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The 8-nitroguanine lesion in DNA is increasingly associated with inflammation-related carcinogenesis, whereas the same modification on guanosine 3',5'-cyclic monophosphate generates a second messenger in NO-mediated signal transduction. Very little is known about the chemistry of 8-nitroguanine nucleotides, despite the fact that their biological effects are closely linked to their chemical properties. To this end, a selection of chemical reactions have been performed on 8-nitroguanine nucleosides and oligodeoxynucleotides. Reactions with alkylating reagents reveal how the 8-nitro substituent affects the reactivity of the purine ring, by significantly decreasing the reactivity of the N2 position, whilst the relative reactivity at N1 appears to be enhanced. Interestingly, the displacement of the nitro group with thiols results in an efficient and specific method of labelling this lesion and is demonstrated in oligodeoxynucleotides. Additionally, the repair of this lesion is also shown to be a chemically feasible reaction through a reductive denitration with a hydride source.

Rapid and sensitive methods to detect DNA lesions are essential in order to understand their role in carcinogenesis and for potential diagnosis of cancers. The 8-nitroguanine DNA lesion, which is closely associated with inflammation-induced cancers, has been characterised for the first time by surface-enhanced Raman spectroscopy (SERS). This lesion has been studied as the free base, as well as part of a dinucleotide and oligodeoxynucleotides (ODNs) at 5 different excitation wavelengths in the range 785-488 nm. All nitrated samples produced distinctly different spectra from their control guanine counterparts, with nitro bands being assigned by DFT calculations. Additional resonance enhancement was observed at the shorter excitation wavelengths, these SERRS measurements allowed the detection of one nitrated guanine in over 1,300 bases. In addition, SER(R)S can be used to detect whether the unstable lesion is covalently attached to the ODN or has been released by hydrolytic depurination.

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group (‘sulfation’) from the enzyme co-factor 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer’s disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

The guanine base in DNA, due to its low oxidation potential, is particularly sensitive to chemical modifications. A large number of guanine lesions have been characterized and studied in some detail due to their relationship with tissue inflammations. Nevertheless, one example of these lesions is the formation of 8-nitro-guanosine, but the NMR data of this compound was only partially interpreted. A comprehensive study of the two possible tautomeric forms, through a detailed characterization of this compound, has implications for its base pairing properties. The target compound was obtained through a synthetic sequence of five steps, where all intermediates were fully characterized using spectral data. The analysis of the two tautomers was then evaluated through NMR spectroscopy and theoretical calculations of the chemical shifts and NH coupling constants, which were also compared with the data from guanosine.

Triplexes formed from oligonucleic acids are key to an umber of biological processes. They have attracted at- tention as molecular biology tools and as ar esult of their relevance in novel therapeutic strategies. The recognition properties of single-stranded nucleic acids are also relevant in third-strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson-Crick (W-C) base-paired DNA duplexes and aH oogs- teen base-paired RNA strand, are reported to be more ther- modynamically stable than those in which the third strand is DNA. Consequently ,s ynthetic efforts have been focused on developing TFOs with RNA-like structura lp roperties. Here, the structura la nd stability studies of such aT FO, composed of deoxynucleic acids, bu tw ith 3'-S-phosphorothiolate (3'- SP) linkages at two sites is described. The modification re- sults in an increase in triplex meltin gt emperature as deter- mined by UV absorption measurements. 1 HN MR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altere db yt he major groove binding of either TFO. However ,t he triplex involving the 3'- SP modifications is more compact. The 3'-SP modification was previously shown to stabilis eG -quadruplex and i-motif structures and therefore is now proposed as ag eneric solu- tion to stabilising multi-stranded DNA structures.

