Studies on the attachment of DNA to silica-coated nanoparticles through a Diels-Alder reaction.

A new method has been investigated for the functionalization of gold nanoparticles with DNA. Silica-coated nanoparticles functionalized with a maleimide have been prepared. These particles are designed to react with modified DNA containing a diene functionality at one end of the molecule. The result would be the formation of a more stable attachment of the DNA to the particle through a Diels-Alder reaction. This covalent attachment would not be susceptible to ligand exchanges, which are known to occur in the conventional DNA functionalization of gold nanoparticles.

Synthesis of the flavour precursor, alliin, in garlic tissue cultures.

The path of synthesis of alkyl cysteine sulphoxides, or flavour precursors, in the Alliums is still speculative. There are two proposed routes for alliin biosynthesis, one is from serine and allyl thiol while the other is from glutathione and an allyl source via gamma glutamyl peptides. The routes have been investigated by exposing undifferentiated callus cultures of garlic and onion to potential pathway intermediates. After a period of incubation of 2 days the callus was extracted, and analysed for flavour precursors and related compounds by HPLC. Standards of alliin, isoallin and propiin were synthesised and their identity confirmed by HPLC and NMR. Putative intermediates selected included the amino acids serine and cysteine, as well as more complex intermediates such as allylthiol, allyl cysteine and glutathione. Both garlic and onion tissue cultures were able to synthesize alliin following incubation with allylthiol, and cysteine conjugates such as allyl cysteine. The ability of the tissue cultures to form alliin from intermediates was compatible with the proposed routes of synthesis of alliin.

The synthesis of, and studies on, 2'-C-modified nucleotides.

The synthesis of uridine monomers containing either a 2'-deoxy-2'-C-methylcyano 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.

Novel macrocycles derived from nucleosides.

A novel nucleoside-derived macrocycle formed from three thymidine 5'-carboxylic acid monomer units is described.

Solid-phase synthesis of oligodeoxynucleotides containing 3'-S-phosphorothiolate linkages.

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.

A second catalytic metal ion in group I ribozyme.

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.

The RuvC protein dimer resolves Holliday junctions by a dual incision mechanism that involves base-specific contacts.

The Escherichia coli RuvC protein resolves DNA intermediates produced during genetic recombination. In vitro, RuvC binds specifically to Holliday junctions and resolves them by the introduction of nicks into two strands of like polarity. In contrast to junction recognition, which occurs without regard for DNA sequence, resolution occurs preferentially at sequences that exhibit the consensus 5'-(A/T)TT/(G/C)-3' (where / indicates the site of incision). Synthetic Holliday junctions containing modified cleavage sequences have been used to investigate the mechanism of cleavage. The results indicate that specific DNA sequences are required for the correct docking of DNA into the two active sites of the RuvC dimer. In addition, using chemically modified oligonucleotides to introduce a hydrolysis-resistant 3'-S-phosphorothiolate linkage at the cleavage site, it was found that, as long as the sequence requirements are fulfilled, the two incisions could be uncoupled from each other. These results indicate that RuvC protein resolves Holliday junctions by a mechanism similar to that exhibited by certain restriction enzymes.

The specific binding of Escherichia coli integration host factor involves both major and minor grooves of DNA.

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.

Thermodynamic stability and drug-binding properties of oligodeoxyribonucleotide duplexes containing 3-deazaadenine:thymine base pairs.

We have used ultraviolet melting techniques to study the effect on stability of incorporating the nucleoside analogue 2'-deoxy-3-deazaadenosine (d3cA) into the duplex 5'-d(CGCAATCG)-3'-d(GCGTTAGC). Our results demonstrate that the successive replacement of dA by d3CA increasingly destabilises the duplex. The destabilising effect of this analogue is considerably enhanced as the pH is lowered and the results are consistent with protonation of 3-deazaadenine (probably at N-1) contributing to duplex destablisation. Surprisingly, the incorporation of d3CA does not significantly affect the binding of distamycin-A.

Sequence- and strand-specific cleavage in oligodeoxyribonucleotides and DNA containing 3'-thiothymidine.

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-diisopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.

Incorporation of a complete set of deoxyadenosine and thymidine analogues suitable for the study of protein nucleic acid interactions into oligodeoxynucleotides. Application to the EcoRV restriction endonuclease and modification methylase.

