkages.

The effect of 3'-S-phosphorothiolate linkages on the stability of RNA·RNA duplexes and G-quadruplex structures has been studied. 3'-Thio-2'-deoxyuridine was incorporated into RNA duplexes and thermal melting studies revealed that the resulting 3'-S-phosphorothiolate linkages increased the stability of the duplex to thermal denaturation. Additionally, and contrary to expectation, a similar effect on duplex stability was observed when the same thionucleoside was incorporated into the RNA strand of a RNA·DNA duplex. A suitably protected derivative of 3'-thio-2'-deoxyguanosine was prepared using an oxidation-reduction strategy and this residue also increased the thermal stability the [d(TGGGGT)](4) G-quadruplex when positioned centrally. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and imply that the previously noted high thermal stability of parallel RNA quadruplexes is not derived from H-bonding interactions of the 2'-hydroxyl group, but can be attributed to conformational effects.

Divalent metals and pH alter raltegravir disposition in vitro.

Raltegravir shows marked pharmacokinetic variability in patients, with gastrointestinal pH and divalent-metal binding being potential factors. We investigated raltegravir solubility, lipophilicity, pK(a), and permeativity in vitro to elucidate known interactions with omeprazole, antacids, and food, all of which increase gastric pH. Solubility of raltegravir was determined at pH 1 to 8. Lipophilicity of raltegravir was determined using octanol-water partition. Raltegravir pK(a) was determined using UV spectroscopy. The effects of pH, metal salts, and omeprazole on the cellular permeativity of raltegravir were determined using Caco-2 monolayers. Cellular accumulation studies were used to determine the effect of interplay between pH and ABCB1 transport on raltegravir accumulation. Samples were analyzed using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) or scintillation counting. Raltegravir at 10 mM was partly insoluble at pH 6.6 and below. Raltegravir lipophilicity was pH dependent and was reduced as pH was increased from 5 to 9. The pK(a) of raltegravir was 6.7. Raltegravir cellular permeativity was heavily influenced by changes in extracellular pH, where apical-to-basolateral permeativity was reduced 9-fold (P < 0.05) when apical pH was increased from 5 to 8.5. Raltegravir cellular permeativity was also reduced in the presence of magnesium and calcium. Omeprazole did not alter raltegravir cellular permeativity. Cellular accumulation of raltegravir was increased independently by inhibiting ABCB1 and by lowering extracellular pH from pH 8 to 5. Gastrointestinal pH and polyvalent metals can potentially alter the pharmacokinetic properties of raltegravir, and these data provide an explanation for the variability in raltegravir exposure in patients. The evaluation of how divalent-metal-containing products, such as multivitamins, that do not affect gastric pH alter raltegravir pharmacokinetics in patients is now justified.

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

Reverse-direction (5'-->3') synthesis of oligonucleotides containing a 3'-S-phosphorothiolate linkage and 3'-terminal 3'-thionucleosides.

The synthesis of oligodeoxynucleotides containing 3'-thionucleosides has been explored using a reverse-direction (5'-->3') approach, based on nucleoside monomers which contain a trityl- or dimethoxytrityl-protected 3'-thiol and a 5'-O-phosphoramidite. These monomers are relatively simple to prepare as trityl-based protecting groups were introduced selectively at a 3'-thiol in preference to a 5'-hydroxyl group. As an alternative approach, trityl group migration could be induced from the 5'-oxygen to the 3'-thiol function. 5'-->3' Synthesis of oligonucleotides gave relatively poor yields for the internal incorporation of 3'-thionucleosides [to give a 3'-S-phosphorothiolate (3'-SP) linkage] and multiple 3'-SP modifications could not be introduced by this method. However, the reverse direction approach provided an efficient route to oligonucleotides terminating with a 3'-thionucleoside. The direct synthesis of these thio-terminating oligomers has not previously been reported and the methods described are applicable to 2'-deoxy-3'-thionucleosides derived from thymine, cytosine and adenine.

A rapid high-resolution method for resolving DNA topoisomers.

OBJECTIVE: Agarose gel electrophoresis has been the mainstay technique for the analysis of DNA samples of moderate size. In addition to separating linear DNA molecules, it can also resolve different topological forms of plasmid DNAs, an application useful for the analysis of the reactions of DNA topoisomerases. However, gel electrophoresis is an intrinsically low-throughput technique and suffers from other potential disadvantages. We describe the application of the QIAxcel Advanced System, a high-throughput capillary electrophoresis system, to separate DNA topoisomers, and compare this technique with gel electrophoresis. RESULTS: We prepared a range of topoisomers of plasmids pBR322 and pUC19, and a 339 bp DNA minicircle, and compared their separation by gel electrophoresis and the QIAxcel System. We found superior resolution with the QIAxcel System, and that quantitative analysis of topoisomer distributions was straightforward. We show that the QIAxcel system has advantages in terms of speed, resolution and cost, and can be applied to DNA circles of various sizes. It can readily be adapted for use in compound screening against topoisomerase targets.

Effects of pesticides on occupationally exposed humans.

Pesticides are known to contain numerous genotoxic compounds; however, genotoxicity biomonitoring studies of workers occupationally exposed to pesticides have produced variable results. In this study, we employed the Comet assay to examine DNA damage in peripheral blood lymphocytes (PBLs) from 64 greenhouse workers from Almería in south-eastern Spain in comparison to PBLs from 50 men from the same area but not engaged in any agricultural work. The results indicated that there were no differences in the basal levels of DNA damage in the two study groups. In addition, exposure of PBL from the workers and controls to hydrogen peroxide or gamma-irradiation led to similar levels of DNA damage; the subsequent repair of the induced DNA damage was also similar for both study populations. Smoking had no impact on any of the responses. The results of this study indicate that the greenhouse workers had no detectable increase in DNA damage or alteration in the cellular response to DNA damage compared to our control population.

Probing the run-on oligomer of activated SgrAI bound to DNA.

SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single base substitution in either the 1st/8th or the 2nd/7th position of the primary recognition sequence. This modulation of enzyme activity via run-on oligomerization is a newly appreciated phenomenon that has been shown for a small but increasing number of enzymes. One outstanding question regarding the mechanistic model for SgrAI is whether or not the activating primary site DNA must be cleaved by SgrAI prior to inducing activation. Herein we show that an uncleavable primary site DNA containing a 3'-S-phosphorothiolate is in fact able to induce activation. In addition, we now show that cleavage of secondary site DNA can be activated to nearly the same degree as primary, provided a sufficient number of flanking base pairs are present. We also show differences in activation and cleavage of the two types of secondary site, and that effects of selected single site substitutions in SgrAI, as well as measured collisional cross-sections from previous work, are consistent with the cryo-electron microscopy model for the run-on activated oligomer of SgrAI bound to DNA.

Investigation of the genotoxic effect of pesticides on greenhouse workers' lymphocytes.

In the present study, the genotoxic effects of commonly applied pesticides were evaluated using the alkaline comet assay (pH > 13). The amount of DNA damage (% DNA in tail) in peripheral lymphocytes of 49 male agricultural workers from Southern Poland were measured and compared to 50 men from the same area who had no previous occupational exposure to pesticides. No statistically significant differences in basal DNA damage were found between the study groups. In addition, exposure of peripheral blood lymphocytes to hydrogen peroxide (100 and 150 microM) or gamma-irradiation (2.5 or 4.2 Gy) led to a similar degree of additional DNA damage and subsequent repair (for 2 hr) for all studied populations. In conclusion, our results indicate that the greenhouse workers who participated in this study had no detectable increased DNA damage or alteration in their cellular response to DNA damage in comparison to our control population.