8-Nitro-2′-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2′-O-methylguanosine, a ribonucleoside analogue of this lesion, which is sufficiently stable to be incorporated into ODNs. Physicochemical studies demonstrated that 8-nitro-2′-O-methylguanosine adopts a syn conformation about the glycosidic bond; thermal melting studies and molecular modelling suggest a relatively stable syn-8-nitroG·anti-G base pair. Interestingly, when this lesion analogue was placed in a primer-template system, extension of the primer by either avian myeloblastosis virus reverse transcriptase (AMV-RT) or human DNA polymerase β (pol β), was significantly impaired, but where incorporation opposite 8-nitroguanine did occur, pol β showed a 2:1 preference to insert dA over dC, while AMV-RT incorporated predominantly dC. The fact that no 8-nitroG·G base pairing is seen in the primer extension products suggests that the polymerases may discriminate against this pairing system on the basis of its poor geometric match to a Watson–Crick pair.

Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA.

Artemisinin-acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2-4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle. (C) 2009 Elsevier Ltd. All rights reserved.

3 '-S-Phosphorothiolate linkages incorporated into an oligodeoxynucleotide have been shown to stabilise duplex formation with a complementary RNA strand, but destabilise a duplex formed with a complementary DNA strand. The four-stranded i-motif structure is also stabilised this modification.

The efficient digestion of nanoparticle assemblies connected by DNA linkers is demonstrated using a number of complementary methods (enzymatic cleavage with EcoRI is shown in the scheme). The efficiency of digestion is dependent on the design of the DNA linker. The ability to manipulate DNA enzymatically is important in the development of hierarchical chemical methods of nanoscale assembly.

Oligodeoxynucleotides containing 2?-C-branched nucleosides with an amide or nitrile appended to either a one or two carbon alkyl chain have been synthesised. The phosphoramidites of the 2?-C-modified nucleosides were prepared and incorporated into the oligonucleotides using automated DNA synthesis. The duplex stability with complementary RNA and DNA was measured by UV melting experiments, in order to assess whether the amide/nitrile function could induce any duplex stability without the presence of the 2?-oxygen. The duplex stabilities of the oligonucleotides containing the 2?-C-modifications were decreased in the absence of the 2?-oxygen.

NMR spectroscopy has been used to investigate the conformational effects of single and two consecutive 3'-S-phosphorothiolate modifications within a deoxythymidine trinucleotide. The presence of a single 3'-phosphorothioate modification shifts the conformation of the sugar ring it is attached to, from a mainly south to north pucker; this effect is also transmitted to the 3'-neighbour deoxyribose. This transmission is thought to be caused by favourable stacking of the heterocyclic bases. Similar observations have been made previously by this group. When two adjacent modifications are present, the conformations of the attached deoxyribose rings are again shifted almost completely to the north, however, there is no transmission to the 3' deoxyribose ring. Base proton chemical shift analysis and molecular modelling have been used to aid elucidation of the origin of this feature. The observation for the dimodified sequence is consistent with our previously reported results for a related system in which spaced modifications are more thermodynamically stable than consecutive ones.

A glyconucleoside containing a thioglycoside linkage, namely 1-(3- S -β- d -ribofuranosyl-2,3-dideoxy-3-thio-β- d -ribofuranosyl)-thymine, has been prepared through condensation of a suitably protected derivative of 3′-thiothymidine with an activated ribose sugar. NMR has been used to study the conformation of the S-disaccharide and the unmodified O-disaccharide. A full pseudorotational analysis showed that for the S-disaccharide, the ribose and deoxy ribose sugars have a preference for the south and north pucker, respectively; which is the reverse of what is seen for the O-disaccharide.

We report here on the fabrication of a three-dimensional array of nanoparticles which bridges the gap between lithographically defined gold electrode contacts separated by 20 nm. The nanoparticle assemblies are formed from about 5 nm gold nanoparticles and benzenedimethanethiol (BDMT) bridging ligands. These assemblies are introduced between the contacts using a layer-by-layer protocol with successive BDMT self-assembly being followed by nanoparticle adsorption until the gap is bridged. The temperature dependent electrical properties of these devices are analysed to establish whether they are consistent with the notion that the networks are built up from molecularly interlinked discrete gold nanoparticles. To aid this analysis the molecular conductance of single bridging molecules is also characterized at room temperature using a recently introduced method based on the scanning tunnelling microscope (STM). From these measurements it is concluded that the room temperature electrical properties of the nanostructured networks are limited by the small interparticle connectivity and the inherent resistance of the linker molecules.