A complete set of dA and T analogues designed for the study of protein DNA interactions has been prepared. These modified bases have been designed by considering the groups on the dA and T bases that are accessible to proteins when these bases are incorporated into double-helical B-DNA [Seeman, N. C., Rosenberg, J. M., & Rich, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 804-808]. Each of the positions on the two bases, having the potential to interact with proteins, have been subject to nondisruptive, conservative change. Typically a particular group (e.g., the 6-NH2 of dA or the 5-CH3 of T) has been replaced with a hydrogen atom. Occasionally keto groups (the 2- and 4-keto oxygen atoms of T) have been replaced with sulfur. The base set has been incorporated into the self-complementary dodecamer d(GACGATATCGTC) at the central d(ATAT) sequence. Melting temperature determination shows that the modified bases do not destabilize the double helix. Additionally, circular dichroism spectroscopy shows that almost all the altered bases have very little effect on overall oligodeoxynucleotide conformation and that most of the modified oligomers have a B-DNA type structure. d(GATATC) is the recognition sequence for the EcoRV restriction modification system. Initial rate measurements (at a single oligodeoxynucleotide concentration of 20 microM) have been carried out with both the EcoRV restriction endonuclease and modification methylase. This has enabled a preliminary identification of the groups of the dA and T bases within the d(GATATC) sequence that make important contacts to both proteins.

Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC).

A set of dA and T analogues suitable for the study of protein DNA interactions have been incorporated into the central d(ATAT) sequence within d(GACGATATCGTC). The individual analogues have one potential protein contact (either a hydrogen-bonding group or a CH3 group capable of a van der Waals interaction) deleted. In general, the modified bases do not perturb the overall structure of the dodecamer, enabling results obtained to be simply interpreted in terms of loss of protein DNA contacts. We have used the modified oligodeoxynucleotide set to study the recognition of DNA by the EcoRV restriction endonuclease [recognition sequence d(GATATC)]. The kcat and Km values for the set have been determined, and a comparison with results seen with the parent oligodeoxynucleotide (containing no modified bases) has been carried out. Three classes of results are seen. First, some analogues lead to no change in kinetic parameters, meaning no enzyme contact at the altered site. Second (this is seen for most of the modified oligodeoxynucleotides), a drop in the kcat/Km ratio relative to the parent is observed. This comes mainly from a decrease in kcat, implying that the endonuclease uses the interaction under study to lower the transition-state barrier rather than to bind the substrate. Analyses of these results show that the drop in kcat/Km is what would be expected for the simple loss of a hydrogen bond or a CH3 contact between the enzyme and the oligodeoxynucleotide. This implies a contact of these types at these sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular recognition in the minor groove of the DNA helix. Studies on the synthesis of oligonucleotides and polynucleotides containing 3-deaza-2'-deoxyadenosine. Interaction of the oligonucleotides with the restriction endonuclease EcoRV.

An improved procedure for the preparation of 3-deaza-2'-deoxyadenosine (d3CA) is described which is suitable for the synthesis of gram quantities of this analogue. Using phosphoramidite chemistry d3CA has been incorporated into the Eco RV restiction endonuclease recognition sequence (underlined) present in the self-complementary dodecamer d(GACGATATCGTC). The modified oligonucleotides have been thoroughly characterised by nucleoside composition analysis, circular dichroism and thermal melting studies. Studies with Eco RV show that incorporation of d3CA into either the central or outer dA-dT base-pair results in a substantial reduction in the rate of cleavage. The two-step conversion of d3CA to 3-deaza-2'-deoxyadenosine-5'-O-triphosphate (d3CATP) via the 5'-O-tosylate is also described. d3CATP is not a substrate in the poly[d(AT)].poly[d(AT)] primed polymerisation for either E. coli DNA polymerase I or Micrococcus luteus DNA polymerase. In a more detailed kinetic analysis d3CATP was shown to be a competitive inhibitor of E. coli DNA polymerase I with respect to dATP.

Synthesis and properties of dithymidine phosphate analogues containing 3'-thiothymidine.

Dithymidine-3'-S-phosphorothioate (d(TspT)) has been prepared from a 5'-O-monomethoxytritylthymidine-3'-S-phosphorothioamidite (7) by activation with 5-(p-nitrophenyl)tetrazole in the presence of 3'-O-acetylthymidine. The resulting dinucleoside phosphorothioite is readily oxidised to the corresponding 3'-S-phosphorothioate using either tetrabutylammonium (TBA) periodate or TBA oxone and has been deprotected under standard conditions to yield d(TspT). This dithymidine phosphate analogue is comparatively resistant to hydrolysis by nuclease P1, but the P-S bond is readily cleaved by aqueous solutions of either iodine or silver nitrate. Dithymidine-3'-S-phosphorodithioate (d[Tsp(s)T]) was prepared in an analogous fashion using sulphur to oxidise the intermediate dinucleoside phosphorothioite. Absolute stereochemistry has been assigned to the diastereoisomers of d[Tsp(s)T] by comparing their physical and chemical properties to those of the dinucleoside phosphorothioates.

Aplastic anaemia associated with organochlorine pesticide: case reports and review of evidence.

Three patients with aplastic anaemia had a history of substantial previous exposure to organochlorine pesticides. The temporal association between chemical exposure and the onset of first symptoms of anaemia was strongly supportive. Organochlorines have the property of lipid affinity and accumulation in adipose tissue. Objective evidence of clinically important concentrations of tissue pesticide residues may be a useful confirmation of previous exposure. In the patients studied the presence of Lindane (gamma hexachlorocyclohexane) was shown using gas chromatography/mass spectrometry with selective ion monitoring of fragments obtained from one heavily exposed patient, with concentrations about five times greater than a matched control. The presence of clinically important tissue concentrations of pentachlorophenol was also confirmed in a second patient exposed to this agent. The long term safety of organochlorine pesticides remains doubtful as they were introduced before adequate toxicological screening tests had been developed. The central registration of possible haematological adverse reactions, however, forms an important epidemiological method in the study of environmental chemical hazards and should be complied with whenever possible.

An approach to the stereoselective synthesis of Sp-dinucleoside phosphorothioates using phosphotriester chemistry.

An approach to the stereoselective synthesis of Sp- dinucleoside phosphorothioates has been investigated which utilizes phosphotriester chemistry. The stereoselectivity of internucleotide bond formation between N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(S2-cyano-e thyl) phosphorothioate (3) and 3'-O-acetylthymidine has been studied using either mesitylenesulphonyl-5-(pyridin-2-yl)tetrazole (MSPy) or 1-mesitylenesulphonyl-3-nitro-1,2,4-triazole (MSNT) as the activating agent. The removal of the cyanoethyl group from the protected dinucleoside phosphorothioate has been studied, and conditions are reported which provide rapid deprotection without concomittant desulphurisation.

Chiral phosphorothioate analogues of B-DNA. The crystal structure of Rp-d[Gp(S)CpGp(S)CpGp(S)C].

The compound Rp-d[Gp(S)CpGp(S)CpGp(S)C], an analogue of the deoxyoligomer d(G-C)3, crystallizes in space group P2(1)2(1)2(1) with a = 34.90 A, b = 39.15 A and c = 20.64 A. The structure, which is not isomorphous with any previously determined deoxyoligonucleotide, was refined to an R factor of 14.5% at a resolution of 2.17 A, with 72 solvent molecules located. The two strands of the asymmetric unit form a right-handed double helix, which is a new example of a B-DNA conformation and brings to light an important and overlooked component of flexibility of the double helix. This flexibility is manifest in the alternation of the backbone conformation between two states, defined by the adjacent torsion angles epsilon and zeta, trans . gauche-(BI) and gauche-. trans (BII). BI is characteristic of classical of B-DNA and has an average C(1') to C(1') separation of 4.5 A. The corresponding separation for BII is 5.3 A. Each state is associated with a distinct phosphate orientation where the plane of the PO2 (or POS) group is alternately near horizontal or vertical with respect to the helix axis. The BI and BII conformations are out of phase on the two strands. As a consequence, on one strand purine-pyrimidine stacking is better than pyrimidine-purine, while the converse holds for the other strand. At each base-pair step, good and bad stacking alternate across the helix axis. The pattern of alternation is regular in the context of a fundamental dinucleotide repeat. Re-examination of the B-DNA dodecamer d(C-G-C-G-A-A-T-T-C-G-C-G) shows that the C-G-C-G regions contain the BI and BII conformations, and the associated dual phosphate orientation and asymmetric base stacking. Different mechanisms are used in the two structures to avoid clashes between guanine residues on opposite strands, a combination of lateral slide, tilt and helical twist in the present structure, and base roll, tilt and longitudinal slide (Calladine rules) in the dodecamer. The flexibility of the phosphate orientations demonstrated in this structure is important, since it offers a structural basis for protein-nucleic acid recognition.