An in-depth study into the incorporation of multiple 3′-S-phosphorothiolate modifications into oligodeoxynucleotides (ODNs) and their subsequent effect on ODN/DNA and ODN/RNA duplex stability. 3′-S-Phosphorothiolate linkages increase the stability of ODN/RNA duplexes and decrease the stability of ODN/DNA duplexes.

The synthesis of N4-benzoyl-5'-O-dimethoxytrityl-2',3'-dideoxy-3'-thiocytidine and its phosphorothioamidite is described for the first time, together with a shortened procedure for the preparation of 5'-O-dimethoxytrityl-3'-deoxy-3'-thiothymidine and its corresponding phosphorothioamidite. The first fully automated coupling procedure for the incorporation of a phosphorothioamidite into a synthetic oligodeoxynucleotide has been developed, which conveniently uses routine activators and reagents. Coupling yields using this protocol were in the range of 85-90% and good yields of singularly modified oligonucleotides were obtained. Coupling yields were also equally good when performed on either a 0.2 or 1 micro mol reaction column, thus facilitating large scale syntheses required for mechanistic studies.

High-resolution NMR spectroscopy has been used to establish the conformational consequences of the introduction of a single 3[prime or minute]-S-phosphorothiolate link in the DNA strand of a DNA : RNA hybrid. These systems are of interest as potential antisense therapeutic agents. Previous studies on similarly modified dinucleotides have shown that the conformation of the sugar to which the sulfur is attached shifts to the north (C(3[prime or minute])-endo/C(2[prime or minute])-exo). Comparisons made between NOESY cross-peak intensities, and coupling constants from PE-COSY spectra, for both non-modified and modified duplexes confirm that this conformational shift is also present in the double helical oligonucleotide system. In addition it is noted that in both the dinucleotides and the modified duplex, the conformation of the sugar ring 3[prime or minute] to the site of modification is also shifted to the north. That this pattern is observed in the small monomeric system as well as the larger double helix is suggestive of some pre-ordering of the sequences. The conclusion is supported by consideration of the (1)H chemical shifts of the heterocyclic bases near the site of the modification. The enhanced stability that these conformational changes should bring was confirmed by UV thermal melting studies. Subsequently a series of singly and doubly 3[prime or minute]-S-phosphorothiolate-modified duplexes were investigated by UV. The results are indicative of an additive effect of the modification with thermodynamic benefit being derived from alternate spacing of two modified linkers.

The 5'-triphosphates of 2'-hydroxymethyluridine (2'-homouridine) and 2'-hydroxyethyluridine were prepared from the corresponding acetyl-protected nucleosides by initial phosphitylation with 2-chloro-5,6-benzo-1,2,3-dioxaphosphorin-4-one. 2'-Acetamidouridine 5'-triphosphate was prepared in an analogous fashion from uridine 2'-C-, 3'-O-gamma-butyrolactone, in which the 3'-hydroxyl group is internally protected as the lactone. Subsequent treatment with ammonia gave the required acetamido triphosphate. All three triphosphates were investigated as substrates for T7 RNA polymerase and a Y639F mutant of this enzyme. 2'-Homouridine triphosphate was found to be a substrate for the wild-type enzyme in the presence of manganese and was specifically incorporated into short RNA transcripts (20 and 21 nucleotides in length). The presence of the analogue within the transcripts was confirmed through its resistance to alkaline hydrolysis. Gel electrophoretic analysis also showed that 2'-homouridine could be multiply incorporated into a transcript with a length of 75 nucleotides. This is the first report of a 2'-deoxy-2'-alpha-C-branched nucleoside 5'-triphosphate acting as a substrate for T7 RNA polymerase. The 2'-hydroxyethyl- and 2'-acetamido -uridine triphosphates were not substrates for the enzymes.

The programmed self-assembly of nanostructures from well-defined units is an important aim in nanoscience.[1], [2] The use of gold nanoparticles stabilized by thiol-modified DNA is a promising approach towards this goal.[3]-[8] The specificity of DNA base-pairing provides a precise means of programming interactions between particles by hybridization with specifically designed linker strands. To introduce an additional level of control, we have developed a general method by which the reactivity of initially latent DNA linking sites can be switched on deliberately. We have adapted well-developed methods of molecular biology to produce a nanoscale analogue of protecting groups. We show that linking sites can be protected by hybridization with complementary strands and deprotected by cleaving these double strands at predetermined sites with restriction enzymes. This results in cohesive ends of single-stranded DNA, which can bind by hybridization to complementary sequences present in the system. In a second enzymatic step the DNA phosphodiester backbones at the hybridization sites are covalently joined using a DNA ligase. This approach represents a generic protocol that will enable multistep nanostructure syntheses.

The ability of 2'-alpha-C-carboxyalkyl nucleosides to undergo an unusual two-step stereocontrolled nucleobase exchange process has been investigated. Upon silylation a protected 2'-deoxy-2'-alpha-C-(carboxymethyl)uridine derivative can undergo intramolecular displacement of the uracil base, by the 2'-carboxylic acid group, to form a pentofuranosyl gamma-lactone. Under identical conditions the homologous 2'-deoxy-2'-alpha-C-(carboxyethyl)uridine derivative does not yield the corresponding delta-lactone, but undergoes elimination of uracil to give the corresponding glycal. The pentofuranosyl gamma-lactone is a good substrate for nucleoside synthesis by the Vorbrüggen procedures and undergoes completely stereoselective ring opening with either pyrimidine or purine silylated nucleobases to give novel 2'-C-carboxymethyl beta-nucleosides in moderate to high yield.

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.

For the first time a fully automated procedure has been developed for the incorporation of a 3'-S-phosphorothiolate linkage into DNA, using phosphorothioamidite monomers. Coupling yields with either of the activators 5-ethylthiotetrazole or 4,5-dicyanoimidazole were in the range of 80-90%. Coupling yields were equally good when performed on either a 0.2 or 1 mumole reaction column, thus facilitating large scale synthesis.

The course of hydrolysis of 3′-deoxy-3′-thioinosylyl-(3′ → 5′)-uridine (IspU) has been followed by HPLC over a wide pH-range. Two reactions of the internucleosidic thiophosphate linkage compete: (i) cleavage yielding thioinosine monophosphates and uridine, and (ii) isomerization to the 2′,5′-isomer of IspU. Under very acidic conditions, even acid-catalyzed depurination of the inosine moiety is observed. The stability of the thiophosphate linkage and the mechanisms of its rupture are discussed.

Peptides of varying length (dimers to octamers) were prepared from nucleoside beta-amino acids and conformational studies, based on NOE observations, show that the beta-peptides form an unusual 8-helix.

2′-Deoxy-2′-α-C-carboxymethyl-3′-O-tert-butyldimethylsilyl-5′-O-dimethoxytrityluridine (11a) and the corresponding 3′-deoxy derivative 2′,3′-dideoxy-2′-α-C-carboxymethyl-5′-O-dimethoxytrityluridine (11b) have been condensed with 5′-amino-5′-deoxythymidine to prepare dinucleotide analogues (5a and 5b) which contain a 2′→5′-amide linkage. These dimer units have been incorporated into deoxynucleotide dodecamers using solid-phase phosphoramidite chemistry. Thermal melting studies show that a single 2′→5′-amide linkage in a deoxyoligonucleotide has a considerable destabilising effect on duplexes formed with both the DNA and RNA complementary sequences. Interestingly, the amide linkage has a significantly greater destabilising influence in the DNA duplexes than in the RNA hybrids.

An efficient route to 2′-homouridine ( 1 ), a new nucleoside analogue, is reported that is based on an ene reaction. This nucleoside has been incorporated into a dinucleoside monophosphate and hydrolytic studies on the dimer show that it does not behave like a ribonucleotide.

The synthesis of two dinucleoside monophosphate analogues is described in which the 3′-O-P-O-5′ phosphodiester linkage is replaced by either an isosteric 3′-C-S-S-5′ or a shorter 3′-S-S-5′ linkage. In both cases the -S-S- bond was formed through a disulfide exchange reaction using an activated S -nucleosidyl S -aryl disulfide and a suitably protected derivative of 5′-thiothymidine. © 1997 Elsevier Science Ltd.

Although only a subset of protein enzymes depend on the presence of a metal ion for their catalytic function, all naturally occurring RNA enzymes require metal ions to stabilize their structure and for catalytic competence. In the self-splicing group I intron from Tetrahymena thermophila, several divalent metals can serve structural roles, but only Mg2+ and Mn2+ promote splice-site cleavage and exon ligation. A study of a ribozyme reaction analogous to 5'-splice-site cleavage by guanosine uncovered the first metal ion with a definitive role in catalysis. Substitution of the 3'-oxygen of the leaving group with sulphur resulted in a metal-specificity switch, indicating an interaction between the leaving group and the metal ion. Here we use 3'-(thioinosylyl)-(3'-->5')-uridine, IspU, as a substrate in a reaction that emulates exon ligation. Activity requires the addition of a thiophilic metal ion (Cd2+ or Mn2+), providing evidence for stabilization of the leaving group by a metal ion in that step of splicing. Based on the principle of microscopic reversibility, this metal ion activates the nucleophilic 3'-hydroxyl of guanosine in the first step of splicing, supporting the model of a two-metal-ion active site.

Five 2′-C-functionalized nucleosides (1–5) have been prepared and incorporated into dinucleoside monophosphates. The effect of the functionality on the stability of the adjacent phosphodiester bond toward hydrolysis by nuclease enzymes and extremes of pH has been assessed.

Uridine 2′-C-3′-O-γ-butyrolactone 5a has been prepared by cyclisation of 2′-α-C-carboxymethyl-2′-deoxyuridine 8 in acetic acid–methanol. Detailed 1H NMR studies on compound 5a clearly demonstrate cis fusion between the γ-lactone and the sugar moiety, and determination of conformationally averaged structural descriptors, i.e. the pseudorotation phase angle and the puckering amplitude, suggests that the dominant conformer of the furanose ring is S. The 2′-C,3′-O-lactone 5a and its 5′-protected derivatives (5b and 5c) readily undergo aminolyses to yield a range of amide derivatives. Reaction with 5′-amino-5′-deoxythymidine gave a 2′–5′-amide-linked analogue of a dinucleoside monophosphate. Under acidic conditions the amides slowly relactonised. The 5′-protected lactones were also shown to react with simple organometallic reagents.

Four functionalized 2‘-deoxy-2‘-α-C-branched nucleosides, namely, 2‘-deoxy-2‘-α-C-(carboxymethyl)uridine, 2‘-deoxy-2‘-α-C-acetamidouridine, 2‘-deoxy-2‘-α-C-(hydroxyethyl)uridine, and 2‘-deoxy-2‘-α-...

A general procedure for the preparation of S -nucleosidyl S -aryl disulfides from the corresponding thioesters is described. This procedure has been used for the preparation of a ribonucleoside disulphide ( 8d ), a key intermediate for the synthesis of oligoribonucleotides containing 3′- S -phosphorothiolate linkages. The regioselectivity of the Arbusov reaction of 8d with phosphites has been examined. The X-ray structure of 3′-deoxy-3′- S -(2-nitrophenyldisulfanyl)thymidine ( 9b ) is also reported.

Artemisinin-based combination therapies (ACTs) are currently the recommended treatment for uncomplicated and severe cases of malaria.[1] Additionally, artemisinins, as well as a number of other sesquiterpene lactones (SLs), are currently in phase I–II clinical trials against breast, colorectal and nonsmall-cell lung cancers.[2] As outlined by the iron-dependent activation hypothesis,[3] the activity of artemisinin (ART) is dependent on the endoperoxide bridge.[4] The peroxide is cleaved by endogenous sources of FeII to generate highly reactive carbon-centred radicals (CCRs), which are believed to react with critical cellular targets.[3] ART demonstrates selectivity towards rapidly proliferating cancer cell lines that possess a high intracellular iron content required to sustain their characteristic high rates of multiplication.[5] Iron activation links this particular potency of ART towards rapidly proliferating cancer cell lines; differentiation between healthy and cancerous cells by variation of iron concentration provides a strategy for selective cytotoxicity by ART and its derivatives.[4] The mechanism by which ART exerts its cytotoxic activity still remains elusive. ART acts by disruption of proliferation,[6, 7] oxidative stress,[8] anti-angiogenesis,[9] NF-kB signalling,[10] apoptosis[4] and interfering with iron uptake and metabolism.[6] ART also induces DNA breakage,[11] and it has been reported that artesunate-mediated DNA damage contributes to its therapeutic efficacy.

The integration host factor (IHF) of Escherichia coli is a small, sequence-specific DNA-binding protein. The specific and nonspecific binding constants of IHF were estimated by gel-retardation assays. The equilibrium association constant of IHF for the H' site in lambda attP is 6.8 x 10(8) M-1 (Kd = 1.5 nM), and the nonspecific binding constant is 5.8 x 10(5) M-1 (Kd = 1.7 microM), giving a selectivity of approximately 1,000-fold for a specific site over random sequences. To study the molecular determinants specifying IHF binding, we used a series of 41 oligonucleotides containing adenine analogues that modified the surfaces of the major and minor grooves of the DNA. Many of the analogue substitutions within the previously defined consensus region caused decreased binding. Replacement with various analogues outside the consensus domain had little effect. Quantifying the binding constants for those sites with reduced affinities indicated an interaction with the minor groove within the consensus sequence. The binding constants of sites with 2-aminopurine and an inosine substitution within the same region suggest that IHF may also interact with the major groove. Thus, the specific interaction of IHF with its H' site likely involves interactions with both the minor and major grooves of the DNA.

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.

The 5′-O-monomethoxytrityl-3′-S-(aryldisulfanyl)-3′-deoxythymidines 7 and 8 have been prepared by the reaction of 5′-O-monomethoxytrityl-3′-thiothymidine with the appropriate arenesulfenyl chloride. These disulfides undergo a Michaelis–Arbusov reaction with simple trialkyl phosphites to yield 5′-O-monomethoxytrityl-3′-thiothymidin-3′-yl O,O-dialkyl phosphorothiolates. More interestingly, 3′-deoxy-3′-S-(2, 4-dinitrophenylsulfanyl)-5′-O-monomethoxytritylthymidine 8 reacts with a variety of thymidin-5′-yl dialkyl phosphites to give dithymidine phosphorothiolate triesters with the phosphorothiolate group protected with either a methyl or a 2-cyanoethyl group.3′-O-(tert-Butyldimethylsilyl)thymidin-5′-yl triethylammoniumphosphonate 17 is converted into the corresponding bis-(O-trimethylsilyl) phosphite by treatment with bis(trimethylsilyl)trifluoroacetamide. in situ Reaction of this phosphate with disulfide 8 gives, after work-up, the dithymidine phosphorothiolate diester directly. Methylation of compound 17 with methyl chloromethanoate, followed by silylation and subsequent reaction with disulfide 8, gives the methyl-protected dithymidine phosphorothiolate triester.

The 3'-S-phosphorothiolate (3'-SP) linkage has proven to be a very useful analogue of the phosphodiester group in nucleic acid derivatives; it is achiral and also shows good resistance to nucleases. Whilst oligonucleotides containing a 3'-SP linkage are best prepared using phosphoramidite chemistry, the corresponding dinucleotides are most efficiently synthesised using a Michaelis-Arbuzov reaction between a nucleoside 5'-phosphite and a nucleoside 3'-S-disulphide. The method described here is for a thymidine dinucleotide and is based on the use of a silyl phosphite, which is more reactive than simple alkyl phosphites and also simplifies the deprotection strategy. Full experimental details and spectroscopic data for the synthetic intermediates and the target dinucleotide are provided.

Since the first unequivocal description of RNA interference (RNAi) in 1998, it has remained one of the hottest topics under investigation, culminating in the award of a Nobel Prize to its discoverers in 2006. Excitement over this technique derives from the ease with which it can be used to switch-off a specific gene in almost any organism, thereby allowing the role of that gene to be identified. More importantly, it offers the potential to treat certain diseases by switching-off the causative genes. Key to the RNAi pathway are the small-interfering RNAs (siRNAs), which at 21–23 nucleotides in length are very amenable to analogue development by chemists. However in comparison to the use of oligonucleotides as antisense agents, an area where many chemists first developed an interest in nucleic acids, the RNAi pathway is exceedingly complex. The literature is also complicated by the fact that the phenomenon has been studied in a wide range of organisms. In this tutorial review we have presented the subject from a more chemical perspective, incorporating a glossary to give a clear explanation of the specialist terms. However, the coverage of the biology remains sufficiently detailed to give the reader the necessary insight that we believe will be essential for the successful design of chemically modified siRNA.

The 3'-S-phosphorothiolate (3'-SP) linkage has proven to be a very useful analogue of the phosphodiester group in nucleic acid derivatives; it is achiral and also shows good resistance to nucleases. Whilst oligonucleotides containing a 3'-SP linkage are best prepared using phosphoramidite chemistry, the corresponding dinucleotides are most efficiently synthesised using a Michaelis-Arbuzov reaction between a nucleoside 5'-phosphite and a nucleoside 3'-S-disulphide. The method described here is for a thymidine dinucleotide and is based on the use of a silyl phosphite, which is more reactive than simple alkyl phosphites and also simplifies the deprotection strategy. Full experimental details and spectroscopic data for the synthetic intermediates and the target dinucleotide are provided.

The synthesis of uridine monomers containing either a 2′-deoxy-2′-C-methy- lcyano or ethylcyano group is described. These monomers are intended for incorporation into oligonucleotides to investigate a proposed duplex-stabilising effect exerted by 2′-tethered amide groups.

The incorporation of 6-N-(2-aminoethyl)-2′-deoxyadenosine 1 into 6-N-(2-aminoethyl)-2′-deoxyadenylyl-(3′-5′)-thymidine 2 using a phosphoramidite intermediate is described. The trifluoroacetyl group is demonstrated to be an ideal protecting group for the amino function whilst the 4,4′-dimethoxytrityl group is utilised to block the 5′-hydroxy function. This study serves as a model investigation for the incorporation of compound 1 into oligodeoxynucleotides to investigate their potential for forming sequency specific interstrand cross-links.

2′-Deoxy-3′-thio-5′-O-(4-monomethoxytrityl)-6-N-benzoyladenosine ( 1a ) has been prepared from adenosine in seven steps and, following conversion of to a 3′-S-phosphorothioamidite, used to synthesize a dinucleoside 3′-S-phosphorothiolate containing 2′-deoxy-3′-thioadenosine. The procedure is compatible with automated methods of DNA synthesis and extends the potential of the recently developed phosphorothiolate method for strand specific cleavage of DNA.

Efficient procedures are described for the synthesis of dinucleoside phosphorothiolates using either a Michaelis-Arbusov-type reaction or a phosphotriester approach.

An intramolecular Michaelis–Arbusov reaction taking place between a 5′-phosphite and a 3′-S-disulfide can be used to prepare nucleoside 3′-S,5′-O-cyclic phosphorothiolate triesters.